Microsoft Word - 02-박만성.doc

Journal of Bacteriology and Virology 2013. Vol. 43, No. 1 p.9 17 http://dx.doi.org/10.4167/jbv.2013.43.1.9 Review Article Cell Culture-based Influenza Vaccines as Alternatives to Egg-based Vaccines Ilseob Lee 1,2, Jin Il Kim 1,2 and Man-Seong Park 1,2 * 1 Department of Microbiology, 2 Center for Medical Science Research, College of Medicine, Hallym University, Chuncheon, Gangwon-do, Korea Influenza viruses have raised public health concerns by seasonal epidemics and intermittent pandemics. Vaccination is considered as the most effective method for preventing influenza infection in humans. Current influenza vaccines are mostly produced in fertile chicken eggs. However, disadvantages of egg-based vaccines, such as egg dependency, labor-intensive manufacturing system, and huddle for large-scale output, allow us to make an alternative method. A cell-culture platform may be a fine alternative for the next generation vaccine technique. Compared with a classical egg-based method, cell-grown vaccines provide stable pipeline even in the pandemic situation with shorter lead-in times. In addition, cell-grown vaccines are flexible for altering production scales because stocked cell batches can be easily sub-cultured in large quantity without worrying avian diseases and a resultant decrease in egg production. By World Health Organization, MDCK, PER.C6, and Vero cells are only recommended for manufacturing influenza vaccines. In this review, we discuss the necessity, immunogenicity, and efficacy of cell-grown influenza vaccines compared with egg-based vaccines. Key Words: Egg, Influenza, MDCK, Vaccine, Vero 서론인플루엔자바이러스는 10~13종류의단백질을발현하는단일음성가닥의 8개의 RNA 분절로구성된바이러스 (single-stranded, negative-sense RNA virus) 로 Orthomyxoviridae 과 (family) 에속한다 (1). 바이러스는일반적으로호흡기를통해감염되어두통, 오한, 근육통과같은전신증상과호흡기증상을유발한다. 인플루엔자바이러스는 A, B, C의세가지형으로구분되는데, 주로 A 형과 B형이사람사이에유행하고있다. 특히 A형인플루엔자바이러스는표면당단백질인 hemagglutinin (HA) 과 neuraminidase (NA) 의항원성차이에의해다시아형 (subtype) 으로나눠지며, 현재까지 17개의 HA 아형과 10 개의 NA 아형이알려져있다 (2). 인플루엔자바이러스는매년겨울철에반복적으로유행하여공중보건학적문제를발생시키고있다. 세계보건기구 (World Health Organization, WHO) 에따르면, 겨울철에발생하는계절성유행성독감 (seasonal influenza) 은매년전세계적으로약 25만 ~50만명의사망자를발생시키고있는것으로보고되고있다 (3). 또한, 예상치못한시기에갑작스럽게발생하는인플루엔자대유행 (pandemic) 은매우심각한사회적, 경제적피해를발생시킨다. 지난 20세기세번의인플루엔자대유행이발생하였는데 (1918년; Received: December 15, 2012/ Revised: February 5, 2013/ Accepted: February 15, 2013 * Corresponding author: Man-Seong Park, Ph.D. Department of Microbiology, Center for Medical Science Research, College of Medicine, Hallym University, Chuncheon, Gangwon-do, 200-702, Korea. Phone: +82-33-248-2632, Fax: +82-33-252-2843, e-mail: ms0392@hallym.ac.kr or manseong.park@gmail.com ** This study was supported by a grant from the Korea Healthcare Technology R&D Project of the Ministry of Health & Welfare, Republic of Korea (Grant No. A103001). CC This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/license/by-nc/3.0/). 9

