Proc. Natl. Acad. Sci. USAVol. 90, pp. 5214-5218, June 1993Immunology

Priming with recombinant influenza virus followed byadministration of recombinant vaccinia virus induces CD8+T-cell-mediated protective immunity against malariaSHENGQIANG LI*, MAURICIO RODRIGUESt, DOLORES RODRIGUEZt, JUAN RAMON RODRIGUEZt,MARIANO ESTEBANt, PETER PALESE*, RUTH S. NUSSENZWEIGt, AND FIDEL ZAVALAt§*Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029; tDepartment of Medical and Molecular Parasitology, New YorkUniversity School of Medicine, New York, NY 10010; and tDepartment of Biochemistry, State University of New York Health Science Center atBrooklyn, Brooklyn, NY 11203

Communicated by William Trager, March 17, 1993 (received for review January 19, 1993)

ABSTRACT Live vectors expressing foreign antigens havebeen used to induce immunity against several pathogens.However, for the virulent rodent malaria parasite Plasmodiumyoelii, the use of recombinant vaccinia virus, pseudorabiesvirus, or SalmoneUa, expressing the circumsporozoite proteinof this parasite, failed to induce protection. We generated arecombinant influenza virus expressing an epitope from thecircumsporozoite protein ofP. yoelii known to be recognized byCD8+ T cells and demonstrated that this vector induced classI major histocompatibility complex-restricted cytotoxic T cellsagainst this foreign epitope. Immunization of mice with thisrecombinant influenza virus, followed by a recombinant vac-cinia virus expressing the entire circumsporozoite protein,induced protective immunity against sporozoite-induced ma-laria. The sequence of immunization appears to be crucial,since a primer injection with recombinant vaccinia virus,followed by a booster injection with recombinant influenzavirus, failed to induce protection. The protection induced byimmunization with these recombinant viruses is mostly medi-ated by CD8+ T cells, as treatment of mice with anti-CD8monoclonal antibody abolishes the anti-malarial immunity.The use of different live vectors for primer and boosterinjections has a synergistic effect on the immune response andmight represent an effective general strategy for elicitingprotective immune responses to key antigens of microbialpathogens.

Recombinant live viruses expressing foreign antigens areattractive delivery systems, designed to elicit protectiveimmunity against microbial pathogens. A number of exper-imental findings provide strong evidence that recombinantviruses, such as attenuated vaccinia virus expressing micro-bial antigens, can induce protective immunity against viraland bacterial infections (1-7). However, the use of recom-binant vaccinia virus expressing distinct plasmodial antigenshad, to our knowledge, failed to generate a significant degreeof protection against the various developmental stages ofmalaria parasites (8, 9).The need for additional live carriers that would induce

efficient protective immunity against certain infectiousagents led to the engineering of influenza viruses expressingmicrobial epitopes. This negative-strand RNA virus had,until recently, not been amenable to genetic manipulation,because of the lack of an appropriate rescue system. Re-cently, a general procedure was described (10, 11) by whichthe genome of influenza virus could be altered, so that theexpression of a cDNA-derived RNA became feasible. Theconstruction of such a recombinant influenza virus contain-

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ing a plasmodial antigen has provided the opportunity todefine its immunogenicity and compare it with that of arecombinant vaccinia virus.The rodent malaria parasite Plasmodium yoelii was se-

lected for these studies, since a number of its protectiveimmune mechanisms and the corresponding antigens havebeen relatively well characterized. Several studies havedemonstrated that CD8+ T cells play a major role in immunityagainst preerythrocytic stages of rodent malaria (12-14). Inthe P. yoelii system, CD8+ T cells recognize the majorhistocompatibility complex (MHC) class I-restricted epitopeSYVPSAEQI present in the circumsporozoite (CS) protein ofthis parasite (15-17). Adoptive transfer, to naive mice, ofCD8+ T-cell clones specific for this epitope inhibits thedevelopment of the liver stages of the parasite and confersextensive protection against sporozoite-induced malaria (17,18).

In the present report, we describe studies in which weimmunized mice with a recombinant vaccinia virus express-ing the entire P. yoelii CS protein and a recombinant influ-enza virus expressing the cytotoxic epitope of this protein.We determined the immunogenicity of these live vectors,administered individually or sequentially, and characterizedthe protective anti-malaria immune mechanisms they induce.

