INFECliON AND IMMUNITY, May 1994, p. 1733-1741 Vol. 62, No. 50019-9567/94/$04.00+0Copyright ©D 1994, American Society for Microbiology

Cell-Mediated Responses of Immunized Vervet Monkeys toDefined Leishmania T-Cell Epitopes

ALLISON J. CURRY,1t ARMANDO JARDIM,' J. 0. OLOBO,2 AND ROBERT W. OLAFSON*University of Victoria, Victoria, British Columbia, Canada V8W 3P6,' and Institute of

Primate Research, Karen, Nairobi, Kenya2

Received 15 October 1993/Returned for modification 17 November 1993/Accepted 2 February 1994

A population of vervet monkeys was immunized with killed parasites and infected with Leishmania majorpromastigotes either by needle or by infected-fly bite. The responses of recovered monkeys to mitogens, killedparasites, and molecularly defined T-cell epitopes were then compared with those of control animals.Peripheral blood mononuclear cells (PBMC) from both naive and recovered animals proliferated strongly inresponse to both B- and T-cell mitogens, although the responses of the recovered animals were less strong thanthose of the naive animals. Cells from recovered vervets, but not those from naive vervets, also proliferated inresponse to parasite antigens and synthetic T-cell epitopes. Likewise, cells from recovered animals releasedgamma interferon and either interleukin 2 (IL-2) or IL-4 into culture media in response to both of theabove-mentioned antigens, whereas cells from control animals did not. The fact that no IL-5 could be measuredfollowing parasite antigen or synthetic T-cell epitope stimulation of PBMC suggested that cells proliferatingin response to these molecules belonged to the Thl subset. Phenotypic analysis of the PBMC showed a markedincrease in T-cell but not B-cell populations in recovered animals. Among this population was an increasednumber of CD45RO0 memory cells. The data from this study are in keeping with the earlier finding that vervetmonkeys provide an excellent model system for leishmaniasis. Further, these data support the contention thatsynthetic T-cell epitopes are prime candidates for molecularly defined Leishmania vaccines.

Leishmaniasis, in both its cutaneous and visceral forms, is adisease of significant socioeconomic importance for whichthere is still no available vaccine. Research using human cellsand both murine and hamster models has shown that resolu-tion of infection is dependent upon a complex set of eventsassociated with cell-mediated immunity. This includes T-cellresponses to parasite antigens (3, 4, 20, 21, 27, 28, 46, 50) andthe production of cytokines such as gamma interferon (IFN-y),interleukin 2 (IL-2), and tumor necrosis factor (4, 28, 50),resulting in macrophage activation and subsequent intracellu-lar parasite destruction. The specific involvement of certainT-cell subsets in disease resolution or exacerbation has alsoreceived considerable attention, particularly with respect to therelative importance of murine ThI and Th2 CD4+ populations(22, 45), the possible significance of CD8+ cells (49), and theability to produce T-cell memory populations (21, 22, 49).Although the division of the murine CD4+ population intoThl and Th2 cells, based upon differential cytokine profiles, isreasonably well understood (22), a comparable distinctionwithin the human system is less well defined. Differencesbetween CD4+ populations are now becoming more apparent,however, with discrimination of Thl- and Th2-type clonesbased upon differential IL-5 expression (51).The most intensively investigated Leishmania antigen is

gp63, a 63-kDa metalloprotease present on parasites in bothlife cycle stages (6, 12) and implicated in promastigote viru-lence (53, 54). Employed as a natural recombinant antigen orsynthetic peptide antigen, this glycoprotein has been shown toprovide immunoprotection against both New and Old World

* Corresponding author. Mailing address: University of Victoria,P.O. Box 3055, Victoria, B.C., Canada V8W 3P6. Phone: (604) 721-7072. Fax: (604) 721-8855. Electronic mail address: OLAFC@JUVVM.UVic.Ca.

t Present address: Department of Pathology, University of Cam-bridge, Cambridge, England CB2 lQP.

cutaneous disease in the mouse model (16, 40, 55) and tostimulate both murine and human IL-2 and IFN-y productionin vitro (16, 41, 55). Characterization of a selection of syntheticgp63 T-cell epitopes has allowed analysis of the potentialmechanisms underlying this protective immunity and has indi-cated that of the epitopes stimulating T-cell proliferation, onlycertain peptides could elicit protective immunity. Others eitherresulted in no apparent effect or exacerbated the disease (16,55). Similar findings were obtained in a recent collaborativestudy involving human subjects in which gp63 epitopes wererecognized by T cells from patients with various diseasemanifestations (41). In addition to a proliferative response, thecytokine profiles expressed by these cells resembled the pat-terns associated with immunoprotection in the murine model(16). It is clear, however, that neither animal protection datanor human lymphocyte proliferation data alone will be suffi-cient to initiate an ethically correct and economically justifiableclinical trial, particularly with respect to the visceral disease.Although outbred hamster populations have been used ininitial vaccine trials against Leishmania donovani infections(unpublished data), immunological evaluations have been se-verely constrained by a lack of immunoreagents. In addition,our studies with inbred mouse strains have demonstrated thatmajor histocompatibility complex (MHC) restriction plays acritical role in peptide recognition (16), adding further com-plexity to the problem of selection of candidate vaccinemolecules. Taken together with the relative phylogenetic dis-tance of rodents and humans, these difficulties have renderedthe mouse and hamster models less than satisfactory to supportour expanding investigations. The data presented in this com-munication support an earlier report that the African greenmonkey, or vervet (Cercopithecus aethiops), is a suitable pri-mate model for cutaneous leishmaniasis, mimicking humaninfection and overcoming many of the problems of rodentmodels (14). In the present report, we describe an investigationof the immunological responses of nonhuman primates to

