Anthony Muyornbwe

Approches moléculaires pour le développement des vaccins vivants

contre le parasite protozoaire Leishmania

Mémoire présenté à la Faculté des études supérieures de l'Université Laval porir l'obtention du grade Maître ès Sciences (M.SC.)

Département de Microbiologie-lmmunulogie Faculté de Médecine Université Laval

Juillet 1997

O Anthony Muyombwe 1997

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Acknowledgrnen ts

Special thanks to Dr. Barbara Papadopoulou who allowed me to work in her laboratory. Her relentless search for scientitic knowledge and perfection was for me a source of motivation and encouragement. I would like CO thank also Dr. Marc Ouellette and Martin Olivier for their time and advices. And finally I would like to acknowledge al1 members of the Service d'fnkctiologi~ and thank them for their encouragement.

v

contents

. S .

Abstract ............................................................................. ri1

............................................................................... Résumé i v .............................................................................. Contents v ...

Tables and figures list ...................................................... viii

Appendix list ...................................................................... ix Lexicon .............................................................................. 75 . . .................................................................... Ab breviations. x

CHAPTER 1: GENERAL INTRODUCTION

1.2. Lrishrna~iia major ................. ...... ........

5 1.4. Leishmania brailiensis ...............................,...............

CHAPTER II: SUBJECT INTRODUCTION

2.1. Current situation. .............................................................. 7

2.2. The regulation of immuni ty to Lrishnitrtiitr ........................ 8

2.2.1 . Lrisl~rnmicl and niricropliagrs. ............................ 8 A) Mettr c-yc2ojiene.si.s.. ......................................... 8 B ) In iwsion.. .......................................... 9

................ C ) Intriicellir km- c.otlll~clrtinentc~Ii~~ltioli.. 1 O D ) Mcrcr-ophtrge clctivution.. ................................. 13

2.3.2. Development of CD4 effector lymphocytes ............. 1 3 ........ A) Lin eagr of' poltr rizrd 7-11 IfTh2 respotises.. - 1 3

................................................ 2.2.3. Immunogenetics.. - 1 5

. . 2.3. Development of vaccines against Izishnianiasis ...................... -18

2.3.1 . Clinical vacciaes ............ .. ....... ..,,...,.. .................... 18 A) Living promczstigote vaccines ........................... 18

............................ B) Killed pi-omzsrigotr vacciirrs 19

2.3.2. Experimental vaccination ..................................... 19 A) Arrrnrcarrd voccines .................................... 19 B) Killrd vaccines .......................................... 20 C ) Fracriona te J vuccines ................................. 21

2.3.3. Mechanism of prophylactic imrnunity ............... 32

2.3 .4 . Molecu Iar vacci nes ................. ,.. .................

2.4. Objectives ...................................................................

CHAPTER I I I : MATERIALS A N D WIETHODS

Cloning and DNA constructs ......................................... A) CIoning qf DNA in e.rprrssinrl vrcrors ................... B ) DNA consrnt crs .................................................

Transfection ...............................................................

Hybridization .............................................................

Total DNA preparation ................................ A) Rapid ~ c h n i c p e .................................................. B ) Classictzl technique ....................................

RNA preparntion ...............................................

Sou thern b lot .....................................................

Northern blot ................................. ... ...............

Rationale of experiments ............................................

3.9. Cell growth assay ......................................................

3.10. MTT test ................................................................

........................................ 3.1 1 . In vitro macrophage assay

3.12. In vivo assay .................... .... .............................

3 . I 2.2. Pharrnacokinetics und toxicit y .srr~dir.s ........ .. A) Pharrnacokinrtics evcllnution ...................

................................. B ) Tissue clistrïbrltion C ) Hrmutnlo,y icd toxiciry ............................

.................................. D ) Stcitisticul cincdÿsis

CHAPTER IV: RESULTS AND DISCUSSION ..........

CHAPTER V: CONCLUSION .....................................

........................................................ BIBLIOGRAPHY

Tables:

Tables and figures list Page

x

1. Pharmacokinetics parameters in B A L B k 50

2. Footpads ganciclovir distribution ..................... 5 1

3. Hematological effects of ganciclovir on BALBfc mice ...................................... 52

Figures: 1. Mechanism of nucleoside analogues siich as

acyclovir and ganciclovir .............................. 53

3. Construction of Le i shnz~~ i~ i~z vectors expressing the HSV- I TK gene ......................... 54

3. Cytotoxic effect of ganciclovir on Lrishinrznicz spp. prornnstigo tes express i ng

the HSV-1 TK gene ....................................... 55

4. T K expression in Leishtirclrii~~ tilczjor prornastigotes and amas ti gotes. ..................... 56

5. Cytotoxic effect of janciclovir on Leislmclniu incyor arnastigotes expressi iig the

HSV- 1 TK gene ...................................... 57

6. MTT test to evalunte gancicluvii- toxiciry to macrophages ..................................... 53

7. Concentration ciirves of ['HI-gnnciclovir i n nuce serurn ......................................... 59

S. Ganciclovir treatment of irift'cted iiiice ............. 60

9-A. Challenge of cured mice with L. t i i ~ ~ j o r wild-type.. ........................................... 6 1

9-B. Challenge of ciired mice with L. ~ricl.jor- wi ld-type .....................................................

Appendix Iist

Page

I . Geographical distribution of leishmaniasis ..................... 83

II- Transmission cycles of Leishmmi ici .......................... 83

III . Life-cycles of Leishmtrn i<z .................... ... .................. 84

IV . Parasites. reservoirs clinical manifestation and geographical dis tribution A . Visceral leis hmaniasis ................................................. 85 8 . Cu taneous leis hmaniasis ............................................... 85 C . Mucocutaneous leishmaniasis ........................................ 86

V . Mechanisms by whicli macrophages becorne activated to kill parasi tes ............................................................... 87

VI . Cytokines that regii late cel 1-niediareci i mrnii ni ty ................ 88

VI1 . Theories for Th 1 Kh2 divergence i n ssperimental L . major infection ........................................................... 89

VIII . Experimental vaccination agninst cutaneous leishmoniasis (L . mïz~or ) in mice ..................................

IX . Chemical stnictures of naturally occiiring nuclroside analogues ........................................................................ 91

X . Action of ganciclovir ........................................................ 92

XI . pSP72 plasmid vector map ........................................ 93

XI . pSP72 vector restriction si tes ............................................ 94

XI1.A . Linear map of the Herpss simplex virus type- 1 ............... 95

X1I.B . Sequence of the thymidine ki nase.-ene ........................ 101

XII1 . SDM-79 medium ....................................................... 103

Abbreviations

AIDS: Acquired immuno-deficiency syndrome ATPase: adenosine triphosphate nuclease bp: base pairs BSA: Bovine serum albumin BTS: B e c h a n Instruments CGRP: calci toni n gene-related peptide CIP: calf intestinal alkaline phosphatase cm: centimeter(s) DTH: delayed- type hypersensi tivity DHFR-TS: dihydrofolate reductase-thymidyliite synthasr DHSa: Escherichici coli strnin genotype F,080dlacZM 15, recA I ,

endA 1, gyrA96, thi- 1 . hsdR 17(rk-, mk'), supE44, relA 1 , deoR, (lacZYA-argF)U 169.

D-MEM: Dulbecco's modified eagle medium DNA: desoxyribonucleic acid DL-GC: delipidated water-soluble glycoconjugrites EDTA: ethylenediarnine tetracetate dGTP: deoxyguanosine triphosphate EC: elimination cui-ve ETOH: ethanol Fc Ig: immunoglobulin fragment c h a h GCV: ganciclovir GM-CSF: granulocyte/mncrophage colony-stimiilati ng factor G418: geneticin Hb: hemoglobin HBSS: hank balanceci salt Hepes: sulfoniniqur ncid 4-(3-11ydroxyetli~~l)- 1 -pipe[--azinrrthrine FI t: hematocri t HSV-1: herpes simplex vii-lis type- 1 IFN-y: interferon gamma IL: Interleukin kb: kilobase(s) LAF: lymphocyte-activating factor LB: LuriaBerthane LPS: lipopolysaccharide LPG: lipophosphoglycan Lsh: Leishmania

M-CSF: macrophage colony-stimiilating factor MHC: major histocompati bili ty complex ml: milliliter(s) MOPS: (3-[N-morpholino]propanesuifoniç acid) mm: miIIirneter(s) mM: rnillimolar(s) MTT: (3-[4,5-dimethylthiazol-2-y l]-2,5-di phen trazolim brornide;

thiazolblue) mRNA: nbonucleic acid messanger MRT: mean residence time NO: nitric oxide NK: natural kiiler PKa: proteine kinase A PBMC: Peripheral blood mononuclear cells PBS: phosphate-buffered sali ne Pgp: P-glycoprotein RBC: red blood ce11 RNA: ribonucleic acid rpm: rotation per minutes SEM: standard error mean SDS: sodium dodecyl sulfate Spp: species SSC: sodium chloride and sodium citrate biiffer TBE: Tris-Borate-EDTA TE : Tris-EDTA TCR:T ce11 receptor Th: T cell helper TK: thymidine kinase Tris: tris-(hydroxymethy 1)ami nomethane mF: microfarad(s) mg: microgram(s) ml: microliter(s)

V/V: ratio voIurne/voli~ me W/V: ratio weight/voiume

CHAPTER 1

GENERAL INTRODUCTION .

Leishmania is a member of the family of trypanosomatidae and order Kinetoplastida. Like most members of t h e family of trypanosomatidae, I.eishmnnia is heteroxenous: during one stage of its live, is present in the blood and or fixed tissues of al1 classes of vertebrrites, and during the second stage, i t

lives in the intestines of blood-sucki ng invertebrates. Thus , Lrishrnczriiu is usually called hernoflagellate. Al1 species are either elongrited wi th a single flagellurn or rounded with a very short, nonprotruding tlagelluni. A11 forms have a single nucleus. The flagellum arises from the kinetosome and i t is responsible for propulsion of the niicroorganism. The flagrlliim niay also mach the organism to an insect host's gut wall or salivary gland rpithelium. Closely associated with the kinetosome is a structure unique to the order, the kinetoplast. The sausage-or- disc-shaped kinetoplast contains the mitochondrial DNA, and the mitochondria arise from it. Electron niicrographs revraleil the DNA fibers running in an anterior-posterior direction whitliin the kinetopliist. Tlie ti-ypanosomatidac- family has many genuses that i ncliidc Lep rotil O I I U . ~ : Ilerl~eroiiloncis, Crithidin, Blasrocrithidia. Phyromonus, however, the inost important geniis that affect human population and their live stocks are Leislriiic~~iicr and TI-yxrriosoincz. For the purpose of this thesis, I will oniy disciiss i n detriils the genus Lrishrritrrii<i.

The life cycle of Leishrnuniu is dividcd in two stages: the pi-omastigotes within the gut of a tly, and the amastigotes witliin niaci-opliages of the tiost. Tlir vertsbrate hosts of Leid~rritrtii<r .spp are priniarily nianimals, althoiigli neiirly n dozrn spscirs Iiave been reported from lizards. Tlie inani riials riios t coiiiriion l y i nfec trd rire humans, dogs, and several species of d e n t s . Sandflirs of the genera Phlehorortiiis and L~ltzomyicr are the invertebrate hosis and hericr the primary vccrors of leishmaniasis. When they suck the blood of ;in infected animal, they ingest amastigote fornis. These forms pnss to the initlgut. wliere tliry trnnsform into promastigotes and multiply by bi nary fissioii. The parasites may attach to the

walls of the fIyFs gut or rernain frre in the lumen. By the fourth or fifth day after feeding, promastigotes develop in the esophagus and pharynx. When Lrish~~zuriirz begin to clog up the esophagus, tlie feeding srindtly pumps its rsophageal contents in and out to clear the obstruction, thereby inociilating promastigotes into skin of a victirn. AH amastigotes i n vertebrate tissues alike are spher&d, usually 7.5 to 5.0 mm wide. In stained preparations, only the nucleus and a very large kinetoplast can be seen, and the cytoplasm apprars vncuolated. Exceptionally, a short axoneme is visible within the cytoplasm iinder the light microscope.

Although, al1 Lkshmnniu spp. exhibit the same morphology, they differ clinically, biologically, and serologically. Even so. these chai-acteristics often overlap. so distinctions between species are not clenr cut. These populations are closely related and are in the process of speciation as the result of recent geographical isolation. It is likely tl-iat the transport of slaves to the Western world from Africa through the Middle East and Asin spread L~islirnunio organisms into previously uncontaminated areas, where they are now rapidly speciatiiig. Nevrrtheless, three major species of Leishamcznici parasites of humans, rach distinguished by its medical symptoms, are recognized:

1. 2. Lkshmania major

Leishmunirz major prodiices a ciitaneoiis ulcer varioiisly known as oriental sore. cutaneous leishmaniasis, Jericho boil, Aleppo bail' or Delhi boil. I t is found i n West-central Africa, The Mediterranean rii-ea, the Middle Erist, and Asia Minor into 'fndia.

A) Mor~hologv and life cycle

The appearance of L. rncijor is identical io thiit of otlier L ~ ~ i . s l ~ r r i t r ~ z i a of humans. Sandflies of the genus Pklehotoi?ii{s are the intzriurdin[e Iiosts and vectors. When the fly takes blood meal containing ninastigotes of L. m ~ j o t - the parasites multiply in the midgot, move to tlie pharynx, and are inoculatcd into the mammalian host. There, they multiply in the reticiiloendothelial systenl ancl lyniphoid cells of the skn .

B) Pathogenesis

The incubation period lasts from a few days to sevrrnl months. The first symptom of infection is a small, red papule at the site of the bite. This may disappear in a few weeks, but usually it develops a thin crust that hides a . spreading ulcer underneath. Two or more ulcers may coalesce to form a large sore. In uncomplicated cases the ulcer will heal within 2 months to a year leaving a depressed, unpigmentrd scar. A variant clinical form of the disease, known as diffuse cutaneous leishmaniasis, occurs in South and Central America and Ethiopia. Several months or years after the initial infection by the sandfly, the rnicroorganisms disseminate widely and cacse nodules and plaque-like lesions under the skin. The face, nrms, the legs are most commonly affected, and the appearance is similar to lepromatous leprosy. Patients wlio contract this form are believed to have a specific type of immune deficiency involving the cell-rnediated mechanisms (H~itt et al., 1973).

C) Diagnosis and control

Diagnosis of the infection depends on finding amastigotes. Scrapings from the side or edge of the ulcer, smeared on a slide and stained with Wright's or Giemsa's stain, will show the parasites in endotl-ielial cclls and monocytes. Antimony compounds are used succesfully to treat oriental sore. Prorective immunity following medical treatment seems to be absolute, and irnniiinity as a result of the natural course of the disease is between 97 to 98 C/o effective (Gerald D. Foundation of Parasitology, 198 1 ).

In 1900, Sir William Leishmiin discovered Id. dotiowrii i n spleen smrnrs of a soldier wlio died of a fever at D~irn-Diiiii, Iiidia. The disriisè wis known locally as Dum-Dum fever or Kala-azar. Three years later, Cliai-les Donovan found the same parasite i n a spleen biopsy. Leishiirculici c l o ~ w r w l i has a widr distri bution. and several varietirs are distinguished on epideniiological iind dinical grounds. The Mediterranean-Middle Asian form is fou nd throughoii t the Mediterranean basin and extends throiigh Southern Russia to Cliinn. Its cornmon vectors are

Phlebotornlls major. P. pericions. P. chir~esis, ~ i n d P. l o ~ i g icirspis. The major reservoirs are dogs, jackals, and foxes and i t primarily infects infants. Another variety is found in Nortliern India and Bangladesh. Its niost important vector is P. argentipes, i t hns no natural reservoir and i t mainly infects adults and adolescents. A clinically similar, but more virulent, variety occiirs in East Àfrica. Its vectors are P. martini and P. orirnrdis, and the organism may use wild rodents as reservoirs. The New World variety is widrspread i n Central and South America. It seems to be rnainly a zoonotic infection rimons dogs and foxes. and its vrctor is P. longipalpis.

A) Morpho10 y and life cycle

Leishrnania donovani cannot be differentiated from othrr species of Lrshmuniu. Its rounded or 0 ~ 0 i d body measures 2 to 3 mm 114th a large nucleus and kinetoplast. It lives within cells of the reticiiloendothrlial systeiii of the viscera, including spleen, liver, mesentei-ic l ymph nodes, i ntesti nt'. iind bonr marrow. Amastigotes have been found in nearly every tissue and tluid of the body. The life cycle parallels that of L. tr-opicn. Once i n the mamnialiaii host, the parasite is imrnediately engulfed by a macrophage, in wliich i t dividrs rapidly by binary fission, killing the host cell. Escaping the dead macrophages, the protozoa are engulfed by other macrophages, which they also kil l . aiid by this mrnns eventually they severely damage the reticuloendothelial systrm. one of the host's primary defense mechanisms against diseases.

The incubation period in humans may be as short as 10 days or as long as one year, but usually is 3 to 4 months. The diseass iisiinlly begins slowly with low- grade fever and malaise and is followed by pi-ogressivz wasting and aneniia. protrusion of the abdomen from enliiiged liver ancl spleen. and finally by death ( i n untreated cases) i n 2 to 3 yenrs. Viscerd Ieislimaniasis niay br viewrd essentially as a disease of the rrticiiloendotlirliril s y s r r m . The pliagocytic cells, which are so important in defending the Iiost agninst invasion. are themselves the habitat of the parasites. Blood-forming oc-gnns, siicli as the spleen and bone marrow, undergo compensatory production of macrophages and other phagocytes to the detriment of red ce11 production (Gerald D. Foiiiidat.of Parasitol., 198 1).

0

A condition known as post-kala-azar dermal leishmanoid could dcvrlop in somc cases (Morgan et al., 1962). I t is rare i n the Mediterranean and Latin America areas but it develops in 5% to 10% of cases in India. The condition usually becomes apparent about 1 to 2 yenrs after inadeqiiate treatment for kalar-azar.

