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Oral Salmonella: malaria circumsporozoite recombinants induce specific CD8+ cytotoxic T cells

Oral Salmonella: malaria circumsporozoite recombinants induce specific CD8+ cytotoxic T cells Oral Salmonella : Malaria Circumsporozoite Recombinants Induce Specific CD8+ Cytotoxic T Cells By Anita Aggarwal," San .ai Kumar,$ Richard Jaff'e,* David Hone,§ Mitchell Gross,11 and Jerald Sadoff* From the 'Department of Bacterial Diseases, Walter Reed Army Institute of Researrh, Washington, DC 20307; the tLaboratory Parasitic of Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892; the §Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland 21201; and the IlDepartment of Molecular Genetics, Smith Kline and French Laboratories, Suvdeland, Pennsylvania 19406 Summary Oral immunization with an attenuated Salmonella typhimurium recombinant containing the full- length Plasmodium berghei circumsporozoite (CS) gene induces against protective immunity P. berghei sporozoite challenge in the absence of antibody. We found that this immunity was mediated through the induction of specific CD8+ T cells since in vivo elimination of CD8+ cells abrogated protection . In vitro studies revealed that this Salmonella-P. berghei CS recombinant induced class I-restricted CD8+ cytotoxic T cells are directed P that against the berghei CS peptide epitope spanning amino acids 242-253 . This is the same peptide that previously was identified as the target of cytotoxic T lymphocytes (CTL) induced by sporozoite immunization. Salmonella-P. fkiparum CS recombinants were constructed that contained either the full-length CS gene or a repeatless gene consisting of CS flanking sequences . Both of these vaccines were able to induce CD8+ CTL directed against P. fakiparum CS peptide 371-390, which is identical to the target of CTL induced by sporozoites and vaccinia CS recombinants. These results directly demonstrate the ability of an intracellular bacteria such as Salmonella to induce class I-restricted CD8+ CTL and illustrate the importance CTL of CD8 + in immunity to malaria . alaria occurs in of repeat hundreds millions ofpeople and kills region ofthe circumsporozoite protein in terms of the M one to two million children every year (reviewed in numbers of sporozoites that can be protected against (9) . reference 1) . A vaccine to prevent malaria needed is because Recent work has shown that in some mouse strains after control measures, including the use of antibiotics and insec- immunization with radiation-attenuated sporozoites, CD8+ ticides, have been ineffective in most parts the T cells of developing (cytotoxic/suppressors) are induced that are involved world . Under experimental conditions, humans have been in protection against malaria . Protection can be eliminated protected from Plasmodiumfkiparum malaria by by in depletion immuniza- vivo of CD8+ T cells (10, 11) . CTL from tion with the bites of hundreds of infected mosquitoes that P. berghei-immunized mice recognize the protein CS on the have been irradiated (2) . Animals can also immunized in surface infected be of hepatocytes and eliminate these cells from this manner or by the direct injection of radiation-attenuated the culture in a genetically restricted manner (12) . P.f kiparum sporozoites (3) . This protection is mediated in part sporozoites by anti- have been shown to induce circumsporozoite pro- bodies, some of which are directed against the repeat region tein-specific CD8+ CTL (13), and the epitope these CTL of the circumsporozoite (CS)l which are protein, covers the directed against has been mapped to amino acid position sporozoite surface (4-7) . Early animal experiments, however, 370-391 . More recently, cloned cytotoxic T cell lines directed also demonstrated that 1A-suppressed mice, which lack B cells against P. berghei CS protein (position 249-260) have been and circulating Igs, could be immunized with radiation- shown to passively transfer protection against challenge (14) . attenuated sporozoites, indicating that T cells alone are We have previously reported that an orally administered sufficient for sporozoite-induced immunity in mice (8) . This live attenuated Salmonella recombinant vaccine, which ex- cellular immunity is more potent than antibody against the pressed the full-length P. berghei circumsporozoite antigen, protected mice against malaria (15) . Because the protection was achieved in the absence of antibody, we postulated that cellular immune mechanisms were involved. We proposed ' Abbreviations used in this paper: CS, circumsporozoite; MF, microfluoro- metry ; RLF, repeatless fragment . that intracellular