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mitogens, parasite antigens, and selected synthetic T-cellepitopes and present an assessment of the ease of applicationof human immunoreagents and the general utility of lympho-cyte proliferation assays in screening experimental animals.

MATERIALS AND METHODS

Animals. Adult vervet monkeys were wild caught in Kenyaand quarantined for 3 months prior to use at the Institute forPrimate Research, Karen, Kenya. During that period they weremonitored by Institute veterinarians for simian immunodefi-ciency virus and Mycobacterium tuberculosis infections, alongwith evidence for both gastrointestinal and parasitic infections.Control animals were disease free by these criteria and showedno evidence of prior exposure to Leishmania antigens asmanifested by a negative delayed-type hypersensitivity (DTH)response to leishmanin skin tests (32). A negative response wasrecorded when the skin induration was less than 5 mm at both48 and 72 h following intradermal injection of 5 x 107formaldehyde-fixed promastigotes into a shaven thorax. DTH-negative animals, utilized as controls, were numbered 1through 13.

Experimental animals were immunized interdermally on thelateral aspect of the thorax on three occasions at 3-weekintervals with 5 x 108 to 10 x 108 merthiolate (1:10,000)-killed Leishmania major (NLB 144) organisms in combinationwith Mycobacterium bovis BCG (human equivalent dose). BCGwas a gift from the Statens Seruminstitut, Copenhagen, Den-mark. One month after the final vaccination the animals werechallenged by intradermal needle inoculation of 107 late-log-phase L. major (NLB 144) promastigotes in salivary glandlysate into the eyelid, the ear, and the nose (animals numbered15 16, 17, 18, and 21) or alternatively by infected-sandfly bite(15 flies, animals 19 and 20). This infection methodology hasbeen described previously (14, 18, 32). All animals developedcutaneous lesions which healed within 3 months. At 5 to 6months after the initial infection the vervets were challengedonce again by intradermal inoculation of promastigotes asdescribed above and found to possess protective immunity withno disease relapse occurring regardless of the prior immuni-zation methodology employed.

Isolation of PBMC. Vervets were anesthetized with ket-amine hydrochloride (10 mg/kg of body weight), and 15 ml ofblood was removed from the femoral vein by using heparinizedsyringes. The influence of stress and adverse anesthetic effectson experimental animals was minimized by limiting suchbleedings to 21-day intervals. Peripheral blood mononuclearcells (PBMC) were isolated by density gradient centrifugationusing Histopaque 1083 diluted 10-fold with RPMI 1640 (bothfrom Sigma Chemical Co., St. Louis, Mo.) to compensate forthe difference between human and vervet lymphocyte densi-ties.

Antigens and mitogens. L. donovani (LD3) and L. major(NLB 144) promastigotes were isolated from stationary-phasecultures grown in Dulbecco modified Eagle medium supple-mented with 10% fetal bovine serum (FBS) as previouslydescribed (18) and used either as live cells or following fixationin 3% formaldehyde. Synthetic L. major gp63 peptides weresynthesized by the Merrifield solid-phase methodology on anApplied Biosystems model 430A automated peptide synthe-sizer as previously described (16). Sequences of the peptidesused were as follows: PT3, YDQLVTRVVTHEMAHA; PT4,TRVVTHEMAHALGFSG; PT7, AARCIDGAFRPKATDG;and PT1 1, GFSGPFEDARIVANVP. These peptides repre-

sented gp63 residues 154 to 168, 158 to 173, 378 to 393, and 170to 186, respectively.Lymphocyte proliferation assays. Freshly isolated PBMC (3