MATERIALS AND METHODSConstruction of a Recombinant Influenza Virus Expressing

the Cytotoxic CS Epitope of P. yoelii. A unique BstEIIrestriction site was engineered into a full-length cDNA ofWSN virus hemagglutinin (HA), immediately upstream oftheantigenic site E (PT3/WSN-HAm) (19). The recombinantMEvirus HA [where ME is the malarial cytotoxic T-lymphocyte(CTL) epitope] was constructed by replacing the BstEII-HindIII fragment of the WSN HA with a PCR product, inwhich the nucleotide sequence encoding the 8 aa between theCys and Trp residues was replaced by the sequence encodingthe plasmodial peptide NEDSYVPSAEQI. Transfection of invitro-synthesized RNA into Madin-Darby bovine kidneycells and rescue of infectious recombinant influenza viruswere done as described (19). The nucleotide sequence en-coding NEDSYVPSAEQI has been confirmed by directsequencing of purified viral RNA. This recombinant influ-enza virus was designated influenza ME.

Construction of a Recombinant Vaccinia Virus Expressingthe Entire P. yoelu CS Protein (PYCS). A Xba I fragment of

Abbreviations: HA, hemagglutinin; CS, circumsporozoite; CTL,cytotoxic T lymphocyte; pfu, plaque-forming unit; ME, malaria CTLepitope; wt, wild type; MHC, major histocompatibility complex;mAb, monoclonal antibody.§To whom reprint requests should be addressed at: 341 East 25thStreet, New York, NY 10010.

5214

Proc. Natl. Acad. Sci. USA 90 (1993) 5215

1.75 kb, containing the gene of the P. yoelii CS protein, wasisolated from the plasmid pBS-PY1993. This DNA fragmentwas blunt-ended by treatment with a large fragment ofEscherichia coli DNA polymerase (Klenow), purified byagarose gel electrophoresis, and cloned into the Sma I site ofthe vaccinia virus insertion vector pSC11 (20). As a result ofthis cloning strategy, we isolated a plasmid, pJR88, contain-ing the P. yoelii CS gene under control of the vaccinia virusearly-late promoter p7.5, the ,3galactosidase gene lacZ underthe control of the vaccinia virus late promoter pll, andflanking regions from the vaccinia virus thymidine kinasegene. To generate vaccinia PYCS recombinants, the insertionvector pJR88 was introduced by homologous recombinationinto the thymidine kinase locus of the vaccinia virus genome.The recombinant virus was selected by l3-galactosidase ex-pression (20) and purified (21).

Cytolytic Assay. P815 target cells (5 x 105 cells) wereincubated with 5 x 106 plaque-forming units (pfu) of influenzaME or wild-type (wt) WSN virus for 1 h at 37°C. Afteradsorption, the target cells were resuspended in fetal catfserum and labeled for 2 h with 200 ,uCi of 51Cr (ICN; 1 Ci =37 GBq). For the chromium release assay, these target cellswere washed, distributed at 2.5 x 103 cells per well, andincubated with the CTL clone YA26, at various effector/target cell ratios. Clone YA26 was generated and maintainedas described (17, 18). This CTL clone recognizes the epitoperepresented by the peptide SYVPSAEQI, in association withMHC class I H-2kd. After 5 h at 37°C, the supernatants werecollected with a semiautomatic harvester (Skatron, Sterling,VA). The percentage of specific lysis was calculated asfollows: (experimental - spontaneous release)/(total -spontaneous release) x 100.The spleen cells of influenza ME and wt virus-immunized

mice were obtained 14 days after aerosol exposure. Thesespleen cells (4 x 107 cells per 10 ml) were restimulated in vitroin the presence of 2.5 x 106 P815 cells pulsed with 1 ,uMNEDSYVPSAEQI plus 1% of the EL-4 supernatant. After 6days in culture, these cells were washed and incubated atvarious ratios with 2.5 x 103 51Cr-labeled P815 cells per well,in the presence or absence of 0.2 ,uM NEDSYVPSAEQI.

