P R E V E N T I O N & C O N T R O L
D N A V A C C IN E S
P r e p a r e d b y P I . B u r a k A k s u , M . D . , P h . D . D e p a r t m e n t o f M i c r o b i o l o g y a n d C l i n i c a l M i c r o b i o l o g y , S c h o o l o f M e d i c i n e , M a r m a r a U n i v e r s i t y , I s t a n b u l , T u r k e y .T r a d itio n a lly , p r o te c tio n a g a in s t in fe c tio u s d is e a s e s h a s re lie d o n th e u s e o f a tte n u a te d o r k ille d v a c c in e s . H o w e v e r, m a n y s u c h v a c c in e s a re in a d e q u a te fo r re a s o n o f e ffic a c y , s a fe ty , a n d c o s t e ffe c tiv e n e s s . L iv e -a tte n u a te d v a c c in e s m a y be im m u n o s u p p re s s iv e , c a u s e d is e a s e if n o t a tte n u a te d s u ffic ie n tly , o r p ro v id e lim ite d im m u n ity if to o m u c h a tte n u a te d . A m a jo r c o n c e rn re g a rd in g th e u s e o f liv e v a c c in e s is th e p o s s ib ility o f o u tg r o w th o f m o re v ir u le n t o rg a n is m s . K ille d v a c c in e s a re o fte n u n a b le to g e n e r a te p r o te c tiv e le v e ls o f im m u n ity fo r re a s o n s o f A g lo a d a n d lo s s o f im p o r ta n t e p ito p e s d u rin g in a c tiv a tio n . In a d d itio n , th e y a re fr e q u e n tly in c o n v e n ie n t, g iv e n th a t re p e a te d im m u n iz a tio n is o fte n n e c e s s a ry to a c h ie v e e ffe c tiv e le v e ls o f im m u n ity . F u rth e r, b e c a u s e k ille d v ira l v a c c in e s d o n o t p ro v id e e n d o g e n o u s ly s y n th e s iz e d p ro te in s , th e y a re in g e n e ra l u n a b le to in d u c e c y to to x ic T c e lls , p o s s ib ly a re q u ire d c o m p o n e n t o f a tru ly e ffe c tiv e v a c c in e . S u b u n it v a c c in e s a re g e n e ra lly s a fe b u t c o s tly a n d p o o rly im m u n o g e n ic . L iv e re c o m b in a n t v a c c in e v e c to rs a re e ffe c tiv e , b u t th e ir re p e a te d u s e in th e s a m e h o s t m a y b e lim ite d b y v e c to r im m u n ity ; fu rth e rm o re , th e y a re s u b je c t to re v e rs io n e v e n ts a n d c a n c a u s e d is e a s e o r d e a th in im m u n o c o m p ro m is e d h o s ts . T h e re is a n e e d fo r b e tte r v a c c in e s , a n d re c e n tly D N A v a c c in e s h a v e b e e n d e v e lo p e d (1). D N A v a c c in e te c h n o lo g y o ffe rs a p ro m is in g n e w a lte r n a tiv e to c o n v e n tio n a l v a c c in e d e liv e r y s y s te m s . D N A v a c c in e s a re e u k a r y o tic e x p r e s s io n v e c to r s th a t c o n ta in th e g e n e e n c o d in g th e a n tig e n o f in te r e s t. W ith in in tr a m u s c u la r in je c tio n , p la s m id D N A e n te rs m u s c le c e lls , d ire c t e x p re s s io n o f th e p la s m id - e n c o d e d a n tig e n , a n d a n im m u n e re s p o n s e to th e a n tig e n is in d u c e d . D N A v a c c in e s h a v e b e e n s tu d ie d in m a n y s y s te m s , in c lu d in g in flu e n z a , h u m a n im m u n o d e fic ie n c y v iru s (H IV ), ra b ie s , h e rp e s v iru s , a n d h e p a titis B, a n d c a n in d u c e lo n g - la s tin g h u m o r a l a n d c e llu la r Im m u n e re s p o n s e s . D N A v a c c in e s a re e a s ily c o n s tru c te d b y te c h n iq u e s c o m m o n to all m o le c u la r b io lo g y la b o ra to rie s , s h o u ld b e in e x p e n s iv e to p ro d u c e , a n d s h o u ld n o t re q u ire re frig e ra tio n ; all e s s e n tia l a ttrib u te s fo r a v a c c in e in te n d e d to be u s e d in th e d e v e lo p in g w o rld . A n o th e r p o te n tia lly u s e fu l fe a tu re o f D N A v a c c in e s is th a t th e y c a n be d e s ig n e d to e x p re s s m o re th a n o n e a n tig e n s im u lta n e o u s ly , e ith e r b y e n c o d in g m u ltip le a n tig e n s (o r e p ito p e s ) o n a s in g le p la s m id o r b y im m u n iz in g w ith a m ix tu re o f p la s m id s , e a c h e n c o d in g a s in g le a n tig e n (2).
