B[y(yC oAU NGHIEN CLfU T^O C H O N G V A C XIN D y TUY^N EV 71 TR^N Te B A O V E R O
Le Thj Ludn'*, Tran Bfch Hanh', Nguyen DSng Hi^n', ^ Nguyen Phirong Yen', Nguyen Phwong Thiio', Nguyin Thj Thanh Th^o
' Truitg tam Nghien eiru Sdn xuat V&c xin va Sinh pham y te. Ha Npi
^ Vifn Pasteur thanh pho Ho Chi Minh TOM TAT
Dd tgo ra chung gidng irng CLP vidn cho sdn xudt vdc xin, chdng tdi dd tidn hdnh nghidn ci>u 10 chung virus EV71 do vidn Pasteur thdnh ph6 Hfi Chi Mmh cung cdp. Tn/dc hdt, 10 chiing dirge nhdn len 3 ldn trgn td bdo vero. ChOng VN06-278NH-RD4 {EV-04) dd phdt tridn t6t vdi hidu gid 6,62 Log10CCID50/0,1ml ndn d w c clipn dd Idch ddng, Sau 3 ldn tdch ddng V04,12.38,76 du'pc xdc djnh Id toi u'u nhdt, Chung nay du'g'c cdy truydn 30 ldn lidn tidp trdn td bdo vero. Kdt qud cho thdy EV04 12,38 76 da thfch nghi tdt tren td bdo vero, tiidu gid thu du'p'c 5,75-7,62 log10CCID50/0,1ml.
Ti> khda: Virut EV71, td b^o vero, tdch ddng vdc xin dy tuydn.
LDATVANDE
EV71 thupc nhom virus duong rupt typ huyet thanh A [1]. EV71 thuang gay ra them cac benh cdnh ve than kinh cap tinh bao gom liet m6m gi6ng bai liet, viem nao, viem mang nao vo khudn. phii phoi. Cac benh do EV71 gay ra thuang d^ lai cac di chirng vh than kinh nghiem trpng va co ty le tir vong cao [1]. Mac dli da CO su biing phat manh dich EV71 tai nhieu quoc gia trong khu vuc chau A Thai Binh Duong, nhung cho toi nay van chua co thuoc khang virus hay 1 vac xin sir dung de dieu tri va phong benh tay chan mieng E V71. Phat tnen vSc xin dugc xem la giai phap tot nhat de phong benh va kiem soat dich. Mpt loat cac vac xin phong benh tay chan mieng da dupc nghien ciiu nhu vdc xin bat hoat, vac xin tieu the dang vims (VLP), protein tai to hgp viing VPI, vac xin ADN viing VPI, vac xm peptide viing tao ra khang the trung hoa, vector vi khuan hay \ irus bieu hien VPI va vdc xin EV71 giam dpc luc thich ling tren te bao vero[2].
Do EV71 da noi len nhu mpt moi nguy hiem v6 site khoe cpng dong tai cdc quoc gia viing chau A Thai Binh Duong, mpt so nha san xuat vac xin tai khu vuc nay da co dugc vac xin cho
thii nghi?m lam sang tuy nhien de kiem soat dupc dich benh EV71 co hieu qua can co su c6 ging va hgp tac gitra nhieu quoc gia tren thfi gioi. Do dich benh de dga chu yeu h-e em tai cac nuoc dang phat trien, nen vac xin EV71 ly tuong can an toan, hieu qua nhung de sii dung, gia thanh re. Do la ly do vi sao chpn hucmg nghien ciiu tao he thong chiing giong cho vac xin phong tay chan mieng song giam dpc lire u6ng. Mpt trong nhimg khau quan trgng nhat de tao ra vac xin do !a nghien cim tao he thong chiing giong. Trong khuon kho bai bao nay POLYVAC dua ra ket qua nghien cim cdy truyen chung goc EV71 tren te bdo vero tii chiing gay benh tay chan mieng phan lap tir benh nhan mac benh Tay Chan Mieng dugc di6u tri tai benh vien Nhi Dong 1 thanh pho Ho Chi Minh.