10 I Lee, et al. H1N1 아형, 1957년 ; H2N2 아형, 1968년 ; H3N2 아형 ), 1918년에는전세계적으로약 5천만명, 1957년과 1967년에는약백만명의사람이인플루엔자바이러스에의해사망하였다 (4). 더불어치사율이약 59%(2012년 8월 10 일기준, WHO) 에이르는고병원성 H5N1 조류인플루엔자 (highly pathogenic avian influenza, HPAI) 도매년지속적으로발생하고있다 (5). 현재다양한항바이러스제가사용또는개발되고있지만 (6), 인플루엔자바이러스감염에대응하는가장효과적인방법은백신을접종하는것으로알려져있다 (7). 현재사용되는계절성인플루엔자백신 (seasonal influenza vaccine) 은 A형인플루엔자바이러스중 H1N1, H3N2 아형항원과인플루엔자 B형바이러스항원으로구성된삼가백신 (trivalent influenza vaccine) 이다. 최근에는 WHO 의권고에따라인플루엔자 B형바이러스의두계통 (Yamagata 계통과 Victoria 계통 ) 항원을모두포함하는사가백신 (quadrivalent influenza vaccine) 의개발도이루어지고있다 (8, 9). 인플루엔자바이러스는항원성의변이가자주일어나매년 WHO에서전세계의역학조사결과 를바탕으로인플루엔자백신생산에사용될백신주를선정하고이를이용하여백신을생산한다 (10). 현재사용되고있는대부분의인플루엔자백신은유정란을이용하여생산된다. 1945년인플루엔자백신이첫사용허가를받은이후약 70여년동안유정란은인플루엔자백신을생산하는데사용되어 (11), 현재대량의인플루엔자백신을생산하기위한최적화시스템이구축되어있다. 하지만유정란을이용하여백신을생산하는경우유정란의안정적인공급및사용후폐기물처리와같은문제점들이지속적으로제기되어왔다. 이에따라세포를인플루엔자백신생산에이용하려는시도는 1980 년대부터시작되었으며 (12~15), 최근들어세포배양관련기술이발달함에따라세포를기반으로한인플루엔자백신개발에관련연구자및산업체들의관심이더욱증폭되고있는실정이다. 현재세포배양을기반으로하여생산된다양한바이러스성감염질환에대한예방백신이사용되고있다 (Table 1). 이러한사실은유정란기반백신생산의문제점을해결하기위한하나의대안으로세포배양기반예방백신 Table 1. Licensed cell-culture based viral vaccines in the United States. Virus Type Trade name Cell line Company Adenovirus Live No Trade Name WI-38 Barr Labs, Inc. Hepatitis A Inactivated Havrix MRC-5 Merck & Co, Inc. Inactivated VAQTA MRC-5 GlaxoSmithKline Biologicals Japanese Encephalitis Inactivated Ixiaro Vero Intercell Biomedical Measles Live-attenuated Attenuvax CEF Merck & Co, Inc. Poliovirus Inactivated IPOL Vero Sanofi Pasteur, SA Rabies Inactivated Imovax MRC-5 Sanofi Pasteur, SA Inactivated RabAvert CEF Novartis Rotavirus Live-attenuated ROTARIX Vero GlaxoSmithKline Biologicals Live-attenuated RotaTeq Vero Merck & Co., Inc. Vaccinia Live-attenuated ACAM2000 Vero Sanofi Pasteur Biologics Co. Varicella-zoster virus Live-attenuated Varivax MRC-5 Merck & Co, Inc. Live-attenuated Zostavax MRC-5 Merck & Co, Inc. MMR Live-attenuated M-M-R II CEF, CEF, WI-38 Merck & Co, Inc. MMRV Live-attenuated ProQuad CEF, CEF, WI-38, MRC-5 Merck & Co, Inc. Data are collected from U.S. Food and Drug Administration (FDA) Measles, Mumps, and Rubella Measles, Mumps, Rubella, and Varicella