P r e c lm ic a l e f fic a c y o f D N A v a c c in e s
In f lu e n z a
T h e firs t d e m o n s tra tio n o f D N A v a c c in e e ffic a c y in a n a n im a l m o d e l w a s u s in g th e in flu e n z a v iru s , w h ic h c o n tin u e s to p ro v id e a u s e fu l s y s te m w ith w h ic h to c h a ra c te riz e im m u n e re s p o n s e s to D N A v a c c in e s . In flu e n z a is o n e o f th e in fe c tio u s d is e a s e ta rg e ts fo r a D N A v a c c in e c u rre n tly b e in g in v e s tig a te d in h u m a n c lin ic a l tria ls . O n e ra tio n a le fo r e ffo rts to d e v e lo p a n in flu e n z a D N A v a c c in e , d e s p ite th e a v a ila b ility o f w id e ly u tiliz e d e x is tin g in flu e n z a v a c c in e s , is th a t c u r r e n t v a c c in e s a re e ffe c tiv e o n ly in a s tr a in - s p e c ific m a n n e r. M u ta tio n s in in flu e n z a v iru s s tra in s m e a n th a t fr e q u e n t r e - e v a lu a tio n a n d re fo rm u la tio n o f th e v a c c in e is n e c e s s a ry . D N A v a c c in e s o ffe r th e a d v a n ta g e o f s tim u la tin g th e g e n e ra tio n o f c y to to x ic T ly m p h o c y te s (C T L s ) a g a in s t e p ito p e s fro m a c o n s e rv e d p ro te in o f th e v iru s . A c o m b in a tio n D N A v a c c in e c o n ta in in g p la s m id s e n c o d in g b o th in te r n a l a n d c o a t p ro te in s o f th e v iru s w a s p re p a re d in a m ix tu re to m im ic th e p ro te in s p re s e n t in th e w h o le v iru s v a c c in e . V a c c in a tio n o f n o n h u m a n p rim a te s g e n e ra te d tite rs o f h e m a g g lu tin a tio n in h ib itin g
Prevention & Control
a n tib o d ie s (u s e d a s s u rro g a te s fo r n e u tra liz in g a n tib o d ie s ) a s h ig h a s a o r h ig h e r th a n th o s e g e n e ra te d b y th e fu ll h u m a n d o s e s o f w h o le in a c tiv a te d a n d s p lit in a c tiv a te d v iru s v a c c in e s , re s p e c tiv e ly . F e rre ts im m u n iz e d w ith th e D N A v a c c in e c o c k ta il s h e d le s s v iru s in n a s a l w a s h in g s a fte r c h a lle n g e w ith a d rifte d (i.e. a n tig e n ic a lly d iffe re n t) s tra in o f v iru s th a n did c o n tro l a n im a ls im m u n iz e d w ith th e a n a lo g o u s c o m m e rc ia lly lic e n s e d w h o le in a c tiv a te d v iru s v a c c in e (3, 4, 5).