IL PHLOfNG P H A P N G H I E N CUU
2.L Nguyen vat lieu
• 10 chiing E V71 dugc cung cdp tir Vien Pas- teur thanh ph6 H6 Chi Minh voi ma s6 h6 so:
VN06-265NH-RD3, VN06-272P-RD4, VN06- 272P-RD4, VN06-278NH-RD4, VN06-285NH -RD3, VN06-287NH-RD3, VN06-309P-RD3,
* Tac gia : Le Thi Luan
Dja chi: TT Nghien ciiu Vdc xin va Sinh phdm Y te Dien thoai: 0904001727
Email: [email protected]
44
Ngay nhan bai: 11/3/2013 Ngay phan bien; 15/3/2013 Ngay dang bai: 6/6/2013
Tap chi Y hgc dir phong, Tap XXIII, s6 3 (139)
VN06-319P-RD3, VNNCO5-5804BV-RD3 va VNNC05-5885NH-RD3;
• Te bao vero ACTT dugc cung cap tir Vien Nghien cihi Bai U^t Nhat ban (JPRl) cho san xudt vdc xin.
• Moi truong DMEM va cac hoa chat, dung cu dil cho nghien ciiu.
2.2.Fhuwng phdp tien hdnh
• Buoc 1. Khuyech dai 10 chiing EV71 tren t6 bdo vero 3 lan Hen ti6p, chudn dp hipu gia bdng phuang phdp vi Iugng tren phi6n 96 giSng;
chpn chiing co hi^u gia cao nhat sau 3 lan cay tmyen cho thuc hi?n buac 2.
• Buac 2. Chiing c6 hieu gia cao nhat chpn 0 buac 1 dupc sir dung tach dong bang phuang phdp nong do giai han de chpn dugc dong vimt EV71 CO hieu gid cao nhat cho thuc hien buac 3.
Phuang phap nong dp gidi han dugc thuc hien nhu sau: pha loang virus EV71 theo bac 5 hoac bac 10, gay nhiem moi nong dp vdo 8 gieng, sau do thu virus theo timg gieng rieng biet, xdc dinh nong dp virus tai moi gieng, lua chpn nhung gieng te bdo co hieu gia cao tai nong do pha loang nhat cay truyen tiep theo.
- Nuoi te bao vero mpt ldp tren chai nhua bang moi trudng DMEM 5% huyet thanh be bdo thai tai nhiet dp 37*'C trong 4 ngay.
- Pha loang mau virus bac 10 tir 10"' den 10"^
bang moi truang duy tri MEM 2% huyet thanh be bao thai.
- Sir dyng phien day phang 96 gieng. sau do cho titng n6ng dp vims vao philn. 0. Iml/gieng;
8 gieng/nong dg.
- Tao hon dich te bao vero 2 x 10^ te bdo/ml bang moi trudng MEM duy tri, sau do cho vao tat cd cdc gieng 0,1ml hon dich te bao, u phien t^i nhipt dp 36"C trong 6 ngay.
- Tai ngay thii 6, quan sat phien te bao tren kinh hifin vi, thu hon dich vims tir nhiing gieng te bao hiiy hoai (CPE) tai pha loang vimt cao nhat, moi gieng dugc coi la 1 dong virus. Ky hieu rieng biet. bdo quan tai nhiet dp -70°C cho tai khi sir dung.
- Sau khi chpn dugc dong virus tai lan tdch dong lan 3 vai day du tieu chuan: khong bi nhiem nam, mycoplasma, vi khuan, hieu gia cao nhat, bao quan tai nhiet do -70''C cho tdi lam budc tiep theo.
• Budc 3.D6ng virut EV71 co hieu gia cao nhat chpn d budc 2 dugc thich nghi lien tiep tren te bdo vero 30 lan, moi lan cay truyen, vimt EV71 dugc xac dinh hieu gid bang phuang phdp chudn dp vi lugng tren te bdo vero. Ddnh gid, nhdn djnh ket qua.