Cell Culture-based Influenza Vaccines as Alternatives to Egg-based Vaccines 11 의생산공정을인플루엔자에적용할수있음을시사하고있다. 이에따라저자들은현재대두되고있는세포배양기반인플루엔자백신에대해고찰해보고자한다. 본론 1. 세포배양기반인플루엔자백신의필요성지난수십년간인플루엔자백신은유정란을이용하여생산되어왔기때문에유정란기반인플루엔자백신생산시스템은상당히안정화된방법으로여겨지고있다. 하지만유정란기반인플루엔자백신생산은많은양의유정란이안정적으로미리확보되어야하고, 생산과정이노동집약적이며다수의복잡한처리과정을거쳐야순수백신을얻을수있다는단점을갖고있다. 유정란기반인플루엔자백신생산에있어대두되는가장큰문제점은필요한만큼의백신을기간내에생산하기위해서유정란을적시에안정적으로확보하여야한다는것이다. 일반적으로 1도즈 (dose) 의인플루엔자백신을생산하기위해서는 1~2개의유정란이필요한것으로알려져있다 (16). 지난 2006~2007 인플루엔자바이러스유행시기에는전세계적으로약 4억 1,300만도즈의삼가백신 (trivalent vaccine) 이생산되었는데 (17), 이러한수치는상당한양의유정란이백신생산개시이전까지반드시준비되어야한다는것을의미한다. 하지만유정란생산시설이위치한지역에조류질병이유행하거나자연재해로인해유정란의공급에차질이빚어질가능성은항상존재한다 (12) 또한예상하지못한시기에갑작스러운인플루엔자대유행이발생하여단기간에백신수요가급격히증가할경우, 유정란의공급이신속하게이루어지기어려워백신생산규모는제한적일수밖에없다 (18). 유정란기반인플루엔자백신생산의또다른문제점은신속한백신생산이어렵다는점이다. 현재충분한양의유정란을준비하는단계부터백신생산이완성되는단계까지는약 5~6개월이소요되는것으로알려져있다 (19). 이러한오랜기간에걸쳐생산되는백신은새로운인플루엔자바이러스에의한갑작스러운대유행발생시신속한백신생산을어렵게하여인플루엔자바이러스를효과적으로방어하지못하게한다. 실제로지난 2009년대유행을일으킨 H1N1 아형의신종인플루엔자바이러스의경우, 첫발생이보고된후 2개월만에전세계적으로전파되어대유행을발생시켰다 (20). 이런인플루엔자대유 행이발생하면단기간에백신에대한수요가 5~10배정도증가하는것으로분석되었으며 (21), 이러한상황하에서유정란기반백신생산체계로는단기간에신속한인플루엔자백신의공급이어렵다 (18). 한편인플루엔자백신생산용 seed 바이러스가간혹유정란에서잘자라지못하는특성을나타내는경우도있다. 이에유정란기반인플루엔자백신의생산성을높이기위해서는유정란에서높은바이러스성장특성을나타내는백신균주 (high growth reassortants, HGR) 가요구되는데, 이를위해유정란에서인플루엔자바이러스를적응 (adaptation) 시켜유정란에서잘자라는백신균주를선택 (selection) 해야한다. 그러나이런과정중에 HGR 바이러스의항원성에변이가올수있는데, 이는유정란기반인플루엔자백신생산에큰문제를야기할수있다. 이전의연구결과를살펴보면인플루엔자 B형바이러스를유정란과 MDCK 세포주에서적응시킨경우, 유정란에적응된바이러스에서만 HA1 부위의 196-198번째아미노산의돌연변이로 HA 단백질의당쇄화 (glycosylation) 변이가발생하여항원성변이가초래되었다 (22, 23). 이러한현상은 H1N1 아형또는 H3N2 아형의인플루엔자바이러스에서도보고되었다. 즉유정란또는 MDCK 세포주유래의다양한 H1N1 아형의인플루엔자바이러스를이용한혈청학적연구결과, 유정란에서유래된바이러스에서만항원성변이가일어남을확인하였다 (24). 그리고임상시료를유정란과동물세포주에서배양한후염기서열을분석한결과, 유정란에서배양된바이러스는원래의임상시료와는다른 (heterogeneous) 유전자염기서열을갖는다는결과가보고되었다 (25, 26). 이러한문제점외에도유정란기반인플루엔자백신은외래성바이러스 (adventitious virus) 나세균에오염될수있는위험성을갖고있으며, 유정란유래단백질이백신에포함될수있기때문에백신을접종받은사람에게과민반응이나발열을유발시킬수있는단점을갖고있다 (27). 이에세포배양기반인플루엔자백신은위와같은유정란기반인플루엔자백신의단점을극복할수있는새로운방법중의하나로여겨지고있다 (28). 세포배양은유정란과는달리필요한시기에대량의세포를안정적으로확보하는것이가능해인플루엔자백신을약 3개월정도의기간안에대량생산할수있는것으로분석되었다 (21). 그러므로이러한백신생산기간의단축은갑작

12 I Lee, et al. Figure 1. A new paradigm for next generation of influenza vaccine. Cell culture-based influenza vaccine is challenging to issues caused by egg-based influenza vaccine. 스럽게발생하는새로운인플루엔자바이러스의대유행또는 H5N1 HPAI의유행에좀더신속히방어할수있어국가위기시좀더효과적인안전망역할을할수있다. 실제로지난 2009년신종인플루엔자바이러스가 2~3개월만에전세계적으로전파되어대유행이일어났다는사실은신속한백신생산이얼마나중요한지깨우쳐주는좋은사례이다. 최근마이크로캐리어 (microcarrier) 를이용하여세포를배양했을경우 1 ml의세포주한바이알 (vial) 을 8주만에 6,000리터 (L) 규모로증폭시킬수있는보고가있었다 (29). 그러므로이렇게개선된세포배양기술을적용할경우세포배양기반인플루엔자백신은더욱신속히효율적인비용으로대량생산될수있어유정란기반인플루엔자백신을대체할수있는가능성이있다. 또한앞에서제시된것처럼인플루엔자바이러스는세포주에서배양될경우항원성변이가유정란에비해상대적으로낮은장점이있다. 그리고세포배양인플루엔자백신은폐쇄된 (closed) 시스템내에서생산되어무균화가가능하고동일한배지를사용하여생산되기때문에세포주간의개체차이가적어균질한효능을가진백신의생산이가능하다는장점도갖고있다 (Fig. 1). 이에이러한장점을가진세포배양기반인플루엔자백신개발의필요성이점점더강조되고있다. 지난 2006년미국보건복지부는약 10억불을세포배양기반인플루엔 자백신개발에지원하였으며 (30), 2009년약 4억 8,700만불을지원하여 Novartis사와함께세포배양기반인플루엔자백신생산시설을구축하기시작하였다 (31). 