TWI413647B - Novel h5 proteins, nucleic acid molecules and vectors encoding for those, and their medicinal use - Google Patents
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本發明係關於醫藥領域,較佳係關於傳染病領域。特定言之,本發明係關於流感蛋白;編碼彼等蛋白質之核酸分子及載體;及疫苗。更特定言之,本發明係關於該等蛋白質、核酸分子、載體或疫苗中之任一者用於治療及預防流感感染,進一步用於預防流感病毒之種內及種間傳染的用途。The present invention relates to the field of medicine, and more preferably to the field of infectious diseases. In particular, the present invention relates to influenza proteins; nucleic acid molecules and vectors encoding the same; and vaccines. More specifically, the present invention relates to the use of any of such proteins, nucleic acid molecules, vectors or vaccines for the treatment and prevention of influenza infections, and further for the prevention of intra- and interspecies infection of influenza viruses.
流感感染仍然為動物及人類之重要感染。流感係由經歷連續抗原性變異/修飾且佔據動物宿主的病毒引起。因此,未來可能發生新的流行病及廣泛性流行病,且難以實現該疾病之根除。流感病毒已熟知於此項技術且詳述於(例如)可供進一步參考之P.Palese,Nature Medicine第10卷,第12期,第S 82至S 86頁(2004年12月) 中。簡而言之,流感A病毒之基因組係由八個單股區段組成,且病毒顆粒在其表面上具有兩種主要醣蛋白:血球凝集素(H)及神經胺糖酸苷酶(N)。在流感病毒之間因存在至少16種不同的血球凝集素(H1至H16)及9種不同的神經胺糖酸苷酶(N1至N9)亞型而存在大量的抗原性變異。Influenza infection remains an important infection for animals and humans. Influenza is caused by a virus that undergoes continuous antigenic variation/modification and occupies an animal host. Therefore, new epidemics and pandemics may occur in the future, and it is difficult to eradicate the disease. Influenza viruses are well known in the art and are described, for example, in P. Palese, Nature Medicine Vol. 10, No. 12, pp. S 82-S 86 (December 2004) , which is hereby incorporated by reference. Briefly, the influenza A virus genome consists of eight single-stranded segments, and the viral particles have two major glycoproteins on their surface: hemagglutinin (H) and neuraminidase (N) . There are a large number of antigenic variations between influenza viruses due to the presence of at least 16 different hemagglutinin (H1 to H16) and 9 different neuraminidase (N1 to N9) subtypes.
已證明H5N1型禽流感病毒之流感病毒可感染禽類、豬及人類。該等病毒亦可自禽類物種直接傳染給人類(Claas等人,Lancet 1998,351:472;Suarez等人,J.Virol.1998,72:6678;Subbarao等人,Science 1998,279:393;Shortridge,Vaccine 1999,17(增刊1):S26-S29 )。已知人類臨床病例之死亡率接近約50%。Influenza viruses of the H5N1 avian influenza virus have been shown to infect poultry, pigs and humans. These viruses can also be transmitted directly from human species to avian species ( Claas et al, Lancet 1998, 351:472; Suarez et al, J. Virol. 1998, 72: 6678; Subbarao et al, Science 1998, 279: 393; Shortridge , Vaccine 1999, 17 (suppl. 1): S26-S29 ). The mortality rate in human clinical cases is known to be close to about 50%.
上一個世紀,豬一直為流感廣泛性流行病之重要媒介。豬、駱駝及海豹(以豬為較佳)可充當禽流感病毒之'混合室'且因此代表一種越過禽類(流感病毒之天然宿主)至哺乳動物之物種障礙之潛在風險因素。此通常發生為易感性動物(例如豬)為已確定哺乳動物(豬)流感病毒與禽流感病毒雙重感染。此雙重感染可形成可導致人類或豬廣泛性流行病的新重組病毒。然而,最近有跡象表明,當前禽類H5病毒株與哺乳動物流感病毒之重組不會形成強毒性重組體。另一方面,禽流感病毒可感染豬且藉由自發突變而變得順應於豬。一旦病毒在豬(或其他哺乳動物)群體內引起橫向感染,則將越過臨界障礙。In the last century, pigs have been an important medium for the pandemic of influenza. Pigs, camels and seals (preferably pigs) can act as a 'mixing chamber' for avian influenza viruses and thus represent a potential risk factor for species barriers that cross the avian (the natural host of influenza virus) to mammals. This usually occurs as a susceptible animal (eg, a pig) that has been identified as a dual infection of a mammalian (porcine) influenza virus and an avian influenza virus. This dual infection can form a new recombinant virus that can lead to a widespread epidemic in humans or pigs. However, there have been recent indications that the current recombination of avian H5 strains with mammalian influenza viruses does not form highly toxic recombinants. On the other hand, the avian influenza virus can infect pigs and become compliant with pigs by spontaneous mutation. Once the virus causes a lateral infection within the swine (or other mammalian) population, the critical disorder will be crossed.
然而,東南亞豬大部分已經來源於鄰近家禽飼養業之禽(H5)流感病毒株感染。由於彼等感染迄今為止尚為亞臨床性,因此其僅可藉由實驗室方法診斷而由此常常被忽視。存在以下高風險:彼等受亞臨床性感染之豬為病毒順應於哺乳動物系統、在豬群體內傳播以及感染人類提供了機會。However, most of the Southeast Asian pigs have been infected with avian (H5) influenza virus strains adjacent to the poultry industry. Since their infections have so far been subclinical, they can only be diagnosed by laboratory methods and are often overlooked. There is a high risk that their subclinically infected pigs provide an opportunity for the virus to conform to mammalian systems, spread within the swine population, and infect humans.
當前流感疫苗包括次單位疫苗(Babai等人,Vaccine 1999,17(9-10):1223-1238;Crawford等人,Vaccine 1999,17(18):2265-2274;Johansson等人,Vaccine 1999,17(15-16):2073-2080 )、減毒疫苗(Horimoto等人,Vaccine 2004,22(17-18):2244-2247 )、DNA疫苗(Watabe等人,Vaccine 2001,19(31):4434-4444 )及滅活流感疫苗(Cao等人,Vaccine 1992,10(4):238-242 ),其中滅活流感疫苗以商業規模最廣泛使用(Lipatov等人,J Virol 2004,78(17):8951-8959 )。Current influenza vaccines include subunit vaccines ( Babai et al, Vaccine 1999, 17(9-10): 1223-1238; Crawford et al, Vaccine 1999, 17(18): 2265-2274; Johansson et al, Vaccine 1999, 17 (15-16): 2073-2080 ), attenuated vaccine ( Horimoto et al., Vaccine 2004 , 22(17-18): 2244-2247 ), DNA vaccine ( Wabebe et al., Vaccine 2001, 19(31): 4434 -4444 ) and inactivated influenza vaccine ( Cao et al., Vaccine 1992, 10(4): 238-242 ), in which inactivated influenza vaccines are most widely used on a commercial scale ( Lipatov et al, J Virol 2004, 78(17) :8951-8959 ).
次單位疫苗、重組性血球凝集素及神經胺糖酸苷酶(Babai等人,Vaccine 1999,17(9-10):1223-1238;Crawford等人,Vaccine 1999,17(18):2265-2274;Johansson等人,Vaccine 1999,17(15-16):2073-2080 )可為頗受關注的滅活疫苗替代物,儘管目前尚未作為商用疫苗投入使用。該等疫苗之製備顯然比滅活疫苗安全。此外,次單位疫苗並不對內部流感病毒蛋白產生抗體反應且從而使經接種之動物與已感染動物之間有區別。(Crawford等人,Vaccine 1999,17(18):2265-2274 )。 Subunit vaccine, recombinant hemagglutinin and neuraminidase ( Babai et al, Vaccine 1999, 17(9-10): 1223-1238; Crawford et al, Vaccine 1999, 17(18): 2265-2274 Johansson et al., Vaccine 1999, 17(15-16): 2073-2080 ) may be a highly inactivated vaccine replacement, although not currently available as a commercial vaccine. The preparation of such vaccines is clearly safer than inactivated vaccines. In addition, the subunit vaccine does not produce an antibody response to the internal influenza virus protein and thus distinguishes between the vaccinated animal and the infected animal. ( Crawford et al., Vaccine 1999, 17(18): 2265-2274 ).
血球凝集素蛋白為流感病毒之受體結合及膜融合醣蛋白以及用於感染性中和抗體之標靶。H5N1之完整血球凝集素蛋白(HA)係由568個胺基酸組成,分子量為56 kDa。HA分子係由HA1及HA2次單位組成,HA1次單位介導與細胞膜之初始接觸而HA2負責膜融合(Chizmadzhev,Bioelectrochemistry 2004,63(1-2):129-136 )。Hemagglutinin proteins are receptor bindings for influenza viruses and membrane fusion glycoproteins as well as targets for infectious neutralizing antibodies. H5N1's intact hemagglutinin protein (HA) consists of 568 amino acids with a molecular weight of 56 kDa. The HA molecule consists of HA1 and HA2 subunits, HA1 subunits mediate initial contact with cell membranes and HA2 is responsible for membrane fusion ( Chizmadzhev, Bioelectrochemistry 2004, 63(1-2): 129-136 ).
桿狀病毒/昆蟲細胞系統已用於表現自禽流感亞型分離之血球凝集素基因(Babai等人,Vaccine 1999,17(9-10):1223-1238;Crawford等人,Vaccine 1999,17(18):2265-2274;Johansson等人,Vaccine 1999,17(15-16):2073-2080);New等人,BMC Mircobiology 2006,6(16):doi:10.1186/1471-2180-6-16 )。然而,彼等重組蛋白在某些情況下似乎不具有保護性,或者僅在最低程度上對某些物種具有較低有效性(Treanor等人,Vaccine 2001,19:1732-1737)。Baculovirus/insect cell systems have been used to express hemagglutinin genes isolated from avian influenza subtypes ( Babai et al, Vaccine 1999, 17(9-10): 1223-1238; Crawford et al, Vaccine 1999, 17 ( 18): 2265-2274; Johansson et al., Vaccine 1999, 17(15-16): 2073-2080); New et al., BMC Mircobiology 2006, 6(16): doi: 10.1186/1471-2180-6-16 ). However, their recombinant proteins do not appear to be protective in some cases or, to a minimum, have low efficacy for certain species (Treanor et al, Vaccine 2001, 19: 1732-1737).
因此,需要增強經改良之疫苗及新疫苗接種方法之可用性以提供控制流感感染的更佳方法及對疾病負荷產生正面影響。Therefore, there is a need to enhance the availability of improved vaccines and new vaccination methods to provide better methods of controlling influenza infection and have a positive impact on disease burden.
在本發明之實施例之前,應瞭解,如本文中及隨附申請專利範圍中所使用,單數形式"一"及"該"包括複數提及物,除非上下文另有明確說明。因此,舉例而言,提及"一種製劑"包括複數種該等製劑;提及該"載劑"係指熟習此項技術者已知之一或多種載劑及其均等物,諸如此類。除非另外定義,否則本文中所使用之所有技術及科學術語具有的含義與普通熟習本發明所屬技術者通常所瞭解之含義相同。除非另外指明或熟習此項技術者另外得知,否則所有給定範圍及值可變化1至5%,因此術語"約"在本說明書中省去。儘管可在本發明之實施或測試中使用與本文中所述之彼等方法及物質相似或相當的任何方法及物質,但較佳之方法、裝置及物質現加以描述。為描述並揭示可配合本發明使用之如公開案中所報導之物質、賦形劑、載劑及方法之目的,本文中所提及之所有公開案以引用的方式併入本文中。不應認為本文中認可本發明無權優先於先前發明之此類揭示案。The singular forms "a", "the", and "the" Thus, for example, reference to "a formulation" includes a plurality of such agents; reference to the "carrier" refers to one or more carriers and their equivalents known to those skilled in the art, and the like. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All ranges and values may vary from 1 to 5%, unless otherwise indicated or known to those skilled in the art, so the term "about" is omitted in this specification. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices and materials are now described. To the extent that the materials, excipients, carriers, and methods disclosed in the disclosure are used in conjunction with the present invention, all of the disclosures herein are incorporated by reference. It is not to be considered as an admission that the invention is entitled
藉由本說明及申請專利範圍中之特徵性實施例獲得上述技術問題之解決方法。The solution to the above technical problem is obtained by the present description and the characteristic embodiments in the scope of the patent application.
本發明係關於流感病毒之H5蛋白,其中該H5蛋白具有胺基酸223N及修飾體328K+,其中H5蛋白之胺基酸位置編號係指如SEQ ID NO:1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置328上插入第二離胺酸(K+)。較佳地,本發明之該H5蛋白及其他任何H5蛋白為經分離之H5蛋白。已驚人地發現,與在位置223及328/329上不具有相應胺基酸的H5蛋白相比,具有上述修飾體之H5蛋白具有更高的抗原性。The present invention relates to an H5 protein of influenza virus, wherein the H5 protein has an amino acid 223N and a modification 328K+, wherein the amino acid position number of the H5 protein refers to an amino acid as exemplified in SEQ ID NO: 1. Position and wherein the modification 328K+ means insertion of a second lysine (K+) at the amino acid position 328 of the H5 protein. Preferably, the H5 protein of the present invention and any other H5 protein are isolated H5 proteins. It has been surprisingly found that the H5 protein having the above modifications has higher antigenicity than the H5 protein which does not have the corresponding amino acid at positions 223 and 328/329.
如本文中所使用之術語"血球凝集素5(H5)"或"禽流感病毒之H5"或"H5蛋白"意謂(但不限於)任何天然存在之H5蛋白及H5蛋白之任何經修飾形式(包括H5蛋白之任何缺失、取代及/或插入突變體),其中彼等H5蛋白具有胺基酸223N及修飾體328K+。The term "hemagglutinin 5 (H5)" or "H5 protein of avian influenza virus" or "H5 protein" as used herein means, but is not limited to, any naturally occurring H5 protein and any modified form of H5 protein. (including any deletions, substitutions, and/or insertion mutants of the H5 protein) wherein the H5 proteins have an amino acid 223N and a modification 328K+.
如本文中所使用之H5蛋白之胺基酸位置之編號係指如SEQ ID NO:1中所例示性指定之胺基酸位置。SEQ ID NO:1代表病毒株鴨/China/E319-2/03之血球凝集素之胺基序列(但缺少胺基末端信號肽)。換而言之,若提及位置223上之胺基酸(胺基酸223),則意謂與SEQ ID NO:1中之胺基酸223對應的胺基酸殘基。然而,此並非意謂本發明之H5蛋白具有與SEQ ID NO:1一致的胺基酸序列。其僅意謂本發明之H5蛋白之對應胺基酸編碼所明確提及之胺基酸殘基。在此情況下,胺基酸223為絲胺酸(S)。術語"223N"或"155N"分別例示性意謂,處於位置223及155(根據SEQ ID NO:1之胺基酸位置編號)上之胺基酸可編碼胺基酸天冬醯胺酸(N)。換而言之,若提及"具有胺基酸223N之H5蛋白",則H5胺基酸分子通常在胺基酸位置223(根據SEQ ID NO:1之胺基酸位置編號)上編碼的胺基酸絲胺酸可經天冬醯胺酸(N)取代。術語"328K+"或"修飾體328K+"意謂,在H5蛋白之胺基酸位置328(根據SEQ ID NO:1之胺基酸位置編號)上插入第二離胺酸(K+)。在位置328及329上天然編碼離胺酸-離胺酸之胺基酸序列之情況下,未插入其他離胺酸(K)。然而,大多數已知H5序列在胺基酸位置328及329上編碼離胺酸-精胺酸。在該等任何情況下,術語328K+修飾體意謂,第二離胺酸(K)應插入位置328上之離胺酸與位置329上之精胺酸之間。於是經修飾之序列應讀成離胺酸-離胺酸-精胺酸(KKR)。The numbering of the amino acid position of the H5 protein as used herein refers to the amino acid position as exemplified in SEQ ID NO: 1. SEQ ID NO: 1 represents the amino sequence of the hemagglutinin of the virus strain Duck/China/E319-2/03 (but lacks the amino terminal signal peptide). In other words, referring to the amino acid (amino acid 223) at position 223, it means an amino acid residue corresponding to the amino acid 223 of SEQ ID NO: 1. However, this does not mean that the H5 protein of the present invention has an amino acid sequence identical to SEQ ID NO: 1. It simply means that the corresponding amino acid of the H5 protein of the present invention encodes an amino acid residue specifically mentioned. In this case, the amino acid 223 is a serine acid (S). The term "223N" or "155N", respectively, exemplarily means that the amino acid at positions 223 and 155 (based on the amino acid position number of SEQ ID NO: 1) encodes the amino acid aspartic acid (N). ). In other words, if "H5 protein with amino acid 223N" is mentioned, the H5 amino acid molecule is typically an amine encoded at amino acid position 223 (numbered according to the amino acid position of SEQ ID NO: 1). The base acid serine can be substituted with aspartic acid (N). The term "328K+" or "modification 328K+" means the insertion of a second lysine (K+) at the amino acid position 328 of the H5 protein (numbering according to the amino acid position of SEQ ID NO: 1). In the case where the amino acid sequence of the lysine-deaminase was naturally encoded at positions 328 and 329, no other lysine (K) was inserted. However, most known H5 sequences encode a lysine-arginine at amino acid positions 328 and 329. In any of these cases, the term 328K+ modification means that the second quaternary acid (K) should be inserted between the lysine at position 328 and the arginine at position 329. The modified sequence should then be read as the amine acid-lysine-arginine (KKR).
因此,本發明係關於H5蛋白及H5蛋白之任何經修飾形式(包括H5蛋白之任何缺失、取代及/或插入突變體),其中彼等H5蛋白具有胺基酸223N及修飾體328K+,其中H5蛋白之胺基酸位置編號係指如SEQ ID NO:1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置328上插入第二離胺酸(K+)。不言而喻,如本文中所提供之任一種H5蛋白具有抗原性,此意謂其在對流感病毒之標準血球凝集素抑制檢定中展示抗原特性。Accordingly, the present invention relates to any modified form of the H5 protein and the H5 protein (including any deletion, substitution and/or insertion mutant of the H5 protein), wherein the H5 protein has an amino acid 223N and a modification 328K+, wherein H5 The amino acid position number of the protein refers to the amino acid position as exemplified in SEQ ID NO: 1 and wherein the modification 328K+ means the insertion of a second lysine at the amino acid position 328 of the H5 protein (K+ ). It goes without saying that any of the H5 proteins as provided herein is antigenic, which means that it exhibits antigenic properties in a standard hemagglutinin inhibition assay for influenza viruses.
