JOSÉ MACIEL RODRIGUES JÚNIOR, KARLA DE MELO LIMA,
ARLETE APARECIDA MARTINS COELHO CASTELO, VÂNIA LUIZA DEPERON BONATO MARTINS, SANDRA APARECIDA DOS SANTOS, LUCIA HELENA FACCIOLI, CÉLIO LOPES SILVA.
Th e DNA va ccin es cu rren t ly u n d er p re- clin ica l a n d clin ica l d evelo p m en t m a y p ro ve t o b e im p o rt a n t t o o ls in co m b at in g in fect io u s d iseases, su ch as t u b ercu lo sis, fo r wh ich n o safe an d effect ive fo rm o f p reven t io n h as yet b een d evelo p ed . In recen t years, several st u d ies h ave aim ed t o d evelo p a DNA vaccin e en co d in g m yco b act erial p ro t ein s su ch a s a n t ig en 8 5 (Ag 8 5 ) a n d t h e 6 5 - kDa m yco b a ct eria l h ea t sh o ck p ro t ein (h sp 6 5 ). Th e la t t er is p ro t ect ive a g a in st viru len t in fect io n wit h Myco b a ct eriu m t u b ercu lo sis (in clu d in g m u lt id ru g - resist a n t st ra in s). Th e hsp 6 5 DNA vaccin e, cu rren t ly u n d er clin ical evalu at io n in Brazil fo r can cer t h era py, is ab le t o in d u ce t h e secret io n o f Th 1 cyt o kin es, su ch as g am m a- in t erfero n , asso ciat ed wit h d isease co n t ro l. Fu rt h erm o re, t h is vaccin e st im u lat es cyt o t o xic CD8 an d CD4 T- cell clo n es t h at can b e ch aract erized as m em o ry cells, wh ich are resp o n sib le fo r effect ive an d lo n g - last in g im m u n it y ag ain st t u b ercu lo sis. Wh en u sed as a t h erap eu t ic ag en t in in o cu lat ed m ice, t h e h sp 6 5 DNA vaccin e p ro m o t es ch an g es in t h e im m u n it y p ro file, t rig g erin g t h e secret io n o f Th 1 cyt o kin es an d est ab lish in g a favo rab le en viro n m en t fo r t h e elim in at io n o f b acilli. Th e resu lt s also d em o n st rat e t h at t h e ro u t e o f ad m in ist rat io n , as well as t h e fo rm u lat io n in wh ich t h e vaccin e is ad m in ist ered , fu n d am en t ally in flu en ce t h e p at t ern an d d u rat io n o f t h e im m u n e resp o n se in d u ced . Takin g all cu rren t ly availab le d at a in t o acco u n t , we ca n co n clu d e t h a t a DNA va ccin e a g a in st t u b ercu lo sis co u ld co n t rib u t e sig n ifica n t ly t o t h e co n t ro l o f t h e d isea se.
Key words: Tuberculosis/epidemiology. Vaccines, DNA/therapeutic use. Heat shock proteins. Auto- immunity.
* St u d y ca rried o u t b y t h e Red e Bra sileira d e Pesq u isa em Tu b ercu lo se (Bra zilia n Tu b ercu lo sis Resea rch Net wo rk) a t t h e Un iversit y o f Sã o Pa u lo Fa cu ld a d e d e Med icin a d e Rib eirã o Pret o (Rib eirã o Pret o Sch o o l o f Med icin e), Rib eirã o Pret o , SP, Bra sil.
Co rresp o n d en ce t o : Célio Lo p es Vieira . Ru a Cló vis Vieira , 2 4 - Ca m p u s d a USP d e Rib eirã o Pret o . CEP 1 4 0 4 9 - 9 0 0 - Rib eirã o Pret o - SP, Bra zil. Em a il: clsilva @ cp t .fm rp .u sp .b r
Fin a n cia l su p p o rt : Red e Bra sileira d e Pesq u isa em TB (Red e- TB)/ Gra n t n o . 6 2 .0 0 5 5 / 01 - 4 - PACDT- Milen io .
Jornal Brasileiro de Pneumologia 3 0 (4 ) - Jul/ Ago de 2 0 0 4
Abbreviations used in this paper:
AIDS – Acq u ired im m u n o d eficien cy syn d ro m e TB – Tu b ercu lo sis
BCG – Ba cillu s Ca lm et t e- Gu érin
RT- PCR – Reverse- t ra n scrip t io n p o lym era se ch a in rea ct io n WHO – Wo rld Hea lt h Org a n iza t io n
h sp 6 5 – 6 5 - kDa m yco b a ct eria l h ea t sh o ck p ro t ein PLGA – Po ly- la ct ic- co - g lyco lic a cid
MHC – Ma jo r h ist o co m p a t ib ilit y co m p lex Mt b – Myco b a ct eriu m t u b ercu lo sis
What a DNA vaccine is
In r e c e n t d e c a d e s , a d va n c e s in va c c in e t echn ology have allowed t he in t rodu ct ion of n ew st rat egies for obt ain in g an d produ cin g an t igen s, as well as for t he opt im izat ion of n ew form s of admin ist erin g an d in t rodu cin g t hese an t igen s in t o immu n e syst em cells. These st rat egies have open ed pat hways t o in n ovat ion , especially in t he con t ext of developing vaccines t hat are safer, more effect ive a n d m o re p o lyva len t . Am o n g t h ese, t h ere a re su bu n it , or secon d- gen erat ion , vaccin es comprised of pu rified or recombin an t an t igen s obt ain ed from in fect io u s a g en t cu lt u res o r t h ro u g h syn t h et ic p ro d u ct io n . Mo re re ce n t ly, g e n e t ic, o r t h ird -gen erat ion , vaccin es have been developed. In DNA va ccin e s, g e n e s o r g e n e fra g m e n t s e n co d in g pot en t ially im m u n izin g an t igen s are delivered by plasmid DNA.