x 106/ml) were cultured in 96-well tissue culture plates withHEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic ac-id)-buffered RPMI 1640 supplemented with streptomycin (100p.g/ml), penicillin (100 U/ml), glutamine (2 mM), 3-mercapto-ethanol (5 x 10-5 M), and 5% heat-inactivated FBS. For eachparasite experiment, three concentrations (1 x 106, 3.3 x 105,and 1 X 105 parasites per ml) of promastigotes were utilizedand optimum responses were reported. Optimum concentra-tions for mitogens were predetermined, and three differentdilutions clustered around the optimum were used (10, 3.3, and1.1 p.g of concanavalin A [ConA] per ml; 2.5, 0.8, and 0.3 pLg ofpokeweed mitogen [PWM] per ml; and 5, 1.7, and 0.6 jig ofphytohemagglutinin [PHA] per ml [Sigma]). Similarly, syn-thetic peptides dissolved in water and diluted in RPMI 1640were tested for optimum response over the range of 50, 16.6,and 2.5 1g/ml. Optimum concentrations were those giving thehighest level of incorporation of tritiated thymidine, and datafor suboptimum responses were not reported. Cultures wereincubated at 37°C in a 5% CO2 atmosphere for 48 h (mito-gens), 96 h (peptides), and 120 h (parasites). Each antigen ormitogen was assayed in triplicate for each of the three differentdilutions on cells from a minimum of two separate bleedings(21-day minimum intervals), and proliferation was detected byincorporation of tritiated thymidine. Results were expressed asthe mean counts per minute ± 1 standard deviation, based ontriplicate cultures.

Cytokine assays. Release of IFN--y in response to stimula-tion was measured by using cell culture supernatants in asandwich enzyme-linked immunosorbent assay (ELISA), pre-viously described for murine IFN--y (9). Murine anti-humanIFN--y (Serotec, Toronto, Ontario, Canada) (5 jig/ml) wasused as the capture antibody. Culture supernatants werediluted in RPMI 1640 containing 5% FBS and assayed intriplicate. Plates were incubated for 3 h at 37°C, and boundIFN--y was detected by using rabbit anti-human IFN-y antiseraraised from recombinant IFN-y (Genentech Inc., SouthSan Francisco, Calif.) in conjunction with a goat anti-rabbithorseradish peroxidase conjugate (Amersham Corporation,Oakville, Ontario, Canada). Standard curves of recombinantIFN-,y (specific activity, 2 x 106 U/mg) were used to calibrateeach plate, and results were expressed in units per milliliter.

Extensive investigations demonstrated that vervet IL-2 couldnot be detected by using either commercially available humanIL-2 ELISA kits or anti-IL-2 specific antibodies. Nonetheless,IL-2 was detected using the CTLL cell line bioassay (13). TheCTLL clone used was shown to preferentially respond tomurine and human IL-2 as opposed to murine IL-4 (data notshown). However, because of the lack of a suitable anti-primate IL-4 antibody for specific interleukin neutralization, itwas not possible to discriminate between levels of IL-2 andIL-4 in culture supernatants. CTLL cells (5 x 104/ml) werecultured in triplicate for 48 h with antigen-stimulated PBMCculture supernatants. Relative proliferation in response tocytokine was detected by using MTT as previously described(26). Interference of serum inhibitors for IL-2 (7) was mini-mized by sixfold dilution of culture supernatants in the pres-ence and absence of autologous bovine serum and FBS.

Production of IL-5 in response to mitogenic or antigenicstimulation was tested by both bioassay and ELISA using cellculture supernatants as described for IFN-,y. For bioassays theIL-5-dependent cell line B13 was used as previously described(35). Three triplicate dilutions of culture supernatants wereincubated for 120 h, and proliferative responses in the pres-

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TABLE 1. Stimulation indices of PBMC from naive and recovered vervet monkeys stimulated in vitro with synthetic peptides and wholeLeislhmania promastigotes

Stimulation index in response to":Animal" Live Killed KilledPT3 PT4 PT7 PTI 1 L. mialjor L. mta(ijor L. doniov'ani

1 1.1 3.3 12.0 1.5 1.1 2.4 1.43 1.1 1.5 0.9 1.1 (.9 2.4 2.75 3.3 3.0) 1.2 1.0 1.1 0.9 1.16 3.3 3.8 6.5 3.7 1.2 1.9 1.27 1.8 1.8 0.6 1.5 1.0 0.8 1.48 1.1 1.1 2.6 1.4 2.5 1.8 2.210 0.9 1.5 0.6 1.2 1.2 1.0 1.01 1 1.7 2.9 1.0 2.4 3.4 1.6 0.915 0.9 14.6 1.0 1.0 2.8 6.2 8.t)16 11.7 42.6 29.6 49.0 16.5 12.1 6.817 0.2 13.8 1.1 0.9 2.4 5.9 2.318 4.1 1.4 3.7 4.9 2.8 11.6 1.119 12.1 1.6 7.1 1.1 2.6 2.6 1.920 12.7 26.0 11.9 29.0 1.4 4.1 1.92 1 8.4 1.1 2.3 2.5 5.6 3.1 2.7

Animals I through 11 were DTH-negative controls, and animals 15 through 21 were experimental animals.The stimulation index was defined as the ratio of tritium (counts per minute) incorporated in the presence of antigen to that incorporated in the ahsence of antigen.

ence of IL-5 were measured using the MTT colorimetric assay(26). The ELISA detection system was that previously de-scribed for human IL-5 using the anti-human IL-5 antibodiesTRFK-4 and TRFK-5 (44). Recombinant human IL-5 (0.5 x10' U/mg; Genzyme Inc., Boston, Mass.) was used to standard-ize assays.