M a l a r i a
A n o th e r m o d e l in w h ic h a c o m b in a tio n D N A v a c c in e w a s re c e n tly e m p lo y e d is m a la ria . In th is d is e a s e th e re a re tw o p ro b le m s w ith v a c c in e s tu d ie s . O n e , b e c a u s e it is u n lik e ly th a t a v a c c in e d ir e c te d a g a in s t a s in g le a n tig e n e x p re s s e d d u rin g o n e s ta g e o f th e life c y c le w ill re m o v e all p a ra s ite s , a v a c c in e w ill p ro b a b ly n e e d to ta rg e t s e v e ra l a n tig e n s fro m d iffe re n t life- c y c le s ta g e s . A D N A v a c c in e a g a in s t m a la ria c o u ld b e d e s ig n e d to e x p re s s a n tig e n s fro m s p ro z o ite , e x o e ry th ro c y tic a n d e ry tro c y tic s ta g e s a n d th e re fo re ra is e im m u n re s p o n s e s a g a in s t m u ltip le life c y c le s ta g e s . S e c o n d , in m a la ria th e re a re re la tiv e ly fe w ta rg e t e p ito p e s d iffe re n t p a ra s ite is o la te s a n d th e v a ria n ts o fte n d o n o t c ro s s re a c t. T h u s , to e lic it e ffe c tiv e im m u n ity a g a in s t m u ltip le w ild is o la te s w ith v a ria n t e p ito p e s , im m u n iz a tio n w ith a v a c c in e w ith c o d in g m a n y a n tig e n s s h o u ld be re q u ire d (2).O t h e r
v i r a l
a n d
b a c t e r ia l
d is e a s e
m o d e ls
A ls o im p o r ta n t fo r b rin g in g D N A v a c c in e te c h n o lo g y c lo s e r to c lin ic a l a p p lic a tio n w a s th e c o n tin u e d e x p a n s io n o f th e b re a d th o f p re c lin ic a l in fe c tio u s a n d n o n in fe c tio u s d is e a s e ta rg e ts s u c c e s s fu lly te s te d in a n im a l m o d e ls . T h e s e in c lu d e d a d d itio n a l v ira l ta r g e ts , a s w e ll as p a r a s itic d is e a s e s , b a c te r ia l d is e a s e s , a n d c a n c e r (a g a in s t w h ic h th e firs t c lin ic a l tria ls fo r D N A v a c c in e s w e re in itia te d d u rin g 1 9 9 5 ). J o in in g th e v ira l d is e a s e ta r g e ts fo r w h ic h im m u n e re s p o n s e s a n d /o r p ro te c tio n h a d a lre a d y b e e n d e m o n s tra te d (s u c h a s in flu e n z a , b o v in e
h e rp e s v iru s , h e p a titis B, H IV a n d ra b ie s ) w e re h e p a titis C v iru s , h e rp e s s im p le x v iru s , p a p illo m a v ir u s , ly m p h o c y tic c h o r io m e n in g itis v iru s a n d fla v iv iru s . T h e lis t o f p a ra s itic d is e a s e s fo r w h ic h p re c lin ic a l e ffic a c y h a s b e e n d e m o n s tra te d w a s e x p a n d e d fro m m a la ria a n d le is h m a n ia s is to in c lu d e s c h is to s o m ia s is (3, 6). It is p o s s ib le th a t b a c te ria l d is e a s e s w e re n o t th e in itia l ta rg e t fo r D N A v a c c in e s b e c a u s e a n u m b e r o f p ro te c tiv e b a c te r ia l a n tig e n s a re n o t p ro te in a c e o u s (e .g . p o ly s a c c h a r id e s ) a n d b e c a u s e le ss is k n o w n a b o u t th e p ro te c tiv e a n tig e n s o f b a c te ria c o m p a re d w ith v iru s e s (i.e. th o s e a n tig e n s th a t in d u c e a p ro te c tiv e im m u n e re s p o n s e ). F o r at le a s t s o m e b a c te ria l ta rg e ts , h o w e v e r, n o t o n ly a re p ro te in s k e y a n tig e n s , b u t th e ty p e s o f c e llu la r re s p o n s e s in d u c e d b y D N A v a c c in e s m a y b e im p o rta n t e le m e n ts o f an e ffe c tiv e v a c c in e . A n e x a m p le o f s u c h a b a c te ria l ta rg e t is M y c o b a c te riu m tu b e rc u lo s is ; th e c e llu la r re s p o n s e s a g a in s t its p ro te in s a p p e a r to p la y k e y ro le s in p ro te c tiv e im m u n ity . R e c e n t w o rk h a s s h o w n th a t tu b e rc u lo s is D N A v a c c in e s in d u c e C T L a n d h e lp e r T ce ll re s p o n s e s o f th e Th1 -like p h e n o ty p e , a s m e a s u re d in v itro u p o n re s tim u la tio n o f s p le e n c e lls w ith a n tig e n (3, 6, 7).