根據另一實施例,本發明亦係關於H5蛋白之任何部分,此部分意謂在標準血球凝集素抑制檢定中展示抗原特性,至少具有胺基酸223N及修飾體328K+的任何肽片段,其中H5蛋白之胺基酸位置之編號係指如SEQ ID NO:1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置328上插入第二離胺酸(K+)。According to another embodiment, the invention is also directed to any portion of the H5 protein, which portion is intended to exhibit antigenic properties in a standard hemagglutinin inhibition assay, at least any peptide fragment having amino acid 223N and modification 328K+, wherein H5 The numbering of the amino acid position of the protein refers to the amino acid position as exemplified in SEQ ID NO: 1 and wherein the modification 328K+ means the insertion of a second lysine at the amino acid position 328 of the H5 protein ( K+).
若H5蛋白在標準血球凝集素抑制檢定(例如,如實例2中所述)中抑制血球凝集,則其展示抗原特性。H5蛋白之該抗原部分通常包含編碼如上所述之經修飾或未經修飾,在如實例2中所述之標準血球凝集素抑制檢定中展示抗原特性之H5蛋白的胺基酸序列中之200、180、160、150、140、130、120、110或最佳105個毗鄰胺基酸。標準血球凝集素抑制檢定例如亦描述於可供進一步參考之Stephenson等人,Virus Research,第103卷,第91-95頁(2004)中。然而,應瞭解,如實例2中所述之HI檢定為配合本文中所述之本發明之所有態樣的相關參考檢定:簡而言之,進行HI檢定以偵測HA特異性抗體之存在。異源H5N2病毒(A/雞/Mexico/232/94)係以四個血球凝集單位[4 HA單位]之濃度用於HI檢定中。隨後在U形底微量滴定板中,將PBS中之連續兩倍血清稀釋液與等體積(25 μL)(含有4 HA單位)之病毒混合,且在室溫(約25℃)下培育30分鐘。將PBS中之濃度為0.5%之雞紅血球添加至含有血清-病毒之孔中且在室溫下培育40分鐘。以觀測到血球凝集抑制的最高血清稀釋度之倒數確定HI效價。If the H5 protein inhibits hemagglutination in a standard hemagglutinin inhibition assay (eg, as described in Example 2), it exhibits antigenic properties. The antigenic portion of the H5 protein typically comprises 200 of the amino acid sequence encoding the H5 protein that exhibits antigenic properties in a standard hemagglutinin inhibition assay as described in Example 2, modified or unmodified as described above. 180, 160, 150, 140, 130, 120, 110 or optimally 105 adjacent amino acids. Standard hemagglutinin inhibition assays are also described, for example, in Stephenson et al., Virus Research, Vol. 103, pp. 91-95 (2004), which is incorporated by reference. However, it will be appreciated that the HI assay as described in Example 2 is a relevant reference assay that cooperates with all aspects of the invention described herein: Briefly, a HI assay is performed to detect the presence of HA-specific antibodies. Heterologous H5N2 virus (A/chicken/Mexico/232/94) was used in the HI assay at a concentration of four hemagglutination units [4 HA units]. Subsequently, serial two-fold serum dilutions in PBS were mixed with an equal volume (25 μL) of virus containing 4 HA units in a U-bottom microtiter plate and incubated for 30 minutes at room temperature (about 25 ° C). . Chicken red blood cells at a concentration of 0.5% in PBS were added to the wells containing serum-virus and incubated for 40 minutes at room temperature. The HI titer was determined by the reciprocal of the highest serum dilution at which hemagglutination inhibition was observed.
值得注意的是,Haesebrouck及Pensaert(1986) 發現"針對激發病毒之HI效價與防禦激發之保護之間可能存在關聯"。Haesebrouck及Pensaert(1986) 亦測定,HI效價>40的豬"完全抵抗激發且在受激發下呼吸道中不發生病毒複製"。因此,在經疫苗接種之豬中形成>40之HI效價將與保護作用有關。(F.Haesebrouck及M.B.Pensaert,1986 )。預先用滅活流感H1N1疫苗免疫之肥育豬之氣管內激發的效應(Veterinary Microbiology ,11(1986)239-249)。須假設等值或至少差不多等值的H5 HI效價亦將對豬產生防禦禽流感病毒的完全免疫保護。較低效價至少引起經接種之動物之血清轉化且產生對彼等動物之部分免疫保護,此亦可大大降低廣泛性流行病之風險。It is worth noting that Haesebrouck and Pensaert (1986) found that "there may be a correlation between the HI titer that triggers the virus and the protection of the defense-inspired." Haesebrouck and Pensaert (1986) also determined that pigs with an HI titer >40 "are completely resistant to challenge and do not replicate in the respiratory tract under challenge". Therefore, the formation of a HI titer of >40 in vaccinated pigs will be associated with protection. ( F.Haesebrouck and MBPensaert, 1986 ). The effect of intratracheal challenge in pigs that were previously immunized with the inactivated influenza H1N1 vaccine ( Veterinary Microbiology , 11 (1986) 239-249). It should be assumed that an equivalent or at least approximately equivalent H5 HI titer will also provide complete immune protection against the avian influenza virus in pigs. Lower potency results in at least seroconversion of the vaccinated animals and produces partial immunoprotection to their animals, which can also greatly reduce the risk of a pandemic.
此外,本發明之H5蛋白之抗原部分包括(但不限於)H5蛋白之缺失突變體,其包含:i.圍繞且包括胺基酸223N之胺基酸序列中之至少35、30、25、20、18、15、13、10、9或最佳8個毗鄰胺基酸;及ii.圍繞且包括胺基酸修飾體328K+之胺基酸序列中之至少35、30、25、20、18、15、13、10、9或最佳8個毗鄰胺基酸;且iii.其中H5蛋白之該抗原部分中之任一者可在如實例2中所述之標準血球凝集素抑制檢定中展示血球凝集素抑制。Furthermore, the antigenic portion of the H5 protein of the invention includes, but is not limited to, a deletion mutant of the H5 protein comprising: i. at least 35, 30, 25, 20 of the amino acid sequence surrounding and comprising the amino acid 223N , 18, 15, 13, 10, 9 or preferably 8 adjacent amino acids; and ii. at least 35, 30, 25, 20, 18 of the amino acid sequence surrounding and including the amino acid modification 328K+, 15, 13, 10, 9 or preferably 8 adjacent amino acids; and iii. wherein any of the antigenic portions of the H5 protein can display blood cells in a standard hemagglutinin inhibition assay as described in Example 2. Lectin inhibition.
較佳地,圍繞胺基酸223N及/或328K+之彼等胺基酸係由SEQ ID NO:1或SEQ ID NO:4編碼。Preferably, the amino acids surrounding the amino acids 223N and/or 328K+ are encoded by SEQ ID NO: 1 or SEQ ID NO: 4.
此外,本發明之較佳H5蛋白為:i.上述具有胺基酸223N及修飾體328K+的彼等H5蛋白中之任一者;ii.上述具有胺基酸94N/223N及修飾體328K+的彼等H5蛋白中之任一者;iii.具有胺基酸223N及修飾體328K+的任何禽源H5蛋白,其中禽源意謂H5序列來源於最初自感染上5型禽流感病毒之禽類分離的病毒分離株;或iv.具有胺基酸94N/223N及修飾體328K+的任何禽源H5蛋白,其中禽源意謂H5序列來源於最初自感染上5型禽流感病毒之禽類分離的病毒分離株;或v.具有胺基酸155N/223N及修飾體328K+的任何禽源H5蛋白,其中禽源意謂H5序列來源於最初自感染上5型禽流感病毒之禽類分離的病毒分離株;或vi.具有胺基酸120N/155N/223N及修飾體328K+的任何禽源H5蛋白,其中禽源意謂H5序列來源於最初自感染上5型禽流感病毒之禽類分離的病毒分離株;或vii.具有修飾體94N/223N及修飾體328K+的任何H5蛋白;或viii.具有修飾體94N/155N/223N及修飾體328K+的任何H5蛋白;或;ix.具有修飾體94N/120N/155N/223N及修飾體328K+的任何H5蛋白;或x.具有修飾體223N、修飾體328K+及選自由以下各者組成之群的以下胺基酸團中之一或多者的任何H5蛋白:a. aa 93-95:GNF b. aa 123-125:SDH c. aa 128-130:SSG d. aa 138-140:GSS e. aa 226-228:MDF f. aa 270-272:EVE g. aa 309-311:NKL;或xi.具有胺基酸223N及修飾體328K+及選自由以下各者組成之群的以下胺基酸團中之一或多者的任何H5蛋白:a. aa 93-95:GNF b. aa 128-130:SSG c. aa 138-140:GSS;或xii.具有SEQ ID NO:4之胺基酸序列的任何H5蛋白。Further, the preferred H5 protein of the present invention is: i. any of the above H5 proteins having the amino acid 223N and the modified 328K+; ii. the above having the amino acid 94N/223N and the modified 328K+ Any of the H5 proteins; iii. any avian H5 protein having an amino acid 223N and a modification 328K+, wherein the avian source means that the H5 sequence is derived from a virus originally isolated from avian infected with avian influenza virus type 5 An isolate; or iv. any avian H5 protein having amino acid 94N/223N and a modification 328K+, wherein the avian source means that the H5 sequence is derived from a virus isolate originally isolated from avian infected with avian influenza virus type 5; Or v. any avian H5 protein having amino acid 155N/223N and modified 328K+, wherein the avian source means that the H5 sequence is derived from a virus isolate originally isolated from avian infected with avian influenza virus type 5; or vi. Any avian H5 protein having amino acid 120N/155N/223N and a modified form 328K+, wherein the avian source means that the H5 sequence is derived from a virus isolate originally isolated from avian infected with avian influenza virus type 5; or vii. Any H5 egg with modified 94N/223N and modified 328K+ Or viii. any H5 protein having the modification 94N/155N/223N and the modification 328K+; or; ix. any H5 protein having the modification 94N/120N/155N/223N and the modification 328K+; or x. having a modification 223N, a modified form 328K+, and any H5 protein selected from one or more of the following amino acid groups of the group consisting of: a. aa 93-95: GNF b. aa 123-125: SDH c. aa 128-130: SSG d. aa 138-140: GSS e. aa 226-228: MDF f. aa 270-272: EVE g. aa 309-311: NKL; or xi. with amino acid 223N and modified 328K+ And any H5 protein selected from one or more of the following amino acid groups of the group consisting of: a. aa 93-95: GNF b. aa 128-130: SSG c. aa 138-140: GSS Or xii. Any H5 protein having the amino acid sequence of SEQ ID NO: 4.
此外,如本文中所提供之較佳H5蛋白包括以下文獻所述之H5蛋白:Hoffmann等人,PNAS,第106卷,第36期,第12915-12920頁(2005年9月6日) ,其中彼等H5蛋白包括如上所述之修飾體中之一或多者,至少包括胺基酸223N及修飾體328K+,其中H5蛋白之胺基酸位置之編號係指如SEQ ID NO:1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置328上插入第二離胺酸(K+)。該參考文獻之揭示內容將以引用方式全文併入本文中。Furthermore, preferred H5 proteins as provided herein include the H5 proteins described in the following literature: Hoffmann et al, PNAS, Vol. 106, No. 36, pp. 12915-12920 (September 6, 2005) , wherein The H5 proteins include one or more of the modifications described above, including at least the amino acid 223N and the modification 328K+, wherein the amino acid position of the H5 protein is numbered as exemplified in SEQ ID NO: 1. The amino acid position of the sex designation and wherein the modification 328K+ means the insertion of a second lysine (K+) at the amino acid position 328 of the H5 protein. The disclosure of this reference is hereby incorporated by reference in its entirety.
此外,如本文中所提供之較佳H5蛋白包括包含含有胺基酸223N及修飾體328K+之肽的H5蛋白,其中H5蛋白之胺基酸位置之編號係指如SEQ ID NO:1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置328上插入第二離胺酸(K+),及:i. SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5或SEQ ID NO:6之胺基酸序列;或ii.具有與i)之多肽之至少85%序列同源性、更佳至少約90%序列同源性、甚至更佳至少約95%序列同源性、甚至更佳至少約97%序列同源性、甚至更佳至少約98%序列同源性且甚至更佳至少約99%序列同源性並在如上所述之標準血球凝集素抑制檢定中展示血球凝集素抑制的任何肽;或iii. i)或ii)之多肽之任何抗原部分,其包含i)或ii)之肽中之任一者的至少35、30、25、20、18、15、13、10、9或最佳8個毗鄰胺基酸。Furthermore, a preferred H5 protein as provided herein includes an H5 protein comprising a peptide comprising an amino acid 223N and a modification 328K+, wherein the numbering of the amino acid position of the H5 protein is as exemplified in SEQ ID NO: 1. a designated amino acid position and wherein the modification 328K+ means insertion of a second lysine (K+) at the amino acid position 328 of the H5 protein, and: i. SEQ ID NO: 1, SEQ ID NO: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 6; or ii. having at least 85% sequence homology to the polypeptide of i), more preferably at least About 90% sequence homology, even more preferably at least about 95% sequence homology, even more preferably at least about 97% sequence homology, even more preferably at least about 98% sequence homology and even more preferably at least about 99 % sequence homology and any peptide exhibiting hemagglutinin inhibition in a standard hemagglutinin inhibition assay as described above; or any antigenic portion of the polypeptide of iii. i) or ii) comprising i) or ii) At least 35, 30, 25, 20, 18, 15, 13, 10, 9 or preferably 8 adjacent amino acids of any of the peptides.
iv. i)、ii)或iii)之任何肽,其具有胺基酸36T、36K、83A、83T、83D、86A、86V、120N、120S、155N、155S、156A、156T、189R、189K、212K、212R、212E、223N、223N或120N/155N。Iv. any of the peptides of i), ii) or iii) having amino acids 36T, 36K, 83A, 83T, 83D, 86A, 86V, 120N, 120S, 155N, 155S, 156A, 156T, 189R, 189K, 212K , 212R, 212E, 223N, 223N or 120N/155N.
v. i)、ii)、iii)或iv)之任何肽,其具有選自由以下各者組成之群的以下胺基酸團中之一或多者:a. aa 93-95:GNF b. aa 123-125:SDH c. aa 128-130:SSG d. aa 138-140:GSS e. aa 226-228:MDF f. aa 270-272:EVE g. aa 309-311:NKL;或vi. i)、ii)、iii)或iv)之任何肽,其具有選自由以下各者組成之群的以下胺基酸團中之一或多者:a. aa 93-95:GNF b. aa 128-130:SSG c. aa 138-140:GSS。v. Any of the peptides of i), ii), iii) or iv) having one or more of the following amino acid groups selected from the group consisting of: a. aa 93-95: GNF b. Aa 123-125: SDH c. aa 128-130: SSG d. aa 138-140: GSS e. aa 226-228: MDF f. aa 270-272: EVE g. aa 309-311: NKL; or vi. Any of i), ii), iii) or iv) having one or more of the following amino acid groups selected from the group consisting of: a. aa 93-95: GNF b. aa 128 -130: SSG c. aa 138-140: GSS.
如本文中所使用之"序列同源性"係指測定兩個序列之相關性的方法。為測定序列同源性,將兩個或兩個以上的序列最佳對齊,且必要時引入空位(gap)。與序列一致性形成對比,在測定序列同源性時,保守性胺基酸取代視為匹配。換而言之,為獲得具有與參考序列之95%序列同源性的多肽或多核苷酸,參考序列中85%、較佳90%、甚至更佳95%之胺基酸殘基或核苷酸必須與其他胺基酸或核苷酸匹配或包含其他胺基酸或核苷酸之保守性取代,或者可將參考序列中全部胺基酸殘基或核苷酸(不包括保守性取代)之至多15%、較佳至多10%、甚至更佳至多5%量的胺基酸或核苷酸插入參考序列中。較佳地,同源序列包含至少一段50個核苷酸、甚至更佳100個核苷酸、甚至更佳250個核苷酸、甚至更佳500個核苷酸。此對齊後,逐位確定序列同源性,例如,若核苷酸或胺基酸殘基在特定位置處一致,則序列在彼位置上"同源"。接著將該等位置一致之總數除以參考序列中核苷酸或胺基酸殘基之總數以得到序列同源性%。序列同源性可容易地藉由已知方法計算,該等方法包括(但不限於)以下文獻中所述之彼等方法:Computational Molecular Biology,Lesk,A.N.編,Oxford University Press,New York(1988),Biocomputing:Informatics and Genome Projects,Smith,D.W.編,Academic Press,New York(1993);Computer Analysis of Sequence Data,第I部分,Griffin,A.M.,及Griffin,H.G.編,Humana Press,New Jersey(1994);Sequence Analysis in Molecular Biology,von Heinge,G.,Academic Press (1987);Sequence Analysis Primer,Gribskov,M.及Devereux,J.編,M.Stockton Press,New York(1991);及Carillo,H.及Lipman,D.,SIAM J.Applied Math.,48:1073(1988),該等文獻之教示內容以引用方式併入本文中。設計序列同源性之較佳測定方法以使所測試之序列之間達成最大匹配。序列同源性測定方法可編程為可公開獲得之測定指定序列之間的序列一致性的電腦程式。該等程式之實例包括(但不限於)GCG程式套件(Devereux,J.等人,Nucleic Acids Research,12(1):387(1984))、BLASTP、BLASTN及FASTA(Altschul,S.F.等人,J.Molec.Biol.,215:403-410(1990))。BLASTX程式可公開獲自NCBI及其他來源(BLAST Manual,Altschul,S.等人,NCVI NLM NIH Bethesda,MD 20894,Altschul,S.F.等人,J.Molec.Biol.,215:403-410(1990),該等文獻之教示內容以引用方式併入本文中)。該等程式利用預設空位權重使序列最佳對齊,以使指定序列與參考序列之間達成最高水準的序列同源性。"Sequence homology" as used herein refers to a method of determining the correlation of two sequences. To determine sequence homology, two or more sequences are optimally aligned and, if necessary, introduced into a gap. In contrast to sequence identity, conservative amino acid substitutions are considered to be matched when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology to a reference sequence, 85%, preferably 90%, or even more preferably 95% of the amino acid residues or nucleosides in the reference sequence The acid must be compatible with other amino acids or nucleotides or contain conservative substitutions of other amino acids or nucleotides, or all amino acid residues or nucleotides in the reference sequence (excluding conservative substitutions) Up to 15%, preferably up to 10%, even more preferably up to 5% of the amount of amino acid or nucleotide is inserted into the reference sequence. Preferably, the homologous sequence comprises at least a stretch of 50 nucleotides, even more preferably 100 nucleotides, even more preferably 250 nucleotides, even more preferably 500 nucleotides. After this alignment, sequence homology is determined bit by bit, for example, if the nucleotide or amino acid residues are identical at a particular position, the sequence is "homologous" at that position. The number of identical positions is then divided by the total number of nucleotide or amino acid residues in the reference sequence to obtain % sequence homology. Sequence homology can be readily calculated by known methods including, but not limited to, those described in the following literature: Computational Molecular Biology, Lesk, AN, ed., Oxford University Press, New York (1988). Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, ed., Humana Press, New Jersey (1994) ;; Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J. ed., M. Stockton Press, New York (1991); and Carillo, H And Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the teachings of which are incorporated herein by reference. A preferred assay for sequence homology is designed to achieve a maximum match between the sequences tested. The sequence homology determination method can be programmed as a publicly available computer program for determining sequence identity between specified sequences. Examples of such programs include, but are not limited to, the GCG program suite (Devereux, J. et al, Nucleic Acids Research, 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, SF et al, J .Molec. Biol., 215:403-410 (1990)). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, SF et al, J. Molec. Biol., 215: 403-410 (1990). The teachings of these documents are incorporated herein by reference. The programs use the preset gap weights to optimally align the sequences to achieve the highest level of sequence homology between the specified sequence and the reference sequence.