Phenotypic analysis of lymphocyte populations. Freshlyisolated PBMC were adjusted to 5 x 10"/ml and stained todetect T- and B-lymphocyte markers as previously described(30). Cells were preincubated in 20% FBS-0.02% sodiumazide in phosphate-buffered saline and then stained with thefollowing antibodies for 45 min at 4°C: FNI8 (anti-primateCD3+) and GM12 (anti-primate B lymphocyte) (gifts from M.Jonker and R. E. Bontrop, TNO Primate Center); anti-CD3-fluorescein isothiocyanate (FITC), Leu-2a FITC-conjugatedanti-CD8, Leu-3a phycoerythrin-conjugated anti-CD4, and an-ti-human immunoglobulin M (from Becton Dickinson, Missis-sauga, Ontario, Canada); and UCHL-1 anti-CD45R0 (a giftfrom P. C. L. Beverley, Imperial Cancer Research Fund,London, England). Unconjugated antibodies were detectedusing FITC-labelled sheep anti-mouse sera (Amersham Cor-poration). Cells were fixed immediately in 2% formaldehyde(30 min, 4"C), washed, and analyzed within 48 h. Analyses werecarried out by forward and side scatter on a Becton DickinsonFACScan Sorter with a minimum of 5,000 gated lymphocytes.The percent positive cells was determined by subtracting thenonspecific binding of FITC-conjugated sheep anti-mouseirrelevant antibody to control cells from the total positive cells.The percents CD4+, CD8+, and CD45R0+ cells were thencalculated with respect to the corrected value for total CD3+ Tcells.

RESULTS

Proliferation of PBMC from cured and naive vervets follow-ing mitogen stimulation. Given that resolution from leishma-niasis is dependent on T-cell-mediated responses, the cell-mediated immunological status of naive and recovered animalswas initially evaluated using the T-cell mitogens ConA andPHA and the T-cell-dependent B-cell mitogen PWM. Use ofboth ConA and PHA reduced the bias of individual mitogensto stimulate specific T-cell populations or specific accessory

cell dependence (10, 36, 37). Figure IA shows the results ofConA stimulation only, as they are typical of results obtainedfor all tested mitogens, which demonstrate that PBMC fromboth naive and recovered animals proliferate in response tothese glycoproteins. These results compare well to literaturedata for other primate studies with vervet (31), rhesus (52),and macaque (47) monkeys as well as baboons (33). It shouldbe noted that the background proliferative responses werehigher in the recovered than in the naive populations, resultingin comparable proliferative responses but reduced stimulationindices, as indicated in Fig. IA and Table 1. Only one animal,no. 8, showed minimal responses to all three mitogens. Thiswas unusual, since the animal appeared to be healthy and wasfound to be free of common infections and also showed noabnormality in cell viability or lymphocyte distribution asrevealed by fluorescence-activated cell sorter (FACS) analysis.The phenotypic analysis of populations is outlined below.

Vervet PBMC proliferation following stimulation with liveor killed parasites. Prior to this study, no in-depth immuno-logical investigation evaluating T-cell activation and cytokineproduction in nonhuman primates which recovered from leish-maniasis had been reported. This investigation has placedparticular emphasis upon proliferative responses of naiveanimal cells to whole parasites, not only for comparison withresponses to molecularly defined epitopes but also to facilitatedevelopment of future vervet screening procedures in whichadministration of antigen during DTH analysis might influencesubsequent immune responses.PBMC from recovered animals proliferated strongly in

response to both live and formaldehyde-fixed L. major, whilenaive control animals demonstrated minimal proliferative re-sponses to parasite antigens (Fig. IB). The early finding thatsome of the candidate control animals responded to antigenwas not unexpected, as all animals were wild caught in an areawhere natural infection of vervets with Leishmania spp. hadbeen reported. Thus the possibility that these animals hadrecovered from a natural infection could not be excluded, andthey were consequently eliminated from further experimentalconsideration. Because all monkeys used in this study werepreviously screened by DTH testing and found to be negative,results obtained here are indicative of the sensitivity advantage

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FIG. 1. Proliferative responses of PBMC from naive and curedvervets. (A) PBMC were stimulated with ConA at 10 to 1.1 pg/ml for48 h. (B) Lymphocytes were stimulated with live or formaldehyde-fixedLeishmania promastigotes for 120 h at three different concentrations:1 x 106, 5 x 105, or 1 x 105 promastigotes per ml. (C) Proliferationof PBMC in response to peptide antigens PT3, PT4, PT7, and PT11.Cells were stimulated with peptide at 2.5 to 50 ig/ml for 96 h. Eachassay was performed in triplicate, and the data represent the optimal[3H]thymidine incorporation for each antigen following a 20-h pulse(error bars show 1 standard deviation). Stimulation indices are indi-cated above the bars in panel A. The average background valuesobtained for the naive and for the recovered vervet monkeys were 324± 300 cpm and 2,790 + 2,159 cpm, respectively.

of T-cell proliferation assays over DTH testing for the selec-tion of potential experimental animals. Lastly, we observedthat a number of cured animals responded strongly to formal-dehyde-fixed L. donovani parasites, underscoring the conser-vation of epitopes in Old World Leishmania species.