Although DNA vaccines are still in the laboratory an d clin ical t est in g phase of developm en t , t hey m ay prove t o be im port an t t ools in com bat in g in f e ct io u s d ise a se s su ch a s h e rp e s, a cq u ire d im m u n o d e ficie n cy syn d ro m e (AIDS), m a la ria , tuberculosis (TB), hepatitis, schistosomiasis, dengue an d ot hers for which n o safe an d effect ive form of preven t ion has yet been developed. Moreover, DNA va ccin e s h a ve sh o wn g re a t p o t e n t ia l fo r t h e p reven t ion an d cu re of specific t ypes of can cer.
Ge n e iso la t io n is a p ro ce ss t h e scie n t if ic com m u n it y can con t rol t oday du e t o sign ifican t advan ces in t he developmen t of molecu lar biology t echn iqu es. In order t o produ ce a DNA vaccin e, gen es t hat are frequ en t ly relat ed t o t he expression of molecu les in volved in viru len ce or pat hogen icit y of in fect iou s agen t s are clon ed an d in sert ed in t o a fragm en t of circu lar DNA called a plasm id. The replicat ion of a plasm id u sed in a gen et ic vaccin e m u st b e b a ct eria l in o rig in , t h ereb y a llo win g efficien t du plicat ion (hu n dreds of copies per cell). In addit ion , t hese plasm ids m u st have a select ion g e n e (wh ich u su a lly co n fe rs re sist a n ce t o a n an t ibiot ic, allowin g clon es of t he cells t hat carry t h e p la sm id t o b e select ed ). Fu rt h erm o re, it is essen t ial t hat su ch plasm ids possess a m u lt iple-clon in g sit e t hat can be cleaved wit h rest rict ion en zym es, allowin g t he in sert ion of t he gen e of in t erest (which, in t he case of DNA vaccin es, is a gen e t hat en codes an an t igen ). Moreover, t hese plasm ids m u st in clu de a prom ot er (u su ally a viral promot er) t hat allows t ran script ion of t he gen e of
in t erest in eu karyot ic cells. Aft er clon in g t he gen e t hat en codes t he vaccin e an t igen in t he plasm id, t h e g en e is in t ro d u ced in t o a h o st b a ct eriu m , u su ally Escherichia coli, u sin g a p ro cess called b a ct eria l t ra n sfo rm a t io n , in o rd er t o p ro d u ce plasm ids on a large scale, providin g a su fficien t qu an t it y of DNA for vaccin at ion(1)
.
These DNA vaccin es m ay be adm in ist ered t o variou s an im al species (an d t o hu m an s) in variou s fo rm s a n d u sin g va rio u s reg im en s. Th e m o st c o m m o n f o r m o f DNA va c c in e d e live r y is in t ra m u scu la r in je ct io n . In a d d it io n , g e n e t ic vaccin es m ay be delivered orally, in t ran asally (as a n a e r o s o l), o r in t r a d e r m a lly b y m e a n s o f bombardmen t wit h gold micropart icles coat ed wit h gen et ic mat erial. Alt hou gh in t ramu scu lar in ject ion is a simple, in expen sive process, it u su ally requ ires a great qu an t it y of plasm ids in order t o st im u lat e a n a p p ro p ria t e im m u n e re sp o n se . Hyp e rt o n ic glu cose solu t ion , which cau ses edema in mu scu lar t issu e(2)
, or a local an est het ic solu t ion , su ch as bu pivacain e, which can in ju re t issu es an d cau se an in flam m at ory react ion(3- 5)
, have been u sed as m ea n s o f in crea sin g a n t ig en t ra n sfect io n in t o macrophages and dendritic cells after intramuscular in ject ion . However, du e t o t heir adverse effect s, t hese procedu res are n ot accept able for u se in hu m an s. In t ram u scu lar in ject ion of plasm ids is a sim ple t echn iqu e, bu t it exposes DNA t o variou s obst acles at t he delivery sit e, su ch as en zym es (n u clea ses) t h a t a re ca p a b le o f d eg ra d in g t h e plasmid DNA, t hereby preven t in g t he in du ct ion of an im m u n e respon se.
prot ein syn t hesis. Therefore, t he delivery of high d o s e s o f p la s m id DNA (f r o m h u n d r e d s o f micrograms t o on e or more milligrams) is requ ired in a st an d ard t reat m en t . Ph arm aceu t ical- g rad e recom bin an t DNA can be obt ain ed by m ean s of in du cin g it s expression in t ran sformed bact eria an d p u rif yin g it in a se rie s o f c h ro m a t o g ra p h ic procedu res, t hereby allowin g it t o be produ ced on a large scale(6).
Perpectives on an anti- tuberculosis DNA
vaccine
Sp e c ia l a t t e n t io n h a s b e e n g ive n t o t h e d e ve lo p m e n t o f m o re e f f e ct ive TB va ccin e s. Main t ain in g t he dist in ct ion of bein g t he in fect iou s disease wit h t he highest worlwide m ort alit y rat e, t he an n u al in ciden ce of n ew TB cases has recen t ly arrived at 10 million , an d t he an n u al mort alit y rat e at 3 m illion people – 18.5% am on g adu lt s from a g e 1 5 t o a g e 5 9 , co rresp o n d in g t o t h e m o st produ ct ive port ion of t he popu lat ion . Accordin g t o t he World Healt h Organ izat ion (WHO), 32% of the global population is infected. In the last decade, 300 m illion people were in fect ed, 90 m illion n ew cases were report ed, an d approximat ely 30 million people died from t he disease. In 1993, t he WHO d e c la re d TB a g lo b a l e m e r g e n c y, a n d M yc o b a c t e r iu m t u b e r c u lo s is (M t b ) is n o w con sidered t he in fect iou s agen t respon sible for m ore adu lt deat hs t han an y ot her.
Variou s vaccin es en list ed t o com bat TB have been st u died. Not able am on g su ch vaccin es are t h o se e n co d in g a n t ig e n 8 5 a n d t h e 6 5 - kDa m ycobact erial heat shock prot ein (hsp65). These are t he t wo most - st u died an t igen s, an d pre- clin ical t rials (which have gen erat ed a great volu me of dat a in t he lit erat u re) have shown promisin g resu lt s. In t he presen t st u dy, we will focus on resu lt s obt ain ed wit h hsp65.