此外,較佳之H5蛋白包括包含如上所述之328K+修飾體及表1中所提供之胺基酸序列的H5蛋白或其任何免疫原部分:
此外,本發明亦係關於至少具有胺基酸223N及修飾體328K+的H5蛋白,其中H5蛋白之胺基酸位置之編號係指如SEQ ID NO:1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置328上插入第二離胺酸(K+),且包含:i.具有以下NCBI寄存編號之序列的肽:AAT65209、CAJ32556、ABC47656、CAF21874、CAF21870、AAC58998、AAC58997、AAC58996、AAC58994、AAC58993、AAC58992、AAC58991、AAC58990、AAC58995、AAS45134、AAN17270、AAN17269、AAN17268、AAN17267、AAN17266、AAN17265、AAN17264、AAN17263、AAN17262、AAN17261、AAN17260、AAN17259、AAN17257、AAN17256、AAN17255、AAN17254、AAA43083、AAA43082、AAB19079、BAE48696、BAE48693、BAE48696、BAE48695、BAE48694、BAE48692、BAE48691、BAE48690、BAE48689、BAE48688、BAE48687、BAE48686、BAE48685、BAE48684、BAE48683、AAC58999、ABC72082、AAV91149、AAP71993、AAP71992、AAP71991、AAP71990、AAP71989、AAP72011、AAP72010、AAP72009、AAP72008、AAP72007、AAP72006、AAP72005、AAP72004、AAP72003、AAP72002、AAP72001、AAP72000、AAP71999、AAP71998、AAP71997、AAP71996、AAP71995、AAP71994、AAF99718、ABF58847、AAG38534、AAC32102、AAC32099、AAL75847、AAC32101、AAC32098、AAC32088、AAC32078、AAR99628、AAC32100、AAM49555、AAL75843、AAL75839、AAD13573、AAD13568、AAF04720、AAF04719、AAC34263、AAR16155、AAD13574、AAD13570、AAD13575、AAD13572、AAD13569、AAD13567、AAD13566、AAK57506、AAG01225、AAG01215、AAG01205、AAG01195或ABD83813,該等序列可以上述方式修飾,此意謂彼等序列包括上述不為野生型序列之部分的修飾體223N及328K+;或ii.具有與i)之多肽之至少85%序列同源性、更佳至少約90%序列同源性、甚至更佳至少約95%序列同源性、甚至更佳至少約97%序列同源性、甚至更佳至少約98%序列同源性且甚至更佳至少約99%序列同源性且在如上所述之標準血球凝集素抑制檢定中展示血球凝集素抑制的任何肽;iii. i)或ii)之肽中之任一者,其具有胺基酸36T、36K、83A、83T、83D、86A、86V、120N、120S、155N、155S、156A、156T、189R、189K、212K、212R、212E、263A、263T或120N/155N;或iv. i)、ii)或iii)之該等肽中之任一者,其具有選自由以下各者組成之群的以下胺基酸團中之一或多者:a. aa 93-95:GNF b. aa 123-125:SDH c. aa 128-130:SSG d. aa 138-140:GSS e. aa 226-228:MDF f. aa 270-272:EVE g. aa 309-311:NKL;或v. i)、ii)、iii)或iv)之任何肽,其具有選自由以下各者組成之群的以下胺基酸團中之一或多者:a. aa 93-95:GNF b. aa 128-130:SSG c. aa 138-140:GSS。Furthermore, the present invention is also directed to an H5 protein having at least an amino acid 223N and a modification 328K+, wherein the numbering of the amino acid position of the H5 protein refers to the amino acid position as exemplified in SEQ ID NO: 1 and Wherein the modification 328K+ means inserting a second lysine (K+) at the amino acid position 328 of the H5 protein and comprising: i. a peptide having the sequence of the following NCBI accession number: AAT65209, CAJ32556, ABC47656, CAF21874, CAF21870 , AAC58998, AAC58997, AAC58996, AAC58994, AAC58993, AAC58992, AAC58991, AAC58990, AAC58995, AAS45134, AAN17270, AAN17269, AAN17268, AAN17267, AAN17266, AAN17265, AAN17264, AAN17263, AAN17262, AAN17261, AAN17260, AAN17259, AAN17257, AAN17256, AAN17255 , AAN17254, AAA43083, AAA43082, AAB19079, BAE48696, BAE48693, BAE48696, BAE48695, BAE48694, BAE48692, BAE48691, BAE48690, BAE48689, BAE48688, BAE48687, BAE48686, BAE48685, BAE48684, BAE48683, AAC58999, ABC72082, AAV91149, AAP71993, AAP71992, AAP71991 , AAP71990, AAP71989, AAP72011, AAP72010, AAP72 009, AAP72008, AAP72007, AAP72006, AAP72005, AAP72004, AAP72003, AAP72002, AAP72001, AAP72000, AAP71999, AAP71998, AAP71997, AAP71996, AAP71995, AAP71994, AAF99718, ABF58847, AAG38534, AAC32102, AAC32099, AAL75847, AAC32101, AAC32098, AAC32088, AAC32078, AAR99628, AAC32100, AAM49555, AAL75843, AAL75839, AAD13573, AAD13568, AAF04720, AAF04719, AAC34263, AAR16155, AAD13574, AAD13570, AAD13575, AAD13572, AAD13569, AAD13567, AAD13566, AAK57506, AAG01225, AAG01215, AAG01205, AAG01195 or ABD83813, Such sequences may be modified as described above, which means that the sequences include the above-described modifications 223N and 328K+ which are not part of the wild type sequence; or ii. have at least 85% sequence homology with the polypeptide of i), preferably At least about 90% sequence homology, even more preferably at least about 95% sequence homology, even more preferably at least about 97% sequence homology, even more preferably at least about 98% sequence homology and even more preferably at least about 99% sequence homology and display blood in a standard hemagglutinin inhibition assay as described above Any of the peptides inhibited by lectin; iii. any of the peptides of i) or ii) having amino acids 36T, 36K, 83A, 83T, 83D, 86A, 86V, 120N, 120S, 155N, 155S, 156A Any of the peptides of 156T, 189R, 189K, 212K, 212R, 212E, 263A, 263T or 120N/155N; or iv. i), ii) or iii) having a composition selected from the group consisting of One or more of the following amino acid groups: a. aa 93-95: GNF b. aa 123-125: SDH c. aa 128-130: SSG d. aa 138-140: GSS e. aa 226-228: MDF f. aa 270-272: EVE g. aa 309-311: NKL; or v. i), ii), iii) or iv) any peptide having a group selected from the group consisting of One or more of the following amino acid groups: a. aa 93-95: GNF b. aa 128-130: SSG c. aa 138-140: GSS.
根據另一實施例,本發明亦係關於編碼上述H5蛋白中之任一者的核酸分子。較佳地,彼等核酸分子為RNA、DNA或複本(c)DNA分子。因此,本發明係關於核酸分子,較佳編碼H5蛋白及H5蛋白之任何修飾形式(包括H5蛋白之任何缺失、取代及/或插入突變體)的cDNA分子,其中彼等H5蛋白具有胺基酸223N及修飾體328K+,其中H5蛋白之胺基酸位置編號係指如SEQ ID NO:1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置328上插入第二離胺酸(K+)。According to another embodiment, the invention is also directed to a nucleic acid molecule encoding any of the above H5 proteins. Preferably, the nucleic acid molecules are RNA, DNA or a replica (c) DNA molecule. Accordingly, the present invention relates to a nucleic acid molecule, preferably a cDNA molecule encoding any modified form of the H5 protein and the H5 protein, including any deletion, substitution and/or insertion mutant of the H5 protein, wherein the H5 protein has an amino acid. 223N and Modification 328K+, wherein the amino acid position number of the H5 protein refers to the amino acid position as exemplified in SEQ ID NO: 1 and wherein the modification 328K+ means at the amino acid position 328 of the H5 protein Insert the second lysine (K+).
根據另一實施例,本發明亦係關於核酸分子,較佳編碼H5蛋白之任何部分的cDNA分子,其意謂編碼在上述標準血球凝集素抑制檢定中展示抗原特性且至少具有胺基酸223N及修飾體328K+之任何肽片段的cDNA分子,其中H5蛋白之胺基酸位置之編號係指如SEQ ID NO:1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置328上插入第二離胺酸(K+)。通常,編碼H5蛋白之抗原部分的該等核酸分子包含編碼上述經修飾或未經修飾且在如本文中所述之標準血球凝集素抑制檢定中展示抗原特性之H5蛋白的核苷酸序列中之600、540、480、450、420、390、360、330或最佳315個毗鄰核苷酸。According to another embodiment, the invention also relates to a nucleic acid molecule, preferably a cDNA molecule encoding any portion of the H5 protein, which means encoding an antigenic property in the above-described standard hemagglutinin inhibition assay and having at least an amino acid 223N and A cDNA molecule of any peptide fragment of modified 328K+, wherein the numbering of the amino acid position of the H5 protein refers to the amino acid position as exemplified in SEQ ID NO: 1 and wherein the modification 328K+ means the H5 protein A second lysine (K+) is inserted at the amino acid position 328. Typically, the nucleic acid molecules encoding the antigenic portion of the H5 protein comprise a nucleotide sequence encoding an H5 protein as described above which is modified or unmodified and exhibits antigenic characteristics in a standard hemagglutinin inhibition assay as described herein. 600, 540, 480, 450, 420, 390, 360, 330 or optimal 315 contiguous nucleotides.
H5蛋白之抗原部分之其他實施例在上文中加以描述。建構任何該等核酸分子(較佳編碼上述H5蛋白之抗原部分的cDNA分子)已為熟習此項技術者所共知。此亦包括(但不限於)建構編碼上述H5蛋白之抗原部分(包括H5蛋白之缺失突變體)的核酸分子(較佳cDNA分子),其包含:i.圍繞且包括編碼胺基酸223N之編碼序列的核苷酸序列中之至少105、90、75、60、48、45、39、30、27或最佳24個毗鄰胺基核苷酸;及ii.圍繞且包括編碼修飾體328K+之編碼序列的核苷酸序列中之至少105、90、75、60、48、45、39、30、27或最佳24個毗鄰胺基核苷酸;且iii.其中H5蛋白之該抗原部分中之任一者在如實例2中所述之標準血球凝集素抑制檢定中展示血球凝集素抑制。Other examples of antigenic portions of the H5 protein are described above. The construction of any of these nucleic acid molecules, preferably cDNA molecules encoding the antigenic portion of the above H5 protein, is well known to those skilled in the art. This also includes, but is not limited to, construction of a nucleic acid molecule (preferably a cDNA molecule) encoding an antigenic portion of the above H5 protein, including a deletion mutant of the H5 protein, comprising: i. encoding surrounding and including the encoding amino acid 223N At least 105, 90, 75, 60, 48, 45, 39, 30, 27 or optimal 24 contiguous amino nucleotides in the nucleotide sequence of the sequence; and ii. coding surrounding and including the coding modification 328K+ At least 105, 90, 75, 60, 48, 45, 39, 30, 27 or optimal 24 contiguous amino nucleotides in the nucleotide sequence of the sequence; and iii. wherein the antigen portion of the H5 protein Either showed hemagglutinin inhibition in a standard hemagglutinin inhibition assay as described in Example 2.
較佳地,圍繞編碼胺基酸223N及/或328K+之核苷酸的彼等核苷酸編碼SEQ ID NO:1或SEQ ID NO:4。Preferably, the nucleotides encoding nucleotides 223N and/or 328K+ of the amino acid encode SEQ ID NO: 1 or SEQ ID NO: 4.
此外,本發明之編碼H5蛋白之較佳核酸分子為:i.上述編碼胺基酸223N及修飾體328K+的彼等核酸分子中之任一者;ii.上述編碼胺基酸94N/223N及修飾體328K+的彼等核酸分子中之任一者;iii.編碼胺基酸223N及修飾體328K+的任何禽源核酸分子,其中禽源意謂H5序列來源於最初自感染上5型禽流感病毒之禽類分離的病毒分離株;或iv.編碼胺基酸94N/223N及修飾體328K+的任何禽源核酸分子,其中禽源意謂H5序列來源於最初自感染上5型禽流感病毒之禽類分離的病毒分離株;或v.編碼胺基酸155N/223N及修飾體328K+的任何禽源核酸分子,其中禽源意謂H5序列來源於最初自感染上5型禽流感病毒之禽類分離的病毒分離株;或vi.編碼具有胺基酸120N/155N/223N及修飾體328K+之禽源H5蛋白的任何核酸分子,其中禽源意謂H5序列來源於最初自感染上5型禽流感病毒之禽類分離的病毒分離株;或vii.編碼具有修飾體94N/223N及修飾體328K+之H5蛋白的任何核酸分子;或viii.編碼具有修飾體94N/155N/223N及修飾體328K+之H5蛋白的任何核酸分子;或ix.編碼具有修飾體94N/120N/155N/223N及修飾體328K+之H5蛋白的任何核酸分子;或x.編碼具有修飾體223N、修飾體328K+及選自由以下各者組成之群的以下胺基酸團中之一或多者之H5蛋白的任何核酸分子:a. aa 93-95:GNF b. aa 123-125:SDH c. aa 128-130:SSG d. aa 138-140:GSS e. aa 226-228:MDF f. aa 270-272:EVE g. aa 309-311:NKL;或xi.編碼具有胺基酸223N、修飾體328K+及選自由以下各者組成之群的以下胺基酸團中之一或多者之H5蛋白的任何核酸分子:a. aa 93-95:GNF b. aa 128-130:SSG c. aa 138-140:GSS;或xii.編碼具有SEQ ID NO:4之胺基酸序列之H5蛋白的任何核酸分子。Furthermore, preferred nucleic acid molecules encoding the H5 protein of the present invention are: i. any of the above nucleic acid molecules encoding amino acid 223N and modified 328K+; ii. the above-described amino acid encoding 94N/223N and modification Any of the nucleic acid molecules of 328K+; iii. any avian nucleic acid molecule encoding amino acid 223N and modification 328K+, wherein the avian source means that the H5 sequence is derived from the original infection with avian influenza 5 virus. Avian isolated virus isolate; or iv. any avian nucleic acid molecule encoding amino acid 94N/223N and modified 328K+, wherein the avian source means that the H5 sequence is derived from a bird originally isolated from avian influenza A virus. a virus isolate; or v. any avian nucleic acid molecule encoding amino acid 155N/223N and modified 328K+, wherein the avian source means that the H5 sequence is derived from a virus isolate originally isolated from avian infected with avian influenza virus type 5 Or vi. any nucleic acid molecule encoding avian H5 protein having amino acid 120N/155N/223N and modified 328K+, wherein the avian origin means that the H5 sequence is derived from a bird originally isolated from avian influenza A virus. Virus isolate; Vii. any nucleic acid molecule encoding a H5 protein having a modified form 94N/223N and a modified form 328K+; or viii. any nucleic acid molecule encoding a H5 protein having a modified form 94N/155N/223N and a modified form 328K+; or ix. Any nucleic acid molecule that modifies the H5 protein of 94N/120N/155N/223N and modified 328K+; or x. encodes the following amino acid group having a modification 223N, a modification 328K+, and a group selected from the group consisting of: Any nucleic acid molecule of one or more of the H5 proteins: a. aa 93-95: GNF b. aa 123-125: SDH c. aa 128-130: SSG d. aa 138-140: GSS e. aa 226-228 MDF f. aa 270-272: EVE g. aa 309-311: NKL; or xi. encoding one of the following amino acid groups having an amino acid 223N, a modification 328K+, and a group selected from the group consisting of: Or any nucleic acid molecule of the H5 protein: a. aa 93-95: GNF b. aa 128-130: SSG c. aa 138-140: GSS; or xii. encoding an amino acid having SEQ ID NO: Any nucleic acid molecule of the sequence H5 protein.
此外,如本文中所提供之較佳H5蛋白包括以下文獻所述之H5蛋白:Hoffmann等人,PNAS,第106卷,第36期,第12915-12920頁(2005年9月6日) ,其中彼等H5蛋白包括如上所述之修飾體中之一或多者,至少包括胺基酸223N及修飾體328K+,其中H5蛋白之胺基酸位置之編號係指如SEQ ID NO:1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置328上插入第二離胺酸(K+)。此參考文獻之揭示內容將以引用方式全文併入本文中。因此,根據另一實施例,本發明亦係關於任何核酸分子,較佳編碼以下文獻所述之該等蛋白質中之任一者的cDNA分子:Hoffmann等人,PNAS,第106卷,第36期,第12915-12920頁(2005年9月6日) ,其中彼等H5蛋白包括上述修飾體中之一或多者,至少包括胺基酸223N及修飾體328K+,其中H5蛋白之胺基酸位置之編號係指如SEQ ID NO:1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置328上插入第二離胺酸(K+)。Furthermore, preferred H5 proteins as provided herein include the H5 proteins described in the following literature: Hoffmann et al, PNAS, Vol. 106, No. 36, pp. 12915-12920 (September 6, 2005) , wherein The H5 proteins include one or more of the modifications described above, including at least the amino acid 223N and the modification 328K+, wherein the amino acid position of the H5 protein is numbered as exemplified in SEQ ID NO: 1. The amino acid position of the sex designation and wherein the modification 328K+ means the insertion of a second lysine (K+) at the amino acid position 328 of the H5 protein. The disclosure of this reference is hereby incorporated by reference in its entirety. Thus, according to another embodiment, the invention is also directed to any nucleic acid molecule, preferably a cDNA molecule encoding any of the proteins described in the following literature: Hoffmann et al, PNAS, Vol. 106, No. 36 , pp. 12915-12920 (September 6, 2005) , wherein the H5 proteins include one or more of the above modifications, including at least the amino acid 223N and the modified 328K+, wherein the amino acid position of the H5 protein The numbering refers to the amino acid position as exemplified in SEQ ID NO: 1 and wherein the modification 328K+ means the insertion of a second lysine (K+) at the amino acid position 328 of the H5 protein.