A wide variety of animals can be infected experimentally, although dogs are the only important reservoir in rnost areas. Canine infection is lcss common in India, where i t is believed that a fly-to-human relationship is rniiintainrd (Grrald D. Foundation of Parasitology, 198 1 ).

C) Diaonosis and control

As i n L. rropica, diagnosis depends on finding (L-D) bodies i n tissue or secretions. Spleen piinctures, blood or nasal smears. bons marrow. and other tissues should be exnmined for the characteristic parasites, and cultures from these and other organs should be attempted. Treatrncnt consists of injections of various antimony cornpounds, together witli good niirsing ciire. .An adrquatr diêt with high protein and vitamins is essential to combat nutrilionid deficiency. Relapses and post-kalar-azar dermal leishmanoid may follow insufticient treatment. -

Leishmtrnicz hrcisilien.sis prodiicrs ri di sease i n hi1 mans various l y known as espundia, iitn. or mucocutaneoiis leishn~aninsis. Ii is Ioiiiid tlirougliout the vast

r 7 area between Central Mexico and Nortlirii AI-grritiiiii. 1 he Jinical niiinifrtstations of the disease Vary along i ts riiiige. Morpliolo;ic;il l y. L. I>t-~l:ilirrisis cannot br differentiated from L. major or L. clonowtzi.

A) Live cycle and pnthogenesis

The life cycle and methods of reproduction are identical to other Leishmmiu with several species of Lutzomyin serving as vectors. The parasite nrver causes a visceral disease biit often develops n secondai-y lesion on some regions of the body. In Mexico and Central America the secondnry lesion is often on the ear,

resulting in considerable destriiction of that orgnn. One spccies is found commonly infecting chicleros forest-dwelling peoples, iind has been given status of a separate s pecies known as L. niexicarici. Several manin~alian resrrvoirs are known in this area, including spiny rats and other forest rodents, kinkajou, dogs, and cats. In Venezuela and Paraguay the lesions more often appear as flat, ulcerated plaques that remain open and oozing. The disense is called pian bois (Gerald D. Foundation of Parasitology, 198 1) . In the more Southern area L. braziliensis have tendency to metastasize, or spread directly from primary lesion to mucocutaneoiis zones. The secondary lesion rnay appear before the primary has healed, or ic may be many years up to 24 before secondai-y symptcms appear. The secondary lesion often involves the nasal system, causing degeneiation of the cartilaginous and soft tissues. Necrosis and secondary bacterial infection are common. Espundia and Uta are the nnmes applied to these conditions. The ulceration rnay involve the lips. palate, and pharynx. lrading to great deformity. Invasion of the infection into t h e larynx, and trachea destroys the voice. The condition rnay last for many yenrs, and death mny resiilt from secondary infection or respiratory complications. A similar condition is known to occur in Ethiopia (Gerald D. Foundation of Parasi tology, 198 1 ).

- B) Diamosis and treatment

Diagnosis is established by finding (L-D) bodies in affected tissues. Treatment is similar to that for kala-azar: antirnonial compounds ripplicd on the lesion or injected intravenously. Miicociitaneoiis lesions rire parriciilarly ret'ractory to treatment and require extensive chemothernpy. Relapses is comrnon, but once cured a person iis~iaily has lifelong immuniry.

CHAPTER II

SUB JECT INTRODUCTION

2. 1. Current Situation

Incidence of leishmaniasis is increasing at the same time the world is experiencing an econornic crisis, the disease mainly affect 83 countries of which 72 are developing or least developed countries. 'Yorld wide, 15 million people are infected. Increase in world traveling and intervention in regional conflicts such as in Persian Gulf war (Magill et al., 1993) has aiigmentrd the incidence of leishmaniasis in non endemic regions. Furtherrnore, i t has became more and more difficult to treat infected individuals becaiise of the ernergence of resistant parasites (Ouellette and Pnpadopoulou, 1993). More importantly, i n immunosuppressive conditions such as in AIDS, Leishmimh appears to be a CO- factor or an opportunistic patliogen (Tremblay et al. . 1996).

As 1 have explained earlier, the treatment of choicr agiiinst Ittishmaniasis is basëd on antimony compounds, notnbly the pentnvalent antimonials sodium stibogluconate and N-niethylglucamine antirnonate. Howrver , these rnrdications have to be administered daily for several weeks and are not effective against cutaneous forms (Liew et al., 1984). New drug designs takr i n consideration the unique features of purine salvage system of Leisl~tmitli<r to cause a selrctive susceptibility to the cytotoxic effects of several pyrazolopyriniidine analogs of the naturally occuring purines hypoxnnthinr and inosine. Leislr~iiciriicl is capable of efficient conversion of the pyrazolopyrimidines to the niiclrotidr Ittvel, thesr metabolites which are isomers of inosiiie nionoplinsphate, are subsequrntly aminated and incorporated as the adenylate anrilog into R N A . Manimalian cells are incapable of tliese metnbolic transformations. Tlirsr drugs have bern extensively tested agai nst Lrisl~rricuiici, and are ef tc t i ve rigai nst sorne, but not necessary al1 parasite stages. Treatment with niiiphotei-iciri B delivered by liposomes has shown sorne effectiveness (Liew er al., 1984).

Meanwhile, control of sandflies hns been difficiilt because of the val-iability and

general inaccessibility of the insect breeding and resting sites. In China, for example chemotherapy coupled withe the tlimination of infected dogs has proved highly effective in eliminating Kala-azar, bii t s u d i niechaniciil drsrruction of animal reservoirs and habitat is not likely to b r gsnrrrilly feasible for al1 Ieishmanias. .

Vaccines remain the best answer to the control of leishrnaniasis. There are many reasons to believe that vaccines against leishmaniasis are frasible, such as :

a) A state of immunity generally follows recovery from cutaneous disease.

b) Indigenous populations living i n highly rndeiiiic zones usuall y suffer the disease only once at a young age.

C) Leishmaniasis in foreigners visiting the endernic regions is usually more severe than in the indigenoiis people.

d) The parasite promastigotes can be grown in axenic culture easily and are therefore amenable to manipulations reqiiired for vricci ne developmen t.

e) The practice of leishmanization by delibernie infection wirh virulent - parasites to prevent multiple lesions allows simple nieans for clinical evaluation of vaccines.

But before we detail different rationales for vaccine, w r would like to give some current iinderstandings of the i mmiinology of leish mmiasis.

2.2. The regulation of immiinity to Leishnzcrnin nzcrjor

2. 2.1. Leisizmarzia niid macrophages

A) Metacvcloeenesis

The promastigotes of the L. mi~jor dcvelop as esplainrd enrlier, i n close approximation to epithrlinl cells i n the insect midgiit. The major surface glycoconjugate lipopliosphogl ycan (LPG) consti tii tes a dense gl ycocaly x that

covers the entire surface of the parasi te i ncliidi ng the flagellum. Structurally, LPG consists of four components: a terminal mannose disaccharide cap structure, a polymer of repeating phosphory lated clisaccharides of galactose and mannosr with substituted side chains, a phosphosaccharide core, and a phosphatidylinositol lipid anchor that embeds the structure in the membrane (Turc6 and Descoteaux, 1992).

Immature organisrns, termed procyclics, express shorter LPG molecules with terminal p-galactose residues that bind to unidentified epi theliai lectin-li ke molecules on the insect gut (Pimenta et al., 1993). During dcvelopment in the insect and under in vitro conditions, LPG undergoes developniental modification, t emed metacyclogenesis, that involves cnpping of terriiinril P -galactose residues with a-arabinose and elongation by incrensing tlir iiurnbers of repeating disaccharide units by two-to three fold. Capping of trriiiiiial residues obscures the epithelial-binding domain, allowing the mature fol-ms, trriiicd rnetacyclics, to be released from the midgut and to migrate to the proboscis for transformation, a process that correlates with enzyniatic damage by the 01-gnnisms to the cardiac valve that norrnally prevents reflux froiu gut to rhe plinryngeal pump and proboscis (Schlein et al., 1992). -

The elongation and chemicnl modifications of the mature nietncyclic LPG are important i n con ferri ng con-iplement resistance. Wliereiis psocyclic organisms from log-phase cultiire are extremely sensitive to compleinrnt-mediated lysis activated through the alternative pathway, nietacyclic 01-ganisrns activare the classical pathway biit are not lysed. LPG serves as the iicceptor moleculc for complement, witli C3b constituting the ninjor fixation pi-odiict (Fuentes et al., 1988). Furthei, passive transfer of rnatiire LPG restorzd the capacity of a niutant to survive in nincrophnges (Handman et al.. 1986).

B) Invasion

The major component of complement f ixed to nietacyclic forrii of the parasite is C3b, as supported by studirs with hunian monocytes denionstrating that CRI constituted the niajor macrophage ligaiid foi- iiintiii-e proiiiastigotrs (Da Silva et al., 1989). Fixation of C3 is reqiiired Soi- s~irvival of prornastigotes i n macrophages, in part by targetinp the organisms to iiiternalization pnthways. thus

avoiding activation .of respiratory biirst (Mosser et al.. 1987). Moreover. additional parasite surface glycoproteins such as gp63 membrane protease and other macrophage receprors such as CR3, mannose-fiicose receptor, advanced glycosylation end-product receptor, fi bri nectin receptor have been i mplicated i n various studies. .

The establishment of the infection in the host can ~ I S O be traced from the role of components of sand-tly salivary-gland material inoculated wi th the organisms. A vasodilatory peptide, rnaxadilan, comprises 1 2 % of the total salivary gland protein and is at least 500 times more potent than the neuropeptide, calcitonin gene-related peptide (CGRP), with which i t shai-es imnimologic cross-reactivity (Lerner et al., 1991). Further, CGRP and snlivary mntei-ial shared the capacity to inhibit macrophage oxidative metabolism and antigen presentation in vitro (Theodos et al., 1991), suggesting a mechanism for the enhancing effect of salivary material in vivo. Although salivary gland material has shown to give shifts in dose-response curve in experimental L. m!jor infection, i r is not required to indiice the fui1 spectrum of disease i n mice. Once internalized,promastigotes transforrn i nto the i ntrncellular amastigotes, a developmental foi-m i n which they remnin i n t h e vertrbrate host. Amastigotes - replicate by binary fission, eventually r up t~ i r i n~ the maci-apliage and spreading to

uninfected cells. The receptors iised by anustigotes are not conipletely known. However, the identification of antibodies coati ng arniisti~otes in vivo suggests that macrophages FcIg and CR3 receptors may contribute to phagocytosis (Guy et al., 1993). A hiph-nffinity, heparin-binding activi ty lias bçrn idc'ntified on arnastigotes that interacts wi th cell-surface heparin-s~ilfate proteo~ly gnns ttiat inay constituir the initial binding step prior to internalization by yet ~inknown additional receptors on macrophages (Love et al., 1993).

Phagocytosis of pnthogenic organisms indiices macrophages ro produce cytokines that coordinnte various aspects of host defeiics. i ncliidi ng clic.motactic factors such as the chemokines, nctivating factors siich as TNF-a, IL- 12 iind IL- 1 , ncutr phase stimulants such as IL-6, modulatory factors scicli as IL-IO. and hematopoietic- stimulating factors such as M-CSF and GM-CSF (Lirw et al.. 1990).

C) intracelliilar con~partmentnliz~ition and nnt i en pi-esen t a t ion

Following binding, organisms are intei-nalizrd into pliagosomes to which

secondary lysosomes are fiised to form the mature parasitophorous vacuole. Metacyclic forrns rapidly transforni i nto smallrr, roiindeci, intracellular amastigotes, a process accomprinied by shedding of pi-ornristigotr LPC. The shed rnaterial can be demonstrated to migrnte to the surface of the infected macrophage; using L. c/oriovu~ii, epi topes contai ned in the diisacharide repeats were present betwcen 2 and 24 hrs after infection (Tolson et al., 1990).

LPG may have several functions during this period, including protection frorn hydrolytic enzymes of the lysosomes and abrogation of intracellular signaling, possibly through chelation of calcium and inhibition of protein C. The latrer results in attenuation of the oxidative burst stiniulated by subsequently ingested particles. Various markers have been used to stiidy the inti-acrllular compartment in which amastiçotes replicate i n macrophages. The pal-asitophorous vacuole becomes acidified witliin 30 min of internalization, consistent with the acidity of proton ATPase (Antoine et al., I W O ) , and lysosorniil proteases are srcreted into the organelle without damage to the parasite (Prina et al., 1990). Additional lysosomal markers such as LAMP 1 , are matures to a late endosomal compartment.

Irnportantly, MHC class II molecules have vacuole (Russel et al., IgW), siiggesting

present, but with time, the vacuole

- been localized to the parasitophorous a mttchnnism by which the immune

response to this i ntracellular orpnnism beconies class 11-and CD?-dependenr (Locksley et al., 1991). Indeed, two stiidirs have <lernonsti-ated that Leishmznicz- infected macrophages had a greatly rediiced abi lit? to pi-esent exopenous antigrns to T ce11 hybridomas (Friith et al.. I993), consistent wi t h the cnpacity of parasite- derived antigens to flood the class I I peptide-loadi ng compartment. The association of this intracellular organisni witli host clnss I I recognition. in contrast to the class 1 recognition of otlier in tracrl l~ i ln i - p;itliogms siich as viruscs, can thus be explained by celliilar compartmentrilizniion. The coevolution of parasite and host for recognition tiirough class I I pathway was ievealed h y rxperiments using transgenic mice without the MHC class I I or Pî--microglobiilin genes that are deficient in MHC class WCD4 cells or M H C class [/CD8 cells. respectively. Whereas MHC class II-deficient mice siifféi-ed fatal, ii~~controlled infection (Locksley et al., 1 W3), MHC class 1-de ficien& niice con ti-01 led infection with L. inujor i n a manner not different from normal littermates (Wang et al.. 1993).

Dl Macrophage activation and coiitrol of narasite replication O

Macrophage activation by T cell-derived cytokines is reqiiired to establish control of inü-acellular infection and progressive disense. Among macrophage-activating cytokines, F N - y has been cri ticall y implicnted. O thei-w ise resistzint su-ai ns of rnice with targeted disruption of either the IFN-y or the IFN-y -receptor genr were unable to restrict growth of L. rnczjor- i n vivo and suffrred fatal infection. In a number of studies, recombinant IFN-y was capable of activatinp infected macrophages from both resistant and susceptible mice to clear L. rncijm- infection in vivo (Wang et al. 1994).

The final common pathway mediating parasites stasis or destruction by murinr macrophages invoives production of nitric oxide (NO) fi-orn NOS. Inhibition of NO production renders macrophages iiniible to restrict L. tlicyol- replication in vitro (Green et al., 1990), and administrottion of NO inliibitors to otherwisè resistant mice abrogates the capncity to conirol infection (Liew et al., I W O ) . Such findings are consistent with the impnired prodiiction of NO by macrophages from IFN-y and IFN-y -receptor gene knockoiit mice (Dalton et al., 1993). The inability of propriate iNOS transcripts following stiniiilntion with LPS and IFN-y suggests that this transcription activator is involved in normal NO production <O

pathogens (Kamijo et al., 1994). The importance of IFN-y i n NO induction was demonstrated by the finding that IFN-y, dont: nmong a ncirnbei- of cytokines, was capable of independently enliiincing iNOS ti-arisci-iption aiid NO relensr from stimulated mouse peri toneal mnci-opl-iages ( Di ng et al., 1 98s).

However, several cytokines enhance NO pi-oclitction i i i a synergis tic manner with IFN-y and are likely involvrd i n niediatiiig parasite control i n vivo. Such u cytokine is the TNF-ci t l~at sy~iergizr's witli IFN-y i n tlie iiidiiction of iNOS and NO production by mnci-ophriges i n 1.i ti-o (Driig et ;il.. 19%). Furthcr. administration of recoii-ibinaiit niw-inr or liiiiiiori TNF-cl 10 1-trsistniit or susceptible strains of mice i nfected wi tli L. t~rïijor rinie lioratai tlie coiii-se of dissasc. although the eventual expression of the disease phrnotype rrnuined iiniiltei-ed (Ti tus et al., i 989).

Additional cytokines that, wi th IFN-y, sy nergistically n~ediate activation of macrophages to clear L. rnc l j o r , include IL-2, IL-4, and IL-7. Synei-gy usually

requires the presence.of infection and either prior or CO-culture with !FN-y. Indeed, reversing the incubation using I L 4 prior to IFN-y abro, oated the subsequent activation of macrophages (Liew et al., 1989). Whsn examined, the mechanism reflected enhanced production of endogenous TNF-a and was

mediated through induction of iNOS (Stenger et al., 199 1 ). [t 1s likrly that this group of cytokines transduce a common signal, reflecting tlicir sharzd use of the IL-2 receptor chain (Kondo et al., 1994). However, mice on a genrtically resistanr background with constitutive IL-4 expression developed persistent infection (Leal et al., 1993). This suggests that expression of IL-4 interferes with normal clearance of the parasites. IL-3 and GM-CSF, which with IL-5 also constitute a faniily of cytokines that shai-e a common signal-transdiicing niolecule (Stahl et al., 1993), were detrimental to L. mczjor infection in vivo or in vitro in various systems (Greil et al., l988), demonstrating the capacity of cytokines to modulate both positively or negatively the coiirse of experimentnl leislimaiiiasis.

A) Lineage of polarized Th 1 /Th2 responses

- The capacity of Lrivhtmzniu specific CD4'T cells to passivcly transfer resistancr or exacerbation of dismse to immunodefjcicnt or sublethally irradiatrd naive hosts correlated with their production of Th I or Th2 cytokines. emphasizing the ability of these conirnited CD4'T cells alonr to confer the entire phenotype of the murine disease (Scott et al., 1988). Althougli certain stiidirs have suggested that some antigens with Th 1 or Th2 type tendencies niplit be identified, the bulk of experirnental data suggests botli tliat mosr riiiive T cells have the potential to mature into either siibset of rffrctor cells riiid tliat criticnl cytokines supplird at the time of T ce11 priniin; mediatr this diffei-entiation (Seder et al., 1991). Using systems siich as liniitinp diluting techniqiies or cells I'roni transgcnic niicr: expressing n single TCR (Hsieh et al., 1992). i t hiis been possible to idmtify the important role for IL-4 i n rnediating Th? developnient and for IL-12 i n mediating Th1 development from naive pieciirsors. Additional cytokines have been incriminated in different systeius in \.i tro, cg . IL- 1 O i n Th? de\dopnient and IFN-y in Th 1 developmen t.