이러한세포배양기반인플루엔자백신개발및생산관련인프라구축을위한정부의적극적인지원은언젠가인류에또다른대유행이올경우막강한효력을발휘할것으로기대된다. 2. 세포배양기반인플루엔자백신개발 2-1. 세포배양기반인플루엔자백신생산에사용되는세포주와이를이용하여개발된백신세포배양기반인플루엔자백신에사용되는세포주는 1) seed 백신균주가생산에충분한역가 (titer) 까지잘성장할수있어야하고, 2) 안전성, 특히발암원성 (tumorigenic) 과무관하다는증명이있어야하며, 3) 세포주가우태아혈청 (fetal bovine serum) 이존재하지않는배지에서도충분히자라야하는등위의세가지조건이모두충족되어야한다 (28). WHO는인플루엔자바이러스의성장특성을기초로 Vero (Africa green monkey kidney cell), MDCK (Madin-Darby canine kidney cell), PER.C6 (Embryonic human retinal cell derived) 의세가지세포주가인플루엔자백신생산에가장적절한것으로분석하였으며 (28), 현재세포배양기

Cell Culture-based Influenza Vaccines as Alternatives to Egg-based Vaccines 13 Table 2. Authorized cell-culture based influenza vaccines. Trade name Viruses Cell line Authorization Company Influvac Seasonal MDCK 2001; The Netherlands Solvay InfluJect Seasonal Vero 2002; The Netherlands Baxter Optaflu Seasonal MDCK 2007; EU Novartis Celvapan Pandemic Vero 2009; EU Baxter Celtura Pandemic MDCK 2009; Germany Novartis PreFluCel Seasonal Vero 2010; Austria Baxter FlucelVax Seasonal MDCK 2012; U.S. Novartis Never commercially distributed The first cell-culture based seasonal vaccine in U.S. 반인플루엔자백신은위의세가지세포주를이용하여개발되고있다 (Table 2). 2-1-1. Vero 세포주 Vero 세포주는현재사람에게접종하고있는다양한백신들을생산하는데가장널리이용되고있는세포주중의하나이다 (Table 1). 1980년대 Montagnon 등이 Vero 세포주를이용하여불활화 poliovirus 백신 (inactivated poliovirus vaccine) 을생산한것을 (32) 시작으로 1985년광견병백신, 1988년 poliovirus 생백신등이 Vero 세포주를이용하여생산되었다 (33, 34). 최근에는일본뇌염백신 (Japanese encephalitis vaccine) (35~38) 및로타바이러스 (rotavirus) 백신생산 (39) 에 Vero 세포주가이용되고있다. 이와같이 Vero 세포주는약 30여년의기간동안사람백신생산에성공적으로사용되었으며, WHO가선정한인플루엔자백신용세포주중유일하게안전성이증명된세포주이다. Vero 세포주가인플루엔자백신생산용세포주로대두되기시작한초기, 인플루엔자바이러스가 Vero 세포주에서는높은역가로잘증식하지못해백신생산에적합하지않은세포주로평가되었다 (40, 41). 왜냐하면다른바이러스질환의백신생산에이용되어아무리안전성이확보된세포주일지라도인플루엔자 seed 백신균주가잘성장하지못한다면필요한양만큼의백신을생산할수없는문제가일어날수있기때문이다. 하지만지속적인연구를통해 1990년대중반 Govorkova 등은 trypsin (TPCKtreated trypsin) 을여러번반복적으로첨가해주는방법을통해 Vero 세포주에서인플루엔자바이러스의성장능을향상시킨결과를보고하였고 (42), Kaverin 등은 trypsin의 활성저하가 Vero 세포주에서바이러스의증식을억제하는원인임을보고하였다 (43). 또한 Govorkova 등은 Vero 세포주에서인플루엔자 A형바이러스뿐만아니라 B형바이러스도 trypsin의반복투여방법으로증폭시킬수있음을보고하였고 (44), Kistner 등은혈청이첨가되지않은배지 (serum-free) 에서배양한 Vero 세포주를이용하여인플루엔자백신을대량생산하는시스템에대해보고하였다 (16). 이런향상된세포배양기술을적용하여현재는 Vero 세포주에서인플루엔자백신의대량생산이가능하게되었다 (29). 현재 Baxter사는 Vero 세포주를이용한인플루엔자백신을개발하고있다 (Table 2). 현재계절성인플루엔자 (seasonal influenza) 바이러스와 2009년대유행을일으킨 H1N1 아형의신종인플루엔자바이러스, H5N1 HPAI 에대한백신이개발되어임상시험중에있다 (28, 45, 46). 2-1-2. MDCK 세포주 MDCK 세포주는인플루엔자바이러스에대한감수성이매우높아세포배양기반인플루엔자백신의생산효율측면에서가장적합한세포주로여겨지고있다 (47~49). 또한 MDCK 세포주는온도나 ph 변화에덜민감하다는특징도가지고있다 (50). 이러한장점을바탕으로 MDCK 세포주를이용한세포배양기반백신개발연구가활발히이루어지고있다. 하지만많은장점에도불구하고, MDCK 세포주는크게두가지의잠재적인문제점을갖고있다. 즉 MDCK 세포주는현재까지백신생산에실제적으로이용된적이없는세포주라는것이다. 사람백신생산용세포주로오랫동안사용된 Vero 세포주와는달리실제로사람에대한안전성분석이이루어지지못했다.