將上述任何修飾體引入核苷酸序列(包括流感病毒之H5蛋白之編碼序列)內的方法已熟知於此項技術。完整流感病毒之基因組序列可根據本發明加以修飾,例如根據可供進一步參考之US 6,951,754中所述之方法加以修飾。Methods for introducing any of the above modifications into a nucleotide sequence, including the coding sequence of the H5 protein of influenza virus, are well known in the art. The genomic sequence of the entire influenza virus can be modified in accordance with the present invention, for example, as described in US 6,951,754, which is incorporated by reference.
此外,可利用此項技術技能範圍內之習知分子生物學、微生物學及重組DNA技術修飾編碼本文中所述之抗原的核酸序列。該等技術已詳釋於文獻中。參見例如Sambrook等人,Molecular Cloning:A Laboratory Manual,第二版(1989)Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.;DNA Cloning:A Practical Approach,第I及II卷(D.N.Glover編,1985);Oligonucleotide Synthesis(M.J.Gait編,1984);Nucleic Acid Hybridization [B.D.Hames & S.J.Higgins編(1985)];Transcription And Translation[B.D.Hames & S.J.Higgins編(1984)];Animal Cell Culture[R.I.Freshney編(1986)];Immobilized Cells And Enzymes [IRL Press,(1986)];B.Perbal,A Practical Guide To Molecular Cloning (1984);F.M.Ausubel等人編,Current Protocols in Molecular Biology,John Wiley & Sons,Inc.1994) 。In addition, nucleic acid sequences encoding the antigens described herein can be modified using conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. These techniques are well documented in the literature. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; DNA Cloning: A Practical Approach, Volumes I and II (DNGlover, ed., 1985); Oligonucleotide Synthesis (edited by MJ Gait, 1984); Nucleic Acid Hybridization [edited by BD Hames & SJ Higgins (1985)]; Transcription And Translation [edited by BD Hames & SJ Higgins (1984)]; Animal Cell Culture [edited by RI Freshney (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); FMAusubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. 1994) .
根據另一實施例,本發明亦係關於包含上述該等核酸分子中之任一者的載體。換而言之,本發明係關於包括上述任何該H5蛋白或其部分之編碼序列的載體。較佳地,該載體為使上述任何該H5蛋白或其部分得到表現的表現載體。本發明之載體為適於在活體外或活體內轉染或感染細菌、酵母或動物細胞的彼等載體。According to another embodiment, the invention is also directed to a vector comprising any of the above-described nucleic acid molecules. In other words, the invention relates to vectors comprising the coding sequences of any of the H5 proteins or portions thereof described above. Preferably, the vector is an expression vector for rendering any of the H5 proteins or portions thereof described above. The vectors of the present invention are those suitable for transfecting or infecting bacterial, yeast or animal cells in vitro or in vivo.
載體及用於製備及/或使用表現載體(或重組體)的方法可依據或類似於以下文獻中所揭示之與DNA表現載體相關之方法:美國專利第4,603,112號、第4,769,330號、第5,174,993號、第5,505,941號、第5,338,683號、第5,494,807號、第4,722,848號、第5,942,235號、第5,364,773號、第5,762,938號、第5,770,212號、第5,942,235號、第382,425號;PCT公開案WO 94/16716、WO 96/39491、WO 95/30018;Paoletti,"Applications of pox virus vectors to vaccination:An update,"PNAS USA 93:11349-11353,1996年10月;Moss,"Genetically engineered poxviruses for recombinant gene expression,vaccination,and safety,"PNAS USA 93:11341-11348,1996年10月;Smith等人,美國專利第4,745,051號(重組性桿狀病毒);Richardson,C.D.(編者),Methods in Molecular Biology 39,"Baculovirus Expression Protocols"(1995 Humana Press Inc.);Smith等人,"Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vector",Molecular and Cellular Biology,1983年12月,第3卷,第12期,第2156-2165頁;Pennock等人,"Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infect Cells with a Baculovirus vector," Molecular and Cellular Biology,1984年3月,第4卷,第3期,第399-406頁;EPA0 370 573;美國申請案第920,197號(1986年10月16日申請);EP專利公開案第265785號;美國專利第4,769,331號(重組性疱疹病毒);Roizman,"The function of herpes simplex virus genes:A primer for genetic engineering of novel vectors," PNAS USA 93:11307-11312,1996年10月;A ndreansky等人,"The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors," PNAS USA 93:11313-11318,1996年10月;Robertson等人,"Epstein-Barr virus vectors for gene delivery to B lymphocytes",PNAS USA 93:11334-11340,1996年10月;Fro1ov等人,"Alphavirus-based expression vectors:Strategies and applications," PNAS USA 93:11371-11377,1996年10月;Kitson等人,J.Virol.65,3068-3075,1991;美國專利第5,591,439號、第5,552,143號、WO 98/00166;已受理之美國申請案第08/675,556號、第08/675,566號(兩者均於1996年7月3日申請)(重組性腺病毒);Grunhaus等人,1992,"Adenovirus as cloning vectors," Seminars in Virology(第3卷),第237-52頁,1993;Ballay等人,EMBO Journal,第4卷,第3861-65頁;Graham,Tibtech 8,85-87,1990年4月;Prevec等人,J.Gen Virol.70,42434;PCT WO 91/11525;Felgner等人,(1994),J.Biol.Chem.269,2550-2561,Science,259:1745-49,1993及McClements等人,"Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B,alone or in combination,induces protective immunity in animal models of herpes simplex virus-2 disease",PNAS USA 93:11414-11420,1996年10月;及美國專利第5,591,639號、第5,589,466號及第5,580,859號;以及WO 90/11092、WO 93/19183、WO94/21797、WO95/11307、WO95/20660;Tang等人,Nature;及Furth等人,Analytical Biochemistry等。亦可參見WO 98/33510;Ju等人,Diabetologia,41:736-739,1998(豆狀病毒表現系統);Sanford等人,美國專利第4,945,050號;Fischbach等人(Intracel),WO 90/01543;Robinson等人,seminars in Immunology第9卷,第271-283頁(1997)(DNA載體系統);Szoka等人,美國專利第號(將DNA插入活細胞內之方法);McCormick等人,美國專利第5,677,178號(細胞病病毒之使用);及美國專利第5,928,913號(用於基因傳遞之載體)以及本文中所引用之其他文件。The vector and methods for making and/or using the expression vector (or recombinant) can be based on or similar to those disclosed in the following literature: US Patent Nos. 4,603,112, 4,769,330, 5,174,993 , PCT Publication No. 5,505,941, 5,338,683, 5,494,807, 4,722,848, 5,942,235, 5,364,773, 5,762,938, 5,770,212, 5,942,235, 382,425; PCT Publication WO 94/16716, WO 96/39491, WO 95/30018; Paoletti, "Applications of pox virus vectors to vaccination: An update," PNAS USA 93: 11349-11353, October 1996; Moss, "Genetically engineered poxviruses for recombinant gene expression, vaccination, And safety, "PNAS USA 93:11341-11348, October 1996; Smith et al., U.S. Patent No. 4,745,051 (Recombinant Baculovirus); Richardson, CD (Editor), Methods in Molecular Biology 39, "Baculovirus Expression Protocols" (1995 Humana Press Inc.); Smith et al., "Production of Human Beta Interferon in Insect Cells Infected with a Baculovirus Expression Vecto r", Molecular and Cellular Biology, December 1983, Vol. 3, No. 12, pp. 2156-2165; Pennock et al., "Strong and Regulated Expression of Escherichia coli B-Galactosidase in Infect Cells with a Baculovirus vector, "Molecular and Cellular Biology, March 1984, Vol. 4, No. 3, pp. 399-406; EPA 0 370 573; US Application No. 920,197 (filed on October 16, 1986); EP Patent Publication No. U.S. Patent No. 4,769,331 (Recombinant Herpesvirus); Roizman, "The function of herpes simplex virus genes: A primer for genetic engineering of novel vectors," PNAS USA 93: 11307-11312, October 1996; Ndreansky et al., "The application of genetically engineered herpes simplex viruses to the treatment of experimental brain tumors," PNAS USA 93:11313-11318, October 1996; Robertson et al., "Epstein-Barr virus vectors for gene delivery to B Lymphocytes", PNAS USA 93:11334-11340, October 1996; Fro1ov et al., "Alphavirus-based expression vectors: Strategies and applications," PNAS USA 93: 11371-11377, October 1996; Kitson et al, J. Virol. 65, 3068-3075, 1991; U.S. Patent Nos. 5,591,439, 5,552,143, WO 98/00166; Nos. 08/675,556, 08/675,566 (both applied on July 3, 1996) (recombinant adenovirus); Grunhaus et al., 1992, "Adenovirus as cloning vectors," Seminars in Virology (Vol. 3 ), pp. 237-52, 1993; Ballay et al., EMBO Journal, Vol. 4, pp. 3861-65; Graham, Tibtech 8, 85-87, April 1990; Prevec et al., J. Gen Virol. 70,42434; PCT WO 91/11525; Felgner et al, (1994), J. Biol. Chem. 269, 2550-2561, Science, 259: 1745-49, 1993 and McClems et al, "Immunization with DNA vaccines encoding Glycoprotein D or glycoprotein B,alone or in combination,induces protective immunity in animal models of herpes simplex virus-2 disease",PNAS USA 93:11414-11420, October 1996; and U.S. Patent Nos. 5,591,639, 5,589,466 and No. 5,580,859; and WO 90/11092, WO 93/19183, WO 94/21797, WO 95/11307, WO 95/20660; Ang et al., Nature; and Furth et al., Analytical Biochemistry et al. See also WO 98/33510; Ju et al, Diabetologia, 41: 736-739, 1998 (soyavirus expression system); Sanford et al., U.S. Patent No. 4,945,050; Fischbach et al. (Intracel), WO 90/01543 Robinson et al., Seminars in Immunology, Vol. 9, pp. 271-283 (1997) (DNA Vector System); Szoka et al., U.S. Patent No. (Method of Inserting DNA into Living Cells); McCormick et al., USA Patent No. 5,677,178 (the use of cytopathic viruses); and U.S. Patent No. 5,928,913 (a carrier for gene delivery) and other documents cited herein.
最好使用例如選自豬疱疹病毒(諸如Aujeszky氏疾病病毒)、豬腺病毒、痘病毒(尤其牛痘病毒、禽痘病毒、金絲雀痘病毒及豬痘病毒)之病毒載體以及DNA載體(DNA質體)來實施本發明。Preferably, for example, a viral vector selected from a herpes simplex virus (such as the Aujeszky's disease virus), a porcine adenovirus, a poxvirus (especially a vaccinia virus, a fowlpox virus, a canarypox virus, and a porcine pox virus) and a DNA vector (DNA) are used. The plastids are used to practice the invention.
根據另一態樣,本發明提供如下製備及/或回收高量重組性H5蛋白之方法:i)使培養物中之易感性細胞經含有H5 DNA編碼序列之重組性病毒載體感染,其中H5蛋白係由重組性病毒載體表現;及ii)爾後自細胞培養物中回收H5蛋白。H5蛋白之高量意謂(但不限於)以每毫升細胞培養物計約20 μg以上、較佳約25 μg以上、甚至更佳約30 μg以上、甚至更佳約40 μg以上、甚至更佳約50 μg以上、甚至更佳約60 μg以上、甚至更佳約80 μg以上、甚至更佳約100 μg以上、甚至更佳約150 μg以上、最佳約190 μg以上。According to another aspect, the present invention provides a method of preparing and/or recovering a high amount of recombinant H5 protein by: i) infecting a susceptible cell in a culture with a recombinant viral vector containing an H5 DNA coding sequence, wherein the H5 protein Expressed by a recombinant viral vector; and ii) the H5 protein is recovered from the cell culture. A high amount of H5 protein means, but is not limited to, about 20 μg or more, preferably about 25 μg or more, even more preferably about 30 μg or more, even more preferably about 40 μg or more, even better, per ml of cell culture. About 50 μg or more, even more preferably about 60 μg or more, even more preferably about 80 μg or more, even more preferably about 100 μg or more, even more preferably about 150 μg or more, and most preferably about 190 μg or more.
根據一較佳實施例,藉由收穫表現H5蛋白之全(亦即完整)SF+細胞來回收H5蛋白。According to a preferred embodiment, the H5 protein is recovered by harvesting whole (i.e., intact) SF+ cells that express the H5 protein.
較佳細胞為彼等易受含有H5 DNA並表現H5蛋白的適當重組性病毒載體感染之細胞。較佳地,該等細胞為昆蟲細胞,且更佳地,其包括以商標SF+昆蟲細胞(Protein Sciences Corporation,Meriden,CT)出售的昆蟲細胞。較佳之細胞培養物具有約0.3×106 -2.0×106 個細胞/毫升之間,更佳約0.35×106 -1.9×106 個細胞/毫升、甚至更佳約0.4×106 -1.8×106 個細胞/毫升、甚至更佳約0.45×106 -1.7×106 個細胞/毫升且最佳約0.5×106 -1.5×106 個細胞/毫升的細胞數。Preferred cells are those which are susceptible to infection by a suitable recombinant viral vector containing H5 DNA and expressing the H5 protein. Preferably, the cells are insect cells, and more preferably, they comprise insect cells sold under the trademark SF+ insect cells (Protein Sciences Corporation, Meriden, CT). Preferably, the cell culture has between about 0.3 x 10 6 and 2.0 x 10 6 cells/ml, more preferably about 0.35 x 10 6 to 1.9 x 10 6 cells/ml, even more preferably about 0.4 x 10 6 -1.8. × 10 6 cells/ml, even more preferably about 0.45 x 10 6 - 1.7 x 10 6 cells/ml and optimally about 0.5 x 10 6 - 1.5 x 10 6 cells/ml.
較佳病毒載體包括桿狀病毒,諸如BaculoGold(BD Biosciences Pharmingen,San Diego,CA),尤其是在製備細胞為昆蟲細胞的情況下。儘管桿狀病毒表現系統為較佳的,但熟習此項技術者應瞭解,其他表現系統可用於本發明之目的,亦即用於使H5表現至細胞培養物之上清液中。該等其他表現系統可能需要使用信號序列以使H5表現至培養基中。Preferred viral vectors include baculoviruses such as BaculoGold (BD Biosciences Pharmingen, San Diego, CA), especially where the preparation of cells is an insect cell. Although a baculovirus expression system is preferred, those skilled in the art will appreciate that other performance systems can be used for the purposes of the present invention, i.e., for rendering H5 into the supernatant of a cell culture. Such other expression systems may require the use of a signal sequence to allow H5 to be expressed into the culture medium.
適當生長培養基亦可由熟習此項技術者確定,較佳生長培養基為無血清昆蟲細胞培養基,諸如Excell 420(JRH Biosciences,Inc.,Lenexa,KS)及類似培養基。Suitable growth media can also be determined by those skilled in the art. Preferred growth media are serum-free insect cell culture media such as Excell 420 (JRH Biosciences, Inc., Lenexa, KS) and similar media.
當用於感染易感性細胞時,含有H5 DNA序列的重組性病毒載體具有較佳約0.03-1.5之間、更佳約0.05-1.3、甚至更佳約0.09-1.1且最佳約0.1-1.0之感染複數(MOI)。較佳地,上述MOI係關於1 mL之細胞培養流體。較佳地,本文中所述方法包含:用含有H5 DNA且表現H5蛋白的重組性病毒載體感染0.35×106 -1.9×106 個細胞/毫升、甚至更佳約0.4×106 -1.8×106 個細胞/毫升、甚至更佳約0.45×106 -1.7×106 個細胞/毫升且最佳約0.5×106 -1.5×106 個細胞/毫升,該病毒載體具有約0.03-1.5之間、更佳約0.05-1.3、甚至更佳約0.09-1.1且最佳約0.1-1.0之MOI(感染複數)。When used to infect a susceptible cell, the recombinant viral vector containing the H5 DNA sequence has preferably between about 0.03 and about 1.5, more preferably between about 0.05 and about 1.3, even more preferably between about 0.09 and about 1.1, and most preferably from about 0.1 to about 1.0. Multiplicity of infection (MOI). Preferably, the MOI described above relates to 1 mL of cell culture fluid. Preferably, the method described herein comprises: infecting 0.35 x 10 6 - 1.9 x 10 6 cells/ml, even more preferably about 0.4 x 10 6 - 1.8 x with a recombinant viral vector containing H5 DNA and expressing the H5 protein. 10 6 cells/ml, even more preferably about 0.45 x 10 6 -1.7 x 10 6 cells/ml and optimally about 0.5 x 10 6 - 1.5 x 10 6 cells/ml, the viral vector having about 0.03-1.5 An MOI (multiplicity of infection) between, preferably about 0.05-1.3, even more preferably about 0.09-1.1 and most preferably about 0.1-1.0.
接著將受感染細胞培育至多十天、更佳約兩天至約十天、甚至更佳約四天至約九天且最佳約五天至約八天。較佳之培育條件包括介於約22-32℃之間、更佳約24-30℃、甚至更佳約25-29℃、甚至更佳約26-28℃且最佳約27℃之溫度。較佳地,接種之後觀測到SF+細胞之桿狀病毒誘導之特徵性變異。此觀測可包括監測感染後期間細胞密度趨向及存活力之降低。已發現,感染後3-5天觀測到峰值病毒效價,且細胞中之H5蛋白表現在第5天與第8天之間達到峰值,且/或在細胞存活力降至小於10%時。The infected cells are then incubated for up to ten days, more preferably from about two days to about ten days, even more preferably from about four days to about nine days and optimally from about five days to about eight days. Preferred incubation conditions include temperatures between about 22-32 ° C, more preferably about 24-30 ° C, even more preferably about 25-29 ° C, even more preferably about 26-28 ° C and most preferably about 27 ° C. Preferably, a characteristic variant of baculovirus induction by SF+ cells is observed after inoculation. This observation may include monitoring the trend of cell density and viability during post-infection. It has been found that peak viral titers are observed 3-5 days after infection and the H5 protein expression in the cells peaks between day 5 and day 8 and/or when cell viability drops to less than 10%.