IIL K E T QUA Bang 1. Hieu gia 10 chiing EV71 khi cay truyen 3 lan lien tiep
Hieu gid (Log CCID50/0.1ml)
Ian1 Idn 3
1 2 3 4 5 6 7 8
VN06/265NH-RD3 VN06-272P-RD4 VN06-277NH-RD4 VN06-278NH-RD4 VN06-285NH-RD3 VN06-287NH-RD3 VN06-309P-RD3 VN06-319P-RD3
EV-01 EV-02 EV-03 EV-04 EV-05 EV-06 EV-07 EV-08
4.62 4.87 4.87 5.5 4.75 4.62 5.75 3.62
5.25 5.37 5.37 6.12 5.87 4.75 5 87 4.25
5.75 5.62 5 75 6.62 6 5 87 6 37 4.62 9 VNNCO5-5804BV-RD3
10 VNNC05-5885NH-RD3
Tap chi Y hoc du phong, Tap XXIII, s6 3 (139)
45
Bang 1 cho thay, k6t qua hi^u gia 10 chiing EV 71 nguon gde tir Vi6n Pasteur thanh phd H6 Chi Minh cung cap. Hipu gid hdu het cdc chiing deu tang sau titng lan cdy truyen, cao nhat sau ian 3. Tuy nhien c6 3 chiing tir EV08-EV10 hipu gid tang it d cdc lan cay truyen sau vd hipu gid sau 3 ldn cay truyen Id thap nhat deu dudi 5,0
IglO CClD50/0,lml. 3 chimg hr EV01-EV03 vd 3 chiing rtr EV05 den EV07 hieu gia vimt tren 5,0 log 10 CCID50/0,lml nhung th^p hem 6,5 log 10 CCID50/0,lml. Chi chimg EV04 hifu gid tang cao nhdt sau 3 ldn cdy huyen vdi hi?u gid 6,62 log 10 CCID50/0,lml dugc lua chpn cho nghien cim tiep theo.
Bdng 2. KM qud tdch d6ng chung EV04
Tdch dbng ldn 1 Tdch d6ng ldn 2 Tdch d6ng lan 3
Ndng d$
pha lodng Ky hidu Hidu gid* Ndng d$
pha loang K^ hi^u Hifu N6ngd$
gid* pha lodng Ky hi$u Hi$u 100
100 100 100
100 EV04 8 EV04 9 EV04.10 EV04.11
EV04.12 4.25 4.75 4.62 3.75 5.87
1.000.000 1.000.000 1.000.000 1.000.000
1.000.000
EV04.9 35 EV04.10.36 EV04.11.37
EV04.12.38 5.87 1.000.000 EV04.12.3e.76 7.25
1.000.000 EV04 12 38.77 5.62
1000 000 EV04.12.38.78 6.62
100 EV04.13 4.37 1.000.000 EV04.13.39 5 12 100 EV04 14 4 62 10.000.000 EV04.14.52 5.25 100 EV04.15 4.12 10.000.000 EV04.15.53 4.62
*rnih theo logW CCIDSO/O.lml
Bang 2 cho ket qua nghien cuu chiing virus EV04. Lan 1 tach dong, da chon dugc 8 dong viiais tai nong dp pha loang 100.000 lan vai dong virus c6 hieu gia cao nhat 5,87 IglO CCID 50/ 0,1ml (ky hieu s6 EV04.12) va thap nhat 3,75 IglO CCID 50/ 0,1ml. 7/8 dong tach lan 1 dugc tach dong Ian 2 va da chgn dugc 5 dong tai nong dg pha loang 1.000.000 liin va 2 dong tai nong do pha loang 10.000.000 liin, hieu gia
cac dong nay deu tang Ien, dong virus co hieu gia cao nhat ky hieu s6 EV04.12.38(5,87 IglO CCID 50/ 0,lml) dugc chgn cho lin tach dong lan 3, dong nay tach dugc 3 dong tai nong dp pha loang 1.000.000 lan vdi hieu gia cao, mgt trong nhirng dong tach lan 3 dugc lira chgn cho nghien ciru tiep theo la dong mang ky hieu EV04 12.38.76 voi hieu gia 7,25 IglO CCID 50/
0,1 ml.