Parasites and Animals. P. yoelii (17X NL strain) wasmaintained as described in ref. 17. Four- to 8-week-oldfemale BALB/c mice, used for the immunological studies,were purchased from The Jackson Laboratory and fromCharles River Breeding Laboratories. Parasite injection wasdone i.v. into the tail vein and parasitemia was determined bymicroscopic examination of Giemsa-stained thin bloodsmears, collected daily from the 3rd to the 14th day afterchallenge.Immunizations and Challenge. BALB/c mice were immu-

nized, by aerosol, with 500 pfu of influenza ME or wt virus.When indicated, a second dose containing equivalentamounts of virus was administered by the same route.Mice immunized with recombinant vaccinia PYCS or wt

vaccinia virus received 5 x 107 pfu, injected i.p. When asecond dose of these viruses was administered, 108 pfu wasused. In mice that received two immunizing doses, consistingof different combinations of viruses, the second dose wasadministered 3 weeks after the first dose.

All immunized mice were challenged 13 days after the lastimmunizing dose. In those experiments in which protectionwas assessed by blood-stage parasitemia, mice were chal-lenged by intravenous injection of 100 sporozoites. Whenprotection was assessed by measuring plasmodial rRNA,mice were challenged i.v. with 5 x 105 P. yoelii sporozoites.

Quantification of P. yoelii rRNA in the Liver of InfectedMice. Quantification of P. yoelii rRNA was performed asdescribed (22). Briefly, total RNA was isolated from thelivers of mice sacrificed 42 h after they had been injected i.v.with 5 x 105 sporozoites of P. yoelii. RNA was prepared by

the method of Chomczynski and Sacchi (23). One-tenth of thewhole liver RNA was precipitated with isopropanol. TheRNA pellet was dissolved in 10 mM Tris HCl, pH 8.0/1 mMEDTA and denatured at 650C in 20x SSC (lx SSC = 150mMNaCl/15 mM sodium citrate) plus 37% (vol/vol) formalde-hyde, 1:1 (vol/vol). This preparation was diluted 1:45 and0.2-ml samples were blotted onto nylon membranes. TheRNA was fixed to the fiters by UV cross-linking. Hybrid-ization was performed overnight in 5 x SSPE (1x SSPE = 180mM NaCl/10 mM sodium phosphate/l mM EDTA, pH7.7)/1% SDS/500 ug of heparin at 42°C, containing three32P-labeled oligonucleotide probes (106 cpm/ml; specific ac-tivity, >2 x 108 cpm/,ug). The probes are described else-where (22). For comparison purposes, a standard curve wasprepared using serial dilutions of purified RNA from P.yoelii-infected erythrocytes and blotted under the same con-ditions.

In Vivo Depletion of CD4+ or CD8+ T Cells. The hybridomasproducing rat IgG anti-CD4 (GK1.5) or anti-CD8 (2.43) werepurchased from the American Type Culture Collection. As-cites were produced in BALB/c nude mice, and the concen-tration of rat immunoglobulin was estimated by RIA usingmouse-adsorbed anti-rat IgG heavy and light chains (Kirke-gaard and Perry Laboratories, Gaithersburg, MD). For 3consecutive days, each mouse received daily doses of 1 mgof anti-CD4 or anti-CD8 monoclonal antibody (mAb). Thesemice were challenged with P. yoelii sporozoites 2 days afterreceiving the last mAb dose. The efficacy of the depletionwas estimated by two-color flow cytometry analysis of pe-ripheral blood lymphocytes, using phycoerythrin-conjugatedanti-THY-i and fluorescein-conjugated anti-CD4 or anti-CD8mAbs (all purchased from Accurate Chemicals, Westbury,NY). We found that the amount ofCD4+ T cells was reducedby 98% in mice treated with anti-CD4 mAb. The treatmentwith anti-CD8 mAb eliminated 95% of the CD8+ T cells.

RESULTSConstruction of Recombinant Influenza and Vaccinia Vi-

ruses and Characterization of Their Immunogenic Properties.A peptide containing the cytotoxic epitope of the P. yoelii CSprotein was expressed in antigenic site E of the HA proteinof the influenza virus A/WSN/33. This recombinant influ-enza virus (ME) was generated by inserting the nucleotidesequence for the peptide NEDSYVPSAEQI into the influ-enza HA gene (Fig. 1A). Earlier studies had shown that theepitope SYVPSAEQI is recognized by MHC class I H-2Kd_restricted CD8+ CTLs (15-17).We also generated a recombinant vaccinia virus expressing