因此,本發明之一態樣提供一種如下製備及/或回收重組性H5蛋白(較佳為上述量)的方法:i)使培養物中之大量易感性細胞(參見上文)經具有如上定義之MOI之重組性病毒載體感染;ii)由重組性病毒載體表現H5蛋白;及iii)爾後自感染後第5天與第8天之間獲得且/或細胞存活力降至小於10%之細胞中回收H5蛋白。較佳地,重組性病毒載體為含有H5 DNA編碼序列的重組性桿狀病毒且該等細胞為SF+細胞。此外,較佳定期檢查培養物受污染之宏觀及微觀跡象或感染後期間細胞形態之異常變化。任何呈現任何污染的培養物應棄去。Accordingly, one aspect of the present invention provides a method of preparing and/or recovering a recombinant H5 protein (preferably the above amount) as follows: i) making a plurality of susceptible cells (see above) in the culture have the above definition Recombinant viral vector infection of MOI; ii) expression of H5 protein by recombinant viral vector; and iii) cells obtained between day 5 and day 8 after infection and/or cell viability reduced to less than 10% The H5 protein is recovered. Preferably, the recombinant viral vector is a recombinant baculovirus containing the H5 DNA coding sequence and the cells are SF+ cells. In addition, it is preferred to periodically check for macroscopic and microscopic signs of contamination of the culture or abnormal changes in cell morphology during infection. Any culture that presents any contamination should be discarded.
為達成將用於免疫原性或免疫組合物(諸如疫苗)中之H5蛋白之回收,較佳包括滅活步驟以便將病毒載體滅活。To achieve recovery of the H5 protein to be used in an immunogenic or immunological composition, such as a vaccine, it is preferred to include an inactivation step to inactivate the viral vector.
"免疫原性或免疫組合物"係指包含至少一種抗原的物質組合,該抗原可在宿主體內引發對受關注之組合物或疫苗之細胞性免疫反應及/或抗體介導之免疫反應的免疫反應。通常,"免疫反應"包括(但不限於)以下效應中之一或多者:特異性針對包含於受關注之組合物或疫苗中之抗原的抗體、B細胞、輔助性T細胞、抑制性T細胞及/或細胞毒性T細胞及/或γ-δ T細胞之產生或活化。較佳地,宿主可呈現治療性或保護性免疫反應,以便增強對新感染之抵抗力及/或降低疾病之臨床嚴重程度。此保護作用表現為受感染宿主通常所呈現之症狀減少或消失、恢復時間加快及/或受感染宿主之病毒效價降低。"Immunogenic or immunological composition" means a combination of substances comprising at least one antigen which elicits immunity in a host against a cellular immune response and/or an antibody-mediated immune response of a composition or vaccine of interest. reaction. Generally, an "immune response" includes, but is not limited to, one or more of the following: antibodies, B cells, helper T cells, inhibitory T that are specific for an antigen contained in a composition or vaccine of interest. Production or activation of cellular and/or cytotoxic T cells and/or γ-δ T cells. Preferably, the host may present a therapeutic or protective immune response in order to increase resistance to new infections and/or reduce the clinical severity of the disease. This protective effect is manifested by a reduction or disappearance of the symptoms typically exhibited by the infected host, an increased recovery time and/or a reduced viral titer of the infected host.
因此,本發明亦係關於一種如下製備及/或回收重組性H5蛋白(較佳為上述量)的方法:i)使培養物中之大量易感性細胞(參見上文)經具有如上定義之MOI之重組性病毒載體感染;ii)由重組性病毒載體表現H5蛋白;iii)將感染後第5天與第8天之間獲得且細胞存活力降至小於10%之細胞中所表現的H5回收;及iv)將重組性病毒載體滅活。Accordingly, the present invention is also directed to a method of preparing and/or recovering a recombinant H5 protein (preferably the above amount) as follows: i) subjecting a plurality of susceptible cells in culture (see above) to having an MOI as defined above Recombinant viral vector infection; ii) H5 protein expressed by recombinant viral vector; iii) H5 recovery in cells obtained between day 5 and day 8 after infection and cell viability reduced to less than 10% ; and iv) inactivate the recombinant viral vector.
較佳地,此滅活係在即將進行過濾步驟之前進行或在過濾步驟剛結束之後進行,過濾步驟之後為較佳滅活時間。任何習知滅活方法可用於本發明之目的。因此,滅活可藉由化學及/或物理處理來進行。在較佳形式中,測定所收穫流體之體積且使溫度介於約32-42℃之間、更佳介於約34-40℃之間且最佳介於約35-39℃之間。較佳滅活方法包括添加環化二元伸乙基亞胺(BEI),此物質濃度較佳為約1 mM至約20 mM、較佳為約2 mM至約10 mM、甚至更佳為約2 mM至約8 mM、甚至更佳為約3 mM至約7 mM、最佳為約5 mM。舉例而言,滅活包括將較佳為約0.4 M之2-溴伸乙基胺氫溴化物溶液(其已在0.3 N NaOH中環化為0.2 M二元伸乙基亞胺BEI)添加至流體中以得到最終濃度約5 mM之BEI。較佳地,接著將流體連續攪拌72-96小時,且可將經滅活之收穫流體在-40℃或-40℃以下冷凍儲存或在約1-7℃之間儲存。滅活完成後,添加硫代硫酸鈉溶液(較佳為1.0 M)以中和任何殘餘BEI。較佳地,硫代硫酸鈉的添加量與滅活之前所添加之BEI的量相當。舉例而言,在添加BEI至最終濃度為5 mM之情況下,添加1.0 M硫代硫酸鈉溶液以得到最終最小濃度5 mM以中和任何殘餘BEI。Preferably, the inactivation is carried out immediately before the filtration step or immediately after the filtration step, after which the filtration step is a preferred inactivation time. Any conventional inactivation method can be used for the purposes of the present invention. Therefore, inactivation can be carried out by chemical and/or physical treatment. In a preferred form, the volume of harvested fluid is determined and the temperature is between about 32-42 °C, more preferably between about 34-40 °C and optimally between about 35-39 °C. Preferably, the inactivation method comprises the addition of a cyclized binary ethylenamine (BEI), preferably at a concentration of from about 1 mM to about 20 mM, preferably from about 2 mM to about 10 mM, even more preferably about 2 mM to about 8 mM, even more preferably from about 3 mM to about 7 mM, most preferably about 5 mM. For example, inactivation comprises adding a solution of preferably 2-0.4 M 2-bromoethylamine hydrobromide (which has been cyclized to 0.2 M binary ethylenimine BEI in 0.3 N NaOH) to the fluid. To obtain a BEI with a final concentration of about 5 mM. Preferably, the fluid is then continuously agitated for 72-96 hours, and the inactivated harvest fluid can be stored frozen at -40 °C or below -40 °C or between about 1-7 °C. After completion of the inactivation, a sodium thiosulfate solution (preferably 1.0 M) is added to neutralize any residual BEI. Preferably, the amount of sodium thiosulfate added is comparable to the amount of BEI added prior to inactivation. For example, with the addition of BEI to a final concentration of 5 mM, a 1.0 M sodium thiosulfate solution was added to give a final minimum concentration of 5 mM to neutralize any residual BEI.
因此,本發明之另一態樣係關於一種如下製備重組性H5蛋白(較佳為上述量)的方法:i)使培養物中之大量易感性細胞(參見上文)經具有如上定義之MOI之重組性病毒載體感染;ii)由重組性病毒載體表現H5蛋白;iii)將感染後第5天與第8天之間獲得且細胞存活力降至小於10%之細胞中所表現之H5回收;及iv)將重組性病毒載體滅活。較佳地,重組性病毒載體為含有H5 DNA編碼序列的桿狀病毒且該等細胞為SF+細胞。較佳滅活步驟為上述彼等步驟。較佳地,滅活係在約35-39℃之間且在2 mM至8 mM BEI之存在下、甚至更佳在約5 mM BEI之存在下進行。Thus, another aspect of the invention pertains to a method of preparing a recombinant H5 protein, preferably in the above amounts: i) subjecting a plurality of susceptible cells in culture (see above) to having an MOI as defined above Recombinant viral vector infection; ii) H5 protein expressed by recombinant viral vector; iii) H5 recovery in cells obtained between day 5 and day 8 after infection and cell viability reduced to less than 10% ; and iv) inactivate the recombinant viral vector. Preferably, the recombinant viral vector is a baculovirus containing the H5 DNA coding sequence and the cells are SF+ cells. Preferred inactivation steps are those described above. Preferably, the inactivation is carried out between about 35-39 ° C and in the presence of 2 mM to 8 mM BEI, even more preferably in the presence of about 5 mM BEI.
根據本發明之另一態樣,上述方法亦包括步驟iv)之後的中和步驟。此步驟v)包含添加中和溶液中之滅活劑的等量試劑。較佳地,若滅活劑為BEI,則較佳添加等量之硫代硫酸鈉。因此,根據另一態樣,當滅活劑為BEI時,步驟v)包含添加硫代硫酸鈉溶液直至最終濃度為約1 mM至約20 mM、較佳約2 mM至約10 mM、甚至更佳約2 mM至約8 mM、甚至更佳約3 mM至約7 mM、最佳約5 mM。According to another aspect of the invention, the above method also includes a neutralization step after step iv). This step v) contains an equal amount of reagent to add the inactivating agent in the neutralizing solution. Preferably, if the inactivating agent is BEI, it is preferred to add an equivalent amount of sodium thiosulfate. Thus, according to another aspect, when the inactivating agent is BEI, step v) comprises adding a sodium thiosulfate solution until a final concentration of from about 1 mM to about 20 mM, preferably from about 2 mM to about 10 mM, or even more Preferably from about 2 mM to about 8 mM, even more preferably from about 3 mM to about 7 mM, optimally about 5 mM.
在較佳形式中且尤其在以免疫原性組合物(諸如疫苗)使用重組性H5蛋白之形式中,將每一批所收穫之H5蛋白藉由於固著依賴性、桿狀病毒易感性昆蟲細胞(諸如Sf9細胞)中繼代來測試滅活。在此測試之一較佳形式中,將150 cm2 之適當細胞培養物單層用1.0 mL經滅活之H5流體接種且在25-29℃下維持14天,至少繼代兩次。在維持期結束時,對細胞單層檢查H5桿狀病毒所特有之致細胞病變作用(CPE)。較佳地,亦使用陽性病毒對照。此等對照可由以下各物組成:一份經未滅活之參考H5桿狀病毒接種之Sf9細胞培養物及一瓶尚未接種之Sf9細胞。培育及繼代之後,經BEI處理之病毒流體中不存在受病毒感染之細胞說明滅活測試令人滿意。經參考病毒接種之對照細胞應呈現H5桿狀病毒所特有之CPE而未接種燒瓶應不呈現任何H5桿狀病毒CPE跡象。或者,在維持期結束時,可收集上清液樣本且將其接種於Sf9 96孔板上,該孔板已裝載Sf9細胞且接著在25-29℃下維持5-6天。接著將孔板固定且用結合FITC之抗-H5抗體或針對桿狀病毒特異性蛋白(亦即gp64)的任何標記抗體染色。經BEI處理之病毒流體中不存在CPE、H5表現或桿狀病毒特異性蛋白(亦即gp64)之表現說明滅活測試令人滿意。經參考病毒接種之對照細胞應呈現CPE及IFA活性,而未接種燒瓶應不呈現任何H5桿狀病毒CPE跡象且不含IFA活性。In a preferred form and especially in the form of a recombinant H5 protein in an immunogenic composition such as a vaccine, each batch of harvested H5 protein is caused by a fixation-dependent, baculovirus-susceptible insect cell (such as Sf9 cells) relay generation to test inactivation. In one preferred form of this test, a 150 cm 2 of an appropriate cell culture monolayer with 1.0 mL of inactivated H5 fluids and maintained at seeded 25-29 ℃ 14 days, at least two subculture. At the end of the maintenance period, the cell monolayer was examined for the cytopathic effect (CPE) characteristic of H5 baculovirus. Preferably, a positive virus control is also used. These controls can be composed of one Sf9 cell culture inoculated with a non-inactivated reference H5 baculovirus and one vial of uninoculated Sf9 cells. After incubation and subculture, the absence of virus-infected cells in the BEI-treated viral fluid indicated satisfactory inactivation testing. Control cells inoculated with reference to the virus should present CPE specific to H5 baculovirus and uninoculated flasks should not present any signs of H5 baculovirus CPE. Alternatively, at the end of the maintenance period, a supernatant sample can be collected and plated on a Sf9 96-well plate that has been loaded with Sf9 cells and then maintained at 25-29 °C for 5-6 days. The well plates were then fixed and stained with FITC-conjugated anti-H5 antibody or any labeled antibody against a baculovirus-specific protein (ie, gp64). The absence of CPE, H5 expression or baculovirus-specific protein (i.e., gp64) in the BEI-treated viral fluid indicates satisfactory inactivation testing. Control cells inoculated with reference to the virus should exhibit CPE and IFA activity, while uninoculated flasks should not exhibit any signs of H5 baculovirus CPE and no IFA activity.
因此,本文中所述之另一態樣係關於用於測定表現H5蛋白之重組病毒載體之滅活有效性的滅活測試,其包含以下步驟:i)使含有重組性病毒載體之培養流體之至少一部分與較佳如上所述之滅活劑接觸;ii)添加較佳如上所述之中和劑以中和滅活劑;及iii)藉由如上所述之檢定測定殘餘感染性。Thus, another aspect described herein relates to an inactivation assay for determining the inactivating effectiveness of a recombinant viral vector expressing an H5 protein comprising the steps of: i) cultivating a culture fluid containing a recombinant viral vector At least a portion is contacted with an inactivating agent preferably as described above; ii) a neutralizing agent as described above is preferably added to neutralize the inactivating agent; and iii) residual infectivity is determined by the assay as described above.
滅活之後,可以多種方式測定樣本中重組性H5蛋白之相對量。較佳量化方法包括SDS-PAGE密度測定法、ELISA及動物接種研究,其使已知疫苗量與臨床結果(血清學等)相關。當利用SDS-PAGE量化時,將含有未知量之重組性H5蛋白的樣本物質連同含有各種已知量之重組性H5蛋白的樣本一起在凝膠上展開。接著可基於已知樣本形成標準曲線,且可藉由與此標準曲線比較來測定未知樣本中重組性H5之量。由於ELISA通常公認為抗原量化之行業標準,因此較佳利用ELISA進行量化。After inactivation, the relative amount of recombinant H5 protein in the sample can be determined in a variety of ways. Preferred methods of quantification include SDS-PAGE densitometry, ELISA, and animal vaccination studies, which correlate known vaccine amounts with clinical outcomes (serology, etc.). When quantified by SDS-PAGE, a sample material containing an unknown amount of recombinant H5 protein was developed on a gel along with a sample containing various known amounts of recombinant H5 protein. A standard curve can then be formed based on the known samples, and the amount of recombinant H5 in the unknown sample can be determined by comparison to this standard curve. Since ELISA is generally recognized as an industry standard for antigen quantification, it is preferably quantified by ELISA.
根據另一態樣,本發明係關於通常包含以下物質的疫苗或醫藥組合物:i.一或多種如本文中所述之H5蛋白;ii.一或多種如本文中所述之編碼任何該等H5蛋白之核酸分子;及/或iii.一或多種如本文中所述之載體,其包含如本文中所述之任何該等核酸分子且編碼如本文中所述之任何該等H5蛋白;及iv.醫藥學上可接受之載劑及/或賦形劑。According to another aspect, the invention relates to a vaccine or pharmaceutical composition that generally comprises: i. one or more H5 proteins as described herein; ii. one or more of any of these encoded as described herein a nucleic acid molecule of H5 protein; and/or iii. one or more vectors as described herein, comprising any of the nucleic acid molecules as described herein and encoding any of the H5 proteins as described herein; Iv. Pharmaceutically acceptable carriers and/or excipients.
如本文中所述之術語"醫藥組合物"、"醫藥/疫苗組合物"包括(但不限於)用於減少或預防感染之疫苗或用於治療及減輕感染之物質組合。The terms "pharmaceutical composition", "pharmaceutical/vaccine composition" as used herein include, but are not limited to, vaccines for reducing or preventing infection or combinations of substances for treating and alleviating infection.
編碼流感血球凝集素之基於核酸之疫苗(較佳cDNA疫苗)的製備例如描述於以下參考文獻中:Deck等人,Vaccine 1997;15(1):71-78;Ulmer等人,Science 1993;259:1745-1749;Ulmer等人,Vaccine 1994;12(16):1541-1544 。任何彼等方法可用於製備編碼如本文中所述之流感H5蛋白之基於核酸之疫苗,較佳cDNA疫苗。The preparation of nucleic acid-based vaccines (preferred cDNA vaccines) encoding influenza hemagglutinin is described, for example, in the following references: Deck et al, Vaccine 1997; 15(1): 71-78; Ulmer et al, Science 1993; : 1745-1749; Ulmer et al., Vaccine 1994; 12(16): 1541-1544 . Any of these methods can be used to prepare a nucleic acid based vaccine, preferably a cDNA vaccine, encoding an influenza H5 protein as described herein.
此外,包含如本文中所述之H5蛋白或其部分的疫苗可藉由習知方法製備,例如藉由重組表現技術或藉由生物化學純化及分離技術製備。重組表現技術(包括在昆蟲細胞中表現)已熟知於此項技術且例如描述於以下參考文獻中:Sambrook等人,Molecular Cloning:A Laboratory Manual,第二版(1989)Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.;DNA Cloning:A Practical Approach,第I及II卷(D.N.Glover編,1985);Oligonucleotide Synthesis(M.J.Gait編,1984);Nucleic Acid Hybridization[B.D.Hames & S.J.Higgins編(1985)];Transcription And Translation[B.D.Hames & S.J.Higgins編(1984)];Animal Cell Culture[R.I.Freshney 編(1986)];Immobilized Cells And Enzymes[IRL Press,(1986)];B.Perbal,A Practical Guide To Molecular Cloning(1984);F.M.Ausubel等人(編),Current Protocols in Molecular Biology,John Wiley & Sons,Inc.1994) 。習用之重組表現系統之其他實例為細菌表現系統,諸如大腸桿菌(E.coli )或枯草芽孢桿菌(B.subtilis );基於酵母之表現系統,諸如釀酒酵母(S.cerevisiae )或粟酒裂殖酵母(S.pombe );或哺乳動物細胞表現系統,諸如基於BHK之表現系統、基於CHO之表現系統及/或基於NS0之表現系統。該等系統已熟知於此項技術且一般可(例如)經由Clontech Laboratories,Inc.4030 Fabian Way,Palo Alto,California 94303-4607,USA購得。其他表現策略例如描述於L schow等人,Vaccine第19期(2001),第4249-4259頁或Veit等人,PNAS第103卷(2006),第8197-8202頁中 。此外,重組性腺相關病毒系統為習用系統且描述於例如可供進一步參考之US 5,436,146或WO200203872中。此外,基於牛痘(痘)病毒之表現系統(例如,如可供進一步參考之US 6,265,183中所述)亦為習用系統且適於製備如根據本發明使用之重組性抗原、抗原組合物。其他適當表現系統利用重組性popova病毒,諸如SV40、禽痘病毒、假狂犬病毒及逆轉錄病毒。Furthermore, vaccines comprising an H5 protein or a portion thereof as described herein can be prepared by conventional methods, for example, by recombinant expression techniques or by biochemical purification and separation techniques. Recombinant expression techniques, including expression in insect cells, are well known in the art and are described, for example, in the following references: Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; DNA Cloning: A Practical Approach, Volumes I and II (edited by DNGlover, 1985); Oligonucleotide Synthesis (edited by MJ Gait, 1984); Nucleic Acid Hybridization [edited by BD Hames & SJ Higgins (1985)]; Transcription And Translation [ BD Hames & SJ Higgins (1984)]; Animal Cell Culture [RI Freshney (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); FMAusubel et al. (ed.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. 1994) . Other examples of conventional recombinant expression systems are bacterial expression systems such as E. coli (E. coli) or Bacillus subtilis (B. subtilis); the yeast based expression systems, such as Saccharomyces cerevisiae (the S. cerevisiae) or the Schizosaccharomyces pombe Yeast ( S. pombe ); or mammalian cell expression system, such as BHK-based performance systems, CHO-based performance systems, and/or NS0-based performance systems. Such systems are well known in the art and are generally commercially available, for example, from Clontech Laboratories, Inc. 4030 Fabian Way, Palo Alto, California 94303-4607, USA. Other performance strategies are described, for example, in L Schow et al., Vaccine, No. 19 (2001), pp. 4249-4259 or Veit et al., PNAS, Vol. 103 (2006), pp. 8197-8202 . In addition, the recombinant adeno-associated virus system is a conventional system and is described, for example, in US Pat. No. 5,436,146 or WO200203872. In addition, a system for the expression of vaccinia (pox) virus (for example, as described in US Pat. Other appropriate expression systems utilize recombinant popova viruses such as SV40, fowlpox virus, pseudorabies virus, and retrovirus.
