Brief communication

Interferon-g-independent CD8þ T cell-mediated protective

anti-malaria immunity elicited by recombinant adenovirus

E.G.RODRIGUES1, J.CLAASSEN1, S.LEE1, J.M.WILSON2, R.S.NUSSENZWEIG1 & M.TSUJI1

1Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, NY 10010 and2Institute for Human Gene Therapy, Department of Molecular and Cellular Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA

SUMMARY

Recombinant adenovirus, expressing the CS protein ofPlasmodium yoelii, AdPyCS, was shown to induce a com-parable degree of T cell-mediated protection againstmalaria as a single dose of irradiatedP. yoelii sporozoites,causing inhibition of liver stage development. We nowreport that differently from sporozoite-induced immunity,interferon (IFN)-g does not mediate the protective immunityinduced by AdPyCS, since a similar degree of protectionwas observed in AdPyCS immunized mice lacking IFN-g–/–

and the IFN-g receptor (IFN-gR–/–) compared to that inwild-type mice. Depletion of CD8þ T cells from theseimmunized mice almost completely abolished the AdPyCS-induced immunity, indicating that the immunization withAdPyCS induces CD8þ T cell-mediated protective anti-malaria immunity, which is independent of IFN-g.

Keywords liver stages,Plasmodium yoelii,recombinantadenovirus, IFN-g, CD8þ T cells, protection

INTRODUCTION

Immunization with radiation-attenuated sporozoites wasshown to induce full protection against malaria challengein rodents, monkeys, and humans (Nardin & Nussenzweig1993). In two rodent malaria models,Plasmodium bergheiandP. yoelii, this protection was shown to be antibody andinterferon (IFN)-g mediated (Nardin & Nussenzweig 1993).However, when a single dose of irradiated sporozoites wasused to immunize the mice, protective immunity wasinduced in the absence of significant amounts of anti-sporozoite antibodies (Tsujiet al. 1995). Furthermore, asingle immunizing dose of irradiated sporozoites failed toelicit protective immunity in mice lacking the IFN-g recep-tor, indicating the major role of IFN-g in the protectiveimmunity induced by a single immunizing dose of spor-ozoites (Tsujiet al. 1995). The immune competent cells thatmediate this protection are CD4þ and CD8þ a/b T cells andpossiblyg/d T cells and NK cells (Schofieldet al. 1987, Weisset al. 1988, Rodrigueset al. 1993, Weisset al. 1993, Seguinet al. 1994, Tsujiet al. 1994, Doolan & Hoffman 1999).

We recently demonstrated that a single immunizing doseof a recombinant adenovirus expressing the circumsporo-zoite (CS) protein ofPlasmodium yoelii, AdPyCS, elicits ahigh degree of resistance to infection mediated primarily byCD8þ T cells (Rodrigueset al. 1997). This led us tocompare the effector mechanism(s) of these two immuno-gens, irradiated sporozoites and AdPyCS. A single immu-nizing dose of these two immunogens elicited similar levelsof protection, resulting in 95–97% reduction of the parasiteload in the liver, as assessed by measuring the amounts ofparasite ribosomal RNA. However, immunization with asingle dose of AdPyCS elicited approximately 10 times asmany CS-specific CD8þ T cells as a single immunizing doseof irradiated sporozoites (Rodrigueset al. 1998). Thepurpose of the current study was to determine and compare

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Correspondence: Moriya TsujiReceived: 28 July 1999Accepted for publication: 15 November 1999

the role that IFN-g plays in the protective anti-malariaimmunity elicited by AdPyCS versus sporozoites.

P. yoelii sporozoites (17X NL strain) were obtained byalternate cyclic parasite passage inAnopheles stephensiandblood transfer in mice (Rodrigueset al. 1993). Sporozoiteswere obtained from mosquito salivary glands approximately

2 weeks after the infective blood meal. A replication-defective recombinant adenovirus encoding amino acids1–356 of the CS protein ofP. yoelii was engineered, aspreviously described (Rodrigueset al. 1997). This recom-binant adenovirus (AdPyCS) contains a small deletion in theE3 region, and its E1 region has been replaced by the gene

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Figure 1 Anti-malaria protection in IFN-g–/– and IFN-gR–/– mice as well as their respective control, wild-type mice elicited by irradiatedP. yoelii sporozoites or AdPyCS. Groups of five IFN-g–/– mice and wild-type BALB/c mice (a) or groups of five IFN-gR–/– mice and wild-type(129/Sv/Ev X B10.D2)F2 mice (H-2d) (b) were immunized with a single s.c. dose of 108 AdPyCS or AdlacZ or i.v. with 2×105 irradiatedsporozoites. Twelve days later, all mice were challenged i.v. with 1×105 viable P. yoelii sporozoites, and the parasite development in the liverswas measured by RT-PCR.