C a n c e r im m u n o t h e r a p y
D N A -b a s e d im m u n iz a tio n is a n a ttra c tiv e n o n v ira l a lte rn a tiv e fo r c a n c e r im m u n o th e ra p y . D N A v a c c in a tio n is a c c o m p lis h e d b y th e e x p re s s io n o f in o c u la te d b a c te ria l p la s m id D N A e n c o d in g th e fo r e ig n g e n e o f in te re s t a c c o m p a n ie d b y a m a m m a lia n p ro m o te r/'e n h a n c e r, a n d o th e r s e q u e n c e s s u ch a s K o z a k is c o n s e n s u s s e q u e n c e a n d le a d e r s e q u e n c e s th a t e n a b le th e g e n e to be e x p re s s e d w ith in m a m m a lia n c e lls u tiliz in g h o s t m a c h in e ry . In th is re p o rt, a h a n d -h e ld h e liu m - p o w e re d d e v ic e w a s u s e d to a c h ie v e th e d ire c t in tr a c e llu la r d e liv e r y o f D N A -c o a te d g o ld p a rtic le s to th e e p id e rm is . F o llo w in g d e liv e ry , th e D N A re d is s o lv e s in th e a p u e o u s e n v iro n m e n t of th e c y to p la s m o r n u c le u s a n d is th e n a v a ila b le fo r e x p re s s io n . A lte rn a tiv e ly , s k e le ta l m u s c le c e lls h a v e d e m o n s tra te d th e a b ility to ta k e up a n d e x p re s s D N A fo r a p p ro x im a te ly th e life tim e o f th e m o u s e w ith o u t a n y s p e c ific d e liv e ry s y s te m . H o w e v e r, e p id e rm a l g e n e g u n im m u n iz a tio n of
M. Burak Aksu
D N A m a y b e m o re e ffic ie n t th a n in tra m u s c u la r im m u n iz a tio n a t e lic itin g s im ila r im m u n e re s p o n s e s (8). B o th D N A -b a s e d a p p ro a c h e s h a v e b e e n s h o w n to s u c c e s s fu lly in d u c e b o th h u m o ra l a n d c e llu la r im m u n ity in m a n y A g s y s te m s . In c a n c e r im m u n o th r e p y , p la s m id c o n s tr u c ts e n c o d in g e ith e r th e fu ll le n g th c D N A fo r c a rc in o e m b ry o n ic A g (C E A ) o r H IV -I e n v e lo p e p ro te in , g p 1 6, h a v e b e e n s h o w n to p ro te c t m ic e fro m s u b s e q u e n t c h a lle n g e w ith s y n g e n e ic tu m o rs e x p re s s in g th e s e m o d e l A g s (8).
D N A d e l i v e r y
D N A v a c c in e s c o u ld b e m o d ifie d b y th e u s e o f a d e liv e ry s y s te m , s u c h a s lip o s o m e s o r p o ly m e rs th a t c a n c o m p a c t D N A a n d e n h a n c e c e llu la r u p ta k e , o r th e in c lu s io n o f p e p tid e s o r p ro te in s th a t c a n fa c ilita te in tra c e llu la r ta rg e tin g o f D N A to th e c y to p la s m a n d n u c le u s . In a d d itio n , D N A v a c c in e s m a y b e ta rg e te d to s p e c ific tis s u e s s u c h a s m u c o s a l s ite s fo r th e in d u c tio n o f m u c o s a l im m u n e r e s p o n s e s . T o th is e n d . S iz e m o re c o n ta in s a n a s d ( a s p a r tic /s e m ia ld e h y d e d e h y d ro g e n a s e ) m u ta tio n th a t d o e s n o t in te rfe re w ith th e a b ility o f th e o rg a n is m to in v a d e c e lls b u t w h ic h c a u s e s it to b u rs t o p e n in s id e th e c e ll, th e re b y re le a s in g e x p re s s io n p la s m id s in to th e c y to p la s m . R e c o m b in a n t S h ig e lla c o n ta in in g p la s m id s e x p re s s in g g a la c tro s id a s e u n d e r th e c o n tro l o f th e h u m a n c y to m e g a lo v iru s (C M V ) e a rly p ro m o te r a n d e n h a n c e r a re n o t th e m s e lv e s a b le to e x p re s s g a la c to s id a s e b u t c a n d ire c t e x p re s s io n o f 3 g a la c to s id a s e in c e ll c u ltu re s , in th e g u in e a p ig e y e , a n d in th e m o u s e lu n g . M ic e im m u n iz e d in th is m a n n e r p ro d u c e d c e llu la r im m u n e re s p o n s e s a n d h ig h le v e ls o f s p e c ific a n tib o d ie s . H IV a n d m a la ria g e n e s h a v e a ls o b e e n e x p re s s e d in th is s y s te m . T h e s e fin d in g s o p e n u p to p o s s ib ility fo r th e r e la tiv e ly in e x p e n s iv e o ra l d e liv e ry o f fu n c tio n a l D N A w ith th e p o te n tia l fo r m a n ip u la tio n o f th e lo c a l im m u n e s y s te m a s w e ll a s fo r p ro d u c tio n o f s y s te m ic re s p o n s e s (3).E x p r e s s io n l i b r a r y im m u n i z a t i o n