14 I Lee, et al. 또다른문제점은발암원성 (tumorigenicity) 에대한것이다. WHO는 MDCK 세포주가 Vero 세포주보다발암원성이높은것으로확인하였다 (28). 하지만최근 Liu 등이 MDCK 세포주의안전성을분석한결과, 인플루엔자백신을생산하는데 MDCK 세포주를이용하여도발암원성은큰문제가되지않을것이라보고를하였다 (51). 이러한결과들로미루어볼때, MDCK 세포주도유정란을대체할수있는효과적인백신생산세포주가될수있을것으로판단된다. 현재 Norvatis, Solvay, GlaxoSmithKline, 및 MedImmune 사가 MDCK 세포주를이용하여인플루엔자백신을개발하고있다. Solvay사가개발한 Influvac 이라는제품은세포배양기반인플루엔자백신으로서최초로허가를받은제품이지만 (Table 2), 실제판매까지이루어지지는못했다 (50). 이후 2007년과 2009년에 Norvatis사에서개발한백신이각각 EU와독일의허가를받았다 (Table 2). 그리고지난 2012년 11월, Norvatis사에서개발한 MDCK 세포주를이용한세포배양기반인플루엔자백신인 FlucelVax 가세계최초로미국식품의약품안전청의허가를받았다 (Table 2) (52). 2-1-3. PER.C6 세포주 PER.C6 세포주를인플루엔자백신생산에이용하는연구는비교적최근에시작되었다. 2001년에 Pau 등은최초로 PER.C6 세포주를인플루엔자백신생산에이용할수있음을보고하였다 (53). PER.C6 세포주를이용한인플루엔자백신생산의경우 Crucell사가특허권을갖고있으며, H7N1 백신을개발하여현재임상시험중에있다 (54). PER.C6 세포주를이용한백신중허가를받은백신은현재까지는없으나, 발암원성 (tumorigenicity) 에대한연구를수행한결과누드마우스와햄스터에서종양원성이나타나지않는것으로확인되었다 (55). 2-2. 높은성장특성을갖는 seed 백신균주의개발세포배양인플루엔자백신에있어중요하게고려되어야할점중의하나는높은성장특성을나타내는 seed 백신균주를개발하는것이다. 이는백신생산에있어항원의양이매우중요한의미를갖기때문이다. 이러한이유로성장특성이높은세포배양용 seed 인플루엔자백신균주를개발하기위한연구가활발히진행되고있다. MDCK 세포주의경우 NA 유전자의 stalk 부위에 H5N1 조류인플루엔자바이러스로 (A/Goose/Guangdong/3/97) 부터유래한 38개의아미노산을삽입하여 MDCK 세포주 에서높은성장특성을나타내는재조합 H5N1 백신주를개발한연구결과가보고되었다 (56). 또한현재 H5N1 조류인플루엔자 seed 백신균주로개발되어있는 NIBRG-14 을 Vero 세포주에서계대배양하여성장능을향상시킨보고가있다 (57). 또다른연구에서는 H1N1 아형의 A/ Puerto Rico/8/34 (PR8) 인플루엔자바이러스를 Vero 세포주에서계대배양하여높은성장특성을나타내는 PR8 바이러스를얻은뒤, 유전자를비교분석하여성장특성에영향을미치는결정인자를발굴하였다. 이를다른 H1N1 또는 H3N2 아형의재조합백신균주에적용하여 Vero 세포주에서높은역가로자랄수있는 seed 백신균주를개발하였다 (58). 3. 세포배양기반인플루엔자백신의효능평가세포배양기반인플루엔자백신의효능은유정란기반인플루엔자백신과비교해거의차이가없는것으로보고되고있다. Szymczakiewicz-Multanowska 등은 MDCK 세포주에서생산한인플루엔자백신효능을유정란기반인플루엔자백신과비교한임상 3상시험결과, 세포배양기반인플루엔자백신의효능이유정란기반인플루엔자백신과매우비슷함을보고하였다 (59). Reisinger 등도백신임상시험결과, 세포배양기반인플루엔자백신은유정란기반인플루엔자백신과비교하여안전성및면역원성에거의차이가없음을보고하였다 (60). Groth 등은 MDCK 세포주를이용하여개발한계절성인플루엔자백신 (Optaflu, Norvatis) 을독일에서임상 1상및 2상시험을수행한결과, 세포배양기반인플루엔자백신은 18~40세사이의성인과 61세이상의노인에서대조군으로사용된유정란유래인플루엔자백신과거의비슷한면역원성을유도한다고보고하였다 (61). 또한 Hatz 등은 18~60세의성인을대상으로 MDCK 세포주에서생산된 2009년 H1N1 대유행인플루엔자백신 (Celtura, Norvatis) 의임상시험을수행하였다. 그결과 7.5 μg(ha 항원양기준 ) 의백신을면역보조제 (adjuvant) 와함께접종하였을때높은면역원성이유도되는것을확인하였다 (62). 한편 Ehrlich 등은 Vero 세포주를이용하여개발된 H5N1 HPAI 백신 (Baxter) 의임상 1상과 2상시험에서 18~ 45세사이의성인에게 7.5 μg과 15 μg을 2회접종하였을경우면역보조제 (adjuvant) 의사용없이도면역원성이유도되는결과를보고하였다 (63). 이러한결과들을종합하여볼때, 세포배양기반인플

Cell Culture-based Influenza Vaccines as Alternatives to Egg-based Vaccines 15 루엔자백신의효능은유정란기반인플루엔자백신의효능과큰차이가없는것으로판단된다. 결론세포배양기반인플루엔자백신은유정란기반인플루엔자백신에비해 1) 단기간에대량으로백신을생산할수있고, 2) 안정적인백신생산세포주의공급이가능하며, 3) 백신생산규모를상대적으로쉽게확대할수있고, 4) 화학적으로조성이정해진 (chemically defined) 배지를사용하기때문에항상일정한양의백신을생산할수있으며, 5) 무균배양이가능하고, 6) 알러지반응을일으키는유정란단백질성분이포함되지않는등의여러장점을갖고있다. 그러므로세포배양기반인플루엔자백신은갑작스러운인플루엔자대유행또는 H5N1 HPAI와같은새로운아형의인플루엔자바이러스가유행하여단기간에백신수요가급격히증가할경우효과적으로대응할수있는강력한도구가될수있다. 