In order t o develop a DNA vaccin e again st TB, Silva et al. in t ro d u ced t h e g en e o f o n e o f t h e m ycobact erial im m u n odom in an t an t igen s, st ress
an im als in ocu lat ed on ly wit h in t raderm al Bacillu s Calm et t e- Gu érin (BCG) or on ly wit h plasm ids n ot con t ain in g t he hsp65 gen e. Two weeks aft er t he t h ir d p la s m id d o s e , p r o d u c t io n o f s p e c if ic an t ibodies was observed in t he seru m of an im als immu n ized wit h hsp65 DNA. In du ct ion of immu n e respon se was det ect ed by a specific t est which measu red t he proliferat ive capacit y of lymphocyt es in t he lym ph n odes when com bin ed wit h hsp65 a n t ig e n s . Im m u n e r e s p o n s e m e d ia t o r s a n d regu lat ors, su ch as in t erleu kin s, were also det ect ed in t he cu lt u re su pern at an t or in t he lym phocyt es of t he im m u n ized an im als u sin g bot h ELISA an d Reverse- t ran script ion polym erase chain react ion (RT- PCR) t echn iqu es. In t hese lym phocyt es, t here was increased liberation of gamma- interferon (IFN-³ ) an d in t erleu kin s (IL- 2 an d IL- 12), which are con sidered cyt okin es t hat st im u lat e a Th1- t ype cellu lar im m u n e respon se, bu t n ot of IL- 4, IL- 10 o r IL- 1 3 , wh ich su p p re ss im m u n e re sp o n se . Therefore, t he pat t ern of induced immune response du e t o immu n izat ion wit h hsp65 DNA in mice was report ed t o be of t he Th1 t ype, which is highly effect ive in t he elim in at ion of in fect iou s agen t s.
DNA vaccine- induced protection
Immunization of mice with hsp65 DNA has been sh o wn t o b e h ig h ly effect ive in p ro t ect in g t h e an im als again st Mt b in fect ion , an d t he prot ect ive effect was very similar t o t hat presen t ed by an imals vaccin at ed wit h BCG(1 0 ). Based on t hese dat a, ot her
Jornal Brasileiro de Pneumologia 3 0 (4 ) - Jul/ Ago de 2 0 0 4
t he gen es of t he im m u n ogen ic prot ein s hsp65, hsp70 an d 36- kDa, com bin ed wit h prom ot ers of cyt om egaloviru s an d hydroxym et hylglu t aryl CoA redu ct ase, con fer sign ifican t prot ect ion again st Mt b in fect ion in m ice(11 ).
Cells involved in protection and studies
on immunologic memory
Silva et al. have carried ou t variou s st u dies in order t o charact erize m ore precisely t he cellu lar s u b p o p u la t io n s c o r r e la t e d w it h c o n t r o l o f in fect io u s p ro cesses, a s well a s t h e m ed ia t o rs in volved (in t erleu kin s), an d t he effect or fu n ct ion s t riggered at t he on set of t he cellu lar act ivat ion p ro ce ss. Th e co m p re h e n sio n o f t h e se fa ct o rs in f o r m s t h e u n d e r s t a n d in g o f t h e im m u n e m echan ism s in volved in TB pat hogen esis, leadin g t o n ew p ersp ect ives o n co n t ro l o f t h e d isease. St u dies evalu at in g an imals in fect ed wit h Mt b an d vaccin at ed wit h hsp65 DNA have been carried ou t in order t o est ablish relat ion ships am on g T- cell su bpopu lat ion phen ot ypes, m ediat ors released by t hese cells, an d t he expression of m olecu les, su ch as CD44, that have been related to cellular migration an d immu n ologic memory.
The T cells of an im als previou sly vaccin at ed w e re t ra n s f e rre d t o n o n im m u n iz e d a n im a ls challen ged wit h Mt b H37Rv. The lat t er an im als sh o wed b et t er co n t ro l o f t h e in fect io n wh en com pared t o t hose on ly in fect ed wit h t he bacilli. The cellu lar phen ot ype correlat ed wit h prot ect ion was charact erized by cells presen t in g high CD44 expression on t heir su rface (CD44h i vs. CD44lo). The
CD44hi cells obtained from immunized animals were
able t o con t rol t he proliferat ion of bacilli bet t er t han t he CD44lo cells from t he sam e im m u n ized
animals, as well as conferring better protection than t he CD44hi an d CD44lo cells from t he in fect ed- on ly
an im als. Am on g t he CD44h i cells, t he CD8+/ CD44h i
cells were m ore efficien t t han t he CD4+/ CD44h i in
in fect ion con t rol, effect ively preven t in g bacillu s mu lt iplicat ion . Wit hin t his cellu lar phen ot ype (t hat provides prot ect ion again st Mt b in fect ion ), it has b e e n sh o wn t h a t CD4 4h i ce lls p re d o m in a n t ly
p ro d u ce IFN- ³ , wh erea s CD4 4lo cells p refera b ly
produ ce IL- 4(7 ,1 2 ).
In order t o defin e which cellu lar phen ot ype p r o vid e d t h e b e s t p r o t e c t io n a g a in s t t h e m ycobact erial in fect ion , clon es of T CD4+/ CD44h i,
T CD4+/ CD44lo, T CD8+/ CD44h i an d T CD8+/ CD44lo
cells were select ed from vaccin at ed an d in fect ed animals. The T CD4+ and T CD8+ cells were obtained
t h ro u g h n eg a t ive select io n a n d sep a ra t ed in t o CD44hi and CD44lo subpopulat ions, using a FACSort
flo w cyt o m et er. Th ro u g h t h e lim it ed d ilu t io n t echn iqu e, 16 T CD4+ clon es an d 16 T CD8+ clon es
were select ed from im m u n ized an im als an d from an imals vaccin at ed wit h hsp65 DNA. These clon es were charact erized for CD44 expression (CD44hi or
CD44lo), IFN- ³ or IL- 4 produ ct ion , an d cyt ot oxic
pot en t ial. The resu lt s show t hat t he fou r CD8+/
CD44h i clon es obt ain ed from im m u n ized an im als
produ ced IFN- ³ an d were cyt ot oxic. These clon es con ferred higher prot ect ion on in fect ed an im als when com pared t o T CD8+/ CD44lo clon es. All fou r
T CD4+/ CD44h i clon es also produ ced IFN- ³ , an d
t h ree were cyt o t oxic. Ho wever, t h eir p ro t ect io n pot en t ial was lower t han t hat of T CD8+/ CD44h i
clon es. As for t he clon es obt ain ed from in fect ed an imals, on ly t wo of t he fou r T CD8+/ CD44hi clon es
produ ced IFN- ³ an d were cyt ot oxic, whereas t he o t h e r t wo clo n e s p ro d u ce d IL- 4 a n d h a d n o cyt ot oxic pot en t ial(7 ,1 2 ,1 3 ).