如本文中所述之相關醫藥/疫苗組合物亦可包含含有如本文中所述之H5蛋白的滅活病毒(包含如本文中所述之H5蛋白之活病毒的非病原形式)、病毒之製劑及/或片段,其中該製劑及/或片段包含如本文中所述之H5蛋白。A related pharmaceutical/vaccine composition as described herein may also comprise an inactivated virus (a non-pathogenic form of a live virus comprising an H5 protein as described herein) containing an H5 protein as described herein, a preparation of the virus And/or a fragment, wherein the preparation and/or fragment comprises an H5 protein as described herein.
熟習該項技術者已知可連同抗原一起包含於該等組合物/疫苗中的其他組分(參見例如Remington's Pharmaceutical Sciences.(1990),第18版,Mack Publ.,Easton )。熟習此項技術者可使用已知的生理學上可接受之無菌可注射溶液。可易於利用等張水溶液(諸如生理食鹽水)或相應血漿蛋白溶液來製備即用溶液。醫藥組合物/疫苗可以凍乾製劑或乾燥製劑(例如作為部分套組)提供,該等製劑可在臨用前、在無菌條件下用已知可注射溶液復水。Other components known to those skilled in the art that can be included in the compositions/vaccines along with the antigen are described (see, for example, Remington's Pharmaceutical Sciences. (1990), 18th ed., Mack Publ., Easton ). Those skilled in the art can use known physiologically acceptable sterile injectable solutions. A ready-to-use solution can be readily prepared using an isotonic aqueous solution (such as physiological saline) or a corresponding plasma protein solution. The pharmaceutical compositions/vaccines may be provided as a lyophilized formulation or as a dry formulation (e.g., as a partial kit) which may be reconstituted with known injectable solutions prior to use, under sterile conditions.
此外,本發明之醫藥/疫苗組合物可包括一或多種獸醫學上可接受之載劑。如本文中所使用,"獸醫學上可接受之載劑"包括(但不限於)任何及所有溶劑、分散介質、衣料、佐劑、穩定劑、稀釋劑、防腐劑、抗菌劑及抗真菌劑、等張劑、吸收延遲劑及類似載劑。Furthermore, the pharmaceutical/vaccine compositions of the present invention may comprise one or more veterinary acceptable carriers. As used herein, "veterinary acceptable carrier" includes, but is not limited to, any and all solvents, dispersion media, clothing, adjuvants, stabilizers, diluents, preservatives, antibacterials, and antifungals. Isotonic agents, absorption delaying agents and similar carriers.
稀釋劑可包括水、生理食鹽水、右旋糖、乙醇、甘油及類似物。等張劑尤其可包括氯化鈉、右旋糖、甘露糖醇、山梨糖醇及乳糖。穩定劑尤其包括白蛋白及乙二胺四乙酸之鹼金屬鹽。Diluents can include water, physiological saline, dextrose, ethanol, glycerol, and the like. The isotonic agents may especially include sodium chloride, dextrose, mannitol, sorbitol, and lactose. Stabilizers include, inter alia, albumin and alkali metal salts of ethylenediaminetetraacetic acid.
如本文中所使用之防腐劑係指抗微生物活性劑,諸如慶大黴素(Gentamycin)、硫柳汞(Merthiolate)及類似物。特定而言,製備多劑量組合物最佳添加防腐劑。彼等抗微生物活性劑係以一定濃度添加以有效防止受關注之組合物受任何微生物污染或抑制受關注之組合物內之任何微生物生長。Preservatives as used herein refers to antimicrobial active agents such as Gentamycin, Merthiolate, and the like. In particular, the preparation of a multi-dose composition is best accomplished by the addition of a preservative. These antimicrobial actives are added at a concentration to effectively prevent the composition of interest from being contaminated by any microorganisms or inhibiting the growth of any microorganisms within the composition of interest.
如本文中所使用之"佐劑"可包括氫氧化鋁及磷酸鋁、皂苷(例如Quil A、QS-21(Cambridge Biotech Inc.,Cambridge MA)、GPI-0100(Galenica Pharmaceuticals,Inc.,Birmingham,AL))、油包水型乳液、水包油型乳液、水包油包水型乳液。"Adjuvants" as used herein may include aluminum hydroxide and aluminum phosphate, saponins (eg, Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL)), water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion.
乳液尤其可基於輕質液狀石蠟油(歐洲藥典類型);類異戊二烯油,諸如角鯊烷或角鯊烯;烯烴(尤其異丁烯或癸烯)之寡聚反應所產生之油;含有直鏈烷基之酸或醇之酯,尤其植物油、油酸乙酯、丙二醇二-(辛酸酯/癸酸酯)、甘油基三-(辛酸酯/癸酸酯)或丙二醇二油酸酯;支鏈脂肪酸或醇之酯,尤其異硬脂酸酯。油可與乳化劑組合使用以形成乳液。乳化劑較佳為非離子型界面活性劑,尤其脫水山梨糖醇酯、二縮甘露糖醇酯(例如脫水甘露糖醇油酸酯)、乙二醇酯、聚甘油酯、丙二醇酯及油酸酯、異硬脂酸酯、蓖麻酸酯或羥基硬脂酸酯(其視情況經乙氧基化);及聚氧丙烯-聚氧乙烯共聚物嵌段,尤其Pluronic產品,尤其L121。參見Hunter等人,The Theory and Practical Application of Adjuvants(Stewart-Tull編,D.E.S.).John Wiley and Sons,NY,第51-94頁(1995)及Todd等人,Vaccine 15:564-570(1997)。適當水包油型乳液之實例為基於Emulsigen之佐劑,諸如EMULSIGEN、EMULSIGEN-D、EMULSIGEN-P、EMULSIGEN-75(MVP Laboratories,Inc.Omaha,NE,USA)。已驚人地發現,包含H5蛋白、較佳如本文中所述之重組性H5蛋白的醫藥/疫苗組合物有效地輔以水包油型乳液佐劑,較佳為該等基於Emulsigen之佐劑,更佳EMULSIGEN及EMULSIGEN-D。The emulsion may especially be based on a light liquid paraffinic oil (European Pharmacopoeia type); an isoprene-like oil, such as squalane or squalene; an oil produced by oligomerization of an olefin (especially isobutylene or decene); A linear alkyl acid or an alcohol ester, especially vegetable oil, ethyl oleate, propylene glycol di-(octanoate/caprate), glyceryl tri-(octanoate/caprate) or propylene glycol dioleate An ester; a branched fatty acid or an ester of an alcohol, especially an isostearate. The oil can be used in combination with an emulsifier to form an emulsion. The emulsifier is preferably a nonionic surfactant, especially sorbitan ester, mannitol ester (such as dehydrated mannitol oleate), ethylene glycol ester, polyglycerol ester, propylene glycol ester and oleic acid. Ester, isostearate, ricinoleate or hydroxystearate (which is optionally ethoxylated); and polyoxypropylene-polyoxyethylene copolymer blocks, especially Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, DES). John Wiley and Sons, NY, pp. 51-94 (1995) and Todd et al., Vaccine 15: 564-570 (1997). . An example of a suitable oil-in-water emulsion is an adjuvant based on Emulsigen, such as EMULSIGEN ,EMULSIGEN-D ,EMULSIGEN-P ,EMULSIGEN-75 (MVP Laboratories, Inc. Omaha, NE, USA). Surprisingly, it has been found that a pharmaceutical/vaccine composition comprising an H5 protein, preferably a recombinant H5 protein as described herein, is effectively supplemented with an oil-in-water emulsion adjuvant, preferably such an Emulsigen-based adjuvant. Better EMULSIGEN And EMULSIGEN-D .
此外,可使用M.Powell及M.Newman所編之"Vaccine Design,The Subunit and Adjuvant Approach"第147頁所述之SPT乳液及該書第183頁所述之乳液MF59。In addition, the SPT emulsion described on page 147 of "Vaccine Design, The Subunit and Adjuvant Approach" by M. Powell and M. Newman and the emulsion MF59 described on page 183 of the book can be used.
佐劑之另一實例為選自丙烯酸或甲基丙烯酸之聚合物及順丁烯二酸酐與烯基衍生物之共聚物的化合物。有益的佐劑化合物為尤其與糖或多元醇之聚乙烯基醚交聯之丙烯酸或甲基丙烯酸之聚合物。該等化合物稱為卡波姆(carbomer)(Phameuropa第8卷,第2期,1996年6月)。熟習此項技術者亦可參考美國專利第2,909,462號,其描述與具有至少3個羥基、較佳不超過8個羥基之多羥基化合物交聯的該等丙烯酸聚合物,至少三個羥基中之氫原子可置換為具有至少2個碳原子的不飽合脂族基。較佳基團為含有2至4個碳原子的彼等基團,例如乙烯基、烯丙基及其他烯系不飽合基團。不飽合基團本身可含有其他取代基,諸如甲基。在Carbopol(BF Goodrich,Ohio,USA)名下銷售的產品尤其適用。其與烯丙基蔗糖或烯丙基異戊四醇交聯。其中可提及Carbopol 974P、934P及971P。最佳為使用Carbopol 971P。在順丁烯二酸酐與烯基衍生物之共聚物中,共聚物EMA(Monsanto)為順丁烯二酸酐與乙烯之共聚物。該等聚合物溶解於水中可產生酸溶液,使該酸溶液中和,較佳中和至生理pH值,以便得到可免疫原性、免疫或疫苗組合物自身可併入的佐劑溶液。Another example of an adjuvant is a compound selected from the group consisting of a polymer of acrylic acid or methacrylic acid and a copolymer of maleic anhydride and an alkenyl derivative. A beneficial adjuvant compound is a polymer of acrylic acid or methacrylic acid, especially crosslinked with a polyvinyl ether of a sugar or polyol. These compounds are known as carbomers (Phameuropa Vol. 8, No. 2, June 1996). Those skilled in the art can also refer to U.S. Patent No. 2,909,462, which describes the use of such acrylic polymers crosslinked with a polyhydroxy compound having at least 3 hydroxyl groups, preferably no more than 8 hydroxyl groups, of at least three of the hydroxyl groups. The atom may be substituted with an unsaturated aliphatic group having at least 2 carbon atoms. Preferred groups are those groups containing from 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. The unsaturated group itself may contain other substituents such as a methyl group. Products sold under the name Carbopol (BF Goodrich, Ohio, USA) are especially suitable. It is crosslinked with allyl sucrose or allyl isopentaerythritol. Among them, Carbopol 974P, 934P and 971P can be mentioned. The best use is Carbopol 971P. In the copolymer of maleic anhydride and an alkenyl derivative, the copolymer EMA (Monsanto) is a copolymer of maleic anhydride and ethylene. The polymer is dissolved in water to produce an acid solution which is neutralized, preferably neutralized to a physiological pH, to provide an adjuvant solution which is immunogenic, immunized or the vaccine composition itself can be incorporated.
其他適當佐劑尤其包括(但不限於)RIBI佐劑系統(Ribi Inc.)、嵌段共聚物(CytRx,Atlanta GA)、SAF-M(Chiron,Emeryville CA)、單磷醯脂A、阿夫立定(Avridine)脂質胺佐劑、獲自大腸桿菌之熱不穩定性腸毒素(重組體或其他形式)、霍亂毒素或胞壁醯二肽。Other suitable adjuvants include, but are not limited to, RIBI adjuvant systems (Ribi Inc.), block copolymers (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphorus a, Afu Avridine lipid amine adjuvant, heat labile enterotoxin (recombinant or other form) obtained from E. coli, cholera toxin or cell wall dipeptide.
較佳地,佐劑以每劑量約100 μg至約10 mg之量添加。甚至更佳地,佐劑以每劑量約100 μg至約10 mg之量添加。甚至更佳地,佐劑以每劑量約500 μg至約5 mg之量添加。甚至更佳地,佐劑以每劑量約750 μg至約2.5 mg之量添加。最佳地,佐劑以每劑量約1 mg之量添加。Preferably, the adjuvant is added in an amount from about 100 μg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of from about 100 μg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of from about 500 μg to about 5 mg per dose. Even more preferably, the adjuvant is added in an amount of from about 750 μg to about 2.5 mg per dose. Most preferably, the adjuvant is added in an amount of about 1 mg per dose.
醫藥/疫苗組合物可進一步包括一或多種其他免疫調節劑,諸如介白素、干擾素或其他細胞因子。醫藥/疫苗組合物亦可包括慶大黴素及硫柳汞。儘管適用於本發明之上下文中之佐劑及添加劑之量及濃度可易於由熟習此項技術者確定,但本發明涵蓋1 ml劑量之疫苗組合物包含約50 μg至約2000 μg佐劑且較佳約250 μg佐劑的組合物。在另一較佳實施例中,本發明涵蓋包含約1 μg/ml至約60 μg/ml抗生素且更佳小於約30 μg/ml抗生素的疫苗組合物。The pharmaceutical/vaccine composition may further comprise one or more other immunomodulatory agents, such as interleukins, interferons or other cytokines. The pharmaceutical/vaccine composition may also include gentamicin and thimerosal. Although the amounts and concentrations of adjuvants and additives suitable for use in the context of the present invention can be readily determined by those skilled in the art, the present invention contemplates that a 1 ml dose of the vaccine composition comprises from about 50 μg to about 2000 μg of adjuvant and A composition of about 250 μg adjuvant. In another preferred embodiment, the invention encompasses a vaccine composition comprising from about 1 μg/ml to about 60 μg/ml of antibiotic and more preferably less than about 30 μg/ml of antibiotic.
因此,根據另一實施例,本發明亦係關於醫藥/疫苗組合物,其包含:i.治療有效量之如本文中所述之任一種流感病毒H5蛋白,其中該H5蛋白具有胺基酸223N及修飾體328K+,其中H5蛋白之胺基酸位置編號係指如SEQ ID NO:1中所例示性指定之胺基酸位置且其中修飾體328K+意謂在H5蛋白之胺基酸位置328上插入第二離胺酸(K+);及ii.如上所述之醫藥學上可接受之佐劑。Thus, according to another embodiment, the invention is also directed to a pharmaceutical/vaccine composition comprising: i. a therapeutically effective amount of any of the influenza virus H5 proteins as described herein, wherein the H5 protein has an amino acid 223N And modification 328K+, wherein the amino acid position number of the H5 protein refers to the amino acid position as exemplified in SEQ ID NO: 1 and wherein the modification 328K+ means insertion at the amino acid position 328 of the H5 protein a second lysine (K+); and ii. a pharmaceutically acceptable adjuvant as described above.
較佳地,佐劑係選自由以下各物組成之群:a)EMULSIGEN:一種水包油型乳液(o/w);b)EMULSIGEN-D:一種具有溴化二甲基二-十八烷基銨(DDA)之水包油(o/w)乳液;c)Polygen:一種共聚物;d)EMULSIGEN-P,一種具有專有免疫刺激劑的水包油型(o/w)乳液;e)Carbigen為一種交聯聚合物;f)EMULSIGEN-75:一種包含具有交聯聚合物之水包油型(o/w)乳液的雙重佐劑;g)ISA 70為一種油包水型(w/o)乳液。Preferably, the adjuvant is selected from the group consisting of: a) EMULSIGEN : an oil-in-water emulsion (o/w); b) EMULSIGEN-D An oil-in-water (o/w) emulsion with dimethyldi-octadecyl ammonium bromide (DDA); c) Polygen: a copolymer; d) EMULSIGEN-P , an oil-in-water (o/w) emulsion with a proprietary immunostimulant; e) Carbigen is a cross-linked polymer; f) EMULSIGEN-75 : A dual adjuvant comprising an oil-in-water (o/w) emulsion having a crosslinked polymer; g) ISA 70 is a water-in-oil (w/o) emulsion.
最佳地,該等佐劑為水包油型乳液,諸如選自由以下各物組成之群的基於emulsigen之佐劑:EMULSIGEN、EMULSIGEN-D、EMULSIGEN-P、EMULSIGEN-75、EMULSIGEN及EMULSIGEN-P。最佳EMULSIGEN及EMULSIGEN-P用於本發明之調配物中。Most preferably, the adjuvants are oil-in-water emulsions, such as emulsigen-based adjuvants selected from the group consisting of: EMULSIGEN ,EMULSIGEN-D ,EMULSIGEN-P ,EMULSIGEN-75 EMULSIGEN And EMULSIGEN-P . Best EMULSIGEN And EMULSIGEN-P It is used in the formulation of the present invention.
根據另一態樣,如本文中所提供之醫藥/疫苗組合物包含一或多種抗原。較佳地,其他抗原為禽類或哺乳動物病原體之抗原。根據另一實施例,其他抗原為其他流感抗原,諸如流感病毒之血球凝集素H3、H7、H9或其他任何血球凝集素。該(等)其他抗原可以純化形式、作為抗原製劑之部分添加,以經滅殺微生物之形式或以經修飾之活微生物之形式添加。According to another aspect, a pharmaceutical/vaccine composition as provided herein comprises one or more antigens. Preferably, the other antigen is an antigen of an avian or mammalian pathogen. According to another embodiment, the other antigen is another influenza antigen, such as the hemagglutinin H3, H7, H9 or any other hemagglutinin of the influenza virus. The (or other) antigen may be added in purified form, as part of the antigen preparation, in the form of a killed microorganism or in the form of a modified live microorganism.