the entire P. yoelii CS protein, by insertion ofthe P. yoelii CSgene into the thymidine kinase locus ofthe viral genome (Fig.1B) (25).We established that target cells infected by influenza ME

virus express, process, and present the cytotoxic CS epitopeofP. yoelii. As shown in Fig. 2A, target cells infected in vitrowith influenza ME virus were lysed by the CD8+ T-cell cloneYA26, which recognizes the SYVPSAEQI epitope (17, 18).This CTL clone did not lyse target cells infected with the wtinfluenza virus or uninfected cells.More importantly, we determined that H-2Kd mice immu-

nized with influenza ME virus, unlike those immunized withthe wt virus, generated CTLs that specifically lysed targetcells coated with the synthetic peptide containing the cyto-toxic plasmodial epitope (Fig. 2B). These findings demon-strate that a foreign epitope expressed in an influenza viruscan be processed and presented in the context ofclass I MHCand can also induce specific cytotoxic T cells in vivo.We obtained similar in vitro results with recombinant

vaccinia constructs (data not shown), known to efficiently

Immunology: Li et al.

Proc. Natl. Acad. Sci. USA 90 (1993)

A67 76

WSN(wt): NPECDSLLPARSWSYI

ME: NPECNEDSYVPSAEQIWSYI

BXbal Xbal

i IM pBS-PY1993PYCS

Xbel/Klenow

pycsP_

pl I p7.5 Sm l~~~~p1 p7 5

i r* pscI IC1TK LeecZ TK

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FIG. 1. Recombinant influenza virus(influenza ME) and recombinant vacciniavirus (vaccinia PYCS). (A) Amino acidsequences ofthe original antigenic site E ofinfluenza A/WSN/33 (wt) virus HA and ofthe "substituted site" in the HA of therecombinant influenza ME virus. Boldfacetype corresponds to the amino acid se-quence containing the cytotoxic epitope ofthe P. yoelii CS protein. Numbering in theWSN virus is according to ref. 24. (B)Introduction of the P. yoelii CS proteingene in vaccinia virus DNA.

induce CD8+ T cells in various systems, including malaria(26-28).

Induction of Protective Immunity Against Malaria by Im-munization with Recombinant Influenza and Vaccinia Viruses.To determine whether these recombinant viruses could in-duce protective immunity against preerythrocytic forms(namely, sporozoites and liver stages of P. yoelii), BALB/cmice immunized with one or two doses of these viruses werechallenged with viable sporozoites. Parasite developmentwas monitored by measuring plasmodial rRNA in the liver ofthe sporozoite-injected mice (22).

Immunization of mice with one or even two doses of theinfluenza ME virus did not appear to affect the intrahepato-cytic development of the parasites. In fact, the levels ofplasmodial rRNA in the liver of mice immunized with influ-enza ME virus were similar to control mice immunized withwt virus (Fig. 3A). In mice immunized with a single dose ofthe vaccinia PYCS virus, we observed a partial decrease(50-55%) of the parasite load in the liver (data not shown). Asecond immunizing dose of this vaccinia recombinant failedto enhance the level of anti-parasite immunity (Fig. 3B).Control mice immunized with wt vaccinia had a parasite loadsimilar to that found in naive mice.

To determine whether the use of these two different livevectors, in a single immunization protocol, would enhancethe protective immune response, mice were inoculated firstwith influenza ME virus and 3 weeks later with vacciniaPYCS virus. These immunized mice were challenged 13 dayslater with P. yoelii sporozoites. We found that this immuni-zation resulted in a 96.3% decrease ofplasmodial rRNA in theliver, revealing a remarkable synergistic effect of the vacci-nation with two distinct live vectors (Fig. 3C). Surprisingly,protective immunity failed to be induced when the tworecombinant viruses were administered in the reverse or-der-namely, a primer injection with the vaccinia PYCSfollowed 3 weeks later by immunization with influenza MEvirus (Fig. 3D).The findings based on measuring rRNA in the liver of

immunized and challenged mice were fully corroborated byexperiments in which we observed protection against thedevelopment of blood infection in mice immunized withinfluenzaME and given booster injection with vacciniaPYCSvirus. When these immunized mice were challenged with P.yoelii sporozoites, 60% were protected against this highlyinfective parasite, never developing patent parasitemia. Fur-thermore, those immunized mice that became infected (40%)did so only after a prolonged prepatent period, reflecting a