Vervet PBMC proliferation following stimulation with syn-thetic gp63 T-cell epitopes. Previous investigations have dem-

onstrated that specific gp63 synthetic peptides could stimulateboth proliferation and lymphokine production in murineCD4+ T-cell populations associated with immunoprotection(16). However, analysis of the proliferative responses ofPBMCfrom cured leishmaniasis patients revealed a spectrum ofpeptides different from those recognized by any of the mouse

strains tested to date (41). Therefore, in order to maximize thelikelihood of observing a response of vervet PBMC to syntheticgp63 T-cell epitopes, we selected a panel of peptides known tostimulate PBMC from recovered human subjects (41) or toprotect mice (16, 55) and evaluated their ability to stimulatePBMC from recovered vervets.PBMC from recovered animals demonstrated an apparent

MHC-restricted proliferative response to all four peptidestested (PT3, PT4, PT7, and PT11) (Fig. 1C). Results rangedfrom those for animals 16 and 20, which responded to all fourpeptides, to those for animals 15 and 17, which responded toonly PT4. It was observed that those animals which recognizedPT3 did not respond to PT4 (animals 18, 19, and 21) and thosethat responded to PT4 did not recognize PT3 (animals 14, 15,and 17). On the other hand, responses to PT7 and PT11showed no clear pattern. Lastly, it was notable that no individ-ual peptide stood out as being a more potent activator of T-cellpopulations than the others. These findings clearly demon-strated a marked response to the synthetic epitopes by recov-ered animal cells which was equal to or greater than that towhole parasites, while naive control cells showed negligibleresponses.

Production of IFN-y following stimulation with mitogens,parasite antigens, or synthetic T-cell epitopes. Mitogen stim-ulation of lymphocyte populations from both naive and recov-ered animals resulted in production of IFN-,y (Fig. 2A),although concentrations of the cytokine were generally lowerin culture supernatants from recovered animals regardless ofthe mitogen employed. Whether this was due to a higherpercentage of non-IFN-y-producing cells in the proliferatingpopulations or suppression of IFN-y production is unclear.IFN-y production by cells stimulated with parasite antigen

was tested next. Figure 2B shows that PBMC from recoveredmonkeys were readily stimulated to produce IFN-,y, suggestingthe presence of protective memory T-cell populations. Al-though there was some variation between animals, IFN--y was

produced in response to all forms of parasite antigens testedwhile PBMC from control animals showed essentially no

response. This was in contrast with the substantial IFN--yproduction by PBMC from naive animals following stimulationwith mitogens. These findings are consistent with those ofsimilar investigations of IFN-y production by PBMC fromrecovered human subjects (41).PBMC cultures were similarly stimulated with synthetic gp63

T-cell epitopes, resulting in substantial amounts of IFN--y inPBMC from recovered animals (Fig. 2C). With the exceptionof one naive animal and one peptide, only the recoveredanimals responded to the peptide challenge. Thus, these dataare analogous to the previous lymphocyte proliferation resultsobtained following stimulation with the synthetic peptideepitopes. Levels of IFN-y varied, however, and while all fourpeptides were stimulatory, there was no individual peptide thatresulted in enhanced cytokine production.

Production of IL-2/11L4 following stimulation with mitogens,parasite antigens, or synthetic T-cell epitopes. The productionof IL-2/IL-4 in vervet cells was evaluated because of the knownrelationship between the release of these lymphokines and theprogress of cutaneous leishmaniasis in mice (22). In light of theabsence of specific nonhuman primate ELISA reagents, we

attempted to measure the vervet interleukins by application of

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FIG. 2. IFN-y production by PBMC isolated from naive vervetsand vervets recovered from cutaneous leishmaniasis. (A) Response tostimulation with ConA, PWM, and PHA. The data represent theoptimum responses from three concentrations of mitogen (10, 3.3, and1.1 .g/ml). (B) Response to stimulation with either L. donovani or L.major promastigote antigens. (C) Response to stimulation with L.major gp63 synthetic peptides. Each value represents the mean IFN-yconcentration based on values obtained with triplicate wells. In allcases, the standard error for each IFN-y value was less than 5%.