이에 WHO에서는 Vero, MDCK, PER.C6 세포주를인플루엔자백신생산에이용하도록선정하였으며, 최근세포배양기술의발달에힘입어유정란기반인플루엔자백신과마찬가지로대량으로백신을생산할수있게되었다. 또한그효능이유정란기반인플루엔자백신과거의다르지않는것으로보고되었다. 이러한사실들을종합하여볼때, 세포배양기반인플루엔자백신은기존의유정란기반인플루엔자백신의문제점을극복하여효과적으로사용될충분한가능성을갖고있는것으로판단된다. 그러므로, 백신주권을자체적으로마련하기위해서는유정란기반백신생산시설뿐만아니라세포배양기반백신생산관련인프라를구축하는것도미래를대비한현명한선택이될수있으리라사료된다. 참고문헌 1) Lee I, Kim JI, Park MS. A Novel PA-X Protein Translated from Influenza A Virus Segment 3. J Bacteriol Virol 2012;42: 368-71. 2) Tong S, Li Y, Rivailler P, Conrardy C, Castillo DA, Chen LM, et al. A distinct lineage of influenza A virus from bats. Proc Natl Acad Sci U S A 2012;109:4269-74. 3) WHO. Fact sheet on influenza. 2009. 4) Klenk HD, Garten W, Matrosovich M. Molecular mechanisms of interspecies transmission and pathogenicity of influenza viruses: Lessons from the 2009 pandemic. Bioessays 2011;33: 180-8. 5) WHO. Cumulative number of confirmed human cases of avian influenza A (H5N1) reported to WHO. 2012. 6) Park S, Kim JI, Park MS. Antiviral Agents Against Influenza Viruses. J Bacteriol Virol 2012;42:284-93. 7) Cox NJ, Subbarao K. Influenza. Lancet 1999;354:1277-82. 8) Kim JI, Park MS. An Universal Approach to Getting Ahead for Influenza B Vaccines. J Bacteriol Virol 2012;42:363-7. 9) Traynor K. First quadrivalent flu vaccine approved. Am J Health Syst Pharm 2012;69:538. 10) WHO. A description of the process of seasonal and H5N1 influenza vaccine virus selection and development. 2007. 11) Nicholson KG WR, Hay AJ. Textbook of Influenza. Oxford: Blackwell Science Ltd., 1998. 12) WHO. Cell culture as a substrate for the production of influenza vaccines: memorandum from a WHO meeting. Bull World Health Organ 1995;73:431-5. 13) Magrath DI. Safety of vaccines produced in continuous cell lines. Dev Biol Stand 1991;75:17-20. 14) Merten OW, Hannoun C, Manuguerra JC, Ventre F, Petres S. Production of influenza virus in cell cultures for vaccine preparation. Adv Exp Med Biol 1996;397:141-51. 15) Tannock GA, Bryce DA, Paul JA. Evaluation of chicken kidney and chicken embryo kidney cultures for the large-scale growth of attenuated influenza virus master strain A/Ann/ Arbor/6/60-ca. Vaccine 1985;3:333-9. 16) Kistner O, Barrett PN, Mundt W, Reiter M, Schober-Bendixen S, Dorner F. Development of a mammalian cell (Vero) derived candidate influenza virus vaccine. Vaccine 1998;16:960-8. 17) PATH. Influenza Vaccine Strategies for Broad Global Access. www.oliverwyman.com/media/vac_infl_publ_rpt_10-07.pdf. 2007. 18) Feng SZ, Jiao PR, Qi WB, Fan HY, Liao M. Development and strategies of cell-culture technology for influenza vaccine. Appl Microbiol Biotechnol 2011;89:893-902. 19) Gerdil C. The annual production cycle for influenza vaccine. Vaccine 2003;21:1776-9. 20) WHO. World now at the start of 2009 influenza pandemic. 2009.