如本文中所使用之術語"抗原"意謂(但不限於)肽、多肽、醣肽或多醣,其能夠與免疫系統之抗原識別分子(諸如免疫球蛋白(抗體)或T細胞抗原受體)特異性交互作用以便在該抗原所投與之宿主中引發、活化或刺激針對該抗原的免疫反應。術語"抗原"亦指核酸分子,較佳為DNA分子或RNA分子,其各自編碼且表現能夠與免疫系統之抗原識別分子(諸如免疫球蛋白(抗體)或T細胞抗原受體)特異性交互作用以便引發、活化或刺激針對由該核酸分子所編碼之抗原的免疫反應的狀、多肽或醣肽。用於製備根據本發明使用之醫藥組合物的抗原為微生物或該微生物之抗原部分及/或製劑。就此而言,如本文中所使用之術語"免疫"意謂(但不限於)免疫反應之任何引起或增強。術語"免疫反應"已於上文中加以描述。The term "antigen" as used herein means, but is not limited to, a peptide, polypeptide, glycopeptide or polysaccharide that is capable of interacting with an antigen recognition molecule of the immune system (such as an immunoglobulin (antibody) or a T cell antigen receptor). A specific interaction is to initiate, activate or stimulate an immune response against the antigen in the host to which the antigen is administered. The term "antigen" also refers to nucleic acid molecules, preferably DNA molecules or RNA molecules, each encoding and expressing a specific interaction with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or a T cell antigen receptor. A trait, polypeptide or glycopeptide that elicits, activates or stimulates an immune response against an antigen encoded by the nucleic acid molecule. The antigen used to prepare the pharmaceutical composition for use in accordance with the present invention is a microorganism or an antigenic portion and/or formulation of the microorganism. In this regard, the term "immunization" as used herein means, but is not limited to, any cause or enhancement of an immune response. The term "immune response" has been described above.
流感疫苗之投藥策略已熟知於此項技術。本發明預期滅活病毒疫苗及減毒病毒疫苗之黏膜接種策略。儘管黏膜可由局部傳遞疫苗標靶導向,但多種策略皆已用於將免疫原性蛋白傳遞至黏膜。The vaccine strategy for influenza vaccines is well known in the art. The present invention contemplates a mucosal vaccination strategy for inactivated viral vaccines and attenuated viral vaccines. Although mucosal membranes can be targeted by local delivery vaccine targets, a variety of strategies have been used to deliver immunogenic proteins to the mucosa.
在一特定實施例中,可將疫苗與霍亂毒素(諸如霍亂毒素B或霍亂毒素A/B嵌合體)混合或作為結合型或嵌合型融合蛋白投藥(Hajishengallis,J Immunol.,154:4322-32,1995;Jobling及Holmes,Infect Immun.,60:4915-24,1992 )。基於使用霍亂毒素B次單位之黏膜疫苗已加以描述(Lebens及Holmgren,Dev Biol stand 82:215-27,1994) 。在另一實施例中,可製備與熱不穩定性腸毒素(LT)之混合物用於黏膜接種。In a specific embodiment, the vaccine can be mixed with cholera toxin (such as cholera toxin B or cholera toxin A/B chimera) or as a binding or chimeric fusion protein ( Hajishengallis, J Immunol., 154:4322- 32, 1995; Jobling and Holmes, Infect Immun., 60:4915-24, 1992 ). Mucosal vaccines based on the use of B-units of cholera toxin have been described ( Lebens and Holmgren, Dev Biol stand 82: 215-27, 1994) . In another embodiment, a mixture with heat labile enterotoxin (LT) can be prepared for mucosal inoculation.
其他黏膜免疫策略包括將病毒囊封於微膠囊中(US 5,075,109、US 5,820,883及US 5,853,763)及使用免疫增強性膜質載劑(WO 98/0558)。經口投藥之免疫原之免疫原性可藉由使用紅血球(rbc)或rbc殘骸(US 5,643,577)或藉由使用藍舌病抗原(US 5,690,938)而得以增強。Other mucosal immunization strategies include encapsulation of the virus in microcapsules (US 5,075,109, US 5,820,883 and US 5,853,763) and the use of immunoenhancing membrane carriers (WO 98/0558). The immunogenicity of an orally administered immunogen can be enhanced by the use of red blood cells (rbc) or rbc residues (US 5,643,577) or by the use of bluetongue antigens (US 5,690,938).
根據另一態樣,本發明係關於一種製備如上所述之醫藥/疫苗組合物的方法,較佳為一種製備包含如上所述之經桿狀病毒表現之重組性H5蛋白之疫苗的方法。通常,此方法包括以下步驟:i)將構築體轉染至病毒內,其中該構築體包含如本文中所述之重組性H5 cDNA;ii)用經轉染之病毒感染生長培養基中之細胞;iii)促使病毒表現如本文中所述之重組性H5蛋白;iv)自培養物回收經表現之H5蛋白;及v)藉由將經表現之H5蛋白與適當佐劑及/或其他醫藥學上可接受之載劑摻混而製備組合物。According to another aspect, the invention relates to a method of preparing a pharmaceutical/vaccine composition as described above, preferably a method of preparing a vaccine comprising a recombinant H5 protein expressed by a baculovirus as described above. Typically, the method comprises the steps of: i) transfecting a construct into a virus, wherein the construct comprises a recombinant H5 cDNA as described herein; ii) infecting cells in the growth medium with the transfected virus; Iii) promoting the expression of a recombinant H5 protein as described herein; iv) recovering the expressed H5 protein from the culture; and v) by expressing the H5 protein with an appropriate adjuvant and/or other medicinal The composition is prepared by admixture of acceptable carriers.
較佳佐劑為上述彼等佐劑。因此,根據另一態樣,用於激發防禦流感感染之免疫反應之抗原組合物(諸如疫苗)的製備方法包含i)製備及回收H5蛋白,及ii)將此蛋白與適當佐劑混合。Preferred adjuvants are the above adjuvants. Thus, according to another aspect, a method of preparing an antigenic composition (such as a vaccine) for eliciting an immune response against influenza infection comprises i) preparing and recovering the H5 protein, and ii) mixing the protein with a suitable adjuvant.
此外,本發明之疫苗組合物亦可包括稀釋劑、等張劑、穩定劑及/或防腐劑。稀釋劑可包括水、生理食鹽水、右旋糖、乙醇、甘油及類似物。等張劑尤其可包括無機鹽或有機鹽(例如氯化鈉)、右旋糖、甘露糖醇、山梨糖醇及乳糖、醣類、海藻糖、甘露糖醇、蔗糖。穩定劑尤其包括白蛋白及乙二胺四乙酸之鹼金屬鹽。適當佐劑為上述彼等佐劑。In addition, the vaccine compositions of the present invention may also include diluents, isotonic agents, stabilizers, and/or preservatives. Diluents can include water, physiological saline, dextrose, ethanol, glycerol, and the like. The isotonic agents may especially include inorganic or organic salts (e.g., sodium chloride), dextrose, mannitol, sorbitol, and lactose, saccharides, trehalose, mannitol, sucrose. Stabilizers include, inter alia, albumin and alkali metal salts of ethylenediaminetetraacetic acid. Suitable adjuvants are those mentioned above.
如本文中所提供之H5蛋白、編碼任何該等H5蛋白之核酸分子、包含任何該等核酸分子(編碼如本文中所述之任何該等H5蛋白)的載體以及包含該H5蛋白、核酸分子或載體中之任一者的任何醫藥/疫苗組合物可用作藥物,較佳用於治療及預防由流感病毒、最佳由流感A病毒引起的感染。如本文中所提供之H5蛋白、編碼任何該等H5蛋白之核酸分子、包含任何該等核酸分子(編碼如本文中所述之任何該等H5蛋白)之載體以及包含如本文中所述之該H5蛋白、核酸分子或載體中之任一者之任何醫藥/疫苗組合物可用於人類之治療或預防且可用於獸醫藥物中。當用於獸醫藥物中時,較佳為治療禽類,較佳鳥、雞、鴨、火雞及類似動物;以及哺乳動物,較佳豬、牛、馬、海豹、駱駝、狗、貓、倉鼠、小鼠及類似動物。An H5 protein as provided herein, a nucleic acid molecule encoding any of the H5 proteins, a vector comprising any of the nucleic acid molecules (encoding any of the H5 proteins as described herein), and comprising the H5 protein, nucleic acid molecule or Any of the pharmaceutical/vaccine compositions of any of the vectors can be used as a medicament, preferably for the treatment and prevention of infections caused by influenza viruses, preferably by influenza A viruses. An H5 protein as provided herein, a nucleic acid molecule encoding any of the H5 proteins, a vector comprising any of the nucleic acid molecules (encoding any of the H5 proteins as described herein), and comprising as described herein Any pharmaceutical/vaccine composition of any of the H5 proteins, nucleic acid molecules or vectors can be used in the treatment or prevention of humans and can be used in veterinary medicine. When used in veterinary medicine, it is preferred to treat birds, preferably birds, chickens, ducks, turkeys and the like; and mammals, preferably pigs, cows, horses, seals, camels, dogs, cats, hamsters, Mice and similar animals.
因此,根據另一態樣,本發明係關於如本文中所提供之H5蛋白、編碼任何該等H5蛋白之核酸分子、包含任何該等核酸分子(編碼如本文中所述之任何該等H5蛋白)之載體以及包含如本文中所述之該H5蛋白、核酸分子或載體中之任一者之任何醫藥/疫苗組合物的用途,其可用作藥物、較佳用作人類藥物及/或獸醫藥物。Thus, according to another aspect, the invention relates to an H5 protein, a nucleic acid molecule encoding any of the H5 proteins, comprising any of the nucleic acid molecules (encoding any of the H5 proteins as described herein). And a use of any of the pharmaceutical/vaccine compositions comprising any of the H5 proteins, nucleic acid molecules or vectors as described herein, for use as a medicament, preferably as a human medicament and/or veterinary drug.
此外,如本文中所提供之H5蛋白、如本文中所述之編碼任何該等H5蛋白之核酸分子、包含任何該等核酸分子(編碼任何該H5蛋白)之載體可用於製備如本文中所述之醫藥組合物,以便預防或治療由流感病毒引起之感染。如上所述,彼等醫藥組合物/疫苗組合物可用於人類之治療及/或預防以及動物之治療及/或預防,該等動物諸如禽類,較佳鳥、雞、鴨、火雞及類似動物,以及哺乳動物,較佳豬、牛、馬、海豹、駱駝、狗、貓、倉鼠、小鼠及類似動物。Furthermore, a H5 protein as provided herein, a nucleic acid molecule encoding any of the H5 proteins as described herein, a vector comprising any of the nucleic acid molecules encoding any of the H5 proteins, can be used to prepare as described herein A pharmaceutical composition for preventing or treating an infection caused by an influenza virus. As mentioned above, their pharmaceutical compositions/vaccine compositions are useful in the treatment and/or prevention of humans, such as birds, preferably birds, chickens, ducks, turkeys and the like, and for the treatment and/or prevention of animals. And mammals, preferably pigs, cows, horses, seals, camels, dogs, cats, hamsters, mice and the like.
如本文中所提供之H5蛋白、如本文中所述之編碼任何該等H5蛋白之核酸分子、包含任何該等核酸分子(編碼任何該等H5蛋白)之載體可用於製備如本文中所述之醫藥組合物,該醫藥組合物適於治療及預防較佳由禽、豬或人類流感病毒或其任何組合或混合之流感病毒感染。A H5 protein as provided herein, a nucleic acid molecule encoding any of the H5 proteins as described herein, a vector comprising any of the nucleic acid molecules (encoding any of the H5 proteins), can be used to prepare a preparation as described herein A pharmaceutical composition suitable for the treatment and prevention of influenza virus infection, preferably by avian, porcine or human influenza virus or any combination or mixture thereof.
根據另一態樣,本發明亦係關於一種治療或預防流感病毒感染之方法,其中該方法包含將治療有效量之如本文中所述之H5蛋白投與需要該治療之受檢者。此外,本發明亦係關於一種治療或預防流感病毒感染之方法,其中該方法包含將治療有效量的如本文中所述之編碼如本文中所述之任何H5蛋白的任何H5核酸分子或載體投與需要該治療之受檢者。此外,本發明亦係關於一種治療或預防流感病毒感染之方法,其中該方法包含將治療有效量之包含如本文中所述之任何該H5蛋白、核酸分子或載體之疫苗投與需要該治療之受檢者。有需要之受檢者可為人類以及動物,較佳為禽類,甚至更佳為鳥、雞、鴨、火雞;或哺乳動物,較佳為豬、牛、馬、海豹、駱駝、狗、貓、倉鼠、小鼠及類似動物。According to another aspect, the invention is also directed to a method of treating or preventing an influenza virus infection, wherein the method comprises administering a therapeutically effective amount of an H5 protein as described herein to a subject in need of such treatment. Furthermore, the invention relates to a method of treating or preventing an influenza virus infection, wherein the method comprises administering a therapeutically effective amount of any H5 nucleic acid molecule or vector encoding any of the H5 proteins as described herein as described herein. With the subject in need of the treatment. Furthermore, the invention relates to a method of treating or preventing an influenza virus infection, wherein the method comprises administering a therapeutically effective amount of a vaccine comprising any of the H5 proteins, nucleic acid molecules or vectors as described herein, in need of such treatment Subject. Subjects in need may be humans and animals, preferably birds, even more preferably birds, chickens, ducks, turkeys; or mammals, preferably pigs, cows, horses, seals, camels, dogs, cats , hamsters, mice and similar animals.
較佳地,當對雞進行接種時,可在1日齡時或1日齡之後(例如在10日齡時,或在1日齡至10日齡時,或在10日齡時或10日齡之後)使用如本文中所述之H5蛋白接種。Preferably, when the chicken is inoculated, it may be 1 day old or 1 day old (for example, at 10 days old, or at 1 day to 10 days old, or at 10 days old or 10 days). After age, vaccination with H5 protein as described herein is used.
可藉由投與任何H5蛋白、編碼該任何H5蛋白之核酸分子或載體或如本文中所述之任何醫藥/疫苗組合物治療的流感感染較佳係由禽、豬或人類流感病毒或其任何組合或混合引起。An influenza infection which can be treated by administering any H5 protein, a nucleic acid molecule or vector encoding the H5 protein, or any of the pharmaceutical/vaccine compositions as described herein is preferably avian, porcine or human influenza virus or any thereof Caused by combination or mixing.
根據另一態樣,本發明係關於部分套組,其包含:i)如本文中所述之該H5蛋白、編碼任何該H5蛋白之核酸分子或載體或包含如本文中所述之該H5蛋白、核酸分子或載體中任一者之任何醫藥/疫苗組合物中的任一者;及ii)指示該H5蛋白、核酸分子、載體或疫苗於治療或預防由流感病毒引起之感染之使用的包裝插頁。當對雞進行接種時,可在1日齡時或1日齡之後使用如本文中所述之H5蛋白接種。According to another aspect, the invention relates to a partial kit comprising: i) the H5 protein as described herein, a nucleic acid molecule or vector encoding any of the H5 proteins or comprising the H5 protein as described herein Any of any of the pharmaceutical/vaccine compositions of any of the nucleic acid molecules or vectors; and ii) a package indicating the use of the H5 protein, nucleic acid molecule, vector or vaccine for treating or preventing an infection caused by an influenza virus insert. When the chicken is inoculated, the H5 protein inoculation as described herein can be used at 1 day of age or after 1 day of age.
根據另一實施例,彼部分套組包含禽類或哺乳動物病原體之至少另一種抗原及指示彼另外抗原之醫藥、人類或獸醫學用途的資訊。According to another embodiment, the partial kit comprises at least one other antigen of the avian or mammalian pathogen and information indicative of the pharmaceutical, human or veterinary use of the additional antigen.
以下實例闡述本發明之較佳物質及程序。然而應瞭解,該等實例僅為說明而提供,且不應視為對本發明之整體範圍之限制。The following examples illustrate preferred materials and procedures of the invention. However, it is to be understood that the examples are provided for illustration only and are not to be construed as limiting the scope of the invention.
建構編碼及表現HA H5抗原的重組性桿狀病毒如下生成含有H5 HA抗原的重組性桿狀病毒:化學合成H5 HA(SEQ ID NO:2)之編碼序列且將其次選殖入轉移載體pVL1392(BD Biosciences Pharmingen,San Diego,CA)內。藉由使用寡核苷酸引子及QuikChange定點誘變套組(Stratagene,La Jolla,CA)生成H5 HA MutK+(SEQ ID NO:4)且將其次選殖入轉移載體pVL1392(BD Biosciences Pharmingen,San Diego,CA)內。接著用DiamondBac(Sigma)桿狀病毒DNA將含有編碼H5 HA抗原(SEQ ID NO:2)及H5 HA MutK+抗原(SEQ ID NO:4)之基因的pVL1392質體共轉染入Sf9昆蟲細胞(BD Biosciences Pharmingen)內以生成含有編碼SEQ ID NO:2之基因H5 HA及編碼SEQ ID NO:4之基因H5 HA mutK+的重組性桿狀病毒。將含有編碼H5 HA(SEQ ID NO:2)及H5 HA MutK+(SEQ ID NO:4)之基因的重組性桿狀病毒進行空斑純化,且將主種子病毒(Master Seed Virus;MSV)於SF+細胞株上繁殖,製成等分試樣且在-70℃下儲存。如由多株血清或單株抗體以間接螢光抗體檢定或西方墨點法所偵測,如上所述經H5 HA桿狀病毒感染(以生成MSV或工作種子病毒(Working Seed Virus))之昆蟲細胞表現H5 HA抗原(SEQ ID NO:2)及H5 HA MutK+抗原(SEQ ID NO:4)。Construction of a recombinant baculovirus encoding and expressing the HA H5 antigen generates a recombinant baculovirus containing the H5 HA antigen by chemically synthesizing the coding sequence of H5 HA (SEQ ID NO: 2) and subsequencing it into the transfer vector pVL1392 ( Within BD Biosciences Pharmingen, San Diego, CA). By using oligonucleotide primers and QuikChange The site-directed mutagenesis kit (Stratagene, La Jolla, CA) generated H5 HA MutK+ (SEQ ID NO: 4) and was subcloned into the transfer vector pVL1392 (BD Biosciences Pharmingen, San Diego, CA). Then use DiamondBac (Sigma) baculovirus DNA co-transfects pVL1392 plastids containing the gene encoding H5 HA antigen (SEQ ID NO: 2) and H5 HA MutK+ antigen (SEQ ID NO: 4) into Sf9 insect cells (BD Biosciences Pharmingen) A recombinant baculovirus containing the gene H5 HA encoding SEQ ID NO: 2 and the gene H5 HA mutK+ encoding SEQ ID NO: 4 was generated. Recombinant baculovirus containing the gene encoding H5 HA (SEQ ID NO: 2) and H5 HA MutK+ (SEQ ID NO: 4) was plaque-purified, and the primary seed virus (MSV) was SF+ The cell lines were propagated, aliquots were made and stored at -70 °C. Insects infected with H5 HA baculovirus (to generate MSV or Working Seed Virus) as described above by multiple serum or monoclonal antibodies by indirect fluorescent antibody assay or Western blot method The cells express the H5 HA antigen (SEQ ID NO: 2) and the H5 HA MutK+ antigen (SEQ ID NO: 4).