T h e id e n tific a tio n o f th e p ro te c tiv e a n tig e n s o f a p a th o g e n is a la b o r io u s a n d s o m e tim e s
p ro b le m a tic p ro c e s s . T h is is p a rtic u la rly tru e fo r p ro te c tio n th a t re q u ire s c e llu la r im m u n ity , s in c e c e rta in ty p e s o f v a c c in e s (e .g . s u b u n it p ro te in s o r w h o le in a c tiv a te d v ir u s e s ) d o n o t g e n e r a lly in d u c e C T L s . F u r th e r m o r e , re s tin g s p e c ific a n tig e n s re q u ire s th a t th e y b e a v a ila b le in a p u rifie d fo rm . W ith D N A v a c c in e s , C T L s a re re a d ily in d u c e d a n d o n e n e e d s o n ly to h a v e th e g e n e e n c o d in g th e a n tig e n . T h e p ro c e s s o f v a c c in e a n tig e n d is c o v e ry m a y b e s im p lifie d b y a re c e n t a n d e x c itin g a p p lic a tio n o f D N A v a c c in e te c h n o lo g y . B a rry e t a l. d e v e lo p e d a m e th o d fo r te s tin g m ix tu re s o f D N A p la s m id s c o n ta in in g fra g m e n ts o f th e g e n o m e o f a p a th o g e n fo r p ro te c tiv e e ffic a c y (te rm e d e x p re s s io n lib ra ry im m u n iz a tio n , o r E L I). In th e ir e x a m p le , v a c c in a tio n s w ith m ix tu r e s c o n ta in in g 3 0 0 0 d is tin c t p la s m id s fro m a M y c o p la s m a p u lm o n is D N A lib ra ry w e re s h o w n to c o n fe r p ro te c tio n in a m o u s e c h a lle n g e m o d e l, in d ic a tin g th a t a t le a s t o n e o f th e p la s m id s e n c o d e d a p r o te c tiv e a n tig e n . B y s u c c e s s iv e fr a c tio n a tio n a n d te s tin g o f th e s e m ix tu re s it m a y b e p o s s ib le to id e n tify th e p ro te c tiv e p la s m id s , a lth o u g h to d a te s u c h fr a c tio n a tio n a n d id e n tific a tio n h a s n o t b e e n re p o rte d . O n e o f th e p o te n tia l d ra w b a c k s o f th e te c h n iq u e in a d d itio n to th e p o te n tia l m a s k in g o f e p ito p e s b y im m u n e in te rfe re n c e is th a t, b e c a u s e fra g m e n ts o f th e g e n o m e a re u s e d , m a n y o f th e p la s m id s w ill n o t e n c o d e a re le v a n t p ro te in . T h is p ro b le m c a n b e o v e rc o m e b y c lo n in g o p e n re a d in g fra m e s in to th e e x p re s s io n lib ra ry a n d , w ith th e b u rg e o n in g file d o f g e n o m ic s , th is s e q u e n c e in fo r m a tio n is r a p id ly b e c o m in g a v a ila b le fo r m a n y p a th o g e n s . S u c h a p p ro a c h e s m a y g re a tly fa c ilita te th e id e n tific a tio n o f v a c c in e a n tig e n s (3).
C o n c lu s io n s
T h e u s e o f D N A v a c c in a tio n h a s g ro w n s u b s ta n tia lly in th e e ig h t y e a rs s in c e it w a s firs t d e m o n s tra te d th a t D N A c o u ld c o n fe r p ro te c tiv e im m u n ity . R e c e n t im p o rta n t a d v a n c e s h a v e b e e n m a d e in s e v e ra l a e a s o f D N A v a c c in e s . T h e s e in c lu d e a n e x p a n s io n o f th e ta r g e ts fo r D N A v a c c in e d e v e lo p m e n t, a g re a te r u n d e rs ta n d in g o f s o m e o f th e u n d e rly in g m e c h a n is m s in v o lv e d in th e in d u c tio n o f im m u n e r e s p o n s e s , th e b e g in n in g s o f a lte rn a tiv e D N A d e liv e ry v e h ic le s th a t c a n ta r g e t m u c o s a l im m u n e s ite s , th e a p p lic a tio n o f D N A v a c c in e te c h n o lo g y to th e
Prevention & Control
d is c o v e ry o f p ro te c tiv e a n tig e n s a n d th e h u m a n c lin ic a l tria ls . T h e s u c c e s s o f D N A v a c c in e s w ill be p re d ic a te d o n th e ir e ffe c tiv e n e s s in h u m a n s .