In light of t his, a hsp65 DNA vaccin e shou ld com prise a grou p of CD4 an d CD8 cell clon es t hat wou ld express high levels of CD44. Su ch CD44h i
clon es cou ld be relat ed wit h immu n ologic memory fu n ct ion s an d possibly wit h accelerat ed migrat ion t o t he in fect ion sit e, where t hey m ight en cou n t er t he appropriat e an t igen - presen t in g cell. Therefore, t hese resu lt s show t hat prior immu n izat ion , u sin g ap p ro p riat e veh icles an d ad m in ist rat io n ro u t es, wou ld be effect ive in t riggerin g specific local an d syst em ic im m u n e respon ses t hat are efficien t in mycobact erial in fect ion con t rol.
Use of carriers in genetic vaccines
against tuberculosis
A st u dy in volvin g DNA vaccin es based on t he hsp65 prot ein , which had previou sly on ly been u s e d a s n a k e d DNA a n d a d m in is t e r e d in t ram u scu larly, was recen t ly con du ct ed(1 4 ). Th e
been an alyzed. However, dat a from com parat ive st u dies have shown t hat in t ram u scu lar in ject ion of DNA preferably in du ces a Th1- t ype im m u n e response, whereas gene- gun bombardment t riggers a Th 2 - t yp e im m u n e r e s p o n s e . It h a s b e e n d em o n st rat ed t h at g en e- g u n ad m in ist rat io n o f hsp65 DNA at a dose level a hu n dred t im es lower t h a n t h a t u sed in co n ven t io n a l in t ra m u scu la r in ject ion has been shown t o st im u lat e bot h t ypes of hu m oral an d cellu lar respon ses. The gen e- gu n admin ist rat ion preferably st imu lat ed su bclass IgG1 a n t ib o d ie s , a n d d if f e r e n c e s w e r e t h e r e f o r e observed bet ween t he levels of t he IgG1 an d IgG2 immunoglobulin subclasses, suggest ing a Th2- t ype response. In addition, BALB/c mice immunized with in t ram u scu lar in ject ion of hsp65 DNA produ ced high levels of IFN- ³ , whereas t he im m u n izat ion u s in g g e n e g u n d o e s n o t s t im u la t e IFN- ³ p ro d u ct io n – a lt h o u g h it d o e s st im u la t e t h e produ ct ion of Th2- t ype cyt okin es, su ch as IL- 10 an d IL- 4. This Th2- t ype respon se m ay be du e t o t he sm all am ou n t of DNA u sed in t he gen e- gu n t echn iqu e. The lower qu an t it y of DNA goes han d in han d wit h a redu ct ion in t he n u m ber of CpG m ot ifs. As a m at t er of fact , m ice im m u n ized wit h in t raderm al in ject ion of hsp65 DNA presen t ed a red u ct io n in t h e b a ct eria l lo a d s in t h e lu n g s, whereas t hose immu n ized u sin g gen e gu n did n ot . Th e re fo re , a lt h o u g h t h e g e n e - g u n t e ch n iq u e t r ig g e r e d t h e s p e c if ic im m u n e r e s p o n s e a t sig n if ic a n t ly lo w e r d o se s o f DNA, it is n o t appropriat e for adm in ist rat ion of t he hsp65 DNA vaccin e again st TB(1 6 ).
A se co n d st ra t e g y t h a t h a s b e e n u se d t o o p t im iz e h sp 6 5 - DNA va c c in a t io n is t h a t o f en capsu lat in g t he DNA in biodegradable polym er microspheres. The resu lt s of st u dies employin g t his t e c h n iq u e h a ve s h o w n t h a t DNA c a n b e en ca p su la t ed in t o p o ly- la ct ic- co - g lyco lic a cid (PLGA) microspheres wit h n o fu n ct ion impairmen t . This m et hod was able t o produ ce part icles of an a p p ro p ria t e siz e (fu n ct io n in g a s ve ct o rs in t o p h a g o cyt ic cells), a s well a s efficien t in DNA
in t ram u scu lar or in t raderm al adm in ist rat ion . The capability to directly transfect professional antigen-in t rodu cantigen-in g cells m ay be antigen-in t erest antigen-in g santigen-in ce several st u d ie s h a ve sh o wn t h a t a n t ig e n ic p e p t id e s, in vo lve d in t h e s t im u la t io n o f c yt o t o xic T lymphocyt es aft er DNA vaccin at ion , are in t rodu ced by m ajor hist ocom pat ibilit y com plex (MHC) class I cells d erived fro m b o n e m a rro w a n d n o t b y t ran sfect ed myocyt es(17- 19). Therefore, when direct ly
t ran sfect ed in vivo, t hese cells t hat express co-st im u la n t m o le cu le s o n t h e ir su rfa ce p re se n t an t igen s t hat provide t he n ecessary sign als for t he s t im u la t io n o f T lym p h o c yt e s . In a d d it io n , following intramuscular injection, the microspheres form a tissue deposit, recruiting antigen- presenting cells t o t h e a d m in ist ra t io n sit e, a llo win g t h e p a r t ic le s t o b e m o r e e a s ily c a p t u r e d a n d , consequ en t ly, facilit at e t he t ran sfect ion of t hese an t igen - presen t in g cells. Alt hou gh m icrospheres are capable of improving antigen presentation, they have n o immu n ost imu lat in g propert ies. Therefore, form u lat ion s wit h m icrospheres con t ain in g DNA have been shown t o st im u lat e specific im m u n e respon se, bu t t his respon se was n ot su fficien t t o prot ect BALB/ c m ice again st in fect ion aft er Mt b challen ge.