Figure 2 Effect of the selective depletion of T cell subpopulations on intrahepatocytic parasite development in IFN-g and IFN-gR mice afterimmunization with a single dose of AdPyCS.Groups of four IFN-g mice (a) and four IFN-gR mice (b) were immunized s.c. with a single dose of108 pfu of AdPyCS. On days 7 and 9 after immunization, the mice were injected i.p. with two doses of 0·75 mg each of anti-CD4þ (GK1·5) oranti-CD8þ (YTS169) or both mAbs. Three days after the last mAb inoculation, the mice were challenged i.v. with 1×105 viable P. yoeliisporozoites, and the amount of rRNA was calculated as described in Brownet al. (1986). The percentage of inhibition was calculated incomparison with the immunized, nondepleted group.

coding for the CS protein under a cytomegaloviruspromoter/enhancer (Davidsonet al. 1993).

IFN-g deficient mice on the BALB/c background (Daltonet al. 1993), BALB/c wild-type controls, B10.D2 and129Sv/Ev mice were all purchased from the JacksonLaboratories (Bar Harbor, ME, USA). Mice with a targeteddisruption of the IFN-gR gene (kindly provided by Dr M.Aguet of the University of Zurich, Switzerland) werederived from the 129/Sv/Ev strain, carrying the H-2b hap-lotype (Huanget al. 1993). Since the only known CD8þ Tcell epitope in the CS molecule is restricted by H-2Kd

(Rodrigueset al. 1991), we generated IFN-gR–/– micecarrying the H-2d haplotype. IFN-gR–/– mice were crossedwith B10.D2 (H-2d) mice, and we obtained IFN-gR–/– micecarrying only the H-2d haplotype. The screening of micehomozygous for the IFN-gR gene knockout was carried outby polymerase chain reaction (PCR) using specific primers(Huanget al. 1993). Screening of mice bearing H-2d/d wasperformed by flow cytometric analysis. Mice which pre-sented peripheral blood lymphocytes (PBL) that stainedwith monoclonal anti-H-2Kd antibody conjugated to FITC,but not with FITC-anti-H-2Kb antibody (Pharmingen, SanDiego, CA, USA), were selected. Wild-type control micewere obtained in a similar manner.

In order to determine the role that IFN-g plays in theprotective immune response elicited by AdPyCS, groups offive IFN-g–/– mice, on a BALB/c background, and fivecontrol wild-type BALB/c mice were immunized with asingle s.c. dose of 108 p.f.u of AdPyCS or i.v. with 2×105

irradiated sporozoites. Twelve days later, all mice werechallenged i.v. with 1×105 viableP. yoelii sporozoites, andthe amount of parasite ribosomal RNA present in the liverwas determined 40 h after the challenge using competitivereverse transcription (RT)-PCR (Brioneset al. 1996). Thepercentage of inhibition of intrahepatic parasite developmentwas calculated in comparison with the rRNA values obtainedby sporozoite inoculation of nonimmunized animals.

A single immunizing dose of AdPyCS elicited a protec-tive response which strongly inhibited parasite developmentin the liver in both IFN-g–/– and wild-type mice, resulting inmore than 95% inhibition, compared to controls (Figure 1a),demonstrating that the protective response elicited byAdPyCS is IFN-g independent. In contrast, as demonstratedearlier, the immunization of IFN-g–/– mice with a singledose of irradiated sporozoites failed to elicit a protectiveresponse (Figure 1a). It is unlikely that antibodies elicited byAdPyCS are responsible for the observed protection, sinceimmunization of IFN-g–/– mice with AdPyCS developedmoderate antisporozoite antibody titres, which are in factcomparable to those elicited after sporozoite immunization,as determined by indirect immunofluorescence assay (IFA)(Figure 1a).