During the first few days after infection witli L. myot- of both resistant and

susceptible rnice, a careful evaluation of cytokine expression in lymph node cells demonstrated a strong mixrd 1-esponse in the CD4' popiilrition consisti ng of IL-?. IL-4, and IL-13. Transcripts for IFN-y were more variable in CD?' cells among different strains and were more prominent in natural killrr crlls (NK). Striking, however, was the magnitude of IL-4 transcription i n cells CDC, which reached levels found i n fully developed Th2 clones i n nll srains of mice analyzed. Such findings were consistent with IL-4 promoter-Herpes thymidine constructs used in transgenic rnice to investigate the lineage of IL-4 and IFN-y -producing cells; in agreement with the findings in leishmaniasis, these experiments identified an obligate Th0 precursor thnt secreted IL-4 during T h I or Th2 maturation in vitro (Kamogawa et al., 1993). Importantly, however, healer strains rapidly downregulated IL-4 transcription, whereas susceptible mice only slightly decreased IL-4 transcrption before reestablishing levels consistent with Th2 cells. This early burst of IL-4 transcription occiirred precisely diiring the period in which immunolopic interventions are effective. Further. wlirn studied, each of these latter manipiilations effectively abrogated IL-4 transcription or protein production, further i mplicati ng the early 1 L-4 burs t in t lie nimiration of Th2 ce11 in leishmaniasis. The inability of niice with disrupted IL-4 gent: to drvelop Th2 responses to helminth infection was consistent with tliis observation (Kopf et al.? 1993).

Such findings have raised speculation tliat interactions of invading promastigotes with the innate inimiinc systeni might tindèrlie the induction of Th 1 responses in healer mice strains. Findings also siiggested thnt the siisceptible mice niay reflcct deficient Th1 indticing responses supplicd by an abnoi-mal acccssory cell cornpartment. IL- 1 . pioduced by maci-ophngrs, and possibly IFN-y, produced by NK cells, are potential candidate cytokines basetl on their known nbility to influence Th1 ce11 developmrnt in vitro in vririoiis systeins (Macatoiiiri et al.. 1993).

Synthesis of these niimerous and various interventions stiggrsts the possibility that the critical early cell reqiiired for the indiictioii of CD4' subset matiii-ation in the L. nzujor system is itself a CD4' ce11 tiiat serves as a soiircc of both I L 4 and IFN- y. The capacity of mice treated with iinti-CD4 at the tinie of infection ro hral correlated wi th abrogation of the I L 4 biii-st. Piesi~rnably, repopiilation T cells would become primed by anmtigotr-indticed IL- 1 2 niicl pi-omote a ctiriiti \Y T h 1

response. The eventual reqiiii-ement for CD4' effector c d 1s is undrrscored by the inability to heal mice given sustainrd anti-CD4 treatment (Titus et al., 1987) or of MHC class II knockout mice. The capncity of CD3 knockoiir mice to heal L. major infection reflected the lack of an absolute requirement for CD4 expression in the development of T helper.

Cytokines conuibiited by naive T cells using an autocrine pathway have been implicated by studies of in vitro priming of cells taken frorn TCR transgenic mice. Although such cells produce relatively low levels of I L 4 during initial stimulation throiigh the TC!?, neutralization of this IL-4 iising nntibody results in differentiation to the Th 1 phenotype; cells i ncubated without an ti-IL-4 matured to Th0 cells (Hsieh et al., 1993). Similnr experiments with anti-IFN-y, however, did not cause differentiation to the Th2 pathway. suggesting that additional signals, particularly the non-T cell-derived cytokiiies. IL- 12. rire reqiiired for Th 1 development. It is also uiilikely that naive CD4' cells alone determine Th2 development in experimental Irishmnniasis.

- Immunological studies of leishmaniasis have been made possible by t h e recognition of strain diversity i n the capacity to develop infection. The classic studies of Bradley and collengues established two levels of L eenetic expression affecting L. clonoiwrii infection: ( 1 ) innatr siiscrptibility bnsrd on the relarivr resistance of the macropliage and determinrd by the Lri.slzt~itrtii<r (Lsh ) grne; and (2) the efficacy of the immune strige for wliicli tlirce regiilatory geiies have bren identified. The Lsh gene hns been niapped to :i position on ~Iiromosome 1 between the Id- 1 marker and the centroniere and is identicnl to Itp and Bcg genes which determine the susceptibility to otlicr obligrite inti-nnincrophngr: pathogen S(dinonrlh yphirnrit-iwrz (Plntn et III.. 1979) r i i i t i hfj-c-ol~(i(-re~-i~c,ri bis (Skamene et al., 1982) respectively. Unlike iniiate siiscrptibility, tlic ininiiins strige of L. donovtrni infection is influenced by the major liistocompntibility (H-2 ) locus on chromosome 17 (Rd-1), H- 1 1- linked p i e ( H - 1 l b) iirid 11-2 linkctd genes (DeTolla et al., 1980). These observations have been implicnted i n resistnncr to L. donovani but are unrelated to control of L. i i z ( ! j o r .

The exquisite sensibility of BALB/c mice is eqiinlled by the BALBIb. and only

slightly !ess so by the BALBIk; the latter crin overcome iising highei- inocula. Although some intermediate strains hiive been identified, siicli as the DBA and PIJ none is as susceptible as BALB/c mice. Evaluations of BALBIc x C57BLhFI crosses, including F? and bot11 BALB/c and C57BL/6 backcrosses. in response to L major demonstrated tliat susceptibility segragated consistent With the effects of a single gene. Although original linkage studies i n recombinant inbred strains of mice suggested a location of the susceptibility locus to chromosome 8, subsequent analysis using a grater number of markers suggested linkage to tlie dista1 end of chromosome I l (Mock et al., 1993). The occurence of the IL-4 and I L 1 2 p40 geres on mouse chromosome 1 1 are of some interest given this initial localization.

The phenotype of BALB/c mice infected with L. major inakes some predictions regarding the nature of the genetic defect based on the large number of studirs. The bulk of studies have demonstrated no consistent defrct BALB/c macruphages in either permitting growth of L. mijot- i n vitro, response to activation stimuli, or response to parasi te invasion. In contrast, the dota ovei-wtieln-iingly suggest a defect in CD4' subset priming such tliat IL-4 is ovei-abimdant during the initial encounter wi th the parasi te.

- In sumrnary, BALB/c strnin carries a delèct exprrssed in CD4'T crlls such that IL-4 production becornes overabundan t du ri ng pri mi ng, prrliaps dur to suppress transcription or to unusual niessage stability (Lirw et al.. 1987). Lrishmirii<r prornastigotes invasion under conditions tliat inininlizr the antagonisr èffects of IL-12 allows CD4' subset devsloprneiit to occur accoi-ding to the undsrlying genetic predisposition. Tlie presence of ; in acldition;il CD4' popiilntion. die N K cells that also express the defect, may hirthsr increasç: the initial LL-4 production, contributing to tlie overprodiiction O t' I L-4 diii-ing pri i i i i ng rhrough both autocrine and exocrine CD4-dependent pathwnys. The rrsult of both nutocrine and exocrine IL-4 production at higli levels, Iiowevrr, is to prime naivr CD-I' T crlls above the threshold at which irre\wsi blc Tl12 di ffei-en tirition occui-S.

The differentiation to Th? effector cells iillows these cells to brconic i-efractory ro the subsequent production of IL-12 in response to iiriiastigotes 1-rlriiseti laier i n infection (Wang et al., 1994). Altliough siich ii mode1 hiis LI nimber of attractions that fit the experimental data, i t reiiiiiiiis plaiisiblz thai BALBlc T c d l s concomitantly express inordinant srnsibility to I L 4 siich that the switch threshold

for Th2 comrnitrnent.is lower due to an intrinsic genetic drficit. Clearly, high doses of exogenoiis IL-4 are tiiiable to establish Ti17 rc.sp»nscs ro L. t~iczjor in resistant mice i n vivo (Sadick et al., 1991). Furthrr, despite induction of IL- 12, amastigotes are conipetent to establish lethnl infection when iised to infect BALB/c rnice. This suggests that the quantity of IL-12 induced by organisrns i n vivo is unable to alter Th2 cornmitment.

The capacity of low numbers of parasites to render B A L B k micr resistant to subsequent challenge with a Iarger inoculum (Bretscher et al.. 1992) may reflect attenuation of the IL-4 burst and efficient priming by IL-12 induced by amastigotes. It is interesting to compare the predictions of this mode1 with observations made using BALB mice in other infections and autoimmune system. In the Trichuris nircris system, i ntestinal infection is resolvrd i n a Th?-dependent rnanner, BALB/k mice rapidly expiilse worms, but the effect is overcorne by neutralization of IL-4. Conversely, the susceptible A K R strain develops a Th1 response to the organism and is tinable to expel the worms, although neutralization of IFN-y promotes woi-m expiilsion. Miirine infection with Borwiiri hltrgdorferi , the rtiologic agent of Lyme disexe. can be establishrd by intradermal inoculation of organisms into BALB/c or C3H/HeJ mice. C3H/HcJ mice contain organisms less well and develop severe ni-thritis of the ankle joints that correlates with a Th1 response, i n contrast, BALB/c drvrlop minimal arthritis, and the C D 4 response is cliasactei-ized by a Th2 response In a niodrl of autoimmune diabetes, nlice with transgeiiic expression of foi-eign antigen in pancreatic p cells were crossed witli M H C class II-rrstricted TCR transgcnic niice specific for the nntigen on BALB/c or 13 10.Dî bnckgi-oiiiids, iiiid the course of disease was nionitoi-ed (Scott et al.. 1994). Altlioiigli. the double-transgenic B 10.D2 mice developed spontaneous autoimmiine disrase, the do11 blr-transgrnic B A L B k mice, did not, an outcorne that correlrited witli the drvelopnient of cither Th1 or Th2 effector celis, respectively. Finrilly, a number of pathogens such as Listeria and My.-ol~acw-icl are no lrss el'ficiently Iinndlttd by Bi\LB/c mice, consistent with the capacity to devrlop appropriate 1-esponses to organisms that provide independent stimiili for strong IL-12-nirdiated priming.

Since the description of CD4' subsets, we \lave seeii aii explosion of information regarding their development and involvement in a n~yriad of animal and human conditions. Such understanding coiipled with the contribiitions made by the L.

major mode1 have beon substnntial and emphnsize the power of infrctious disease models to dissect crucial aspects of normal immiinè physiology, but more importantly has allowed rational approachcs, based on these insights, to study the deveiopment of vaccination against leishmaniasis. .

2. 3. Development of vaccines against leishmaniasis

A) Living promastioote vaccines: In order to produce a lesiori in a selected part of the body and sparing the face from disfigtiring s c x , foi- centuries inhabitants of the Middle East would expose the bare bottoms of the infants to the bites of the sandfly vector. Materials were also taken from active human lesions and inoculated into the skin of the arm and thigh (Grreiiblatt et al.. 1980). Later. cultured promastigotes were iised and injected i n a low dosage (0.1 - 10 X 10' of organisrns). The process which follows iisually miriiics the natiiral infection. Large-scale field trials were carried out in Russiri (20,000 recipients) and in Israel (>5,000 recipients) using ciiltiind L. mc!jor proniiistigotes. I t is genrrally accepted that if the vaccination is followed by a typicnl iilcsrated lssion, the recipient is protecred for life. However. i f a patient is siiffrring from immunosuppression througli medical intervention wi t h steroids or cytotoxic agents or with a genetic iminune disoi-der, tl-ien 1-eciiri-rnce of the disease often happens.

Complications accompanyinç live prornnstigote vaccine are niiniei-011s and serious. The lesions induced persist Ior a niiiiinium 01.4 io 5 iiiontlis biir conipletè httaling can take much longer. On average, 2 to 3 C / o of iiidividiiiils dsvrlop large or nonhealing lesions reqiiiri ng ti-eatnieiit (Modabbrr 1 987 ). Anotliri- pi-oblcni \chic11 may arise i n the vnccinated popiilation is secoiidni-y infection of tlir lssion. This was noted i n 2 5 4 of vaccinees in one stiidy (lariniiklirirnr~lov 197 1 ). .A f i m k r complication exists i n those individtials witli a tendrncy to dei-nintologic problems. There is a clear relationship between leishnianization ancl the recriidescencc of Iaten psoriasis or the appeni-ance of new cases (Iorniiiktianiedov 197 1 ). Furthermore, i t has been noted tliiit after i nocii lritioii ugai nst Le i s l l t ~ i c i r i i ~ r the immune response of children receiving tlieir boosrer to triple vxcinè (diphtheria, pertussis, and tetanus) is depresseci for periotls of iip to 6 rnontlis (Sri-ebryakov et

al., 1972). Because ofits inherent morbidity and variable effects, i ive Lrishnimiiu vaccine was only iised as a last resort for control i n higti endeniic regions.

B) Killed ~rornastioote vaccines .

The earliest record clinical trials wi th killed prornastigote Lrislzrnuniu vaccines were that of Salle Gomes and Pessoa and Pestana in South America. The protection rate in these studies was impressive. The infection rate in one case was 18% of the control group of 600 and 3.7% of' 527 vnccinees. In contras, a study carried out in Middle East involving 32 subjects given phenol killed L. rropiccl intramuscularly in varying schedules showrd no rvidçnce for protection (Berberian, 1944). More recently, a crude parasite-extract polyvalent vaccine made up of few South Arnerican strnins was resteil i n Brazil (Mayrink et al., 1979). This crude parasite-extract vaccine showed no iintoward side effects in the trials. However, the incidence rate was too low in the control groiips to warrant a conclusion on the vaccine efficacy.

A) Atteniiated vaccines

Two spontnneous avirulent clones (A12 and AS?) of prornastigotes were derïved by limiting dilutions from an L. triczjor isolate L 137. None of the clone caused

7 lesions after cutaneous injection to niice. Iiiti-npri-i toiieal iiijrctioii of 1 to 1 X 1 O living prornastigotes of the nviriilrnt clone iiitliiced coniplrte protection i n the resistant C57BL/6 micr when cl~rillenged by ciirrineoiis injection of parhogènic parental isolate. Siniilai-ly, i mmunized siiscepti ble BALRlc iiiicr tlcivrlopr'd only partial resistance to the challenge infection (blitcliel l et ; i l . . 1 984). A\-il-den[ clones of L. rnexicc~tltr were also pi-odiiced by trttntiiièiit of parental parasite stock with miitagen N-methyl-Nt-nitro-N-niti-osogiianidinr ;iiitl selection for growth at 19" C. These clones have a clear preference for infection and prowth in vitro at 19" C rather than the normal 28 " C. BALB/c niicr iriiiniiiiizrd i.p. with the avirulent clones developcd significant resistancr to ri clinllenge infection with the virulent parental isolate cornparrd to controls (Coi-czyiiski 1985). However, the mechanisms of atteniiation in boiti cases are i l 1 dtttined. The clone A52 was found to be infectious when the parasites were ciiltiired for an extended period of I I

days (Sacks et al., 1987). Of mice injected with niiitngenized 19" C selectrd clones, 16% developed Irsions, fiirthermore, the i .p. roi1 te nppenred mandatory for the induction of protection and s.c. routes of inimcinizatioil have been totally without protective e f f e t (Mitchell et al., 1984). The clinical application of thrse attenuated vaccines thiis nppears iinli kel y to siicceed.

B) KilIed vaccines

In view of the discoui-aging experience in clinical vaccination against leishmaniasis with killed parasi tes, various attempts have been made in experimental immunization. Until recently, relevant data are sparse and have not been particularly encouraging. lmrnunization with ulti-nsonicated promastigotes (Preston et al., 1976) or crude antigen-antibody complexes (Handman et al., 1977) have induced some protection agiiinst L. t~itrjot- i n the resistant CBA and C3H mice. Mice imrnunized i.v. with killed promiistigoies plus glucan as adjuvant became sliglitly more resistant to L. dowvï i r i i infection tliat thosr gi ven glucan alone i n one out of tliree short-term experiments (Hobi-ook et al.. 198 1 ). Sorne amelioration of severity of L. mcijor infection was observed in the highly susceptible BALB/c mice previously iiijected with frozrn-thawed infect- macrophages plus Cor\wehectet-iiirri ptrriwtu as adjil vaiit (Mi tchrll et al.. 198 1 ).

A 60 Krad y-irratiated L. rncyor prornastigote pi-epariition injectai in to C B A mice induced significant protection agai nst L. niex. trzeïicwici i nfecrion (.4Icxandrc 1982). Paradoxically, the imniiinized anin~nls werr not protected against the homologoiis L. im!jot- infection. Using 1.50 Krad-irradiatèd or kat-kil led L. major promastigotes, i t has been denionstrnted thnt strong and persistent protection c m be induced in the susceptible BALB/c mice against a challenge infection of L. rrttrjor, L. tiws. m e x i c m tr, L. i>ier. ~ i t ~ i c i z o r i r ~ i s i s . and L . hrïzzi~irn.siv pinczmrn.si.s but not agni nst ~inrelrited i nkctions.

Substantial protection can also be induced by immunization with heterologous irradiated L. clonov~rni promasrigotes. Altlioiigh this i nimiini ty does not extend to i .v. challenge wi th homologo~is L. clonov<riii amnst igotes, the possi bi l i ty of more relevant protection agninst homologous prornnstigores inti-oduced via the cutaneous route cannot be tested in niice. I t is iinportiint to note thrit i n niicc, the route of immunization appenrs critical. The i.v. route is siiprrior to i.p. whereas

the S.C. route and i.m. routes, even with a range of adjiivnnts, is totally ineffective (Howard et al., 1984). Furthermore, the s.c. route of immunization can block the prophylactic effect of i.v immunization. Thrsr findings, if cxtended to clinical leishmaniasis, would form a formidable obstacle to iiiass vaccination against the di sexe. .