Th e a d d it io n o f a d ju va n t s w it h immunostimulant properties and carriers employing m icroen capsu lat ion t echn ology has been u sed in order t o im prove t he im m u n ogen ic capacit y of m icrosphere form u lat ion s. A n ew form u lat ion has been devised t hat con t ain s hsp65- DNA vaccin e an d an im m u n ost im u lan t agen t , t rehalose dim ycolat e (TDM), b o t h e n c a p su la t e d in b io d e g ra d a b le m icrospheres(2 0 ).
Jornal Brasileiro de Pneumologia 3 0 (4 ) - Jul/ Ago de 2 0 0 4
1 5 d a ys p r io r w it h a s in g le d o s e o f DNA en capsu lat ed in microspheres. However, in an imals immunized with three doses of naked DNA, no such e xp re ssio n wa s o b se rve d in t h e se se co n d a ry lym phoid organ s (where lym phocyt e st im u lat ion o ccu rs).
Re g a rd in g t h e im m u n e re sp o n se it se lf , a sign ifican t chan ge in t he qu alit y of an t ibodies has b e e n o b s e r ve d a f t e r im m u n iz a t io n w it h m icro sp h eres co n t ain in g h sp 6 5 - DNA/ TDM. Th e h u m o r a l r e s p o n s e in t h e s e a n im a ls w a s ch aract erized b y h ig h levels o f Ig G2 a, wh ereas an im als im m u n ized wit h n aked DNA presen t ed a mixed profile, wit h high levels of IgG1 an d IgG2a. The cellu lar im m u n e respon se was charact erized b y h ig h levels o f IFN- ³ . Th irt y d a ys a ft er t h e c h a lle n g e , t h e a n im a ls im m u n iz e d w it h m icro sp h eres co n t a in in g h sp 6 5 - DNA a n d TDM presen t ed high levels of IFN- ³ in t he lu n gs(2 1 ). This
profile may have been promot ed by TDM. In 2001, Ryll et al. report ed t hat TDM m ay st im u lat e an im m u n e respon se, eviden ced by a proliferat ion of NK cells, an d t hat TDM m ay also provoke an early im m u n e respon se t hrou gh t he release of IFN- ³ , leading to macrophage activation and upregulation of t he expression of MHC class II m olecu les an d
CD1(2 2 ). Th erefo re, TDM m a y p ro vid e excellen t
co n d it io n s f o r t h e in it ia t io n o f a n im m u n e re sp o n se , w h ic h m a ke s it a g o o d Th 1 - t yp e st im u la t in g co m p o n en t fo r u se in TB va ccin e formu lat ion s u n der developmen t .
Sign ifican t prot ect ion again st in fect ion wit h H3 7 Rv Mt b w a s a c h ie ve d w h e n m ic e w e r e vaccin at ed eit her wit h in t ram u scu lar in ject ion of n aked hsp65 DNA or wit h hsp65 DNA an d TDM e n ca p su la t e d in m icro sp h e re s. Ho w e ve r, t h e microsphere formu lat ion was able t o st imu lat e t he prot ect ive im m u n e respon se aft er a sin gle dose, whereas t hree doses of t he in ject ion form u lat ion we re re q u ire d in o rd e r t o p ro vid e t h e sa m e prot ect ion . The vaccin e based on microspheres also allowed each plasm id dose t o be redu ced t o on e-t en e-t h of e-t hae-t of e-t he n aked DNA vaccin e. These dae-t a showed t hat , in t his part icu lar m ou se st rain , t he immu n e respon se t o a specific an t igen was direct ly in f lu e n c e d b y t h e m e t h o d o f DNA va c c in e adm in ist rat ion . The developm en t of form u lat ion s in volvin g a combin at ion of adju van t s wit h variou s act ion m echan ism s m ay be an alt ern at ive for t he developm en t of m ore effect ive vaccin es.
Exploring the prime- boost strategy
Th e d u ra t io n o f p ro t e ct io n p ro vid e d b y a vaccin e has been relat ed t o t he perm an en ce of t he an t igen in t he organ ism . Therefore, in som e va ccin a t io n re g im e n s, t h e a d m in ist ra t io n o f m u lt iple doses m ay be n ecessary for m ain t ain in g t h e im m u n it y st im u la t e d b y t h e in it ia l d o se . Recen t ly, t h e p rim e- b o o st p rin cip le h a s b een e xt e n sive ly e xp lo re d wit h t h e co n t e xt o f t h e het erologou s boost con cept . In t his con t ext , in it ial an d su bsequ en t doses in t rodu ced in t o t he immu n e syst em co n t a in t h e sa m e a n t ig en , a lt h o u g h in d ifferen t fo rm u la t io n s. It is essen t ia l t h a t t h e form u lat ion u sed in t he in it ial dose in du ce t he n e c e s s a r y r e s p o n s e f o r t h e p r o t e c t io n o f im m u n iz e d in d ivid u a ls . Th e m o s t c o m m o n prot ocols u se DNA or su bu n it vaccin es in t he in it ial d o se , a n d livin g ca rrie rs, su ch a s viru se s o r re co m b in a n t b a ct e ria e xp re ssin g t h e sp e cific an t igen , in t he followin g doses. Alt hou gh som e resu lt s have been prom isin g, t his t ype of prot ocol revert s t o t he u se of livin g vect ors, t he avoidan ce o f w h ic h w a s t h e m o t iva t io n b e h in d t h e developm en t of DNA an d su bu n it vaccin es. The p rim e- b o o st st ra t eg y in vo lves t h e u se o f t wo d if f e re n t va ccin e s, e a ch e n co d in g t h e sa m e an t igen , adm in ist ered at in t ervals of a few weeks. This st rat egy has been shown t o in crease cellu lar im m u n e resp o n se in severa l a n im a l m o d els o f in fect ion(2 3 ). As previou sly men t ion ed, mostprime-b o o st p ro t o c o ls u n d e r d e ve lo p m e n t in vo lve in du ct ion of t he im m u n e respon se wit h a DNA vect o r, su ch a s p la sm id DNA, u sin g live- viru s vect ors(2 4 ). The u se of t he prime- boost st rat egy for
TB preven t ion has been evalu at ed, com bin in g t he u se of DNA in t he in it ial dose for t he in du ct ion of t he im m u n e respon se wit h t he u se of at t en u at ed BCG o r reco m b in an t p ro t ein s t o m ain t ain t h at resp o n se(2 5 ,2 6 ). Th ese p ro t o co ls t yp ica lly req u ire
t hat m ore t han on e DNA dose be adm in ist ered in order t o in du ce t he in it ial im m u n e respon se, an d boost er doses are adm in ist ered su bsequ en t ly.