A group of five IFN-gR–/– mice was also immunizedwith a single dose of AdPyCS or irradiated sporozoites.When these IFN-gR–/– mice were immunized with a singledose of AdPyCS, followed by challenge with viablesporozoites, we observed a strong inhibition of parasitedevelopment in the liver. The degree of inhibition wasvery similar to that observed in immunized wild-type mice(Figure 1b). In contrast, immunization with irradiatedsporozoites failed to induce protective immunity in IFN-gR–/– mice (Figure 1b). As shown in Figure 1(a,b), theparasite development in the livers of both IFN-g–/– andIFN-gR–/– mice was similar to that of respective controlwild-type mice infected with sporozoites. In addition, asexpected, a single immunizing dose of a recombinantadenovirus expressingEscherichia coli lacZ, AdlacZ,failed to inhibit the parasite development in the livers inboth BALB/c and 129×B10.D2 wild-type mice (Figure1a,b). Similar titres of antisporozoite antibodies were devel-oped in the sera of IFN-gR–/– mice immunized withirradiated sporozoites compared to those elicited afterAdPyCS immunization (Figure 1b).

The T cell subpopulation primarily responsible for med-iating the protection induced in IFN-g–/– mice, immunizedwith AdPyCS, was identified by depleting these mice ofCD4þ, CD8þ or both T cell subsets. Briefly, a group of fourIFN-g–/– mice were immunized s.c. with a single dose of108 pfu of AdPyCS. On days 7 and 9 after immunization, themice were injected i.p. with two doses of 0·75 mg each ofanti-CD4þ (GK1·5) or anti-CD8þ (YTS169) or both mAbsas described (Rodrigueset al. 1997). Three days after thelast mAb inoculation, the mice were challenged i.v. with1×105 viable P. yoelii sporozoites, and the amount ofplasmodial rRNA was determined (Brioneset al. 1996).The depletion of CD8þ T cell subpopulation abolishedalmost completely the protective immunity induced byAdPyCS in IFN-g–/– mice, while the depletion of CD4þ Tcells did not affect their level of protection (Figure 2a). Thedepletion of both CD4þ and CD8þ T cell subsets fromAdPyCS-immunized, IFN-g–/– mice diminished the protec-tive response to a similar extent than that observed in micedepleted of CD8þ T cells. These results corroborate themajor role of CD8þ T cells in mediating the protectionelicited by AdPyCS, also in the absence of IFN-g, andsupport the idea that humoral response does not play asignificant role in mediating AdPyCS-induced protectiveimmunity. Similar results were obtained in AdPyCSimmunized IFN-gR–/– mice (Figure 2b).

It has been shown that protective immunity induced byirradiated sporozoites is dependent on IFN-g, and that CD8þ

as well as CD4þ T cells mediate this immunity (Schofieldet al. 1987, Weisset al. 1988, Rodrigueset al. 1993, Weisset al. 1993, Seguinet al. 1994, Tsujiet al. 1995, Doolan &

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Hoffman 1999). Although both AdPyCS and sporozoitesinduce an immune response that is targeted against the liverstages of malaria parasites, our current studies demonstratethat the protective immunity induced by AdPyCS is, in sharpcontrast to that induced by sporozoites, independent ofIFN-g. Depletion studies indicate that AdPyCS-inducedprotective immunity is primarily mediated by CD8þ T cells.These suggest that the CD8þ T cells that mediate AdPyCS-induced protective immunity can display their antiparasiticactivity in the absence of IFN-g. It is likely that the CD8þ Tcells induced after AdPyCS immunization utilize perforin, Fasor tumour necrosis factor, as effector molecules to exert theirantiparasitic activity. This issue is currently elucidated usingmice lacking the respective molecules.

A possible explanation for the different antiplasmodialeffector mechanisms between sporozoites and AdPyCS isthat while the predominant T cell subset that mediatesAdPyCS-induced protection comprises CD8þ T cells, pro-tective immunity induced by sporozoites is mediated bymore than one subset of T cells, including CD4þ and CD8þ

and possiblyg/d T cells and NK cells (Rodrigueset al. 1993,Weiss et al. 1993, Tsujiet al. 1994, Doolan & Hoffman1999). Therefore, in view of the evidence provided by manystudies showing that IFN-g is one of the major effectormolecules of CD4þ T cells, it is plausible to assume thatsporozoite-induced immunity is more dependent on IFN-g

than that induced by AdPyCS.

ACKNOWLEDGEMENTS

We thank Dr F. Zavala for reviewing the manuscript. Thiswork was supported by a grant AI-40656 from the NationalInstitute of Health. E.G.Rodrigues was supported by afellowship from the Brazilian National Research Council.

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