targeting of the Salmonella recombinant or- 1083 The Journal of Experimental Medicine " Volume 172 October 1990 1083-1090 ganisms led to expression of CS antigen on the cell surface Immunization Protocol. BALB/c (H2d) or B10.BR (H2t) female mice, 5-6 in association with class I MHC molecules wk old, were immunized with Salmonella-P. berghei or with the subse- Salmonella-P. falciparum CS recombinants, respectively. Mice were quent induction of specific CTL . To test this hypothesis, we immunized orally with three doses of 10' Salmonella CS recom- constructed Salmonella typhimurium-P berghei CS and S. typhi- binants on alternate days . Salmonella were cultured from liver murium-P . falciparum CS recombinants, immunized mice, and homogenates for up to wk . tested for CS-specific CTL. We now report that orally ad- Challenge. wk after primary immunization with Salmonella- ministered Salmonella-CS recombinants induce CS-specific P. berghei CS recombinants, mice were injected intravenously with CTL . In vivo depletion experiments demonstrate that these 1,000 NK65 P . berghei sporozoites . Thin blood films were made CS-specific CTL are responsible for the protection against every day beginning 5 d after challenge, Giemsa stained, and scanned P berghei . for parasites in 50 oil immersion fields . Mice were considered pro- tected ifno parasites were detected by day 21 after challenge. Serum was collected from individual mice, and anti-CS antibody was de- Materials and Methods tected using an ELISA (15) . Depletion ofT Lymphocytes. Groups of BALB/c mice immunized Plasmids . pADE171 (16) was kindly provided by Dr. David either withWR 4024/pMGB2 or WR4024, or unimmunized mice, Hone, University ofMaryland (Baltimore, MD), and pMGB2 (15), were depleted of CD8' T cells or CD4 ` T plasmid csp, cells by the method AROR-16 and plasmid AR58 repeatless/NS (Gross, of Weiss 4 et al. (10) . Briefly, wk after the immunization, mice M., D.R . Sylvester, G. Sathe, andT Theisen, manuscript submitted were injected intraperitoneally with ofanti-CD8 antibody for publication) were from the Department of Molecular Genetics, 300,ug anti-Lyt-2 .2 hybridoma clone 19/178 (mouse IgG 2a) (25), or anti- Smith Kline Laboratories (Swedeland, PA) . CD4 antibody, anti-L3T4 clone GK 1.5 (rat IgG 2b (26) for four Bacteria. S typhimurium r - S . LB 5010 m*, typhimurium WR4024 successive days . These antibodies were purchased from Bioproducts trp R- were the generous gifts of Dr. Louis Baryon, Walter Reed for Science, Inc. (Madison, WI), and were kindly purified by . Army Institute Dr of Research (Washington, DC) . Walter Weiss, Naval Medical Research Institute, Rockville, MD. Construction ofSalmonella-P. berghei CS Recombinants . The full- 3 d after the last depletion dose, spleen cells from antibody-treated length CS gene of berghei strain was P . NK65 cloned in the Stul mice were stained with FITC-conjugated goat anti-mouse IgG2a restriction site of the PL-based plasmid expression vector, pMG27 or goat anti-rat IgG2b (Kierkegaard & Perry Laboratories, Inc ., NSterm, as described elsewhere in detail Escherichia coli, (17, 18) . In Gaithersburg, MD). Depletion was quantitated microfluorom- by pMGB2 expresses full-length CS protein with six additional amino etry (MF) . Mice were challenged with 1,000 NK65 P berghei acids at its NH2 terminus (Met, Pro, Trp, Arg, Lys) Plasmid Asp, . sporozoites intravenously the fourth day . on of depletion These pMGB2 was purified (19) and transformed into Salmonella typhimu- mice received additional injections of 300 of anti-CD8 or anti- rium WR 4024 trp R- . This strain is a derivative of WR4017, Ftg CD4 antibody every third day for 21 d after challenge . A control which was previously known as strain M206, an avirulent strain group of mice received the same dose of a mouse IgG2a on the with impaired ability to multiply within macrophages (20, 21) . same schedule. Ampicillin-resistant colonies were examined for the expression of for CTL Assay P. berghei Experiments. TheCTL assay was per- P . berghei CS antigen . formed as described by Townsend et al. (27) . Spleen cells from of . CS . Construction Salmonella-P fakiparum Recombinants R16 WR4024/pMGB2, WR4024, or sporozoite-immunized BALB/c CSP, containing repeats of the CS repeat region fused 16 to the mice (haplotype H-2d) were stimulated in vitro in the presence of full-length CS gene ofP .falciparum, strain 7G8, and NS 181 repeat- various concentrations of the five different P . berghei CS peptides less fragment (RLF), containing the first 81 amino acids of influenza listed in Table 2 . Cells were harvested after 6 d in culture, counted, NS 1 protein fused