con t ain in g recombin an t prot ein (also en capsu lat ed an d admin ist ered in con ju n ct ion wit h t he DNA) has been evalu at ed. However, in a form u lat ion wit h microspheres con t ain in g bot h DNA an d prot ein , it is essen t ial t hat t he DNA be released prior t o t he p ro t ein sin ce t h e DNA st im u la t es a Th 1 - t yp e re sp o n se . Th is is n o t a co n ce rn wh e n u sin g m icrospheres con t ain in g recom bin an t hsp65. In a d d it io n , co - e n ca p su la t in g TDM in o rd e r t o p r o m o t e im m u n o s t im u la t io n f a vo r s t h e developm en t of a Th1- t ype respon se sin ce TDM in du ces t he secret ion of cyt okin es su ch as IL- 12, t hereby con t ribu t in g t o t he est ablishm en t of a favorable microen viron men t for t he differen t iat ion of Th1 lymphocyt es du rin g an t igen presen t at ion(20).
Recom bin an t hsp65 was in it ially en capsu lat ed in 75:25 PLGA m icrospheres, which can provide the release of the protein for 90 days. This enhanced du rabilit y is du e t o t he great er n u m ber of lact ic-acid m on om ers in t he polym er chain , m akin g it le ss h yd ro p h ilic a n d re d u cin g it s h yd ro lyt ic degradat ion when compared t o DNA en capsu lat ion in 50:50 PLGA m icrospheres. The com bin at ion of 50:50 PLGA m icrospheres con t ain in g hsp65 DNA a n d 7 5 : 2 5 P LGA m ic r o s p h e r e s c o n t a in in g recom bin an t (prim e boost ) hsp65 produ ced high levels of an t i- hsp65 an t ibodies. Seru m levels of t hese an t ibodies rem ain ed elevat ed 90 days aft er va ccin a t io n . In crea sed p ro d u ct io n o f IFN- ³ b y sp le n ic ce lls in va ccin a t e d a n im a ls wa s a lso report ed du rin g t he st u dy period (90 days). These resu lt s su ggest t hat prime- boost syst ems based on n o n - livin g ve ct o rs sh o w g re a t p o t e n t ia l f o r in creasin g t he du rat ion of t he im m u n e respon se an d decreasin g risks relat ed t o t oxicit y.
Gene therapy for tuberculosis: the
thera-peutic action of the hsp65- DNA vaccine
Un t il recen t ly, t here was n o expect at ion t hat t he TB bacilli cou ld be elim in at ed in m an y ou t of t he great n u mber of in dividu als in fect ed (on e- t hird of t he global popu lat ion ). The bacilli u su ally reside in t he lu n gs for an in defin it e len gt h of t im e, bu t
t he in dividu al m ay in fect ot hers. The disease m ay cau se severe lesion s t hat persist even aft er t he elim in at ion of t he bacilli, cau sin g a great deal of su fferin g for t he pat ien t .
There is a t ren d t oward great er TB let halit y t hat m u st be expedit iou sly in vest igat ed in t he com in g years. Therefore, it is absolu t ely n ecessary t hat appropriat e m odels be devised for com bat in g an d con t rollin g TB.
The results of experiments performed in a recent st u dy showed t hat t he adm in ist rat ion of hsp65-DNA vaccin e in an im als previou sly in fect ed wit h viru len t Mt b n ot on ly preven t s t he developm en t of the disease but eliminates the infection as well(29).
Th is is ext rem ely releva n t if we co n sid er t h a t a p p ro xim a t e ly t wo b illio n p e o p le a re a lre a d y in fect ed wit h TB bacilli. In Brazil, t he n u m ber of people in fect ed is approxim at ely 60 m illion . In addit ion t o t hat , t he vaccin e can also cu re even when admin ist ered in in dividu als wit h more severe p r e s e n t a t io n s o f t h e d is e a s e o r a f t e r t h e d isse m in a t io n o f t h e d ise a se t h ro u g h o u t t h e organ ism. In fect ion wit h Mt b preven t s t he immu n e syst em s of an im als from respon din g again st t he p at h o g en ic ag en t , allo win g t h e b acilli t o g ro w rapidly an d t he disease t o becom e en t ren ched. Th ese au t h o rs d em o n st rat ed t h at g en e t h erap y u sin g h sp 6 5 - DNA va ccin e s p ro vo ke s ra d ica l, p ro fo u n d ch a n g e s, t ra n sfo rm in g t h e im m u n e respon se from t he Th2 t ype t o t he Th1 t ype an d helpin g t he organ ism com bat bacilli an d cu re t he disease, even if an t im ycobact erial chem ot herapy is n o t a d m in is t e r e d(2 9 ). Th is w a s t h e f ir s t
demon st rat ion of t he pot en t ial of gen et ic vaccin es for cu rin g in fect iou s diseases.
Immunotherapy for multidrug- resistant
tuberculosis
Jornal Brasileiro de Pneumologia 3 0 (4 ) - Jul/ Ago de 2 0 0 4
com bin at ion s of t wo, t hree or even of all t hem simu lt an eously. Pat ien t s in fect ed wit h mu lt idru g-resist an t st rains can expect few, or somet imes n o, t reat m en t alt ern at ives. Recen t st u dies have also sh o wn t h a t a n im a ls in fect ed wit h m u lt id ru g -r e s is t a n t b a c illi w e -r e c u -r e d t h -r o u g h t h e adm in ist rat ion of gen et ic vaccin es(2 9 ).