C ) Fractionated vaccines

A glycolipid purified from detergent-soliibilized L. nitrior promastigotes by affinity chromatography and immobilized specific nionocional antibody WIC-79.3 was used to vaccinare genetically resistant or susceptible mice against L. t n a ~ o r infection (Handrnan et al., 1985). Resisunt C3H mice werr totnlly piotrcted from cutaneous disease with the glycolipid iiijrcted i-p. togetlier wirh C. purwni as adjuvant. A high but not absoliite levrl of resistnnce wris also induced i n the susceptible BALB/c mice. No protection was ob~iii lied wi th the carbohydratc fragment of the glycolipid nlone or by injectioii of the glycolipid in the absence of adjuvant.

Intraperitoneal imrnunization wi th a soliiblz. mzmbi-anr-îi-ee parasite extract was - found to induce protection against L. t ~ i c ! ; o r - cliallengr rcliial to ttiat obtainrd with whole organisms (Scott et al.. 1987). Iniiii~inizrd niicr pi-odiicctd anti bodies against two major metabolically labeled proteins of 30 and 50 kDn but failrd to stimulate a detectable humoral response ngainst prornristigote surface antigrns. A moleciilarly more-defined antigen, the cross-1-crictive proinristigote surface glycoprotein, gp63, lias bren assessed as a canditate vaccine. The gp63, an N- linked glycoprotein? is a major glycoprotein prrsrni ovri- tlir entire promastipotr of al1 species of Lrishmtrnici exaniined (Riissell et al.. 1986) iind is known to br involved in the artachnirnt of the parasite to liost ceIl, the macrophage. I t is. Iiowever, n minor conlponent of the arniis[igoie siii-1'xr and is noi r e l r a s d into the macrophage. The glycoprotein crin be isolateci froni a detergrnt rxtract of a

promastigote-crude membrane fraction by lectin affini ty or anion exchange chromatography (Russell et al., 1986) or by sprcific monoclonal antibody. The glycoprotein alone induced little or no protection when injected i.p. into susceptible or resistnnt mice. When incorporated into liposome, hoivever, the glycoprotei n i nduced signi fican t protection agai nst L. m ~ j o t - or L.- tlirx. tw-riccititi

(Russell et al., 1988). Unfortiinately. the i . v . or i .p . routes again nppenr

mandatory for the induction of protection by both the glycolipid and the glycoproteins. The S.C. route consistrntly failzd to induce protective immunity and often elicited exacerbated disease in BALB/c niice.

The use of a fraction is an improvernent over iising the w h o k parasite both i n terms of standardization and reducing unwaiited side efkcts. Howèver, the contents of the fraction must be identified to Fiditate standardization, becausr one of the major requirements for a new vaccine is that its composition must be consistent.

Before we introduce the role of biotechnology as new tool in vaccine development, we would like to siimmarize the different types of vaccination by outlining a mechanism of immunity in tlie animal mode1

2. 3. 3. Meclzanism of proplzy lactic irir ni rr iziiy

The immunity featiires of mice protected by i-epeatcd i.v. injection of killrd promastigotes are s trikingly different fi-oin tliose cliiii-acrei-is tic of convalescent - immunity. Unlike mice recovered from infection. prophylactically i.v. immunized mice show no specific delayed-type liyprrsrnsit ivity (DTH) 1-eactivity whether challenge be with antigen fractions or viable pi'ornastigotes (Liew et al., 1984). Antibody responses to repeated i.v. immiiniznrion are Iiiglitsr thnn those inducrd by the disease itself. Impairment of the huniornl response by pi-ior spleenectorny does not influence tlie induction of pi-otrctiw ininiiinity and repeated passive transfer of large quantities of varioiis fractions of thrsr hyper-immune fail to control the infection (Howard et al., 1984). i i i contrasr. iriiiiiunity is trnnsferable with spleen cells and can be amplifird by pi-ioi- siiblethal irradiation of the recipient. Both positive and neçati ve ce1 1 seleciioii espei-i~nrnis rstablishttd thar this was rittribuable to T cells of the Lyt- I - 1 ' siibser. Altlioiigli sirch T cells from i x immunized mice (Ti) perforni a helper fiiiictioii foi- rintibody responses i n mixed ce11 transfer experirnents, yet consistent witli the donnors, they fail to transfer DTH (Liew et al., 1985). Furtherrnore. Ti cells are capable of inhibitinp the induction and expression of specific Jone-Mote type of delayed hypersensitivity i n mice injected iiitrndermall y or S.C . with leishrnanial antisens. Ti cells are also capable of secreting IL-? i n i-espoiise to concanavalin-A, IFN-y

and MAF when stimulated with Lrish~rrclriio nntigens in vitro (Cillari et al, 1986).

According to Liew, an s.c. route of immunization wi th killed promasrigotes or antigenic fraction is not only inrfkctive, but i t frequently exacerbates subsrquent disease development following infection. Furtherrnore. 'mice prior S.C.

immunization fail to develop prophylactic iriiniuiiity w hm injrctrd i . v . wi th the sarne antigen preparation. He points out that there are four types of T cells inducible during Leishmnniu infection and i mmunization, these are Tr cells recovered from infection, Ts cells from T suppressor from mice with progressive disease, Ti cells from i.v. immunized micr and Tsc from mice given S . C .

immunization. Tr and Ti are host protective. whereas Ts and Tsc cells are counterprotective. He further asserts thnt there is a strict correlation between protection and the ability of the T cells to prodiice IFN--y /MAF, leading to

activation of infected macrophage to eli nii nate in triicel l u Iar arnastigotes.

However, the mechanism leading to the preferentiiil induction of the different subpopulations of T cells is still not well known. I t is iinclrar also whether the protective and disease-promoting T cells nct intleprndently of each other or interact at the cellular level. An interesting hypotliesis has bren put forward by - Michel1 and Handmann (1986) basrd on tlieir work with lipid-containing glycoconjugate (L-GC). I t is proposed tlia[ L-GC, w h r n anchorrd by lipid and orientated in relation to class 11 niajor liistocoiiip~itibility con~plzx niolecules on macrophages, is both an inducer and a tni-get toi- certiiin cliiss Il-rrsri-icted T cells. On the other hand, d d i pidated water-soliible g lycocon jiigiites (DL-CC) bound to macrophage via specific receptors and iiiiass«ciatrd witli M H C is thought to activate the disease promoting T cells. Supporting tliis hypotl-iesis is the finding that L-GC when injected i .p. con ferred pi-otection n w i L. nst L. r r z c i j o r i nkction i n susceptible and resistaiit inice, wlierras iiiiiiiiiiiizatioii witli DL-GC Ird CO diseasé exacerbation. However, there is no evidence h r an association of glycoconjugare with MHC molecules, nor for glycolipid heiiig [lie oiily oi- indeed. a major protective antigen.

2.3.4. Molecrrlar vaccines

Although much has been learned aboi] t the ii~echiiiiisn~ of' protection against Ieishmaniasis, some major challenges still i-eitiriiit. In the coiirse of th&- evoliition,

Leislzmanin have developed nwltiple strategies for tlieir survival, for example they make use of several different receptor molecules i n order to bind to and enter the host macrophages. The sirnplistic approncli that block the recrptor would be sufficient to prevent entry is therefore no longer valid. In short, after dissecting the parasite into single molecules, the reqiiiremrnt fo ra combination of several molecules is forcing to put the parasite b x k tocerher. - I F premunition is indesd an important consideration for lifelong immuiiity. construction of a stable live parasite using the molecular biology to remove or add genes becomes an imperative strategy for vaccination developrnen t agai nst lei shmaniasis.

Our ability to introduce DNA i n parasites has revolutionized research on Lrish.rnaiiia. Al1 major human parasites crin now be triinsfscted by electroporation (Borst and Ouellette, 1995), the DNA c m eitlier reniain rpisomnl or if double stranded breaks are introduced, i t c m integrate into the grnome exclusively by homologous recombination (Criiz et al., 199 1 ). .4s Lci.shtlz<rtiitr is a diploid organism and since no rnating is available, two successi\re rounds of targetinp are necessary to inactivate a single copy gene. The development of this new technology of gene-tarpeting Iias provided a powr fu l tool to study the importance of a given gene which crin be removed or addrd selectivrly.

- Recently Beverley and colleagiies from Harvard U n i versi ty produced a conditionai auxotrophic mutant of L. ,n<ijar (dhfj--rs) by rargeted drlrtion of an essential metabolic gene dihydrofolatr retliictrisr-th y initly lm sy nthasr (dhfr-rs). The growth of tliis mutant depends on the availability of thymidine within the medium, as i t is incapable of synthesizing i t . The initial chfi--tas knockout L. mcijor was made with neomycin resistrint gene (IICO-R) whicli \vas iised for selection purpose, however, it was rendei-ed nonreverrible aiisoti-ophic by removal of the neo-R gene. The clhfi--ts-, when inocuinted by tlir i -v. . s.c., or i.m. routes into mice elicited siibstnntial resistance to a siibsecprnr chnlleiige with virulent L. major.

Howevei-, this sti-ategy could present a problriii b y w11ic.h ihc parasite would undergo a genomic renrrangernent i n orclrr to espress ils èssèntinl metabolic gcnç. To circumvent tliis aspect of the problem, iiiy iliesis woi-k lias rvolvcd in creating mutant parasi tes by transfecting heterologoiis genet ic information to render parasites susceptible to drug trraiment. The cure, lollowing drug treatment, should induce o protective immtinç respoiisz.

We have used the ability o f the negative selrctiiblr rnorkrr (LrBowi tz et al., 1992) to induce a cytotoxic effect on Leisf~~ncirz ic i . The Thymidine Kinase, (TK) an enzyme of the Herpes simplex virus type-1, ( H S V - 1 ) is capable of phosphorylating analogues of nucleosides siich as ganciclovir aiid acyclovir to its 5'-mono nucleotide form, which is furtlier phosphorylnted by cellular kinases to the triphosphate form. This form competitively inhibits incorporation of dGTP into DNA irnpeding and prematurely terminating its elongntion and hence leading to cell death. The high specificity of the viral TK for these nucleoside analogues make it possible to selectively kill HSV- 1 TK-expi-essiiig cells among a population of dividing cells. TK expression has been used to study linragr formation i n cultured cells and transgenic animnls to achieve conditioiial ablation of targeted ce11 types in transgenic mice and as gene theri~py to t r rat seiectively several types of tumor cells and or to control secondary infrcrion i n AlDS patients. Ganciclovir, a 2'-deoxyguanosine nnnlogiir with thri-aprutic activity against human cytornegalovirus infection, was tirst reviewetl in Dt-ti,ys in 1990 (Faulds et al., 1990).

A number of important trials evaliiating the iise of gnnciclovir in this indication have subsequently been published and the driig has been widely used in clinical practice. The dru= prolongs also tiine to progi-rssion i n patients with acquired immunodeficiency syndrome (A1DS)-relntrd cytori~égaloviriis reti nitis although life-long maintenance thernpy is 1-eqiiired. C;aiiciclc>vii- is Aso effective when niven prophylactically or as ex1 y treatnienr loi- iisy riipioiiiatic i nkction in bone c'

mnrrow transplant recipients. The rrcoiiiiiiriidd dosiigct 1-ctgiinèii for ganciclovir as trentment for patients with cyton~rgiilovii-11s wti iiitis iinci iiorrnal i-enal fiinction is Srnglkg (as a constant intravenous infusioi~ ovri- 1 hoiir) evcry 12 hours for 14 to 3 1 days. If reqiiired, maintenance doses of 5ing/kg/diiy. 7dayslwrek, or 6mglkglday 5 daydweek, c m be giveii followiiig tlir iiiitial regirnrn and are recommended for the prevention of cytonieg;ilo\~iriis disrase i n transplanr recipitnts. The driig should bt: i i i t'used over n 1 -iioiii- pcriod. Siibcutanrous or intrarnuscdar administration is iiot reconin~riidetl. Iiiii-avitrral gnnciclovir is generally given at a dose of 200 to 400 pg oiicc oi- rwicr wttrkly during inducrion therapy, weekly maintenance doses are t l i rn given. 'The most coinmont adverse event during ganciclovir therapy is hematoloçiciil toxicity but this appears to be readily reversible on discontiniiation of the dnig.

- 2. 4. Objectives

The main objective of my tliesis is to generate Leishitzuni~z expressing heterologous genes by gene transfection i n ordrr to i ndiice rapid elirnination of the parasite wi thin the host macrophage while developinç a cellular immune response important for an efficient vaccination. Our specific objectives are:

1) To create mutants of Lrishintrnici that cire capable of expressing the Thymidine Kinase (TK) gene in both the promastigote and amastigote stages.

2) To study pharmacokinetics of ganciclovir into animal mode1 (BALBk) mice in order to evaliiate the best route for drug delivery and to determine parameters such as elimination rate constant (K,,), half-lit? (11;2) and t h e niean residence tirne (MRT) of ganciclovir.

3) T o vaccinate BALB/c siiscepti ble niice w i th tlie TU-etpnssi ng Lrishrmit~itr followed by ganciclovir treatrnent and eval ~ ia te a n y ncqtiired i mmunr resistance.

-

MATERIALS AND METHODS

CHAPTER III

3. 1. Cloning and DNA constructs

A) Clonino of DNA in plasmid expression vectoi-s

In principle cloning i n plasmid vectors is done simply by c1ea:ing the plasmid DNA with a restriction enzyme and joined Inter i n vitro to a foreign DNA. The resulting recombinant plnsmids are t h r n used to transform bacteria. Depending on the nature of the DNA ends, several diffei-ent sti-ategies could be used. For example fragment carrying noncon~plemrntnry protruding termini due to the digestion wi th two different restriction enzymes wi 11 req~ii re directional cloning, a process by which the vectoi- is digesred witli rwo difïri-enr restriction enzymes and ligate to a foreign segment of D N A rhüt contains cohesive ends termini compatible with those generated by the digestion of the vector. An other example is the fragment carrying identical termini (blunt-ended) i n wliiçh foreign D N A must bc cloned into a linearized plasmid vzclor beril-iiig conipatiblr ends with the foreign DNA (blunt-ends). The last exaniple is the niore difficult beca~isr the fragment carries blunt ends. Cloning of siicli fragment i-ttqiiires much Iiighrr concentration both of bricteriophage T? DNA ligasr and sprcific tènipèraturr and buffer conditions.

in case that is impossible to f ind n suitahle niarcli betwern rtisrriction sites i n the plasmid and thosr at the ends of foi-eigii DNA. syntlieiic lirikers can bcr ligarcd io the termini of the linearized pirisniid aiitl loi- fi-ngiiierit of foi-eigii D N A or DNA fragments with recessecl 3' tei-niiiii crin b r piil-tinlly filleil by K.;lctnow fragiiisnt of E. coli DNA polyniernse 1. l'liis pi-ocetliire wi l l ;rneritte conipleriientiiry trrniirii from restriction sites that are otherwise incompatible.

To avoid recircularization of the vrctor. drphosphorylation with caif intestinal alkaline phosphatase (CIP) is used to seniove the 5'-phosphate groups from iinear DNA. CIP is then removed by digestion witli 3 sniall rimount of protrinase K i n

the presence of EDTA, and the dephosphoi-ylatrd D N A is then purifiêd by phenoI/chloroform extraction and etl~nnoi precipitiition. Afcer CIP rreatment, the fragment and the vector are ligated and trnnstgrniation in E. coli is performed in order to obtain recombinant plasmids.

Transformation of plasrnid DNA is usually done in Escl~rrichitr coli type DH5a. This strain is rendered competent by treatment with rubidium salt (RbC1). One colony of DH5a is inoculated into 5 ml of LB mediiim. The logarithmic preculture is used to inoculate 200 ml of LE3 culture for 7 hoiiis to reach the 4- 7x10' viable cells/rnl. After centrifuga~ion (2500 rpm for 5 niinutes) cells are collected and resuspended in RF1 solution (1/3 of initial ciilture) thereafter iced for I hour. After n second centrifiigation. cells are resiispended i n RF2 solution (1/12.5 of initial culture) and iced for 2 hours. The new cells becamr competent and were stored at -SO0 C. Typically 100 pl of competent cells are iised for a transformation. The mixtiire of 100 pI conqxtrnt cells wi th the ligation solution is iced for 30 min and incubated at 42°C for 2 min. Theraftrr. 800 pi of LB solution is added to the mixture. and inciibated with agitaiion toi- 1 hour at 37" C. A volume of 300 pl of the culture is spread ovei- a LB solid medium with a selective pressure (ampicillin SOyg/nil) and incubated for ovrr night at 37" C. -

-RF1 Solution

100 mM RbCl 50 rnM MnC13.4H20 30 mM KAc 10 rnM CaCl2.2H20 1 5 8 w/v plycerol pH 5.8 adjusted with 0.2M ilcetic acid Sterilized by filtration (0.22 jm)

-RF? Solution

1 O rnM MOPS 10 mM RbCl 75 rnM CaC122H20 15% wlv glycerol

pH 6.8 adjustedwith NaOH sterilized by filtration (0.22 p rn)

-DHSa: Escherichici coli strain genotype F-,080dIiicZM 15. 1-ecA 1, endAl, gyrA96, thi- 1. hsdR 17(rk-, mk'), s~ipE4-l. 1 - 1 1 , deoR, (1acZYA- argF)U 169.

B) DNA constructs

The most basic eukaryotic expression module contains a promoter elrment ro mediate transcription of foreign DNA seqiiences and signais rrqtiired for efficient polyadenylation of the transcript. Additional elements or iirw elements, as in case of Leish~nania , i n which a promoter element lm not yet been drscribed, includrs a strech of polypyrimidines with functional splice accèptor sires.

During the expression of eu kai-yotic genes, R N A polymerase I I transcri bes through the sites where polyadenylation wi I l occui-. Consequently, the 3' terminus of mature m R N 4 is formrd by site-specific posti;~iisu-iprionril clravage and polyadenylation. Two distinct seqiience elemrnts arc: reqiiired foi- nccurate and - efficient polyadenylation: ( 1 ) GU or U-rich sequelices loc:itrd downstream from the polyadenylation site and (2) a highly conservecl seqiirnçr of six nucleotides A A U A A A , located upstream, which is necessary bu t not sufficiént for postranscriptional clenvage anci poiyadenylntion. The practical implication of these observatons is that seqiiences downstremii froiii rlie polyidciiylation si te must be included in eiikaryotic expression vectoi-s io rnsiii-e efficient polyadenylation of the niRNA of interest.