46 Tap chi Y hpc dtt phong, Tap XXIII, s6 3 (139)
:9
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 25 27 28 29 30 L i n cav nmren Hinh 1. Ket qua nhdn dong tip chung EV04 .12.38.76 F tren te bao vero Hinh 1 cho thay, 10 lan cay truyen dau tien,
hieu gia virus Ian sau cao hon Ian trudc va tdi ldn thli 10 dat cao nhdt 7,62 IglO CCID 50/
0,1ml. 10 lan cay tmyen tiep theo hieu gid virus cac lan tang, lan giam so vdi hieu gid lan cay tmyen thii 11. 10 ldn sau cimg hieu gia giam dan nhung den ldn thit 30 lai tang len mpt chiit dat 6,62 IglO CCID 50/0,1ml.
IV. BAN LUAN
Vims dudng rupt typ EV71 (EV71) thupc chi cdc vims dudng mot thugc hp Picomaviri- dae. Chi cac vims dudng mpt dugc chia thanh 4 nhdm gen tit Ad6n D, EV 71 thupc nhom gen A Cling vdi CoxsackieA (CA) typ 2 dgn typ 8.
CAIO. 12.14 va 16 nguyen nhan gay benh tay chan mieng [3]. Tai nudc ta benh tay chan mieng cung da xdy ra nhiing nam trudc day, dac biet la khu vuc Mien Nam Viet Nam, cdc vu
dich xay ra nam 2003,2005,2008,2011, nguyen nhan do EV71 {42%)va CAI 6 (52%) gay ra[3].
Trong trudng hgp do EV71 c6 29.5% ca benh cd bieu hien lam sang tram trpng, 3 trudng hgp tii vong. Vims EV71 chi c6 1 typ huyet thanh nhung bao gom nhieu kieu gen khdc nhau (genotype), hien nay da xdc nhan dugc 3 nhdm gen tu nhdm A den nhdm C, moi nhdm gen lai bao gom cac dudi nhdm tit A1-A2. B1-B5 va C1-C5. Cac nhom va dudi nhdm gen da luu hanh va gay dich benh tay chan mieng d cac nudc khac nhau. Do tinh chat tram trpng cua benh va kha nang biing phat benh dich ldn ciia EV71, do su luu hanh va gay benh ciia virus EV71 d mdi nudc vdi kieu gen khac nhau can thiet phai cd vaccin phong benh ciia timg nudc rieng biet, do la ly do tai sao chpn chung vims EV71 tai Mien Nam la ddi tugng nghien cim tao chiing cho san xuat vdc xin phdng benh nay tai nudc ta.
Tap chi Y hoc du phong, T^p XXIII, so 3 (139)
47
Tir ket qua nghien ciiu cho thay rang nhiing chiing vims EV71 gay benh tay chan mipng thu dugc tai thdnh phd Ho Chi Minh cd dac tinh thich nghi tuang ddi tot tren te bdo vero, cy the 7/10 chiing c6 hi?u gid >5 log 10 CCID50/ml sau 3 ldn cay truyen. Mgt chiing nhan len tot nhat tren te bao vero dugc sir dyng cho tach ddng dd hi cliiing EV04 (VN06-278NH-RD4).
Thuc hipn tach ddng 3 lan va da chgn dugc ddng iru vipt nhat vdi hipu gia cao nhat tren te bao vero dd la ddng EV04.12.38.76. Ddng ndy nhan len trong 10 lan dau tien lien tiep rat tot, nhung sau dd chua dn djnh bieu hipn hipu gia dao dpng cao, thap xen ke. Muon ddng ndy dn dinh can tinh sach ddng bang phuang phap tao dam hoai tir [5] de chpn ra mpt ddng cd day dii dac tinh: Hieu gia cao, dn djnh tren liit ca cac ddi cay truyen.