Immunotherapy for latent tuberculosis
In correlation with the great number of infected in dividu als, an ot her problem is how easily t he TB bacilli adapt t hemselves t o hu man s. The in fect ion u su ally resu lt s fro m b acilli b ein g in h aled an d en t erin g t he defen se cells of t he host organ ism. From within the macrophages, which are cells with h ig h m icro b icid a l p o t e n t ia l, t h e b a cilli ca n deactivate the defense systems of the macrophage, surviving and reproducing t herein. In humans, t he immu n e defen se syst em iden t ifies t he presen ce of the bacilli and establishes a response, characterized by a chronic granulomatous inflammatory reaction designed to circumscribe and delimit the infection. Un der t hese con dit ion s, t he bacilli may su rvive in a latent or dormant state for years, and the infected in d ivid u a l m a y n o t p resen t sym p t o m s o f t h e d isea se. If t h is m u t u a l rela t io n sh ip b eco m es u n balan ced, a sit u at ion t ypically associat ed wit h su ppression of t he immu n e respon se, t he disease m a y m a n if e s t it s e lf . Th e m o s t c o m m o n immunosuppression conditions seen concomitantly with TB are, among others, patients who are taking immu n osu ppressan t dru gs (su ch as AIDS pat ien t s a n d p a t ie n t s su f f e rin g f ro m st re ss- re la t e d sympt oms), dru g addict s (su ch as alcoholics) an d malnourished pat ient s.Gene therapy has also been used as a means of analyzing the state of latency or dormancy of the mycobacteria, which can become reactivated and manifest t he disease during immunosuppressive states. An experimental mouse model was developed t h a t m im ics t h e co n d it io n s o b se rve d in t h e development of the human form of the disease in immunosuppressed individuals. In the control group an im als, wh ich were n o t vaccin at ed b u t were infected, given antibacterial drugs (to establish a s t a t e o f la t e n c y) a n d t h e n t r e a t e d w it h co rt ico st ero id s t o in d u ce im m u n o su p p ressio n , reactivation and establishment of the disease was observed. In the study group animals, which were treated with the DNA vaccine, no reactivation or
development of the disease was observed, especially when three doses of the vaccine were administered29.
The ability of DNA vaccines to eliminate dormant bacteria may be of considerable benefit in the control, or even eradication, of TB.
Gen et ic vaccin es have been u sed for t reat m en t , which is con cept u ally differen t t han t he m an n er in which t radit ion al vaccin es are u sed (on ly t o preven t diseases). These DNA vaccin es cu re in fect ion , cu re t he disease an d preven t disease react ivat ion , while main t ain in g t heir prophylact ic n at u re. In n u merable pract ical an d st rat egic ben efit s are derived from t he developmen t of t his t herapeu t ic vaccin e again st TB. It is safe an d efficaciou s, an d it can be admin ist ered in a sin g le d o se, st im u la t in g a n a m p le im m u n e respon se. It has a lon g- last in g prot ect ive effect an d may sign ifican t ly con t ribu t e t o decreasin g in ciden ce of t he disease.
Reducing the tuberculosis treatment period
with the use of drugs and DNA vaccine
in n u m e ra b le , m a kin g t h e s e va c c in e s h ig h ly d esira b le in t h e co n t ext o f t h e p u b lic h ea lt h p ro b lem s exist in g in d evelo p in g co u n t ries. Th e impact on in fect iou s disease con t rol will probably be on e of t he most import an t aspect s of mast erin g t his n ew t echn ology, sin ce man y serious in fect ious d isea ses m a y even t u a lly b e p reven t ed t h ro u g h gen et ic im m u n izat ion . The developm en t of n ew vaccin es t hat m ay soon preven t t he u n con t rolled in crease in t he dissemin at ion of in fect iou s diseases is of fu n damen t al import an ce for man kin d. A DNA vaccin e again st TB cou ld con t ribu t e sign ifican t ly t o t he con t rol of t he disease.
REFERENCES
1 . Silva CL. Va c in a s g ê n ic a s . Bio t e c n o lo g ia - Ciê n . Desen vo lv. 1 9 9 7 ; 3 : 3 2 - 4 .
2 . Davis HL, Wh alen RG, Dem en eix BA. Direct g en e t ran sfer in skelet a l m u scle in vivo : fa ct o rs a ffect in g efficien cy o f t ra n sfer a n d st a b ilit y o f exp ressio n . Hu m a n Gen e Th er. 1 9 9 3 ; 4 : 1 51 - 6 .
3 . Vit a d e llo M, Sc h ia f f in o MV, P ic a rd A, Sc a r p a M, Sc h ia f f in o S. Ge n e t ra n s f e r in g e n e ra t in g m u s c le . Hu m a n Gen e Th er. 1 9 9 5 ;5 :11 - 2 .
4 . Wan g B, Ug en K, Srikan t an V, Ag ad jan yan MG, Dan g K, Ra fa eliu J , et a l. Gen e in o cu la t io n g en era t es im m u n e re sp o n se s a g a in st HIV- 1 . P ro c Na t l Ac a d Sc i USA. 1 9 9 3 ; 9 0 : 41 5 6 - 6 0 .
5 . We lls DJ . Im p ro ve d g e n e t ra n sfe r b y d ire ct p la sm id in je c t io n a s s o c ia t e d w it h r e g e n e r a t io n in m o u s e skelet a l m u scle. FEBS Let t ., 1 9 9 3 ;3 3 2 :1 7 9 .
6 . Ferreira GNM, Mo n t eiro GA, Pra zeres DMF, Ca b ra l J MS. Do w n s t r e a m p r o c e s s in g o f p la s m id DNA f o r g e n e t h e r a p y a n d DNA va c c in e a p p lic a t io n s . Tib t e c h . 2 0 0 0 ; 1 8 : 3 8 0 - 7 .