The DNA seqiiences coding for a eiikaryotic protein arc I-arcly contigiious. but are usually separated in the çenome by intervening iionsodiii~._ sccliicnces thar may Vary i n size froni tens to innny tlioi~siinds ot' i~ii'iroiidzs. Following polyadenylation of the primary transcript. the iiiti-ons rire rtinovcd by splicing to oenerate the mature mRNA, wliich tlien traiisportrd froiii the iiiiclsi~s to the b

cytoplasm (Maniatis et al., 1 988). I t is tliei-ctbrr i r i to incliide splicing acceptor and donnor sites in the expi-ession vecioi-. In Lci.slrtirltriitr, no iiiirons havé

been described and niatiiration of the message occiii-s post-trnnscriptioiialy and requires the presence of a splice ricceptoi- site AG.

The entire coding sequence for the thymidine kinase gene of Herpes simplex virus type-1 was obtnined from vector ptk svlt-liyg (a kind gift fi-oni Dr. M. Audette, Molecular Endocrinology, CHUL), and i r was used as template for PCR amplification wi th oligonucleotide primers that contai ned 5'-.CTT G T A G A A TTC GGT ATG GCT TCG T-3' and 5'-TGT CTC CAT CGA TCT TTC AGT TAG C-3' and C M and EcoRl restrictions sites arc rrspectively underiined. To obtain pneotk-1, 1 ~ i g of the PCR product was digesred wi th appropriate buffers in a total of 20 u1 with 0.5 ml of each EcoRI-Cltr I for 1 Iiour at 37" C. The 1.14 kb was isolated by electrophoresis on agarose gel (0.5- 1 % ) i n 1 XTBE (90 mM Tris-borate pH 8.0, 2 mM EDTA). The size of the fragment wns determined by CO-migration of the digestion with 1 Kb commercial ladder of known size (12.3; 11.2; 10.2; 9.2; 8.1; 7.1; 6.1; 5.1; 1.1; 3.1; 2.0; 1.6; 1.0 Kb; 516; 394; 298; 220; 200; 154; 142; 75 pb).

The 1.14 Kb band was removed from the gel and 0.3 M NaCl was added in proportion of 3 volumes. The agarose was thrn heatcd at 65" C for 15 minutes. To recover DNA, two successive extractions of phenol v / ~ and plienol/chloroforrn v/v were performed. The DNA wns then precipitnied by I voliime of isopropanql and washed with 75% of ethanoi (500 P I ) ; dried in speed vuccurn for 10 minutes and resuspended in 20 p1 of TE (10 m M Tris pH 6.0, 1 nihl EDTA). Ligation was

done (8-12 hours nt 15°C) with CM-EcoRI digesrrd pSPY2 1 vector containing a 92 bp synthetic polypyrimidine stretch and splicr: riccrptoi- site AG (Y92 A G ) (Papadopoulou et al., 1994) to yield pSPY2 1 - t k i n which the TK genr was cloned downstream of the Y93 AG. The recombinant plasniid wiis riansfoi-mcd i n Escherichici coli D H S a on nmpici l line (50~ig/ml) LB plates.

In the second step the neomycin phosphoti-ansferase genr (neo) iilong with the Y92 AG motif fi-om vector pSPYneo (Papadopoiiloii et al.. 1994) was subcloned as a 0.9 kb XhoI-EcoRV fragment into the X h o l - P i d sites iipstieam of the TK gene to yield vector pneotk-1. Vector pneotk-2 was iiiade by s~ibcloning a 280 bp SalI-BnrizHI fragment from the tetracycline resistance genr of vçctor pBR327 (Watson et al.,l9SS) into the respective sites of vectoi* pneotk- 1 . Part of the tetracycline resistance gene was used in osder to provide ttnoiigh sequrnce for the correct maturation of the upsti-eam neo gene.

Expression vector pneptk-3 was construcred by siibcloning a 1.6 kb EcoRI-BamHI fragment from pGEM3-neo filled-in with Klenow polymrrase into the EcoRV sire of the pSPYî-1-tk vector. pGM3-neo vector (Papadopoulou et al., 1994) contains the intergenic region of the a-tubilin gene of L. ~nrirrrii (Laban et al., 1990) and the neo gene cloned downstream. . After transformation, the DNA was isolated from E. coli D H S a by the alkaline lysis technique (Maniatis e t al, 1989). DHSa cells were centrifuged (3 000 rpm, 5 min., 4" C) and the pellet was resuspended in 200 pl "quick mix" (25 mM Tris pH 8.0, 50 mM glucose, 10 rnM EDTA) with 400 pl of freshly made 0.2 N NaOH-1% SDS for 10 min. at room temperature to cornpiete lysis of the cells. Thereafter, 200 pl of 3M KAc pH 4.8 were added at 4" C for I O min. The viscous solution was vortexed and centrifuged at 14 000 rpm for 5 min. The supernatant was collected and extracted by phenol-chloroform. To recover the DNA, isopropanol (vlv) precipitation was perforrned; the isolnred DN A was washed twice in 70 % (500 pl) ethanol, dried in speed vacciini and 1-eslispendrd i n 20 pl of TE 1X (TE: lOmM TrisCl pH 8.0, I rnM EDTA). R N x e treatrnent (20 pg/ml. 30 min. at 37" C) was done in order to eliminrite the R N A coiitaminants.

Al1 the Lrishhmnnic strains used are classsified iindrr the Aniei-ican Type Culture Collection [ATCC] and were cultiired in SDM-79 mrdiiirn siipplemenced with - 10% fetal bovine serum (FBS) (Multicell,Wiscnt Inc.) and 5 pl/ml of hernin at arowth optimal temperature of 25-29" C. 0

L. major S F - 1864 L. tropica S F - 1576 L. rnexictrric~ ~nexic~rmr SF- 19 1 1 L ck>novuni rionovclrii MHOWIINIBOIDDB SF-22 1 1 .

The method of tronsfection used i n rny stiiditts is prinsipnlly the electroporation technique. The application of brief, high-voltiige rleciric pulses to a variety of mammalian and plants cells leads to the formation of nanometer-size l ik r pores i n the plasma membrane. DNA is tnken directly into the ceIl cytoplasm rither through these pores or as a conseqiience of the rrdisti-ibution of membrane components that accompany closure of the pores. Elzcri-oporation can be

extremely efficient and can be used both for transirnt expression of a cloned gene and or for establishment of ce11 Iines that crirry integratrtl copies of the gene of interes t.

To facilitate the identification of cells that cnrry the transfected-cells, specialized elements such as genes encoding selectable markers rire cotninfected with genes encoding the protein of interest. These genes have genernlly an enzymatic activity that confers resistance to an antibiotic or drugs. Most of thrse genes are dominant positive selectable markers, otliers as described for the TK gene, are dominant negative selectable markers. The mostly widely used dominant selection system utilizes the bacterial gene encoding neomycin phosphotransferase that confers resistance to nminoglycoside antibiotic neomycin (G41S) which inhibits protein

r - synthesis in both prokaryotic and eukaryotic cells. 1 i-rinsfecrrd cells will grow well in presence of (3418, hence selecting cells thai Iiiive bzen as well transfected with the gene of intrrest. Wild type promastigotes i n logaritlimic phase were washed in HEPES-NaCl (21 mM HEPES pH 7.05, 137 nibl NaCl, 5 niM KCI, 0.7 mM Na2HP04, 6 mM glusose) and resuspended i n the same buffer at 1x108 cells/ml density. Approximately 15 pg of pneotk- 1 , pneotk-:! and pneork-3 were mixed with 500 pl of resuspended cells into elrcrroporaiion cuvette (0.2 c m Biorad). One electric pulse of 0.45 KV, 500 pF w x nppIird witli Biorad genë pulser. Electroporated cells were cultured i n 5 iiil of S D M nirdium wirhour selective pressure.

After 48 hours, 5 ml of fresh medium was added with 40 pg/ml of G41S (Geneticin, Gigco-BRL) as selective pressiire. A fiirther iiiciibarion of 24 hours was allowed before ttrnnsferring 300 p1 of the cult~ii-c: iiito 5 ni1 of frrsh mediuni with G4 18 selection to isolate prornastigotes exprttssi 11: 7'K.

In order to localize the particular seqiience eithei- witliin tlir genoniic D N A or RNA, total Lrishinonici DNA, i n case of Soiithern, is digssted with a particular enzyme, or total R N A preparations in case of Nortliriii, ai-c. separated according to size by electroplioresis throiigh an agarose gel. The D N A or R N A is then denattireci i n situ and transferred from tlir gel to a solid support iisiially a nitrocellulose filter or nylon menibrime. The i-rlativr posiiion of fragments are

preserved during their transfer to the filter. The attached fragments are hybridized to radiolabeled DNA or RNA probes, and autoradiography is used to locate the positions of bands complementary to the probe. in relarion to the position of fragments of DNA or RNA ladciers. . 3. 4. a ) Total DNA Preparation: Rapid technique

Leishmanin-TK and Lrishrnania-pSPYneo were centrifuged (2 700 rpm, 10 min. at 4" C) and washed i n HEPES-NaCI buffer (5 ml). The pellet was resuspended i n an eppendorff tube with 1 ml of the same buffer and 500 pl of TEL-T solution and incubated for 10 min. at room temperattire. The DNA was extracted with phenol:chloroforrn, precipitated with 2 volumes of 99 C/o of ethanol, washed twice in 70 % of ethanol and resuspended in 300 pl of T E after RNase treatment.

TEL-T:

50 mM Tris-HCI pH 8.0 102.5 mM EDTA pH 9.0 2.5 M LiCI 4 % V/V Triton X-100

3. 4. b ) Total D N A Preparatiorz: Clussical rechrziqlre

Leishrnnnia-TK and Leiskmnnici-pS PY ne0 ciiltiires were centi-i fuged (2500 rpm, 10 min., 4" C) and the pellet was washrd i n HEPES-NaCI buffer v/v. and resuspended in 5 ml of 100 niM EDTA. 100 niM NaCI. 10 mM Tris pH 8.0 solution. The parasites were then lysed by 500 ul of 10 '3 SDS and irnmediately proteinase K (50 pglml) was addeci. The viscoiis solution was then incubated at 37" C for 30 minutes. Two phenol extractions (3000 1-pni, 3 min. . 4" C) were followed by of 99 % etlianol (2 voliimes) precipirrition. The Iiigh rnolrcuiar weight DNA is fished out with an inoculating loop and rrsiisprnded in TE (200 pl) with 30 pg/ml of RNase and incubateci at 37°C for 30 minutes. Before two second phenol extractions, 0.1% of SDS and 50 pg/rnl of proteinase K were added for 30 minutes at 37" C. The DNA is precipiwred, fislied out and resuspended i n TE (200 pl).

21 rnM HEPES pH 7.05 137 rnM NaCl 5 mM KCI 0.7 mM Na2HP04 6 rnM glucose

3. 5. RNA Preparatiojz

Leishrnania amastigotes were isolated from infected J774 rnurine nilcrophages following 72 hours infection wi th Lri.sl~~izu~iict-TK and Lri.sh/~iatziu-pS PY neo control. Infected macrophages were taken off from the chnmber slide and resuspended in 5 ml of D-MEM medium. The siispension wiis liomogenizzd with a Pyrex tissue grinder (corning) and tninsfei-i-ed to ;i 50 ml Falcon tube. Three successive centrifugations of 10 min. tricii, the first bein; at 1300 rpm and the two others at 3000 rpm were performed in order to isolate amastigote-TK and amastigote-pSPYneo. The amastigote pellets were collected after 7 min. ar 3000 rpm centrifugation. hornogenized with 1 ml of Trizol (through a syringe). After 5 min. of incubation at room temperature, 200 pl of chloroform solution wa3 added, vortexed and let to rest for 2-3 min. nt 1-oom temperature. The supernatant was collected following centrifugation at I-lOOO 1-piii t'or 20 niin. at 4" C and mixed with 500 u1 of isopropanol for 10 min. ar rooiii rsrnprratiirr. The pellet (15 rnin.,lil 000 rpm, 4" C) was twice washed with 500 pl of 70 5b ethanol. The isolated RNA was dried i n speed vacciim (10 min.) and resiispendrd in 20 pi of sterile water and incubatrd for 10 min. at 56" C. DNast: rreatnient was applied to remove DNA contamination. Once phenol/clilorofoi-ni extniction and isopropanol precipitation were perforrned the recovri-ed R N A was 1-rsuspended i n water diethyl pyrocarbonnte (water- DEPC O. I 5%) t rca [rd.

CAUTI0N:Glovrs must be worn to avoid any çontariiination with RNases.

Leishmcinia-TK and Lrishrnnnicl -pSPY nro were digested with BR/!! to linearalize the pneotk plasmids and, separated by electroplioresis 1111-oiigh an agarose gel (0.7

% gel, 1X TBE containing 0.5 pg/ml of ethidiuin bromidr). The gel was run slowly (25 V overnight) and before rnakinç the photograph. a transparent ruler was placed alongside the gel so that the distance that any band of DNA has rnigrated can be read directly from the photographie image.

\

The gel was soaked in 0.25 N HCI for 15 min. and rinced i n deionized water and then denatured by an alkaline treatment ( 0 5 N NaOH, 1.5M NaCl) for 45 min with gentle shaking. The DNA was then neutralized by soaking the gel in 0.5M Tris HCI (pH 7) and 3M NaCl for 45 min. With the help of VaccuGENE (LKB

TM -N Pharmacia), DNA was transferred to the nylon membrane (Hybond ,

Amersharn) in 20XSSC (LXSSC= 0.15M NaCl, 0.0 1 SM sodium citrate pH 7.0) transfer/buffer for 2 hours, Tbe transferred D N A was heat-fixed for 2 hours at 80" C or 12 hours at 65" C.

Hybridization of radiolabed probes to immobilizrd niicleic ncid was performed and to eliminate the nonspecific attachement of the probe to the surface of the filter, the membrane was prehybridized with a blocking agent containing 6 X SSC. 5X Denhardt's 1-eagent , 0.5% SDS, and 100 pg/ml of drnatiired fragrnented salmon sperm DNA for about 2 hoiirs. Labeled D N A TK probe was synthesizec using random oligonucleotides.

Oligonucleotides can serve as primers for initiation of DNA synthesis of single- stranded templates by DNA polymerasr (Klenow Frasnient of E. c d i DNA polymerase 1). If the oligonucleotides are heterogrnroiis i n sequencr, they will form hybrids at many positions, so that every niiclrotitle o f the trmplnte will br copied at eqiial fieqiiency into the product. By using [o.-'?PI dNTPs as precursors. DNA probes can be rndioliibeled to very high specific iicriviry. Labding was donc as follows: 1 pl of TK (PCR fragment) D N A ( 100 rig) wirh 19 pl of water wri-r

denatui-ed for 5 min. nt 100" C. A mixture soliition of' dcnatiirrd TK DNA. 3 pl of a-? dATP, 6 pl of oligo labeling bufSer (OLB) aiid 1 pl of Klenow (1 unitdpl) was inciibated at 37 " C for 60 min. and the I-eiiction was stoppèd by adding a stop mix solution (100 mM Ti-is, 12.5 mM EDTA, 0.5% SDS). The labeled DNA was then purified throiigh a coliinin of Srphadex G-50. Before hybridization (16 hours at 65" C) of the menibi-ilne, tlir probe lias to be drnaturrd by boiling for 8 min.

After hybridization, the membrane wns washrd, at 65" C, 2 rimes for 15 min in 3X SSC. 0.1% SDS, and 2 times in O. I X SSC O. 1 C/o SDS. Ar rh t end of washes the membrane was covered in a sheet of Siii-an Wrap and rxposrd tiom 7 to 16 hours to X-ray film (Kodak) [O obtnin an autoradiographic image. .

Prehvbridization solution:

for 100 mi of solution add:

15 ml 20X SSC 10 ml Denhardt 50X 100 ml denatured salmon sperrn 10 ml NaPi 0.5 M H20 to 100 ml

Ficoll 5 g Polyvinylpyrrolidone 5 g BSA 5 g

40 to 500 ml

-0ligo labeli ng biiffer:

Sol. O: 1.25 M Tris.HC1 pH 8.0 0.125 M MgCl2

Sol. A 1 mI so1.O 18 pl 3-mercapto EtOH 5 pl dATP 100 mkII 5 pl dGTP 100 mM 5 1.11 dTTP 100 mPvl 5 pl dCTP 100 mM

Sol. B 2M HEPES pH 6.6 (ajusted with NnOH)

Sùl. C pol(N)&90 O.D. iinits/ml

OLB: mix A:B:C in proportion of 100:250:150

. 3. 7. Northeriz blot

The RNA of Leishrnania-TK and Le isl~trztrn i t l - pS PY neo was separated by electrophoresis through an agarose gel ( 1 5% , 16.5 ml forrnaldehyde 3 7 8 , 5 ml migration buffer 20X completed up to 100 ni1 with sterilr wnter). Before transfer the gel, we incubate i t first in NaOH 50 mM-NaC1 100 mM for 45 min., at room temperature; secondly in SSC 20X for 60 min., rit room temperature and we transfer to a nylon membrane in SSCXî-O buffer wi th the help of VaccuGENE. As for the Southern blot, the RNA was fixed by heat, prehybridized and hybridized for 16 hours at 42" C. First washing was done for 30 min., at room temperature in solution A and second and t h - d were in 2 series of 5 niin., at 65" C in solution B and C, respectively.

Probe labeling was done as for Soiithern blot. The a- tubul in gene of Trypanosorna britcri was used as an interna1 control to evaluate the gel. -

-Migration buffer 20X:

20 mM MOPS 5 mM Sodium acetate 1 mM EDTA

-Prehybridization biiffer:

formamide SSPE 20X SDS 10% Denhardt 50X

H2O

NaCl a 175.3 g NaH3P04 27.6 g EDTA 7.4 g

H*O up to IL

-Hybridization buffer: same as prehybidization in which Denhardt is replaced by water.