16 I Lee, et al. 21) Pandey A, Singh N, Sambhara S, Mittal SK. Egg-independent vaccine strategies for highly pathogenic H5N1 influenza viruses. Hum Vaccin 2010;6:178-88. 22) Robertson JS, Naeve CW, Webster RG, Bootman JS, Newman R, Schild GC. Alterations in the hemagglutinin associated with adaptation of influenza B virus to growth in eggs. Virology 1985;143:166-74. 23) Oxford JS, Schild GC, Corcoran T, Newman R, Major D, Robertson J, et al. A host-cell-selected variant of influenza B virus with a single nucleotide substitution in HA affecting a potential glycosylation site was attenuated in virulence for volunteers. Arch Virol 1990;110:37-46. 24) Oxford JS, Corcoran T, Knott R, Bates J, Bartolomei O, Major D, et al. Serological studies with influenza A(H1N1) viruses cultivated in eggs or in a canine kidney cell line (MDCK). Bull World Health Organ 1987;65:181-7. 25) Katz JM, Webster, RG. Amino acid sequence identity between the HA1 of influenza A (H3N2) viruses grown in mammalian and primary chick kidney cells. J Gen Virol 1992;73:1159-65. 26) Katz JM, Wang M, Webster RG. Direct sequencing of the HA gene of influenza (H3N2) virus in original clinical samples reveals sequence identity with mammalian cell-grown virus. J Virol 1990;64:1808-11. 27) James JM, Zeiger RS, Lester MR, Fasano MB, Gern JE, Mansfield LE, et al. Safe administration of influenza vaccine to patients with egg allergy. J Pediatr 1998;133:624-8. 28) WHO. Use of Cell Lines for the Production of Influenza Virus Vaccines: An Appraisal of Technical, Manufacturing, and Regulatory Considerations. 2007. 29) Barrett PN, Mundt W, Kistner O, Howard MK. Vero cell platform in vaccine production: moving towards cell culturebased viral vaccines. Expert Rev Vaccines 2009;8:607-18. 30) U.S. Health and Human Services. HHS Awards Contracts Totaling More Than $1 Billion To Develop Cell-Based Influenza Vaccine. 2006. 31) U.S. Health and Human Services. HHS Awards $487 Million Contract to Build First U.S. Manufacturing Facility for Cell- Based Influenza Vaccine. 2009. 32) Montagnon BJ, Fanget B, Nicolas AJ. The large-scale cultivation of VERO cells in micro-carrier culture for virus vaccine production. Preliminary results for killed poliovirus vaccine. Dev Biol Stand 1981;47:55-64. 33) SanofiPasteur. Our vaccines: a history of innovation. www.sanofipasteur.com/index.jsp?coderubrique=14&lang= EN. 34) Montagnon BJ. Polio and rabies vaccines produced in continuous cell lines: a reality for Vero cell line. Dev Biol Stand 1989;70:27-47. 35) Schuller E, Jilma B, Voicu V, Golor G, Kollaritsch H, Kaltenböck A, et al. Long-term immunogenicity of the new Vero cell-derived, inactivated Japanese encephalitis virus vaccine IC51 Six and 12 month results of a multicenter follow-up phase 3 study. Vaccine 2008;26:4382-6. 36) Tauber E, Kollaritsch H, von Sonnenburg F, Lademann M, Jilma B, Firbas C, et al. Randomized, double-blind, placebocontrolled phase 3 trial of the safety and tolerability of IC51, an inactivated Japanese encephalitis vaccine. J Infect Dis 2008; 198:493-9. 37) Tauber E, Kollaritsch H, Korinek M, Rendi-Wagner P, Jilma B, Firbas C, et al. Safety and immunogenicity of a Vero-cellderived, inactivated Japanese encephalitis vaccine: a noninferiority, phase III, randomised controlled trial. Lancet 2007; 370:1847-53. 38) Lyons A, Kanesa-thasan N, Kuschner RA, Eckels KH, Putnak R, Sun W, et al. A Phase 2 study of a purified, inactivated virus vaccine to prevent Japanese encephalitis. Vaccine 2007; 25:3445-53. 39) Dennehy PH. Rotavirus vaccines: an overview. Clin Microbiol Rev 2008;21:198-208. 40) Lau SC, Scholtissek C. Abortive infection of Vero cells by an influenza A virus (FPV). Virology 1995;212:225-31. 41) Nakamura K, Homma M. Protein synthesis in Vero cells abortively infected with influenza B virus. J Gen Virol 1981; 56:199-202. 42) Govorkova EA, Kaverin NV, Gubareva LV, Meignier B, Webster RG. Replication of influenza A viruses in a green monkey kidney continuous cell line (Vero). J Infect Dis 1995; 172:250-3. 43) Kaverin NV, Webster RG. Impairment of multicycle influenza virus growth in Vero (WHO) cells by loss of trypsin activity. J Virol 1995;69:2700-3. 44) Govorkova EA, Murti G, Meignier B, de Taisne C, Webster RG. African green monkey kidney (Vero) cells provide an alternative host cell system for influenza A and B viruses. J Virol 1996;70:5519-24. 45) Baxter. Baxter Receives European Commission Approval for CELVAPAN H1N1 Pandemic Influenza Vaccine. 2009. 46) Baxter. Baxter Receives EMEA Positive Opinion for