human reagents. Although several monoclonal and polyclonalanti-IL-2 antibodies were investigated for use in an ELISAdetection system, neither these nor commercially prepared kitswere capable of detecting vervet IL-2. Nonetheless, detectionwas possible with the IL-2- and IL-4-dependent cell line,CTLL. The proportional contribution of IL-2 and IL-4 toproliferation in this assay is normally determined by specificneutralization of either lymphokine by the appropriate anti-body (13). However, because the anti-human IL-2 antibodiestested did not recognize vervet IL-2, discrimination of therelative contributions of IL-2 and IL-4 to CTLL proliferative

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FIG. 3. IL-2/IL-4 production by PBMC isolated from naive vervetsand vervets recovered from cutaneous leishmaniasis, as determined bythe stimulation of the CTLL cell line. Results are expressed in units ofIL-2 per milliliter relative to an IL-2 standard curve. Production ofIL-2/IL-4 by cells stimulated with the mitogens ConA, PWM, and PHA(A), with Leishmania promastigote antigens (B), and with peptidesPT3, PT4, PT7, and PTI 1 (C) is shown. Each value represents themean cytokine concentration based on results from triplicate wells.The standard error for the values was less than 10%.

responses was not possible. This limitation notwithstanding, wewere able to observe CTLL proliferative responses to thesupernatants from mitogen-stimulated vervet cells (Fig. 3A). Itwas notable that regardless of the mitogen employed and withfew exceptions, the naive PBMC produced more IL-2/IL-4than cells from cured vervets. Thus, like IFN-y, these interleu-kins were released by cured vervet lymphocytes on exposure tomitogens but at a lower level than interleukins released byPBMC from control animals.

Results of experiments to determine levels of IL-2/IL-4following stimulation with parasite antigens showed a markedsimilarity with those reported above for T-cell proliferation

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and IFN-,y production in the presence of these antigens. Withonly one exception, production of IL-2/IL-4 was observed onlyin recovered animal cells (Fig. 3B), and levels of productionwere comparable when live L. major, killed L. major, and killedL. donovani parasites were used. Considerable variability wasfound, however, from animal to animal, although all recoveredvervets responded to at least one of the complex antigens.Finally, it was noted that the mean level of production ofinterleukin from recovered-animal cells stimulated with para-site antigens was approximately fivefold higher than levelsobtained with the same cells stimulated with mitogen.The studies of antigen-stimulated release of IL-2/IL-4 were

concluded with an investigation of the response of vervetPBMC to the gp63 T-cell epitopes. Results of this experimentwere very similar to those of experiments with whole parasites.Although each of the peptides was able to elicit a response insome of the animals, only PT7 was capable of stimulatingIL-2/IL-4 production in all animals tested (Fig. 3C). This resultwas in contrast with the proliferation and IFN--y studies, inwhich there was no clear bias in favor of any one peptide.

Production of IL-5 following stimulation with mitogens andsynthetic T-cell epitopes. In order to further characterizeT-cell populations proliferating in response to parasite anti-gens, we assayed for the production of vervet IL-5 using anELISA with human reagents. Results from this study gave noevidence of IL-5 production from recovered-animal cells inresponse to in vitro stimulation with either mitogens orantigens. However, because of the low-level production of IL-5reported for human PBMC (1) and the possible complicationsby the low cross-reactivity of human anticytokine antibodiesfor primate cytokines, as observed with IL-2, the B13 cell linewas used to evaluate IL-5 levels in culture supernatants. Cellsfrom all naive and recovered vervets stimulated with ConA,PHA, and PWM secreted detectable levels of IL-5 rangingbetween 60 and 130 ng/ml. However, in marked contrast, noIL-5 production could be detected in PBMC supernatantsstimulated with either parasites or synthetic epitopes.

Phenotypic analysis of lymphocyte populations. FACS anal-ysis of lymphocyte populations is shown in Fig. 4. Becauseprogressive Leishmania infections have been associated withpolyclonal B-cell activation and increased antibody production(42, 43), it was interesting to observe that cured animalsshowed no increase in the percentage of B lymphocytes.Recovery from infection was, however, associated with adramatic increase in the percentage of circulating T cells (Fig.4). In addition, we observed a marked increase in the expres-sion of the CD45R0+ isoform of the leukocyte commonantigen CD45 among the T cells from recovered animals: theproportion of T cells expressing this marker was fourfoldgreater in the recovered vervets (3.1% ± 1.1%) than in thenaive control animals (0.7% ± 0.7%). Although an increasednumber ofT lymphocytes was found in cured vervets, there wasno change in the CD4+/CD8+ ratio (Fig. 4). In humanpopulations, this ratio typically ranges from 2 to 2.5 (19, 23),whereas the ratios seen here, in both cured and naive vervets,were less than 1.