-Washing solutions:

Solution A: SSC 20X 12.5 ml SDS 10% 2.5 ml H 2 0 up to 250 ml

Solution B: SSC 20X 2.5 mI SDS 10% 2.5 ml H 2 0 up to 250 ml

Solution C: SSC 20X 1.25 ml SDS 10% 2.5 ml

H 2 0 up to 250 ml

Once transfectants Lri.sl~inciriitr-T and Leisl~nttrii itr-pSPY neo were avai Iiible, we proceeded to test the expression of TK gent! by a growth curvc test. This experiment did give details of TK expi-essiori i n the promastipotr stage of Lrishnînrzin. However, our iiltiinrite interest resided into the infection of macrophages (murine) by the transfectants so thnt the expression of the TK can bè measured in the arnnstigotes stage. To wuliiate possible intri-fttring eftacts of the ganciclovir on macrophages, a MTT test on J771 niiirinr cells linci and human macrophages was carried out.

J774 ce11 l ine was adapted to cultiire fsom a tiimoi- whicli arose in fernale B A L B k

mouse in 1968. The ce11 propagates in Dulbecco's Modified Erigle's Medium, (D- MEM) 90%; fetal bovine serum 10%. Its growth is inhibited by destrnn sulfate. PPD and LPS. This ce11 line is capable of synthesizing large nniounrs of lysozyme and exhibits minor cytolysis but predominantly antibody-dependent phagocytosis. IL-1 also known as lymphocyte-activating tictor (LAF): is synthesized continously by this ce11 iine. In addition, these cells have been shown to carry cell- bound receptors for immunoglobulin and complement.

Human macrophages were also included, i n order to evaluate the infrctivity of the transfectants and also the effect of ganciclovir drug on these srlls. To prepare human macrophages by adhesion, whole blood was collected directly [rom healthy donor individuals. For Ficoll gradient, 25 ml of fresh blood was carefully added to 20 ml of Ficoll-Paque solution in a Sarsted tube then centifuged at 1 500 rpm for 30 min., at 18 to 20" C. With a sterile pipette PBMC the cells were collected into a sterile tube. The cells were then washed twice (12 000 rpm,iO min.) with 30 ml of Hank balanced salt (HBSS) and slisprnded in 30 nil of RPII.11 IO% human serum, 5 % L glyt, Pan Strep., as final concentration of 3 X 10" crlls/rnL. Aftrr 5 days of incubation at 37°C and at 5 % CO2 i n watei--jacketed incubator (from Forma Scientific) fresh medium was addrd. Most of monocytes have prolifered into mature human macrophages and have adhered to the wall of rhr flask.

-HBSS solution

Measure out 5% of deionized distilled watrr into powder mzdiiim at room temperature with gently stirring. Rinse oiit insidr package to i-enmvc iill triices of powder. Add 0.35 g of NaHC03 per litsi- of medium. Strrilization through membrane fiItration.

Following the infection of macrophages by Leisl~iiirir~itr and the TK expression. the transfectants were used as i nocu Iiirn to inlrcr intravrneousl y and subcutaneously BALB/c female niice. Fiirtliei-more, the pharmacokinetics of ganciclovir into BALBlc niice were perfornied in order to rvaluare the best route for drug delivery and also to determine othrr phxmocokinrtic parameters. Thesr pharrnacokinetic studies were coupled wi th Iiematological toxicity and drug distribution in the BALB/c mice.

Animals were then treated with appropriate doses of ganciclovir to test whether Leishrnarzia infection has been eliminated. Cured mice were challenged with wild- type Leishmania strain to evaluate any ncquired in~mune resistance.

3. 9. Ce12 growth assay

The cytoxicity of the nucleoside Leishmaniu was determi ned by

.

analogue ganciclovir (Cytovene from Syntex) to ~ising a ce11 growth assny. Lrish~nczIiici TK and

Leishmania -pSPYneo transfectants were seeded nt low concentration (3 X 10' cells per culture dish) in 5 ml of SDM-79 medium with various concentrations of ganciclovir. After 77 ho~irs ce11 viability was obtained by measuring the absorbante at 600 nm on an aiitomated microplate reader (Reader 5 10 from Organon Tecknika Inc., Austria). The EC,,, was clilculnted as bring 50 8 of inhibition of celliilar growth i n presence of differrnt concentrations of ganciclovir. At those different concentrations, the expression of thymidine kinase gene will result in death of the Leishnzcirtia expressing T K .

3. 10. MTT Test -

MTT is water soluble tetrazoliiim salt yirldinz a yrllowish solution when prepared in media or salt solutions lncking phrnol r d . Dissolved MTT is converted to an insoluble piil-ple formizan by clctii~iigt: of ille teti-azolium ring by dehydrogenase enzymes. This water insoluble Soin~azan caii bc soliibilized using isopropanol or other solvents and the dissolved mntei-ial is nieasurcd with a spectropliotonirter yielding iibsoi.b;ii~cr us a fiirictioii of coiicciitriition of converted dye. The cleavnge and conversion of rlir soliiblc yellow dye to the insoluble piirple forrnrizan has brsn iiscd to drvrlop an wsay sysiriii alrrrnative to the conventional ' ~ - t h ~ r n i d i n c iiptake iind otliri- iissays ti,r iiirasunnxnt of crll proliferation. Active n~itochondrial tleliydi-ogencises of living cell will cause this conversion to occur. Dead celis however. do not have the enzyme to cause this change. This has been applied i n mensurenient of interieiikin-2 activity i n a rnultiwell assay. Modification has iniprovrd the sensitivity, and other uses such as measlirement of cytotoxicity and ceIl iiiiiiiber have iilso been dtveloped. The solution is filtered through 0.2 nim filter and stol-ed at 4" C for fi-eqiient use or frozen for extended periods. MTT may nlso be iised to score hybridomn development or clona1 development. Cloiies will convert the dye and becorne

3. 12. In vivo assuy

Female, inbred BALBlc mice were purchased from the ~ha r lgs River Breeding Laboratory (Saint-Constant Qc, Quebec, Canada). I n order to rninimize contamination of leishmanial lesion with bacterial pathogens, animals were kept in sterile cages. BALWc mice are susceptible mice, that develop cutaneous infection by L. major which usually undergo fatal visceral dissemination.

3. 12. 2. Pharrnacokiizetics and toxicity s t~rdies of rlie garzciclo vir- drug delivery in tire animal niodel

Most of the pham~acokinetic properties of the ganciclovir were evaluated from studies done in human and i t was found that inti-avenous administration of 1 to 5 m g k g doses of ganciclovir producrs linearly i ncrensing peak plasma concentrations, while intravitral administration of ganciclovir produces high concentrations i n vitra1 fliiid wi th minimal, if any, systemic absorption. Concentrations of ganciclovir i n cerebrospinal fluid were Iower than those reported in serum after intravenous ndmini stration. Ganciclovi r has a stcady-state volume of distribution of approximately 32 to 44.5 L. Almost 100 C/o of an intravenously ridministered dose of ganciclovir is excreted i n the urine of patients with normal rend fuiiction. The drug hns an elirniniitioti lialf-life of brtwrrn 2 and 4 hours after intravenous administration of I to 5mg/kg doses.

Following this previoiis review. we set to present an overvirw of the pharmacokinetic profile of ganciclovir i n tlie animal mode1 BALB/c mice. A single dose of ganciclovir was administri-ed i n mice intraperitoneally (i.p.) or subcutaneously (s.c.) in the iipper back beiow the neck. Eiich animal recrivèd bolus dose of 50 mg of ganciclovir/Kg contnining 0.02 pCi '[HI-ganciclovir (Moraveck), pmoles ganciclovir and the injection volume was about 0.3 ml. At each defined times (O, 1 , 2, 4, 8, 16, 30. 60, 120 min.), 40 to 50 pl of blood was

collected via the retro-orbital sinus.

The pharmacokinetic parameters were determined for each mouse using a noncompartimental model. The elimination rate constant (K,,) was estimated from the slope of the terminal elimination phase. The half-life in plasma of the terminal elimination phase (t"') was calculated as 0.69i/KC,. Area under the plasma concentration-time curve (AUCO-) and nrea iinder the f i rs t moment of the concentration-time curve (AUMCO-) were calculated using the log-trapezoidal rule with the terminal portion extrapolated to infinity. The systemic clearance was calculated as dose/AUCO- and the mean residence t i me (MRT) was calculated as AUMCO-/AUCO-. The maximum dnig concentration in plasma (C max) and the tirne where C max was evaluated by visual inspection of each mouse.

Previous studies in humans indicated that the conceiitrations of ganciclovir i n major organs from patients receiving the dnig inti-nvcnotisly were approximarsly equal. However, it becarne imperative to stiidy the distribution of ganciclovir on infected and non infected footpnd of mice, sincr the infection with L. major- rernains initially at the site of infection, thiis the importance to rvaluatr the effect of ganciclovir at that site. - A single dose of ganciclovir was adrninistered s.c. to mice infectrd and uninfected as described above. At specific time points, the animais wri-r saci-ificrd and rhr footpad tissues were removed and washed i n PBS, and weighted. The tissues were then treated with Beckman Tissue soliibilizer (BTS-450: Beckman Instruments, Inc., Irvine, California, USA) and colored i n water. Drug concentration i n al1 samples was monitored by scintillation coiintinç.

As described earlier, the most common adverse effecr in patients ri-eated with ganciclovir is the inhibition of growth of Iiiiiiian bone nm-row colony-forming cells. This inhibition entails a neutroprnia, defined as inferior to 50 percent decrease in absolute neutrophils count from baseline. Other adverse rffects wrrr reported in human patients such as central nervous systern adverse events that include confusion, seizures, headache and somnolence.

To evaluate pattern of Leivhmcinici infection and the effect of ganciclovir on mice,

hernatological toxicity was studied in both infected and non infected mice. Mice were treated with 1 , 3.5 and 5 m g k g of ganciclovir daily for 7 days. At the end of treatment, the uncoriggulated whole blood samples were collected via the retro- orbital sinus for total red blood ce11 counts (RBC), hemogIobin(ffb) and hernatocrit (Ht). Sarnples were counted in the clinical hernatoiogy laboratory at the Centre Hospi talier de I'Universite Laval (CHUL) iising a coulter counter mode1 T-890.

Results were compared using a one way anriiysis of variance, and group comparison was performed using the Fisher protectetl least significant different test. Al1 data are presented as means SEM.

3. 12. 3. Parasites ar2d animal irroculutioir

L major-pneotk-3 strain was obtained frorn the L. niujor LV 39 line by episomal transfection for the expression of the HSV 1 -TK genr. Both ce11 lines (conrroi and TK-transfectant) were passed in mice to ensure vint Iencr. Promastigotes were obtained from stationary-phase culture, wrished twicr i n SDM and resuspended to a final concentration of 5x10" prornastigotes for 15 p l of inoculation. Subcutaneous injections were delivered i n the hind footpad, and intraveneous inoculations into the tail vein. Subseqiient trentnient with ganciclovir was performed on 1 , 4 and 14 days post-infection. Mice thnt did exhibit no lrsion were challenged with virulent wiid-type sti-iiiii of t. i r i < l j o r - LV39. Protective index to reinfection wns rnonitored by weekly mensiii-in= the diameter of lesions with a metric caliper.

CHAPTER IV

RESULTS AND DISCUSSION

The rationale of t h e development of live vaccines is to be able to create by transfection technologies, live Leishmmiu that have an i mpended ability to survive in vivo. A transfectant containing an exogenoiis genetic information such as the T K gene, should be eliminated in presencr of n specific drug ganciclovir. The infection of BALB/c animal mode1 by such a strain followed by treatment with ganciclovir will provide an experimental vaccine rnodel i n which cured animals could be challenged with wild-type Leishmonia to test the level of acquired irnrnunity.

i t was essential to confirm first the toxicity effect of the ganciclovir on transfected promastigotes that stably express the thymidine kinase (TK) gene. These transfectants and control cells that do not express T K were subjected to various concentration levels of ganciclovir. At 10 FM ganciclovir, for most species of Leishnzunicl. 50% were killed, establishing the EC,, at 10 pM af ganciclovir, and alrnost 100% cellular death occured when 100 pM of ganciclovir were used. The control cells remnined resistant to giinciclovir concentration up to

1 mM (Fig. 3). The expression of thymidine kiiirise is thus. capable of inducing a concentration-dependent toxici ty to TK' promastigotes upon addition of ganciclovir. Surprisingly, acyclovir, another nucleoside analogue with a very good affinity for the HSV-1 TK did not exhibit any cytotoxic activity on Leislimania expressing T K ( results not shown).

The expression of TK gene in the intracelluliii foi-m of the parasite needed to be established. Total RNAs of both L. nwjot- pi-oriiastigotes i n culture and amastigotes hai-vested from i nkcted miiri ne maci-oplinges werr isolated and analyzed on Northern blot hybridized to a TK specific probe. Results indicated that T K was equally expressed i n the pronxistigote and i n nrnristigote stages of Leishrnania, since no difference in the expression lrvel wns detected (Fig. 4).

To further investigate the effect of TK expression on amnstigotes within either

murine or human macrophages, 10\tationary phase L. major-pneotk-2 (Fig. 2) (TK') and L. major-pSPYneo (TK') were iised to infect macrophages for 6 hours. After washes, cells were treated with ganciclovir. The outcome of the infection in vitro was followed by microscopic examination. The intracellular survival of L. major amastigotes expressing TK significantly drcreased 48 hoÙrs following the infection upon a single addition of 40 pM (4-fold the EC5,,) of ganciclovir added 6 hours post-infection. When a second 40 pM dose of ganciclovir was added 24 hours post-infection, the number of parasites found inside rniirine macrophages after 72 hours of infection felt further to O.S/crlI compared to the unueated cells where 9 to 10 parasites/ceil were observed (Fig. 5 , left panel). Values were also expressed as the percentage of macrophages that were infected by L. major expressing or not the TK gene. Only 8-10 % of murine macrophages that were infected by L. major TK, when treated with 80 pM of ganciclovir in cornparison to 90-95 8 for the control untreated cells (Fig. 5 , right panel).

The results obtained with the infection of human macrophages were even more irnpressive as only 0.15 arnastigotes/cell were obtained following treatment with only one dose of 40 y M of ganciclovir (Fig. SA). Similnr 1-esults were obtained with L. donovani amastigotes expressing T K were iising both murine and huma0 macrophages (data not shown).

At this point it became clear that the TK was being expressed in both promastigotes and amastigotes and that conseyiirntly gaiiciclovir rrduces the viabili ty of amastigotes inside macrophages at doses tliiit hrid no harmfu 1 effect to either murine or human macrophages as iiidicated by [lie M T T test (Fig. 6).

Before testing the effect of ganciclovir on LeisA~nclni<l expressing TK in animals, we initiated studies to evaluate the pharmricokinrtics in our animal model, the BALB/c mice. The mice were infected witli L. rrzcljor--pneotk-2 nndL. rritzjor- pSPYneo, transfectants.

For the appropriate route for ganciclovir delivei-y into BALBlc mice, we asseyed both ip and SC injection. The concentration-timr ciirve of ganciclovir i n serum was monitored with a radioactive tracer (.'[HI-PFA) and did produce no discernment of potential metabolites or exchanges of labelrd tracers. Although, there was a minor diffei-ence i n the level of sci-ilni in the two routes, the dope that

represents the elirnina~ion part of ganciclovir and the systemic clearance remained similar in both groups (Table 1 and Fig. 7) . To identify any difference that may occurred with infected mice, similar experitnces were conducted with a set of infected animals yielding comparable results (data not shown). Therefore, the pharmacokinetic evaluations indicated clearly that both routes' for ganciclovir administration were sui tabIe.

Since Our principal objective was to eradicate ille cutaneous infection caused by L. major on the footpad of mice, we set to evaluate the concentration level of ganciclovir on infected and uninfected footpad. The dis tri bu tion pro fi le was clearly and significantly different between the infected and the uninfected footpad. The ganciclovir concentration in the infected footpad was found to be 1.63 fold higher than in the uninfected one (Table 2). These resiilts are consistent with the observation that there is an aggregation of blood cells at the site of infection, thus recmiting more of the circulating free driig. This observation increases the chance of eradication of parasites expressing TK at the si te of infection.

To test the toxicity effect of ganciclovir on mice, we used concentrations equivalent to those achieved clinically by in hi bi ting the growth of red blood cells. Erythropenia defined as less that 50 per cent decrease in absolute count ;S therefore, the most common adverse event i n patients treated with ganciclovir. The red blood cells (RBC), and other parameters such as hemoglobin (Hb) heamatocrit (Ht) at different concentrations ren~ained signi ficüntly similar to the control indicating no toxkity of ganciclovir i n the fernales BALB/c mice for the period of 7 days (Table 3).

We have previously shown that the L. mujor expressing T K in vitro experiments was eliminated upon addition of increasing concentrarion of ganciclovir. Following these experiments and the phamacokinetic evaliiations of ganciclovir, we infected subcutaneously at the level of the footpad of fernale BALB/c with L. major-pneotk-2 strain and L. mijor pSPY neo. The concentration of 5~ loh promastigotes of both strains per mouse, induced cutaneous lesions after two to four weeks following infection. Several experiments were conducted to set the time for commencement of ganciclovir delivery. Intraperitoneal treatment with 7.5 mgkg of ganciclovir starting 1 and 4 days nfter infection and lasted for 14 days showed total eradication of Leishtncinici expressing TK since no lesion was

detected after 10 weeks post-infection. However, treatment that started 14 days after infection showed a regression of piirasitic load for 2 weeks that lasted the treatment period and the progression of the infection initiated once the treatment was terminated (Fig. 8). The fact that during the first week of infection the expansion of the T helper 1 , CD4' healer phenotype take~'.~lace and it is overtaken in the second week by T helper 2, a non healer, phenotype, would indicate the failure to cure mice after 14 days of infection pr~bably due to the prominence of the non healer p heno type.