to the CS gene containing no repeat sequences and incubated for 6 h at E/T ratios from 2:1 to 50 :1 with 5,000 (RLF), were digested by Bg1II and Sall from plasmids AR 13R-16 5'Cr-labeled P815 (haplotype H-2d) or EL4 (haplotype H-2b) target csp and AR 58 repeatless/NS, respectively (Gross, M., D.R . Syl- cells in the presence of each individual peptide. Controls included vester, G. Sathe, andT Theisen, manuscript submitted for publi- incubation with 5'Cr-labeled cells the absence of peptide. After in cation) . The gel-purified inserts R16 CSP and NS 181 RLF were 6 h of incubation at 37 °C, the plates were centrifuged, and 100 blunt-end the site plasmid pADE171(16), which ligated into PstI of ul of supernatant was removed to measure chromium release . Per- carries the his OGD region ofS. typhimurium, resulting in plasmids cent lysis determined (experimental - was as 100x cpm medium containing the malaria gene flanked by S. typhimurium his OGD control cpm)/(detergent-released cpm - medium control cpm) . sequences (Fig . 1, Table 1) . Plasmids pADE171 R16 and pADE171 CTL Assays for P. fakiparum Experiments The methods used RLF were purified and used to transform strain LB 5010, which for cytotoxicity assays and to identify peptide target epitope(s) in is an r-m* S. typhimurium strain . Spectinomycin-resistant colonies P. fakiparum CS protein were essentially the same as described by were for the expression of CS gene. examined Kumar et al . (13) . Spleen cells (5 x 106 ) from B10.BR (H-2t) mice Plasmid pMGB2, pADE171 R16, and pADE171 RLF were trans- immunized orally with WR4024/pADE171 R16, WR4024/ formed into S. typhimurium WR4024 trp R- by a modification pADE171 RLF, WR4024, or recombinant vaccinia (V-71) were in- (22) of standard methods (23) Transformants WR4024/pMGB2 cubated with 2 x 10 5 CS-transfected L cells or with 2 x 105 un- expressing P . berghei CS were identified by mAb 3.28 1 (24) . transfected L cells. Cells were harvested after 6 d in culture, counted, Salmonella transformants WR4024/pADE171 R16 and WR4024/ and incubated for 6 h at various E/T ratios with 5,000 "Cr- pADE171 RLF were examined for the expression of P . fakiparum cells . labeled CS-transfected L cells or labeled untransfected L Per- using 49 .41) 9.1 and rabbit antirepeatless anti- CS antigen mAb cent specific lysis was determined as described above. body 579.15, respectively . mAb 49.4D 9.1 (IgG) was made against To identify the CTL epitope, spleen cells from immunized mice the full-length CS gene by Dr. K. Esser, Walter Reed Army Insti- were in L cells for d . stimulated vitro with CS-transfected 6 Puta- . raised against the repeat- tute of Research Polyclonal rabbit sera CS tive CTL were then incubated with untransfected L cells at an E/T less molecule NS 1 81RLF 9 was a kind gift of Dr. D.M . Gordon ratio of 40 :1 for 6 h in the presence of various concentrations of (Walter Reed Army Institute of Research) . 1084 Salmonella Induces Protective Immunity against Malaria peptide 371-390 (DELDYENDIEKKICKMEKCSS) . Peptide res- nization provided protection in 55% of these animals, whereas idue 291-310 was used as control. Killing was measured in 100 all of the unimmunized and 94% of the mice receiving the pl of supernatant as described above. WR4024 carrier strain were infected (Table 3) . No antibody To demonstrate that CS-specific CTL were CD8+ cells, CS- in the serum of individual mice could be demonstrated against specific CTL were incubated with anti-CD8 19/178) mAb (clone recombinant CS protein using ELISA techniques (data not (25) followed by complement . Washed cells were then stained with shown) . Mice immunized intravenously with 50,000 irradi- FITC-conjugated goat anti-mouse IgG2a, and depletion was quan- ated sporozoites were fully protected . titated by microfluorimetry. Cells were incubated with 5,000 s'Cr- To determine the role of CD8+ T cells in protection, labeled transfected L cells or untransfected L cells for 6 h at various mice were depleted of CD8' T cells by intraperitoneal in- E/T ratios . The percent lysis of target cells bycomplement-treated or CD8-depleted CTL was determined. jections of anti-CD8 mAb 4 wk after immunization . This procedure resulted in removal of >98% of the CD8+ T cells from the spleens of these animals (data not shown) . The deple- Results tion was confirmed by surface phenotyping of CD8+ T cells For construction ofSalmonella-P. berghei CS recombinants, from the spleen by single-color indirect fluorescent staining expression plasmid pMGB2 containing the full-length P. ber- and monitored by microfluorometry. Elimination of CD8+ ghei CS gene was transformed into S. typhimurium WR4024, T