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FIG. 2. Influenza ME-infected target cells process and present the cytotoxic CS epitope. (A) Chromium-labeled P815 target cells infectedwith influenza ME virus (-), control wt WSN virus (o), or uninfected P815 cells (A) were incubated with CD8+ CTL clone YA26, at variouseffector (CTL)/target cell ratios. (B) BALB/c mice were immunized with 500 pfu of influenza ME or control wt WSN virus, by aerosol; 14 dayslater their spleen cells were collected, restimulated in vitro in the presence of irradiated P815 cells pulse-labeled with 1 ,uM NEDSYVPSAEQI.After 6 days in culture, these cells were assayed for the presence of peptide-specific cytolytic T cells as described (15). Spleen cells of miceimmunized with influenza ME virus displayed considerable lysis of P815 target cells in the presence (m) but not in the absence (o) ofNEDSYVPSAEQI. Spleen cells of mice immunized with wt WSN virus incubated with P815 target cells in the presence (A) or absence (A) ofpeptide showed no specific lytic activity.

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5216 Immunology: Li et al.

Proc. Natl. Acad. Sci. USA 90 (1993) 5217

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decrease of their preerythrocytic parasite load (Table 1). Incontrast, all the nonimmunized mice and the mice immunizedwith the wt viruses developed patent blood infections.

Protective Anti-Malaria Immune Mechanisms Induced byImmunization with Recombinant Viruses. Mice immunizedwith these two recombinant viruses were injected with anti-CD8 or anti-CD4 mAb to identify the mechanisms mediatingthe protective immunity induced by priming with influenzaME virus, followed by a booster injection with vacciniaPYCS virus. As shown in Fig. 4, treatment with anti-CD8mAb almost completely ablated the inhibition of liver stagedevelopment induced by immunization, whereas no effectwas observed upon treatment with anti-CD4 mAb. Thesefindings clearly indicate that protective immunity induced bythese recombinant viruses is mostly mediated by CD8+ Tcells.

DISCUSSIONThe present findings demonstrate that protective immunityagainst the highly virulent rodent malaria parasite P. yoeliican be induced by inoculation of recombinant live virusesexpressing the CS protein or portions of it. This protectiveimmunity is greatly enhanced when two distinct live vectorsare used for primer and booster injections.

T B

FIG. 3. Immunization with recombinant influ-

enza ME followed by vaccinia PYCS viruses inhib-its the development of liver stages of P. yoelii.BALB/c mice received two immunizing doses ofdifferent viruses. The second immunizing dose wasadministered 3 weeks after the first immunization.All mice were challenged 13 days after the second

immunizing dose. Immunized and control micewere challenged i.v. with 5 x 10i P. yoelii. Theamount (ng) of plasmodial rRNA of each group ofmice obtained from the livers of five mice is ex-pressed as the mean ± SEM. (A) Mice immunized

2 3 twice by aerosol with 500 pfu of influenza ME orcontrol wtWSN virus. Bars: 1, none; 2, wt virus; 3,

D influenza ME. (B) Mice immunized with two dosesofvaccinia PYCS or control wt virus. The first doseand the second dose consisted of 5 x 107 and 108pfu, respectively, administered i.p. Bars: 1, none;2, wt virus; 3, PYCS. (C) Mice immunized byaerosol with 500 pfu of influenza ME and subse-quently given a booster injection i.p. with 5 x 107pfu of vaccinia PYCS or control wt vaccinia virus.Bars: 1, none; 2, influenzaME plus vaccinia PYCS;3, influenza ME plus wt vaccinia virus. (D) Miceimmunized i.p. with 5 x 107 pfu of vaccinia PYCSvirus or wt vaccinia virus, and subsequently givena second immunizing dose, by aerosol, with 500 pfuof influenza ME virus. Bars: 1, none; 2, vacciniaPYCS plus influenza ME; 3, wt vaccinia plus influ-

2 3 enzaME.