V. KET LUAN
- Sau khi cay truyen 10 chimg EV 71 tren te bao vero, da chpn dugc chimg VN06-27 8NH- RD4 (EV-04) vdi hieu gia 6,62 log 10 CCID50/0,lml cho tach ddng;
- Sau khi tdch ddng 3 ldn chung EV-04 da chpn duac ddng virus EV04.12.38.76 toi uu nhat cho cay truy6n tren te bao vero;
- Da cdy tmyin ddng EV04.12.38.76 30 lan
tren t^ bdo vero cho thdy virut da thich nghi tdt va hipu gid thu dugc tii 5,75 den 7,62 logl0CCID50/0,lml.
T A I L I $ U THAM K H A O
1. Nguyen Thi Hien Thanh va CS. Dich te hpc phan tli chiing vimt dudng rupt typ 71 phan ldp nam 2003 t^i tinh Ha Tay. Tap chi Yhpc Dg phdng, 2004, Tap XIV, sd 4 (68), tr 12- 18.
2. Nguyen Thi Hien Thanh va CS. Nghien cuu tuyen chpn ddng te bdo nhay cam de phan lap virus dudng rupt. Tap chi Y hpc Du phdng, 2005, Tdp XV sd 1 (73) phy ban, tr 188-193.
3. Phan Van Tu, Nguyen Thi Thanh Thao, David Perea, and et al. Epidemiologic and Virological Investigation of Hand, Foot and Mouth Disease, Southem Vietnam, 2005.
EID Joumai Home, Volume 13, Number 11, November 2007, P1-7.
4. S.S.Y.Wong,C.C.Y.YiPS.K.P.LAUandK.
Y.YUEN Human enterovirus 71 and hand, foot and mouth disease. Epidemiol.Infect, 2010,138,1071-1089.
5. Marie L. Landry and G.D Hsiung. Primary Isolation of Viruses. Clinical Virology Man- ual. Chapter 3. page 55, 1992.
48 Tap chi Y hpc dir phong, Tap XXIII, s6 3 (139)
INITIAL DEVELOPING A VACCINE CANDIDATE VIRUS FOR EV71 IN VERO CELL LINE
Le Thi Luan', Tran Bich Hanh', Nguyen Phuong Yen', Nguyen Phuong Thao', Nguyen Thi Thanh Thao^ and Nguyen Dang Hien'
' Center for Research and Production of Vaccines and Biologicals, Hanoi
^ Pasteur Intitule ofHochiminh Ciiy.
To develop a vaccine candidate vims for EV7!, we have used ten EV71 viruses were pro- vided by IP Hochiminh First step, three times of viral propagation were perfonned by using Vero cells, resulting in a highest dter virus was se- lected (VN06-278NH-RD4 (EV-04)/6,62 log 10 CCID50/ml). Next step is cloning by dilution- base method for three times and the EV04.
12.38.76 virus was selected as a candidate vac- cme virus for EV71. Last step is checking stabi- lization of antigen by a serial passages method (30 times), we found that the EV04.12.38.76 virus is introducing a titer ranger from 5 .75 to 7.62 loglO ccid50/ml. Our results might provide an adequate procedure for vaccine candidate
vims selection for EV71 or others
To develop vaccine seed viruse of EV71, we used 10 strains of EV71 provided by Pasteur Intitute of Hochiminh City. Beginning the viruses were propagated in vero cell three times.
The VN06-278NH-RD4 (EV-04) reached titer 6,62 log 10 CCID50/ml was selected for cloning.
After 3 times cloning, EV04.12.38.76 clone was chosen for next step. The virus strain were passage 30 times in vero cell. The result showed that virus propagated well with the titer range fi-om 5.75 to7.621oglOCCID50/ml.
Key words: EV 71, vero cells, vaccine, vac- cine candidate, cloning.
Tap chi Y hpc du phong, Tap XXIII, so 3 (139)