7 . Bonato VLD, Lima VMF, Tascon RE, Lowrie DB, Silva CL. Identification and characterization of protective T cells in hsp65 DNA- vaccin at ed an d Mycobact eriu m t u bercu losis in fect ed mice. In fec Immu n ol. 1998;66:169- 75. 8 . Lowrie DB, Tascon RE, Colston MJ, Silva CL. Towards a DNA
vaccine against tuberculosis. Vaccine. 1994:12:1537- 40. 9 . Lo wrie DB, Silva CL, Co lst o n MJ , Rag n o S, Tasco n RE.
P r o t e c t i o n a g a i n s t t u b e r c u l o s i s b y p l a s m i d DNA.Va ccin e . 1 9 9 7 ; 1 5 : 8 3 4 - 8 .
1 0 . Lo wrie DB, Ta sco n RE, Silva CL Va ccin a t io n a g a in st tuberculosis. Int Arch Allergy Immunol. 1995;108:309- 12. 11 . Lo wrie DB, Co lst o n MJ , Ta sco n RE, Silva CL. DNA e n co d in g in d ivid u a l m yco b a ct e ria l a n t ig e n s p ro t e ct s m ice ag ainst t u b ercu lo sis. In : Bro wn F, Bu rt o n D
tuberculosis. Immunol. Cell Biology unol. 1997;75:591- 4. 1 5 . Mo rein B. No vel ad ju van t s an d vaccin e d elivery syst em s.
Ve t . Im m u n o l Im m u n o p a t o l. 1 9 9 6 ; 5 4 : 3 7 3 - 8 4 . 1 6 . Lim a KM, d o s Sa n t o s SA, Sa n t o s J r. RR, Bra n d ã o IT,
Ro d ríg u e z J r. J M, Silva CL. Ef f ica cy o f DNA- h sp 6 5 va ccin a t io n va ries fo r t u b ercu lo sis va ries wit h m et h o d o f DNA in t ro d u ct io n in vivo . Vaccin e. 2 0 0 3 ;2 2 :4 9 - 5 6 . 1 7 . Do e , B. In d u c t io n o f c yt o t o xic T lym p h o c yt e s b y in t r a m u s c u la r im m u n iz a t io n w it h p la s m id DNA is fa cilit a t e d b y b o n e m a rro w- d e rive d ce lls. Pro c Na t l Aca d Sci USA. 1 9 9 6 ;9 3 :8 5 7 8 - 8 3 .
1 8 . Akbari O, Panjwani N, Garcia S, Tascon R, Lowrie D, Stockinger B. DNA vaccination: transfection and activation of dendritic cells as key events for immunity. J Exp Med. 189:169- 77 1 9 . Klin m an DM. Co n t rib u t io n o f cells at t h e sit e o f DNA
va c c in a t io n t o t h e g e n e r a t io n o f a n t ig e n - s p e c if ic immu n it y an d memory. J Immu n ol. 1999;160:2388- 92. 2 0 . Lima VMF. Role of t rehalose Dimycolat e in recru it men t of cells an d modu lat ion of produ ct ion of cyt okin es an d NO in t u b ercu lo sis. In fec Im m u n . 2 0 01 ;6 9 :5 3 0 5 - 1 2 . 2 1 . Lim a KM, San t o s AS, Lim a VMF, Co elh o - Cast elo AAM,
Ro d rig u e s J r J M, Silva CL. Sin g le - d o se o f a va ccin e b ased o n DNA en co d in g m yco b act eriu m h sp 6 5 p ro t ein p lu s TDM- lo a d ed m icro sp h eres p ro t ect s m ice a g a in st a viru len t st ra in o f Myco b a ct eriu m t u b ercu lo sis. Gen e Th e r. 2 0 0 3 ; 1 0 : 6 7 8 - 8 5 .
2 2 . Ryll R. Mycobacterium cord factor, but not sulfolipid, causes d ep let io n p f NKT cells an d u p reg u lat io n o f CD1 d 1 o n mu rin e macrophages. Microbiol In fect . 2001;3:611- 9. 2 3 . McSh a n e H. Prim e- b o o st im m u n iza t io n st ra t eg ies fo r
in fect io u s d isea ses. Cu rr Op in Mo l Th er. 2 0 0 2 ;4 :2 3 - 7 2 4 . Ro b in so n , H.L. P rim e b o o st va ccin e s p o we r u p in
p eo p le. Na t Med . 2 0 0 3 ;9 :6 4 2 - 3 .
2 5 . Skin n er MA, Bu ddle BM, Wedlock DN, Keen D, de Lisle GW, Tascon RE, et al. A DNA prime- Mycobact eriu m bovis BCG b o o st va c c in a t io n st ra t e g y f o r c a t t le in d u c e s prot ect ion again st bovin e t u bercu losis. In fect Immu n ol. 2 0 0 3 ; 71 : 4 9 01 - 7 .
2 6 . Vo rd erm eier HM, Lo wrie DB, Hewin so n RG. Im p ro ved i m m u n o g e n i c i t y o f DN A v a c c i n a t i o n w i t h m yco b a ct e ria l HSP6 5 a g a in st b o vin e t u b e rcu lo sis b y p ro t ein b o o st in g . Vet Micro b io l. 2 0 0 3 ; 9 3 : 3 4 9 - 5 9 . 2 7 . Lu n sfo rd L, McKeever U, Eckst ein V, Hed ley ML. Tissu e
d ist rib u t io n a n d p ersist en ce in m ice o f p la sm id DNA e n ca p su la t e d in a PLGA- b a se d m icro sp h e re d e live ry veh icle. J Dru g Ta rg et . 2 0 0 0 ;8 :3 9 - 5 0 .
2 8 . Brio n es M, Sin g h M, Ug o zzo li M, Kazzaz J , Klakam p S, Ot t G, O’Ha g a n D. Th e p rep a ra t io n , ch a ra ct eriza t io n , a n d e va lu a t io n o f c a t io n ic m ic ro p a rt ic le s f o r DNA va ccin e d elivery. Ph a rm Res. 2 0 01 ;1 8 :7 0 9 - 1 2 . 2 9 . Lo wrie DB, Tasco n RE, Bo n at o VLD, Lim a VMF, Faccio li