Earlier studies had used a recombinant Salmonella ex-pressing the entire CS protein to generate protective immu-nity against the rodent malaria parasite P. berghei (29).However, protection against P. yoelii, a much more infectiveplasmodial species, failed to be induced by immunizationwith recombinant vaccinia, pseudorabies virus, or Salmo-nella (8, 30). In fact, protection against P. yoelii has so faronly been obtained upon immunization with large numbers oftransfected tumor cells expressing certain sporozoite anti-gens (31).We have currently determined that a certain degree of

protective immunity against this parasite can be induced bya single immunization with vaccinia PYCS virus but that asecond dose of the same recombinant virus fails to enhancethis immunity. These results are, in all likelihood, due to avigorous primary immune response that rapidly neutralizesthe second dose of the virus. In contrast, the immunizationwith vaccinia PYCS, after priming the mice with the influenzaME virus, enhances the effectiveness of the anti-parasiteimmunity, apparently by expanding the influenza virus-induced CD8+ T cells.The sequence in which these vectors are used for immu-

nization appears to be crucial. Protective immunity can onlybe induced by a primer injection with influenza ME virus,followed by a booster injection with vacciniaPYCS virus, butnot when the reverse protocol is followed. Since vaccinia,

Table 1. Immunization with recombinant influenza virus followed by recombinant vaccinia virus confers protectionagainst sporozoite-induced P. yoelii malaria

Immunization dose No. mice protected/ % Prepatent period,First Second no. challenged protection days

Influenza ME Vaccinia PYCS 9/15 60 5.2*Influenza wt Vaccinia wt 0/10 0 4.2None None 0/10 0 4.2

Protocol of immunization was as described in Fig. 3C. Challenge dose consisted of 100 P. yoelii sporozoites per mouse.This dose represents at least five times the minimum dose required to infect 100% of normal BALB/c mice. The protectedanimals remained negative, but all the control mice developed patent parasitemia.*Prepatent period in this group refers to the six animals that were not protected.

Immunology: Li et al.

Proc. Natl. Acad. Sci. USA 90 (1993)

AI-11823 to P.P., AI-32361 to M.E., and AI-27458 to F.Z.) and theAgency for International Development.

T

FIG. 4. Protective immunity induced by immunization with re-

combinant influenza and vaccinia viruses is mediated by CD8+ cells.Except for the controls, all mice were immunized (+) with influenzaME and subsequently given a booster injection with vaccinia PYCSvirus, as described in Fig. 3C. Control mice were not immunized (-).As indicated, two groups of immunized mice were treated for 3consecutive days with anti-CD4 or anti-CD8 mAb. Another group ofimmunized mice was left untreated. Immunized and control micewere challenged with P. yoelii sporozoites 2 days after the lastantibody dose. The amount of plasmodial rRNA obtained from thelivers of four mice is expressed as the mean SEM.

unlike influenza virus, is known to infect liver cells (32), a

possible explanation for the observed difference of results isthat a booster injection with vaccinia might increase themigration of CTLs to this target organ, including thoseinduced by influenza ME virus. When the sequence ofimmunization is reversed (i.e., immunization with vacciniafollowed by influenza), CTLs might be recruited away fromthe liver to the lung. If corroborated experimentally, thiswould suggest that localization of the CTLs in the liver,before sporozoite infection, is essential to achieve an efficientprotective response against the intrahepatocytic stages ofmalaria parasites.We have shown that the protection induced by immuniza-

tion with these recombinant viruses is mostly mediated byCD8+ T cells. It should, therefore, be possible to furtherenhance this protection by eliciting high titers of anti-sporozoite antibodies and CD4+ T cells, known to havestrong anti-parasite effects (33, 34).

Finally, it is worth emphasizing that the present resultsdemonstrate that recombinant influenza viruses provide anefficient approach to the induction of CTLs directed againstintracellular microorganisms. This finding and work demon-strating that immunization with recombinant influenza virusinduces antibodies against foreign epitopes (19) indicate thatrecombinant influenza viruses should be considered as can-

didates for the development of vaccines against infectiousintracellular pathogens, including malaria parasites. Our find-ing that immunity against malaria can be greatly enhanced bythe successive use of two distinct live vectors may lead toadditional strategies for the development of vaccines againstmicrobial pathogens.

S.L. and M.R. are both first authors of this study. We thank ChuiNg, Rita Altszuler, and John Hirst for excellent technical assistance.We also thank Dr. M. A. Davitz for the P. yoelii CS gene. M.R. issupported by fellowships from the Cancer Research Institute andPEW foundation. D.R. and J.R. are recipients of a fellowship fromthe Ministerio de Education y Ciencia of Spain. This work was

supported by grants from National Institutes ofHealth (AI-24460 and

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