To test the protective index in rnice that showed no lesion after ganciclovir treatment, we infected the same footpad of those cured mice with virulent L. major wild-type. After 10 weeks of observation, vaccination had induced a varied level of immunity. Whilst the group of cured mice from 2 weeks treatment post- infection had an increase lesion thickness up to 7th week then decrease to about 1/10 of the control thickness (Fig. 9A), mice from 4 days post-infection treaternent, dernonstrated a very h igh level of immunity since no lesion was detectable (Fig. 9).

To confirm that the lack of immunity on the 14 days post-infection treated mice- - was not due to the incomplete eradication of the L. nlujor-pneotk-2, we isolated amastigotes from the footpad and cultured them to pet promastigotes. By Southern blot analysis with a TK probe, we did not detect any TK sequences on isolated (data not shown). The infection after challenge was mounted by L. major wild-type only and was noi due to an unsuccessfiil eradication of L. major-pneotk- 2.

To assess the importance of the route of initial infection on the overall protective index to reinfection, we infected mice intraveneously by the tail and started the treatment after 4 days. After 10 weeks of incubation, the limiting dilution assay for enumerating the parasitic burden in lymph node showed no parasites on treated mice. These mice were then chalienged su bcu taneousl y on the footpad with virulent L. major wild-type yielding similar level of immunity with mice subcutaneously infected (Fig. 9A and B).

Most of the previous studies have reported that i n i t i a l i.v. infection with Leishmania parasi tes was crucial in m o u n t i ng siiçficirnt immune response against

- re-infection. Howevei, our data on l ive vaccination support that complete eradication of initial infection either be i.v. or s.c. inoculation have no apparent relevance, since they both generated simi lar level of immuni ty.

Table 1. Pharmacokinetic parameters of intraperiteonal (i-p.) and subcutaneous (s.c.) administration of ganciclovir in BALBlc mice. Phmacolcinetic parameters in B ALBlc mice d e r i-p. and S.C. administration of 5 mgkg of ganciclovir. Each of these parameters was compared to the two groups using the nonparametric Mann-WhÎtney rank sum test. Abbreviations: Cmax., maximum drug concentration; Tmax, time taken to reach Cmax; t1/2. elirnination plasma half-Me; Kel, elimuiation rate constant; AUC0-a area under the semm concenfration- time curve from zero to infhity; Cl, total body clearance; MRT, mean residence time. Values are means t SEM for five animals at c .05.

Weight ratio Dnig concentration @g/mI) infected/uninfected infected pad uninfected pad infecteduninfected

Table 2. Ganciclovir distribution in footpads. A single dose of radiolabeled ganciclovir (5 mglkg) was i ntrnperi toneal l y (ip) admi nistered to- femaie BALB/c mice. At 120 min., animals were sacrified and footpads were removed, washed in PBS and weighed. The tissue was then treated with Beckman Tissue solubilizer and decolorized in H,O,. Drug IeveIs i n al1 samples were monitored by scintillation counting. Data represent the mean of five mice SEM, P<0.05 compared to uninfected footpad using non-parametric Mann-Whitney test.

Table 3. Hematological effects of ganciclovir on BALB/c mice. Effect of increasing doses of ganciclovir on various hematologic parameters on mice. Three groups of five mice each, were treated respectively with 1 , 2.5, 5, mgkg of ganciclovir for 7 days. The whole blood sample was collected from each mite for measurement of red blood ce11 count (RBC), a n important hematologic parameter for toxicity studies of ganciclovir on animal model. Furthermore, hemoglobin (Hb), platelets, hematocrit (Ht) were also measured.

Acyclovir (ACV)

Viral f h ymidine

Kinase -

ACV-MP

ceci Enzymes xk -cc

O P-PP-0 i "'" " J

- I ACV-TP

dATP .Viral ONA dGTP Potymerase

Replicating Viral - DNA

Fig. 1. Mechanisrn of nucleotide analogues sucti as acyclovir and ganciclovir action. Ganciclovif is phosphorylnted within virus-infected cells to form an active nucleotide, ganciclovir 5'-monophosphate. After its formation, gancic1o;rir 5'-monophosphate is fiirther phosphoryliited by cellular kinases to di- and triphosphate forms. The rictivity of ganciclovir against humam cytornegaloviriis results primarily from i n h i bition of vil-al DN A synthesis by ganciclovir triphosphate by competitively inliibiting the incorporation of dGTP into DNA and thiis impeding and prematurely termiriating its elongation. The levels of tliis active nucleotide i n infected cells are lower than ganciclovir triphosphosphate levels, suggesting thnt the initial pliosphorylation of ganciclovir is the rate lirniting'step. Early research suggested tlint an induced host-encoded deoxyguanosine kinase facilitated t h e prodi~ction of ganciclovir monophosphate in cytomegalovinis-infected cells. However, more recent evidence suggests that the UL97 open reading frame of human cytomegalovirus encodes a protein capable of regulating the phosphorylation of ganciclovir. -

Y92AG -\

YgzAG - r

neo tk - 92 bp

Fig. 2. Construction of Leislimanin vcctors expressing the HSV-TK gene. The plasrnid pneotk-l is n pSP72 (Promega) bacterial vector in whicli the rzeo and TK genes were tnndemly cloned as decribed i n Materials and niethods. The cross-hatch box corresponds to a 92 bp synihetic polypyridine strech wi th an AG splice si tes. A 280 bp fragment derived froin the tetracycline I-esistance gene of pBR322 (Watsbn 1988) represented by the blrick box in pneotk-2 and the a- tubulin intergenic region of L. rnrieirii in vector pneotk-3 were inserted between the TK and nen genes. Arows indicate the orientation of transcription of the genes present in the expression vectors.

Ganciclovir (PM)

Fig. 3. Cytotoxic effect of ganciclovir on L e i s h m a n i a spp. promastigotes expressing the HSV-1 T K gene. Promastigotes of L. mujor, L. donovani and L. nzexicurta transfected with pneotk-2, pneotk-3 and pSPYneo as a control were gmwn in the presence of various concentrations of ganciclovir. After 72 hours of incubation, the survival of parasites was obtained by measuring the absorbance at 600 nm. AL. donovuni-pneotk-3; A L.donovani pSPYneo; L. mexicana-pneotk-2; O L. mexicanu-pSPY neo; . L. major-pneo tk-2; O L. major-pSPYneo. Curve represents four independant experiments.

a-tu bulin

Fig. 4. TK expression in Leishmuniu major promastigotes and amastigotes. A) Northern blot of total RNAs isolated from the promastigote and amastigote stages of L. major-pSPY neo and L. rizcljor-pneotk-2 and hybridized to a TK specific probe. Each track contains 10 pg of total RNA. Lanes 1 . L. mujor- pSPYneo, 2, L. major-pneotk-2 from promastigotes and 3, L. majur-pneotk-2 frorn arnastigotes. B) The same blot was stripped off and rehybridized with the T. brucei a-tubulin probe to monitor the amount of RNA layered in each iane.

Q I O Z 4 4 1 7 2

Timc (bours)

0 1 e

* 2 4 4 1 7 2

17mc (hours)

Fig. 5. Cytotoxic effect of ganciclovir o n Leish rnania major amastigotes expressing the HSV-1 TK gene. L. mujor expressing the HSV- 1 TK gene were harvested fro stationary phase and counted with the Neubauer improved counting chamber. Murine or humnn macrophages ( 5 ~ 1 o4 celllwell) were incubated with L. major parasites (20: 1, parasites-to-cells ratio) for 6 hours. After this initial incubation, free parasites were washed and fresh media containing or not 40 pM ganciclovir (GCV) was added and cells were further incubated for an extra 24-48 hours. At fixed time points cell cultures were dried and stained with Diff Quick i n order to determine the level of infection. A) Infection with human monocytes di fferentiated i nto macrophages and B) 1 nfection with J774 murine macrophages cell line. The Ieft panels i n A and B correspond to the total number of amstigotes within 100 macrophage cells and the right panels to the percentage-f infected macrophages with corresponding time. . L. rnujor- pSPYneo without GCV; O L. major-pneotk-3 without GCV; L. mujor- pSPYneo in presence of 40 pM of GCV; IJ L. rnu~or-pneotk-3 in presence of 40 p M GCV; A L. rnujor-pneotk-3 with 2x40 FM of GCV. The average and standard deviation of four independen t experiments are shown.

t lluman macrophages

Q Murine macrophages J774

Fig. 6. MTT test to evaluate ganciclovir toxicity to macrophages. Murine or human macrophages (200 pl/well) were incubated into wells of ce11 culture, two wells were pretreated with 40 y M and 80 y M of GCV, respectively and the last untreated well was used as control. After 72 hours of incubation. 10 pl of M~(3-[4,5-dimethylthiazol-2-yl]-5-dipheny1tetrzoium bromide; thiazol bleu-5mg/mI PBS) was added to the wells and fiirther incubated for 4 hours. Following incubation, the medium was removed and the convrrted dye was solubilized with 150 p1 of acidic isopropanol. Absorbnnce of converted dye due to the activity of active rnitochondrial dehydrogenase in living cells was measured at 570 nm.

T

-0- i.p. administration

+ SC. administration

50 100

Time after injection (min)

Fig. 7. Concentration-time curves of [3H]-ganciclovir in mice serum. Following the intraperitoneal (0) and subciitaneous (+) administration of a 5 mgkg ganciclovir dose. Values represent the menn ( SEM) obtained from five animals per group per time point. In order to avoid presence of metabolites. final time point was reached at 120 minutes.

Weeks post infection

Fig. 8. Ganciclovir treatment of infected mice. lnability of TK expressing L. major to initiate infection into BALBIc following ganciclovir treatrnent. 5x10" promastigotes of T K expressing and wild-type L. mcijor- were injected i n rnice footpad. Treatrnent with 7.5 rng/kg of ganciclovir and placebo (PBS) for 14 days commenced after 1 , 4, 14 days post infection on a11 rnice. O L. nrajor Wt (PBS), O L. major Wt (GCV), A L. mu~or-pneo-tk (PBS), A L. nru~or-pneo-tk (GCV) I day, L. major-pneo-tk (GCV) 4 days, L. iimjor-pneo-tk (GCV) 14 days post infection. Infection was measured by the thickness of footpad.

8 - OC

- a u -

-

O e 4 6 8 10 12

Time (Wks)

Fig. 9. A. Challenge of cured mice witli L. major wi ld- type . Lesion development of five mice after challenged su bcu taneously wi th L. major wild- type following subcutaneous (s.c.) inoculation to a non immunized mice used as control, A L. major-pneo-tk with 14 days post infection GCV treatment and . 4 days post infection GCV treatment. A intraveneous (i.v.) inoculation of mice with L. major-pneo-2 with 4 days post infection GCV treatment challenged with L. major wild-type. In both experiments intravenous ( i-v.) and subcutaneous (s.c.) induced same protective response. Al1 results are representative of three experiments. -

\

Fig 9. B. Challenge of cured mice witli L. major wiId-type. Actual picnire showing mice initially S.C. infected and untreated (A) and challenged with wild-type L. major and (B) treated and ciired mice chollenged. The later has rnounted a significant immune response. -

CONCLUSION

CHAPTER V

My thesis work axed principally on the generation of a suicide vector system based on the expression of HSV- 1 TK gene byLrishnzania where subsequent concentration of a nucleoside analogue such as ganciclovir inhibits the intracellular survival of arnastigotes within host macrophages. We were able using this system, to demonstrate its practical usefulness in an experimental live vaccine development. Moreover, during the course of the studies some novel observations were made.

For example, the episomal polycistronic expression in Leishmania does not required only leishmanial sequences since to yield the vector pneotk-2 a bacterial sequence of approximately 280 bp denving from the tetracycline gene of pBR322 has been used. This sequence was inserted downstream the neo gene and upstream the putative T K splice acceptor site. pneotk-3 transfectants, where a specific Leishrnania sequence corresponding to the 800 bp intrrgenic region of the a- tubilin gene was included, ensuring TK pre- messenger maturation, showed similar sensitivity to varying ganciclovir concentration.

In addition, this system gonfirnls earlier observation that transfection does not altered the virulence or the infectivity of a strain and that episomal expression vectors are maintained stably inside the mice macrophages without drug selection pressure. Indeed, L. major pneotk-3 pnssed several times in animal, retained its ability to infect and to be susceptible to ganciclovii- upon expression of its episomal TK.

However, the integration of T K i n the genome of Lrishrnaniu will be more preferable in order to avoid, in time, any possible loss of the expression vector due to lack of selection pressure. Nevertheless, this system provides a window for understanding the host immune response nt specific stages during infection, since we can eliminate parasites at convenient t h e and elucidate at thnt particular tirne the state of the immune system. The system, however, has its shortcornings, i t is

for example not suitable for controlling infection in humans since the processus of infection and treatrnent will naturally present some ethical questions, however, the system can be transfered to animals that are the liurnan reservoir of the parasite such as dogs. Their immunization can contribute significantly to the control of infection in humans.

Meanwhile, this system can be enhanced by the addition of some modulators of the host immune response following Lrishmuniu infection.Cytokine~ such as IL- 10, INF-y are responsible for the expansion of Th I that modulate the host system for the regression of Lrishmania infection. Transfectants that will comprise TWIL-2 or GM CSF (Granulocyte Macrophage Colony Stimulating Factor) with multipotential hernatopoietic function, involved i n monocyte proliferation, mobilization and tissue influx will be an ideal combination for suicide gene system. Yet other techniques such as inactivation of some essential Leishrnaniu genes by gene targeting could proved to be an additional element to the TK suicide gene system.

Dismption of the P-glycoprotein p g p A gene i n Lrishrnania causes an hypersensitivity to low pH suggesting that this m u t a n t will have a low rate of survival in the acidic environment of the host n~acroplirigr. The single allerè inactivation of the trypanothione reductase (TR) gene induces parasite elimination more rapidly from macrophages.

Attempts were made to integrate TK into the Lrivhmuniti genorne without success, but other negative seleitable markers can be used insread of TK. Another unrelated gene to TK, the U937 gene product of cytomegalovirus is also capable to confer sensitivity to ganciclovir and could be used i n conjunction with TK. Another potential dominant negative marker is the cytosine deaminase gene (CD) This gene is present i n prokaryotes and fiingi biit nor i n mainmalian cells. I t

dearninates cytokine to uracil and confers n non toxic drug 5-fl uorocytosine i nto highly toxic metabolite 5-fluo-uracil (FU). Attempts to integrate this gene can be also made.

Our studies have clearly demonstrated a readily and feasible system of live vaccine, opening up more insighfiil questions on the mechanism of the immune system following Lrishrncinict infection. More studirs wi I l be therefore required in order to understand this disease that infects millions of people around the world.

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Lexicon

AUG : The rnost comrnonly used translation initiation CoPpn that also acts as a codon for methionine within mRNA coding sequences

Epitope: Antigenic determinant. The structure on an antigen rnolecule that interact with the combining site of an antibody or T cell receptor as a result of molecular complementary. Protein epitopes recognized by iintibodies may be continuous or discontinuous depending on whether the amino acide residues forming the epi topes are in continuous peptidé linkage or are i n spatial proxirnity to

each other as a consequence of the tertiary or quaternary structure of the molecule. These epitopes recognized by T ce11 receptors are peptides generated by enzymatic degradation of the protein molecule and presented on the ce11 surface in association with class 1 or I I MHC molecules.

Gene rearrangement: FunctionaI- Pirected rearrangement of DNA

occurs in somatic cells of eukaryotes i n a srnall number of cases. Directed gene rearrangernent is involvrd i n the assembly of functional immiinoglobulin and T cell receptor genes in vertebrates, the mating-type switch i n yeast and antigenic variation in trypanosomes. Directed gei~omic rearrangements are also known in proknryotes

Gene therapy: is the term used to described the correction of a disease

by genetic manipulation. It is still at an eni-ly stage of development although a great deal cf work has been done on the transfer of genes to experimentnl animals

Giemsa banding: The pattern of dark and light bands seen on

eukaryotic metaphase chromosomes su bjected to treatment \-

with Giemsa ~tain. The pattern is characteristic for each

chromosome and can be used diagnostically for identification

Glycans: This general term includes polymers composed wholly or

largely of siigars and tkeir simple derivatives (e.g amino sugars and sugar alcohols) and the carbohydrate parts of other types of molecules to which siigars are covolently li nked, sticli as monosaccharides, O ligosaccharides or polysaccharides, to form gl ycoconj iigntes. Tlie l i n kage of sugar is normally as glycoside and the 1-einainder of ihe molecule to which the sugar is linked is the aglycone. Glycan is found at the ce11 surface in association with bot11 the membrane and the ce11 wall of bacterin, cyanobacteria and related pro karyotes

Heat shock: Cells have different stress responses. Eacli is triggered by one or more kinds of environmental insiilt or stress, the main consequence of i ts induction bei ng increrisetl siress tolerance levels. Tlie lieat shock reponse is one of the hest characterized of these stress responses. I n 1963 Ritossa reported that exposiire to Iiigli bii t i:oiiletlinl teiilpci-ntui-es and to certain chemicnls causes the appcni-ance of new piiffs on the salivary gland polytene cl.iromosoiiies 01' the St-iii t

Drosophilti bitsckii.The puffs, clearl y visible i II [lie liglit microscope, represent high triinscriptional octivity on lieat shock-inducible genes. More recently a wide range of organisms. from bacteria ro the higlier vertebrnres, lins been shown to display sirnilar drainatic changes in heat shock gene expression

Hybridonia: Clone of cells produced by the tiision of a somatic cell witli

a ccll l ine tliat Iias the capaciiy to siirvive niid prolifei-ntcjq vit[-o. Hybridomas derived froiri the fiisioii of an nritibody- producing B ceil Sroni n specilically iiiiiiiiinizcd iiiiiinal witli ii

plasmacytoiiin ce11 line. Tliey were origiiinlly developcd foi- the production of inonoclonal antibodies of deli ncd specifici ty.