Cell Culture-based Influenza Vaccines as Alternatives to Egg-based Vaccines 17 CELVAPAN, the First Cell Culture-based Pandemic Flu Vaccine. 2008. 47) Minor PD, Engelhardt OG, Wood JM, Robertson JS, Blayer S, Colegate T, et al. Current challenges in implementing cellderived influenza vaccines: implications for production and regulation, July 2007, NIBSC, Potters Bar, UK. Vaccine 2009; 27:2907-13. 48) Tree JA, Richardson C, Fooks AR, Clegg JC, Looby D. Comparison of large-scale mammalian cell culture systems with egg culture for the production of influenza virus A vaccine strains. Vaccine 2001;19:3444-50. 49) Liu J, Shi X, Schwartz R, Kemble G. Use of MDCK cells for production of live attenuated influenza vaccine. Vaccine 2009; 27:6460-3. 50) Genzel Y, Reichl U. Continuous cell lines as a production system for influenza vaccines. Expert Rev Vaccines 2009;8: 1681-92. 51) Liu J, Mani S, Schwartz R, Richman L, Tabor DE. Cloning and assessment of tumorigenicity and oncogenicity of a Madin-Darby canine kidney (MDCK) cell line for influenza vaccine production. Vaccine 2010;28:1285-93. 52) Novartis. Novartis receives FDA approval for Flucelvax, the first cell-culture vaccine in US to help protect against seasonal influenza. 2012. 53) Pau MG, Ophorst C, Koldijk MH, Schouten G, Mehtali M, Uytdehaag F. The human cell line PER.C6 provides a new manufacturing system for the production of influenza vaccines. Vaccine 2001;19:2716-21. 54) Cox RJ, Madhun AS, Hauge S, Sjursen H, Major D, Kuhne M, et al. A phase I clinical trial of a PER.C6 cell grown influenza H7 virus vaccine. Vaccine 2009;27:1889-97. 55) Ledwith BJ, Lanning CL, Gumprecht LA, Anderson CA, Coleman JB, Gatto NT, et al. Tumorigenicity assessments of Per.C6 cells and of an Ad5-vectored HIV-1 vaccine produced on this continuous cell line. Dev Biol (Basel) 2006;123:251-63. 56) Zhang W, Xue T, Wu X, Zhang P, Zhao G, Peng D, et al. Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine candidate viruses caused by insertion of 38 amino acids into the NA stalk. Vaccine 2011;29:8032-41. 57) Tseng YF, Hu AY, Huang ML, Yeh WZ, Weng TC, Chen YS, et al. Adaptation of high-growth influenza H5N1 vaccine virus in Vero cells: implications for pandemic preparedness. PLoS One 2011;6:e24057. 58) Murakami S, Horimoto T, Ito M, Takano R, Katsura H, Shimojima M, et al. Enhanced growth of influenza vaccine seed viruses in vero cells mediated by broadening the optimal ph range for virus membrane fusion. J Virol 2012;86:1405-10. 59) Szymczakiewicz-Multanowska A, Groth N, Bugarini R, Lattanzi M, Casula D, Hilbert A, et al. Safety and immunogenicity of a novel influenza subunit vaccine produced in mammalian cell culture. J Infect Dis 2009;200:841-8. 60) Reisinger KS, Block SL, Izu A, Groth N, Holmes SJ. Subunit influenza vaccines produced from cell culture or in embryonated chicken eggs: comparison of safety, reactogenicity, and immunogenicity. J Infect Dis 2009;200:849-57. 61) Groth N, Montomoli E, Gentile C, Manini I, Bugarini R, Podda A. Safety, tolerability and immunogenicity of a mammalian cell-culture-derived influenza vaccine: a sequential Phase I and Phase II clinical trial. Vaccine 2009;27:786-91. 62) Hatz C, Cramer JP, Vertruyen A, Schwarz TF, von Sonnenburg F, Borkowski A, et al. A randomised, single-blind, dose-range study to assess the immunogenicity and safety of a cellculture-derived A/H1N1 influenza vaccine in adult and elderly populations. Vaccine 2012;30:4820-7. 63) Ehrlich HJ, Müller M, Oh HM, Tambyah PA, Joukhadar C, Montomoli E, et al. A clinical trial of a whole-virus H5N1 vaccine derived from cell culture. N Engl J Med 2008;358: 2573-84.