用適量重組性桿狀病毒(分別為H5 HA及H5 HA MutK+)接種後,接著將含有SF+細胞(Protein Sciences,Inc.,Meriden,CT)的旋轉瓶在27±2℃下培育7天且在彼期間以100 rpm攪拌。該等旋轉瓶使用通氣蓋以使空氣流動。收穫含有經桿狀病毒感染之SF+細胞的原全細胞培養物及各培養物之細胞培養上清液。After inoculation with appropriate amounts of recombinant baculovirus (H5 HA and H5 HA MutK+, respectively), the rotating flask containing SF+ cells (Protein Sciences, Inc., Meriden, CT) was then incubated at 27 ± 2 ° C for 7 days and at Stir at 100 rpm during the period. These rotating bottles use a venting cap to allow air to flow. The original whole cell culture containing the baculovirus-infected SF+ cells and the cell culture supernatant of each culture were harvested.
製備包含HA H5抗原之醫藥組合物(疫苗)收穫在昆蟲細胞中由基於桿狀病毒之表現系統表現的原全細胞H5 HA蛋白及H5 HA Mutk+蛋白。將桿狀病毒在5 mM環化二元伸乙基亞胺(BEI)(最終濃度)之存在下、在約32℃與39℃之間滅活72至96小時。滅活完成後,添加0.3 M硫代硫酸鈉溶液直至最終濃度為5 mM,以中和任何殘餘BEI。中和後,添加各種佐劑且生成以下疫苗/醫藥組合物。A pharmaceutical composition (vaccine) comprising the HA H5 antigen is prepared to harvest the original whole cell H5 HA protein and the H5 HA Mutk+ protein expressed in insect cells by a baculovirus-based expression system. The baculovirus was inactivated in the presence of 5 mM cyclized binary ethylenimine (BEI) (final concentration) between about 32 ° C and 39 ° C for 72 to 96 hours. After the inactivation was completed, a 0.3 M sodium thiosulfate solution was added until a final concentration of 5 mM to neutralize any residual BEI. After neutralization, various adjuvants were added and the following vaccine/pharmaceutical compositions were produced.
對豬進行接種以防禦禽流感1.引論 此研究之目的係測定含有重組性H5血球凝集素(HA)抗原之粗提取物之實驗性疫苗在豬體內誘導血球凝集抑制(HI)效價的能力。用H5 HA抗原評價各種佐劑。Inoculation of pigs against avian influenza 1. Introduction The purpose of this study was to determine the hematopoietic agglutination (HI) titer in pigs by measuring an experimental vaccine containing a crude extract of recombinant H5 hemagglutinin (HA) antigen. ability. Various adjuvants were evaluated using H5 HA antigen.
此研究中評價含有來自習知H5 HA或H5 HA MutK+之抗原的HA H5原型。習知H5 HA係源自A/鴨/China/E319-2/03,而H5 HA MutK+由習知H5 HA組成,其經工程化以在S120N、D150N、S223N及328mutK+上含有三個特定胺基酸變異。其亦含有胺基酸94N。H5 HA Mut K+中之特定胺基酸變異產生更接近類似於A/HK/213/03之HA的H5 HA。目前認為A/HK/213/03之H5 HA之胺基酸組成有助於H5 HA之抗體識別。The HA H5 prototype containing the antigen from the conventional H5 HA or H5 HA MutK+ was evaluated in this study. The conventional H5 HA line is derived from A/Duck/China/E319-2/03, while H5 HA MutK+ consists of the conventional H5 HA, which is engineered to contain three specific amine groups on S120N, D150N, S223N and 328mutK+. Acidic variation. It also contains the amino acid 94N. The specific amino acid variation in H5 HA Mut K+ produces H5 HA that is closer to HA similar to A/HK/213/03. It is currently believed that the amino acid composition of H5 HA of A/HK/213/03 contributes to the antibody recognition of H5 HA.
2.研究設計:
研究開始時,仔豬為3週±5日齡。研究開始時,仔豬在臨床上為健康的。在研究第0日、第21日及第35日獲得血樣。At the beginning of the study, piglets were 3 weeks ± 5 days old. At the beginning of the study, the piglets were clinically healthy. Blood samples were obtained on Study Days 0, 21, and 35.
在研究第1日至第35日每日觀測全部研究動物之一般健康狀況。在每次接種後七天,每天查看注射部位且記錄可見反應。在研究日第35日動物研究期結束時,對全部動物施以無痛致死術。The general health status of all study animals was observed daily from study day 1 to day 35. Seven days after each inoculation, the injection site was viewed daily and a visible response was recorded. At the end of the animal study period on the 35th day of the study day, all animals were given euthanasia.
3.疫苗將如實例2中所述之疫苗501至514用於豬接種研究。3. Vaccines Vaccines 501 to 514 as described in Example 2 were used for pig vaccination studies.
4.血球凝集素抑制檢定在第0日及第21日用含有H5 HA之原型對豬進行接種。在第0日、第21日、第35日收集豬血清以便藉由血球凝集抑制(HI)檢定進行評價。進行HI檢定以偵測HA特異性抗體之存在。異源H5N2病毒(A/雞/Mexico/232/94)係以四個血球凝集單位[4 HA單位]之濃度用於HI檢定中。隨後在U形底微量滴定板中,將PBS中之連續兩倍血清稀釋液與等體積(25 μL)(含有4 HA單位)之病毒混合,且在室溫(約25℃)下培育30分鐘。將PBS中之濃度為0.5%之雞紅血球添加至含有血清-病毒之孔中且在室溫下培育40分鐘。以觀測到血球凝集抑制的最高血清稀釋度之倒數確定HI效價。4. Hemagglutinin inhibition assay Pigs were inoculated on day 0 and day 21 with a prototype containing H5 HA. Pig serum was collected on day 0, day 21, and day 35 for evaluation by hemagglutination inhibition (HI) assay. A HI assay was performed to detect the presence of HA-specific antibodies. Heterologous H5N2 virus (A/chicken/Mexico/232/94) was used in the HI assay at a concentration of four hemagglutination units [4 HA units]. Subsequently, serial two-fold serum dilutions in PBS were mixed with an equal volume (25 μL) of virus containing 4 HA units in a U-bottom microtiter plate and incubated for 30 minutes at room temperature (about 25 ° C). . Chicken red blood cells at a concentration of 0.5% in PBS were added to the wells containing serum-virus and incubated for 40 minutes at room temperature. The HI titer was determined by the reciprocal of the highest serum dilution at which hemagglutination inhibition was observed.
5.結果HI測試使用Mexican政府法定之H5N1抗原(A/雞/Mexico/232/94)[4 HA單位],接種方案為第0日及第21日1×1 mL。5. Results The HI test used the Mexican government's legal H5N1 antigen (A/chicken/Mexico/232/94) [4 HA units], and the vaccination schedule was 1 x 1 mL on days 0 and 21.
BIV H5(來源於流感A病毒(A/鴨/China/E319-2/03(H5N1))BIV H5 K+(突變之BIV H5,包括S120N、D155N、S223N,且增添328K+)結果證明大部分疫苗組合物在接種豬中引發免疫反應。特定言之,大部分疫苗組合物引起血清轉化,其意謂大部分接種豬產生針對HI檢定中所使用之禽流感病毒的特異性抗體。總而言之,結果清楚且無疑地證明本發明創見極其奏效。藉由用禽流感病毒之相關抗原對豬進行接種可大大降低豬(第二物種之動物)經禽流感病毒(第一物種之病原體)廣泛流行性感染之風險。此已得到清楚證明。此外,依據此接種概念,禽流感病毒對哺乳動物(包括人類)之傳染性及順應性大大降低。豬為禽病原體(包括禽流感病毒)之最重要宿主之一。若病毒在豬體內之複製且因此禽流感對豬之順應性之風險大大降低且得以控制,則禽流感病毒對人類之任何順應性之風險亦大大降低。在投與抗原產生較低HI效價(意謂效價低於30)之情況下,需要用抗原進一步加強以進一步改良HI效價且增強接種豬體內之免疫保護。因此,效價低並不意謂無法獲得保護,其僅教示似乎需要進一步加強以改良免疫反應。接種豬體內可量測到免疫反應證明作為本發明依據之本發明創見極其奏效。換而言之,本文所提供之實驗清楚且無疑地證明本發明之創見可奏效。BIV H5 (derived from influenza A virus (A/Duck/China/E319-2/03 (H5N1)) BIV H5 K+ (mutated BIV H5, including S120N, D155N, S223N, and added 328K+) results demonstrate most vaccine combinations Initiating an immune response in vaccinated pigs. In particular, most vaccine compositions cause seroconversion, which means that most vaccinated pigs produce antibodies specific for the avian influenza virus used in the HI assay. In summary, the results are clear and It is undoubtedly proved that the present invention is extremely effective. By vaccinating pigs with the antigen associated with avian influenza virus, the risk of widespread epidemic infection of avian influenza virus (pathogen of the first species) by pigs (animals of the second species) can be greatly reduced. This has been clearly demonstrated. In addition, according to this concept of vaccination, the infectivity and compliance of avian influenza viruses to mammals, including humans, is greatly reduced. Pigs are one of the most important hosts for avian pathogens, including avian influenza viruses. If the virus replicates in pigs and the risk of avian influenza compliance to pigs is greatly reduced and controlled, the risk of any compliance with avian influenza virus is also greatly reduced. In the case where administration of the antigen produces a lower HI titer (meaning that the titer is less than 30), further enhancement of the antigen is required to further improve the HI titer and enhance the immune protection in the vaccinated pig. Therefore, the titer is low and It does not mean that protection cannot be obtained, and it only teaches that further strengthening seems to be needed to improve the immune response. The immune response can be measured in vaccinated pigs to prove that the present invention, which is the basis of the present invention, is extremely effective. In other words, the experiments provided herein are clear. And it is undoubtedly proved that the inventive concept of the present invention can be effective.
對鳥進行接種以防禦禽流感1.引論 此研究之目的係測定含有重組性H5mutk+血球凝集素(H5 HA mutk+)抗原之粗提取物之實驗性疫苗在雞體內誘導血球凝集抑制(HI)之能力。此外,習知重組性H5抗原(H5 HA)以及滅活疫苗VolvacAI(Boehringer Ingelheim Vetmedica,Mexico)用於對照。此外,用H5 HA抗原評價多種佐劑。Inoculation of birds to protect against avian influenza 1. Introduction The purpose of this study was to determine an experimental vaccine containing a crude extract of recombinant H5mutk + hemagglutinin (H5 HA mutk+) antigen to induce hemagglutination inhibition (HI) in chickens. ability. In addition, conventional recombinant H5 antigen (H5 HA) and inactivated vaccine Volvac AI (Boehringer Ingelheim Vetmedica, Mexico) was used for the control. In addition, various adjuvants were evaluated using the H5 HA antigen.
2.研究設計:將SPF鳥(15-25隻)在1日齡或10日齡時,獨立地用0.5 ml不同實驗性疫苗在頸背部藉由皮下途徑接種;實驗期間將所有鳥供養於隔離器中。不限量提供饋食及水。用H5N2高病原性禽流感病毒株接種後第31日或第32日進行激發。2. Study design: SPF birds (15-25) were independently inoculated with 0.5 ml of different experimental vaccines in the neck and back by subcutaneous route at 1 day or 10 days of age; all birds were kept isolated during the experiment. In the device. Unlimited feeding and water. Excitation was performed on the 31st or 32nd day after inoculation with the H5N2 high pathogenic avian influenza virus strain.
藉由在接種後第15日、第30日自鳥頸靜脈放血獲得血清樣本。為獲得抗體效價,在進行如實例3中所述之血球凝集抑制(HI)測試之前,將所得血清在4℃下儲存。Serum samples were obtained by exsanguination from the jugular vein on the 15th and 30th day after inoculation. To obtain antibody titers, the resulting serum was stored at 4 °C prior to performing the hemagglutination inhibition (HI) test as described in Example 3.
3.疫苗及激發病毒:獨立評價四種不同調配物:1.習知油乳液H5HA Mut k+: 基於Boehringer Ingelheim Vetmedica程序將H5 HA mutk+抗原調配於油乳液(弗氏不完全佐劑(Freund incomplete adjuvant))中。3. Vaccine and challenge virus: Independent evaluation of four different formulations: 1. Conventional oil emulsion H5HA Mut k+: H5 HA mutk+ antigen was formulated on oil emulsion based on Boehringer Ingelheim Vetmedica program (Freund incomplete adjuvant) ))in.
2.Seppic H5HA Mut k+: 基於供應商建議,用非習知之佐劑(ISA 206,W/O/W,獲自Seppic)調配H5 HA mutk+抗原。2. Seppic H5HA Mut k+: H5 HA mutk+ antigen was formulated with a non-known adjuvant (ISA 206, W/O/W, obtained from Seppic) based on supplier recommendations.
3.H5HA習知油乳液: 基於Boehringer Ingelheim Vetmedica程序將H5 HA抗原調配於油乳液(弗氏不完全佐劑)中。3. H5HA conventional oil emulsion: The H5 HA antigen was formulated in an oil emulsion (Freund's incomplete adjuvant) based on the Boehringer Ingelheim Vetmedica program.
4.Seppic H5HA: 基於供應商建議,用非習知之佐劑(ISA 206,W/O/W,獲自Seppic)調配H5 HA抗原。4. Seppic H5HA: H5 HA antigen was formulated with a non-known adjuvant (ISA 206, W/O/W, obtained from Seppic) based on supplier recommendations.
將禽流感Boehringer Ingelheim Vetmedica油乳液疫苗用作對照VolvacAI(Boehringer Ingelheim Vetmedica,Mexico)。Avian influenza Boehringer Ingelheim Vetmedica oil emulsion vaccine was used as a control Volvac AI (Boehringer Ingelheim Vetmedica, Mexico).
藉由鼻內途徑,每隻鳥用0.2 ml含有106.7 CEID之H5N2激發病毒接種來於經接種及未經接種之雞體內進行激發。激發後,記錄病徵及死亡率。接種後第十日,根據動物實驗程序將所有存活雞施以無痛致死術。Each bird was challenged with inoculated and unvaccinated chickens by intranasal route with 0.2 ml of H5N2 challenge virus containing 10 6.7 CEID. After the challenge, the symptoms and mortality were recorded. On the tenth day after inoculation, all surviving chickens were subjected to euthanasia according to the animal experiment procedure.
4.結果:結果描述於下表中:
根據OIE標準將陽性血清效價以log2 4考量。基於此標準,血清學結果為陰性,但與基線相比,觀測到某些陽性值。在1日齡或10日齡接種之鳥體內觀測到用油佐劑及H5HA Mut k+抗原調配之疫苗具有最佳的血清學效價。經觀測,用Seppic及H5HA抗原調配之原型具有最低的血清學效價。在激發研究中觀測到,疫苗原型,尤其H5HA Mut k+抗原調配之習知油乳液疫苗,賦予保護作用。激發研究中觀測到Seppic H5HA具有最低的保護作用,死亡率為68%。相比之下,與在一日齡接種之鳥相比,在10日接種之鳥體內觀測到最高的血清學效價。Positive serum titers were considered in log 2 4 according to the OIE criteria. Based on this criteria, serological results were negative, but some positive values were observed compared to baseline. Vaccines formulated with oil adjuvants and H5HA Mut k+ antigens were observed to have optimal serological potency in birds vaccinated at 1 day or 10 days of age. The prototypes formulated with Seppic and H5HA antigens have been observed to have the lowest serological titers. It has been observed in the challenge study that the vaccine prototype, especially the conventional oil emulsion vaccine formulated with H5HA Mut k+ antigen, confers protection. Seppic H5HA was observed to have the lowest protective effect in the challenge study with a mortality rate of 68%. In contrast, the highest serological titer was observed in birds vaccinated on day 10 compared to birds vaccinated at one day old.
<110> 美商百靈佳殷格輸家畜藥品公司<120> 新穎H5蛋白質、編碼彼等之核酸分子及載體及其醫藥用途<130> Case 1-2150 <140> 096140489 <141> 2007-10-26 <150> 60/863142 <151> 2006-10-27 <160> 6 <170> patentIn version 3.3 <210> 1 <211> 551 <212> PRT <213> 禽流感病毒<400> 1 <210> 2 <211> 567 <212> PRT <213> 禽流感病毒<400> 2 <210> 3 <211> 568 <212> PRT <213> 禽流感病毒<400> 3 <210> 4 <211> 568 <212> PRT <213> 禽流感病毒<400> 4 <210> 5 <211> 263 <212> PRT <213> 禽流感病毒<400> 5 <210> 6 <211> 290 <212> PRT <213> 禽流感病毒<400> 6 <110> American Bailingjia Yinge Transmissible Livestock Pharmaceutical Company <120> Novel H5 protein, nucleic acid molecule and vector encoding the same and its medical use <130> Case 1-2150 <140> 096140489 <141> 2007-10- 26 <150> 60/863142 <151> 2006-10-27 <160> 6 <170> patentIn version 3.3 <210> 1 <211> 551 <212> PRT <213> Avian influenza virus <400> 1 <210> 2 <211> 567 <212> PRT <213> Avian Influenza Virus <400> 2 <210> 3 <211> 568 <212> PRT <213> Avian influenza virus <400> 3 <210> 4 <211> 568 <212> PRT <213> Avian influenza virus <400> 4 <210> 5 <211> 263 <212> PRT <213> Avian Influenza Virus <400> 5 <210> 6 <211> 290 <212> PRT <213> Avian Influenza Virus <400> 6
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Also Published As
Publication number | Publication date |
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CN101553248B (en) | 2012-09-19 |
CN101553248A (en) | 2009-10-07 |
UA99117C2 (en) | 2012-07-25 |
CL2007003102A1 (en) | 2008-04-18 |
AR063427A1 (en) | 2009-01-28 |
TW200825099A (en) | 2008-06-16 |
ZA200902023B (en) | 2010-05-26 |
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