DISCUSSION

In the experiments described in this report, we have dem-onstrated the utility of the vervet monkey in a cutaneousleishmaniasis model and have begun to analyze host-parasiteinteractions at the molecular level. We demonstrate thatPBMC from recovered vervets respond to either live or killedparasites in a manner not unlike that of PBMC from curedhumans and that immunological parameters such as cytokine

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T CELLS B CELLS CD4 CD8FIG. 4. FACS analysis of lymphocyte phenotypic populations in

control (CNTRL) and recovered (RCVRD) vervet monkeys. T cellswere stained with the anti-CD3+-FITC monoclonal antibody FN18; Bcells were stained with GM12 and anti-primate B-lymphocyte mono-clonal antibody; CD4+ cells were labelled with the Leu-3a-phyco-erythrin direct conjugate anti-CD4 monoclonal antibody; and CD8+cells were stained with the anti-CD8 monoclonal antibody Leu-2a-FITC direct conjugate. For T and B cells, each point represents thepercentage of total PBMC from an individual animal expressing T- orB-cell markers. In the case of CD4+ and CD8+ cells, each pointrepresents the percentage of total T cells from an individual animalexpressing the CD4 or CD8 antigen.

quantitation and cell phenotyping can be assessed by adaptinghuman reagents to the vervet model (30). In addition, we haveshown that vervet PBMC respond to the same T-cell epitopesas did cells from patients infected with both Old and NewWorld species of Leishmania (15, 41). The vervet cutaneousleishmaniasis model is an important new tool, not only for bothfundamental and applied immunological studies but also forproviding a basis of comparison for the newly establishedvisceral leishmaniasis model (5).

In this study we have modified immunological methods andreagents routinely used to measure activation of human T-cellpopulations and applied them to investigations with vervets.We have shown that while conventional ELISA detectionsystems could be used to measure vervet IFN--y, levels of IL-2,IL-4, or IL-5 could be detected by employing bioassays whichrelied on the cytokine-dependent cell lines CTLL and B13.However, although this particular CTLL line responded pref-erentially to IL-2, we were unable to discriminate between IL-2and IL-4, because of a lack of suitable antibodies against thevervet cytokines. Similar difficulties arising from poor cross-reactivity of human antibodies to vervet markers have beenreported to occur during FACS analysis (23, 30). However, inthis investigation we have shown that use of phycoerythrin,

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rather than FITC, overcomes these problems, facilitating phe-notypic evaluation of specific vervet lymphocyte populations.

T-cell proliferative responses to both live and fixed L. majorparasites were found for all recovered vervets in this study,while naive control animals were essentially unresponsive. Thisresult is of practical importance for screening of wild-caughtvervets, as lymphocyte proliferation responses to parasiteantigens appear to identify animals with prior exposure todisease with greater sensitivity than traditional DTH screeningand avoid exposure of naive animals to parasite antigens. Ofgreater basic interest, however, was the observation thatrecovered vervets responded to these complex antigens withthe production of substantial amounts of IFN-y and IL-2/IL-4.Although it was not possible to discriminate accurately be-tween IL-2 and IL-4, it is likely that IL-2 was the predominantcytokine produced since no IL-5 was detected from recovered-animal cells regardless of the antigen employed, suggestingthat the peptides investigated here stimulated Th I- as opposedto Th2-type responses. However, it must be noted that the IL-5data represent results from a single experiment and must beregarded as preliminary. It was also of interest that prolifera-tive and lymphokine responses to L. donovani antigens werefound in cells from vervets recovered from L. nmajor infection.This cross-reactivity is an important consideration for selectionof candidate vaccine molecules which might be suitable forbroad-spectrum protection. Several collaborative studies usingdefined T-cell epitopes and human PBMC (23a, 55) haveshown that this cross-reactivity in primate cell-mediated im-munity also extends to New World species of Leishmania.These data take on additional significance with the recentestablishment of a vervet model for L. donovani-derivedvisceral leishmaniasis (5).

In addition to responding to complex parasite antigens,recovered vervet PBMC also responded to individual T-cellepitopes with proliferation and cytokine production. The fourgp63 T-cell epitopes utilized were selected on the basis of theirability to stimulate protective responses in mice (16) or PBMCproliferation in humans (41). PBMC from all recovered pri-mates recognized one or more of the T-cell epitopes, asindicated by either T-cell proliferation or lymphokine produc-tion and in many cases by both. It is noteworthy that human Tcells from cured patients demonstrated a similar variation inresponse when challenged with these epitopes (41), a phenom-enon assumed to be attributable to MHC restriction. As aconsequence, it has been argued that if synthetic peptides areto be employed in vaccine development, rather than wholemolecules, a more effective formulation would include severalepitopes capable of stimulating immunoprotective responses.While we agree with such reasoning, we have noted thatcertain T-cell epitopes do not appear to exhibit strict MHCrestriction. For example, PT7 stimulated IL-2/IL-4 productionin all recovered vervets and IFN-y production in the majorityof these animals. Moreover, an earlier study showed that PT7was capable of stimulating proliferation and IFN-y productionin PBMC from patients who had recovered from cutaneous,visceral, and mucocutaneous infections (41). Another peptide,PT6, has also been shown to stimulate lymphocytes from awide range of murine haplotypes (16). Thus, careful charac-terization of candidate epitopes may reduce the minimumnumber of peptides required for broadly based protection.Analysis of the T-cell proliferative responses of the presentvervet population to the selected panel of peptides demon-strated that these animals could be split into two groups: (i)those that responded to multiple peptides-in some cases, toall four, and (ii) those that responded to individual peptides.Of the latter, two animals were found to respond to PT4 only.