Ison~cr: One of 2 or more chernical compoiinds e;;rli having the same

kind and number of atoms biit differing in arraiigeiiient of the atoms and in pliysical and clleiniciil propcrties

Lysosomes: #

are intracellular organelles wi th ail acid i iitei-ioi-(-pH 5) bounded by a single membrane and contai i i i ng niariy Iiyclrol ytic enzymes whicli are optirnally active ni ni1 ncitlic pH. Tliey are present in alniost d l ariiinal cells, and i r i piaiit cells, the vaciiole is the q u i valent orgaiicl lc. Lysasoii~cs were i ni rial l y identified niid cliaractcrizcd diii-i iig ilic developrnerit of subcelliilar Praciionaiioii tcchiiic~iies by de Drive arid CO-workers i n the I9SOs. 'The Simction of iysosoriies i s the ino-acelliilai- digestiori of al 1 iiini i i c1;ir;scs of biological niacrornolcciiles. Material faor dey 1-;idritioii I il lysosornes caii be delivered vin eidocyiosis. Lysosoiiii~l eiizymes and riiernbrriiic proteiiis are syii~liesizetl oii niebrarie-boiiiid polysomes aiid dclivci-ed to 1ysosc)iiics nlicr passage ~Iirougli the endoplnsniic irticiiliiiii and ilic Golgi complex.

Transfection: nie genetic modificatioii of cil Itiii-cd niiiinnl tells by ilic

iiptake of DNA froni the cultiii+e iiiecliwii. Tlic I N A 10 bc trnnsfected is iisunlly in the form of ic~coiiibiiiniit pliisriiids or otlier types of D N A vectors coniai iiiiig tIic gcncs 01-

interest. Ti-aiisfection is analogoiis io iIic pi-ocess of

transformation O whereby bacterial cells c m tnke iip exogenously added DNA. Tranfection of DNA into plants cells may also be achieved by first removing the ce11 wall to produce a protoplast. Genes can be introduced in to cells by transfection either transiently or stably. In transient gene- transfert, the expression of the recombinant gene product can be detected within the animal ce11 fi-orn 12 hours up to 80 hours post-transfection, and generally the introduced DNA does not become integrated into the ce1 1's chromosomes. In other cases the DNA becomes perrnanently integrated into the cellas chromosomes to form stably transfected ce11 lines. As the spontaneous entry of DNA into the animal ce11 and its subsequent expression in the nucleus is very inefficient, several methods have been developed to facilitate the process, including the use of polycations siich as DEAE-dextran. calcium phosphate coprecipi tation, electroporation, lipofection, retrovirus vectors and microinjection

Transgenic mice: Numerous strains of laboratory mice carrying an

introduced transgene, or in which an endogenous gene has been specifically inactivateci-They are of use in studies of the regulation of gene expression, gene i-eari-angement i n irnmunoglobulin genes and T ceIl receptor genes, oncogenesis, and in the study of developrnent

Appendix 1

Geographical distribution i f leislirnaniasis. (AFIP photograph neg. no. 68- 1805-2)

Appendix I I œ

Oomatic amimat

trammission cycle 4

Transmission cycles of Leislrrnania. Animal reservoir hosts include cats, dogs, jackals, rodents, hyraxes, wolves, and racoons. ( Adapted from Modabber, F., Tropical Disease Rescarch, u ~ l o h a l Pczrtner.ship, TDR, Maurice, J. and Pearce, A. M., Eds., World Health Organisation, Geneva. 1987. 99).

Appendix 111

- Life-cycle of Leishmania. OnIy the femnle sandfly is able to transmit the parasite when it bites an infected animal or person. It injects the infective stages of the parasites, flagellated promastigotes ( 1 ) with its saliva. The promastigotes change rapidly into rounded, aflagellated form, amnsstigotes (2) which are taken up into scavenging phagocytes of the host. Insidr the rnacrophages(3) the parasites rnultiply until the host ce11 is filled ~ i p and lysed (4). The released amastigotes infect new macrophages ( 5 ) of the skin or deep organs such as spleen, liver, and lymph nodes. Finally, some macrophage containing amastigotes (6) are taken by another fernale sandfly when taking ablood meal. The parasites change back into the promastigote form once they enter the stomach of the fly (7). These move forward and mach themselves to the front end of the esophagus where they rnultiply. Finally, about 1 week after entering the sandfly. the promastigotes move into the pharyn and proboscis from where they are ready to be infected into a new animal or man (8).

\

Appendix I V L

A. Viscerai leishmaniasis (from WHO technical report, 1990).

B. Old world cutaneous leishmaniasis (from W H O technical report, - 1990).

- LL. (k.) tmpicu Homme Caicn. rat üiartr nrtma sas De l'Inde m Muoc rigicms wngry 1903 vcha

Fu- cuunécs ~hio~ i : . Kcnm YCmca diffaCs (ré@m d par par sa) Ukims ùumidcs h i c ccnmlç Moyen Chicm.

Afrique du Nord. S a M

Appendix I V (next) 0

C . Mucocutaneous leishrnaniasis (from WHO technical report, 1990).

-.

PARASITE R&ER- *$EX MANIFES~ATIONS . PEPART~T~ON VOIR CLINIQUES G~!OCRAPHIQUE

Appendix V

S E P 81: (LYMPHOKINE WWCTIOH) STEP a: (UACROPHAGE ACTIVATION]

I

0 Parr- @.- . .% .4 -. w- . @? @..--

Mechanisms by which macrophages become activated to kill parasites. Parasites interact with accessory cells and induce a series of monokines (IL- 12. TNF-a, IL-1) that promote the differentiation and triggering of Th1 CD4', C D ~ ' lymphocytes, and NK cells, which, i n turn, secrete the lymphokines IFN-y and TNF-a necessary for macrophage activation. The end resul t of macrophage activation in the mouse and other species is the production of toxic metabolites. Of these, nitrogen oxides (NO) appear ro be primarily responsible for the killing. Parasites that in their interaction with nccessory cells stimulate high levels of IL- 12 synthesis uregulnte this pathway of cell-medinted immunity. In contrat, other parasites stimulate the production of cytokines (IL-4, IL- 1 O) that downregulate the process, inhibiting ei ther lymphokines production and /or the abili ty of lymphokines to activate macrophages.

Appendix

Activity Name Major ccllular sources Pnnciptc mcchmisms of action

Upregulatory IFN-y Th 1. CD8' T cells. NK cclls

Th 1. CD8' T cclls. B ceIls(?)

T cclls. macrophap

Macrophages. 8 cclls. ncutrophils

Th2 cclls. m m cclls. basophils

Th2 cclls. B cclls. macrophagcs. mast cclts

Many cc11 types

Th2 cclls. othcrs(?)

Acrivaics effccfor cc11 microbicidal functions; limits prolifcration of Th2 cclk

Tel1 growh factor

Costimulus for rnacrophagc and NK ce11 activation

Siimulaccs diffcrentiation of Th 1 and CD8' celis. NK cc11 I M - y production

Promotcs Th2 diffcrentiation; inhibits rnacrophagc activation

Inhibits 1 FN-y production frorn T and NK cclls; bIocks monokioc synthesis. maaophagc effector function

Inhibits lymphocyte proliferorion. monokine synthuis. macrophage and NK ce11 tffcctor function

Inhibits monokine synrhesis. aaivation of bone marrow-derivcd

macrophages

Cytokines tha t regulate cell-mediated immunity a

Appendix VI1

L. CS7 more T h l - l i e Th 1

Autocrine T Ccll Differrnccs Under / Neutra1 Primlng Condidons

BALg mare Tht-ï~kc

Swah-spedfic T CelI Oifferences Ourfng Primfng by II-4 Plus 11-1 2

Theories for Thl/Th2 divergence in experimental L. major infection. A . Distinct antigenic peptides underlie stimiilation for divergents subsets. B. Abnormal cytokine or cofactor production by accessory cells in the innate immune system underlies divergent responses. C. Cytokines produced by naive CD4' T cells dunng priming do influence development in an autocrine fashioii. D. Cooperative effects of cytokines produced by Th0 cells and an additional CD4' population together wi th IL- 1 2 from non-CD4' sources provoke divergent T ce11 responses.

CBA

Inirnunogcns (L. major) Rautc

Sonicated pmnia-tigvics S.C. Wcakly pn~tcciivc Excrcrary l ac io r -~n i i kdy corn- i-p. Wezkly. vxriiblc potcc-

plct i.m. [ive

MK- i rnd ia icd arn;~~l ig i fcs S.C. ~ru t& t i vc xgainst L. rra.

nic.r;cwnrc but nit( L. nw-

Susceptible strains

OALBfc. BAL8.b. 150K-imdiïtcd. kat-k i l lcd nr I.V.

BAL8.k. SWR sonicatcd pn>tiisi>rigola i . p. Subsrmtinl prtircçtioii HALHIc Frcctrc-t hawcd inrcctcd cilacru- I.V.

phage + CC. prvum 1.p. Signikant prutcction

BALD/c Nonpaihogcnic cloncslmu~ants i - v .

'.p. Sub>t;i<iiid proccctivn BAU3.k. UALBIÇ Glyculipid + C. purvum/r€~ i.p. Substantial protcc~ion BALBIc Soluble. mcmbrrne-frrc pcurnas- i.p. Substantial prorcciion

tigotc cxtract + C. punwm

Nort: K A = Fmnd's coniplctc adjuvant; i-m. = iritcamuscular. i.p. = intrapcritancal: i.v. = intravcnaus; and s.c, = subcurancous.

Expcrinientnl vaccinntioii ngaiiist ctitaiitoiis Icisliiiiariiasis (L. major) in niice.

Appendix IX 0

Endogenous nudeasides

Thymidine

Chernical struc'iures of naturally occuring nucleoside analogues. 2'- deoxythyrnidine and 2'-deoxyguanosine, and the system antiviral nucleoside analogues zidovudine, stavudine, acyclovir, ganciclovir and ribarivin.

Appendix X 9

Summary of the in vitro activity of ganciclovir and other agents against human cytomegalovirus measured by plaque reduction assay

(updated from Faulds and Heel). -.

Rderence Vira( srtain Cerl culture Concentration achieving 50% inhibition ( J J ~ ~ R P GCV PFA ACV

Andrei et aiP1 A0169 MRC-5 1-92 23.2 120

HEL 2.8 63.2 64

Clinid tsdares MRC-5 1.92 61.6 51.6 (n = 17)

HEL 232 81.6 76.4

Binxi et al.W A0169 HFF 1 7 90

Cde & Balfouri4 A0169 HFF 3.1 60.5 M e et al.Isl AD 1 69 MRC-5 4 5

Oavis 4.3 Konno et J.m Clinicai isaiates NU 0.73

(n = 52)

Rush & Miltslsl A0169 HFF 0.43 100

%@?ta et al.PI A0169 MRC-5 3.1 25 - NS-15 3.1 25

NS-84 3.1 25 Smee et aldW AD r 69 MRC3 7 95

Sroedr et ai.ttal AD169 HEL 273 Davis 1.95

Taykr et al.('n Kerr HEF 0.6

Tdrnan et al.lr41 AD 169 MRC-5 3 1 O

a Resuits reporcied in mgR were corrverred ro pmoUL. Results were reported as the drug concentratiori inh~inting viral plaques. DNA synthesis. amigen expresson or virus-indu& cylogarhogeniaty by 50%.

AbkRiiaiians: ACV = aadov;r. GCV = ganOdovir; HEL = human ernbtyo lung f iWasts; HEF = human embryo rtbrcblasts; HFF = huf foreskio fiorob(asts; MRGS = human embryo lung œüs. NR = not reporte& PÇA = foscarnet

Appendix XI 9

pSP72 piasmid vector map, promoter and multiple cloning site sequences. (From protocols and applications guide Promega).

\

pSP72 plasmid (2462bp)

Figure 37. pSP72 vecfar circle map.

SP6 Transcription Start

r--+

-

xho I p, Il Hmd III Sph I Pst 1 Sa! 1 Acc l xba I BahH 1 Smal Kpn 1 Sac i EcoR I C& 1 EcoR V Bgl il -

S.. ;AT-~A GGTGA CACTA TÀGM CTCGA GCAGC TGAAG C ~ G C ATGCC TGCAG SP6 Promoter uuuuu

Xho I PvuiI HindIII SphI Pst 1

GTCGA CTCTA GAGGA TCCCC GGGTA CCGAG CTCGA AnCA TCGAT U U I I

Sa1 1 Xba 1 BamH I Sma I ' Kpn I I u u U Sac 1 EcoR 1 Cla 1

T7 Transcription Start

Y GATAT CAGAT CTGCC GGTCT CCCTA TAGTG AGTCG TATTA . . -3' UU ~7 Prornoter EcoRV BgliI

Appendix XII (A) O

Linear map of Herpes simplex virus type l.(strain SC16) gene for thymidine kinase. Length: 113 1 bp

TQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLI

WQRPGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQS

NAGPRPHIGDTLFTLFEiAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPA GCRDALLQLTSGMIQTHVTTPGSIPTICDLARTFAREMGEAN"

misc - f e a t u r e 151. ,189 / n o t e = " p u t . ATP b i n d i n g s i t e "

m i s c - f e a t u r e 502 . , 5 2 8 / n o t e = " p o t . n u c l e o s i d e - b i n d i n g site"

m u t a t i o n 502 . -504 /note="GCC ( A h ) is ACC * ( ~ h r ) i n B3 mutant"

m u t a t i o n 526. -528 /note="CGA (Arg) i s CAA (Glu) i n T r 7 m u t a n t "

mutation 1006 , , 1 0 0 8 /note="TGC ( C y s ) is TAC (Tyr) i n S1 mutant"

BASE COüNT 197 a 398 c 342 g 194 t O R I G I N

X03764 tength: 1131 August 29, 1996 16:32 Type: N Check: 6060

1 ATGGCTTCGT ACCCCGGCCA TCAGCACGCG TCTGCGTTCG ACCAGGCTGC

51 GCGTTCTCGC GGCCATAGCA ACCGACGTAC GGCGTTGCGC CCTCGCCGGC -.

101 AGCAAGAAGC CACGGAAGTC CGCCCGGAGC AGAARATGCC CACGCTACTG

Appendix XII (B) 9

Sequence of Herpes simplex virus type I(strain SC16) gene for thymidine kinase. Length: 1131

CGGGTTTATA TAGACGGTCC CCACGGGATG GGGAAAACCA CCACCACGCA

ACTGCTGGTG GCCCTGGGTT CGCGCGACGA TATCGTCTAC GTACCCGAGC

CGATGACTTA CTGGCGGGTG CTGGGGGCTT CCGAGACAAT CGCGILACATC

TACACCACAC AACACCGCCT CGACCAGGGT GAGATATCGG CCGGGGACGC

GGCGGTGGTA AXACAAGCG CCCAGATAAC AATGGGCATG CCTTATGCCG

TGACCGACGC CGTTCTGGCT CCTCATATCG GGGGGGAGGC TGGGAGCTCA

CATGCCCCGC CCCCGGCCCT CACCCTCATC TTCGACCGCC ATCCCATCGC

CGCCCTCCTG TGCTACCCGG CCGCGCGATA CCTTATGGGC AGCATGACCC

CCCAGGCCGT GCTGGCGTTC GTGGCCCTCA TCCCGCCGAC CTTGCCCGGC

ACAAACATCG TGTTGGGGGC CCTTCCGGAG GACAGACACA TCGACCGCCT

GGCCAAACGC CAGCGCCCCG GCGAGCGGCT TGACCTGGCT ATGCTGGCCG

GGCGGCGGGT CGTGGCGGGA GGATTGGGGA CAGCTTTCGG GGACGGCCGT f

GCCGCCCCAG GGTGCCGAGC CCCAGAGCAA CGCGGGCCCA CGACCCCATA

TCGGGGACAC GTTATTTACC CTGTTTCGGG CCCCCGAGTT GCTGGCCCCC

AACGGCGACC TGTACAACGT GTTTGCCTGG GCCTTGGACG TCTTGGCCAA

ACGCCTCCGT CCCATGCACG TCTTTATCCT GGATTACGAC CAATCGCCCG

CCGGCTGCCG GGACGCCCTG CTGCAACTTA CCTCCGGGAT GATCCAGACC

CACGTCACCA CCCCAGGCTC CATACCGACG ATCTGCGACC TGGCGCGCAC

GTTTGCCCGG GAGATGGGGG AGGCTAACTG A

Appendix X l I I O

SDM-79 (Brun & Schonengerger, Acta. Trop., 36, modified by S teiger) k

- 7.0 g de poudre "Dulbecco's mai5cd Eagle" (Gibco 430-1600) Per midium liter:

- 2.0 g de poudre " d e u 199" (Gibco E-12)

- 8.0 ml de solution acides aminés MEM (Gibco-113)

- 6.0 ml de solution acides aminés non-essrntiels MEM (Gibc-1 14)

- 1.0 ,r de glucose (Anhydride)

- 8.0 g a'ziepes (Sigma H 3375)

- 5.0 g de MOPS (Sigma M 1254)

- 2.0 g M C 0 3

- 0.2 g L-Nanine (Merck 1007)

- 0.1 g GArginine HCL (Sigma A 5 13 1)

- 0.07 g L-Methionine (Sigma M 9625) - 0.08 g L-Phenylalanine (Sigma P 2126)

- 0.6 g L-Rohe (Sigma P 0380)

- 0.06 g L - S e ~ e (Sigma S 4500)

- 0.16 g Taurine (Sigma T 0625)

- 0.35 g L-Thréonine (Sigma T 8625)

- O. 1 g L-Tyrosine (Sigma T 3754)

- 10 mg Adénosine (Sigma A 925 1)

- 10 mg Guanosine (Sigma G 6752)

- 50 mg D(t)Glucosamine HCL (Sigma G 4875) - 4 mg Acide Folique (Sigma F 7876) - 2 mg Acide p-Aminobenmique (Sigma A 0254)

- 0.2 mg Biotine (Merck 245 14)

Dissolve in 1/4 of final volume, add solutions of nmino acids MEM and antibiotics (Ampicilline 50 mg/ml, Kanamycine 100 mg/ml). Adjust the pH at 7.3 with NaOH. Then complete the final volume and sterilize by tiltration. For a complete medium add 2 ml/L of hemine (2.5 mglml i n 50niM NaOH) and 5-10 % v/v of Fetal bovine semm inactivated at 56 OC for 30 minutes.

I MAI; t LVALUATION TEST TARGET (QA-3)

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