Similarly, it has been shown that in humans, although PT4 wasrecognized by lymphocytes from a high proportion of patients,these lymphocyte populations did not recognize PT3 (41).These data are interesting, not only because PT3 and PT4 haveoverlapping sequences, but also because the sequence is asso-ciated with the gp63 zinc-binding site, which is highly con-served throughout Leishmaniia species (17). The uniqueness ofthese two peptides lies in their respective nonoverlappingregions-the N-terminal five residues of PT3 and the C-terminal five residues of PT4. Both contain, or overlap, uniqueRothbard and Taylor-predicted T-cell epitopes (38, 39) andare prime vaccine candidates.The responses of recovered-animal cells to the synthetic

epitopes are particularly interesting, as the same cells demon-strated lower-level mitogen responses than cells from naiveanimals. Similar findings have been previously reported forvisceral but not cutaneous leishmaniasis and may reflect adetrimental effect of infection on antigen-presenting-cell func-tion and costimulator production as suggested by other work-ers (8, 29, 34). This observation appears unlikely to be theresult of the BCG immunization, as two saline-control animalshad responses similar to those of vervets receiving BCG (datanot shown). Although these apparent suppressive effects didnot eliminate the response to synthetic peptides, it is conceiv-able that selection of appropriate adjuvants during futurevaccine trials could result in even larger responses.

Results of the phenotypic characterization of T cells re-sponding to mitogens and antigens demonstrate a number ofpoints worthy of comment. Firstly, lymphocytes freshly isolatedfrom recovered vervets showed an increased expression of theCD45R0+ isoform of the leukocyte common antigen. Thelatter has been associated with CD4+ memory T-cell popula-tions (2, 11, 25), and it could explain the high proliferativebackground levels observed within the infected animals andfurther substantiates the similarity of the vervet model with thehuman infection, in which recovery is associated with lastingimmunity. Secondly, our observations of increased numbers ofT cells but not B cells are in keeping with the concept of T-cellactivation in the cure response and B-cell expansion in diseaseprogression (42, 43). The lack of B-cell expansion may alsocorrelate with the inability to stimulate antigen-specific IL-5production and would be further explained if the high levels ofIL-2/IL-4 observed with the T-cell expansion were due to IL-2.Throughout these experiments the recovered animals showedhigh background levels of both proliferation and IL-2/IL-4, butnot IFN-y. Similar observations have been made in previousstudies (31). It is possible that the increased numbers of T cellsobserved in the present study, in conjunction with the elevatedIL-2/IL-4 production, could contribute to this situation in theabsence of stimuli. Attributing the odd T-cell ratio to highproliferative and IL-2/IL-4 background levels, as has beendone by previous investigators (25), cannot be done here, asbackground levels in naive animals were normal. Lastly, al-though initially surprising, the dramatic difference between theCD4+/CD8+ ratio observed in vervets and that seen in humanshas also been noted in studies with other primates, such asbaboons (0.5 to 0.58) and rhesus monkeys (0.69 to 0.9), and inother studies using vervets (0.35 to 0.4) (23, 25, 30, 48). Thenormal CD8+ component in humans is approximately 21 %.The 79% average seen here therefore seems high but is inkeeping with previous studies in which values of 75% forvervets and 65% for baboons have been reported (11, 23, 25,52). Whether infection and cure result in activation of theCD8+ subset is unclear and poses an important question forfurther study. It is interesting, however, that generation ofanti-PT7 human T-cell clones resulted in both CD4+ and

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1740 CURRY ET AL.

CD8+ populations (41). Whether these peptides are processedwithin the cytoplasm of antigen-presenting cells or representunprocessed peptides bound at the surface (23) is still contro-versial, but it is clear that small synthetic peptides wouldfacilitate antigen binding to the cell surface MHC molecules.Although we cannot conclude from our data the degree towhich CD4+ or CD8+ T cells participate in the observedpeptide responses, it is evident from the strong proliferationand IFN-y responses that these peptides stimulate effectorfunctions which have been demonstrated in other models to beimportant in resolution of the parasitic infection.

ACKNOWLEDGMENTS

We acknowledge S. Kielland and D. Hardie of the TripartiteMicroanalytical Center for their excellent technical assistance andL. S. D. Anthony for critical review of the manuscript.

This work was supported in part by the B.C. Health ResearchFoundation and the Natural Sciences and Engineering ResearchCouncil of Canada.

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