cells abrogated the protection after immunization with the an avirulent strain . Ampicillin-resistant transformants were Salmonella recombinant vaccine WR4024/pMGB2 . Protec- examined for the expression of the CS protein . One of the tion was reduced from 55% to 5% (Table 3) . CD8+ transformants, P WR4024/pMGB2, expressing berghei CS cell-depleted control mice were all infected after challenge. protein, was selected for further studies (Table 1) . Mice depleted of T CD4 cells did not lose their immunity For construction . of Salmonella-P falciparum CS recom- (data not shown) . These results indicate that CD8+ T cells binants, plasmid pADE171 R16 containing the full-length are the mediators of protection induced by Salmonella recom- P. falciparum CS gene and plasmid pADE171 RLF containing binant vaccines . However, in contrast to immunity induced the flanking regions without the repeats were constructed by Salmonella recombinant vaccines, immunity achieved by (Fig. 1) . The plasmids were Typhimurium transformed into S. immunization with radiation-attenuated sporozoites could WR4024, and spectinomycin-resistant transformants were not after be eliminated even complete depletion of CD8+ examined for expression of CS antigens . of these Two trans- T cells (Table 3) . formants were picked for further studies (Table 1) . To further characterize the CD8+ T cells induced by Protection studies were performed by immunization of Salmonella-P. berghei CS recombinant immunization, in vitro BALB/c mice with Salmonella-P. berghei CS recombinants . T cytotoxic cell assays were performed. For these assays, we Mice were immunized orally with WR4024/pMGB2 or used the methods ofTownsend et al. (27), where target cells WR4024 and were intravenously challenged 5 wk later with are made by incubation of histocompatible cells with short 1,000 P . berghei infectious . This (NK65) sporozoites immu- synthetic peptides that associate with class I molecules . We Table 1 . Salmonella-CS Recombinant Vaccines Immunogen Plasmid Gene Species WR4024/pMGB2 pMGB2 Full-length CS P. berghei WR4024/pADE171 R16 pADE171 Full-length CS P. falciparum WR4024/pADE171 RLF pADE171 Flanking regions P. falciparum WR4024 None None Carrier Table 2 . Plasmodium berghei CS Peptides Sequence Region Residue ADAPEGKKNEKKNEKIERNN NHz terminus 69-78 (DPPPPNPN)3 Repeat 102-124 NDDSYIPSAEKI COON terminus 242-253 SYIPSAEKILEFVKQQIRDSITEEWSQ COOH terminus 245-270 CFVKQIRDSITEEWSQCNVTCG COOH terminus 256-276 1085 Aggarwal et al . Bglll BamH[ The spleen cells from mice immunized with WR4024/ pMGB2 caused 24%D specific lysis of P815 (H-2d) R11 ANCHOR target cells APL I RIB RI (NANP)37(NVDP)4 R-16CSP incubatedwith peptide 242-253 at a concentration of50 NAg/ml and an E/T ratio of 40 :1 (Fig . 2) . Spleen cells from WR4024/ pMGB2-immunized mice had no Cytolytic activity without 99111 BaMH1 SalVmol in vitro peptide stimulation . In vitro stimulated cells did not show activity if the target cells were not incubated with pep- NS181 R1 R11 APL tide cells 242-253 . Spleen from unimmunized mice or mice NS1 81RLF immunized with the carrier Salmonella WR4024 and stimu- lated with the peptide 242-253 had cytolytic activity, <4%D ECORI Hn0111 ECORI indicating that the CTL arose after immunization and were Aalil Pa11 (E 0 not vitro due to in peptide stimulation. Specific Cytolytic ac- pADE171 tivity of34%D was measured with peptide 242-253-stimulated spleen cells from mice immunized with radiation-attenuated sporozoites. None of the other four peptides stimulated spleen cells from mice immunized either with Salmonella recombinants or Psll/SaII radiation-attenuated sporozoites . These peptides also failed to generate P815 cell targets. There was no specific lysis of P815 cells after incubation with any of the five peptides at RECOMBINANT the concentration used, demonstrating that they were not directly toxic to cells . The CTL lytic activity generated by Construction of WR4024/phDE171 R16 and WR4024/ Figure 1 . WR4024/pMGB2 immunization was genetically restricted . pADE171 RLF. The plasmid containing R16 full-length or NSI repeatless peptide After stimulation of spleen cells with 242-253, no gene P . fakiparum was digested with Bg1II and Sall . The gel-purified CS of specific lytic activity was observed when EL4(H-26) cells in- which insert was blunt-end ligated into the Pstl site of plasmid phDE171, cubated with the same peptide were used as targets carries his OGD region of Salmonella, resulting in the plasmid containing CS gene flanked by Salmonella his OGD . We asked whether Salmonella recombinant vaccines could also induce CTL responses to the human malaria parasite Pfalciparum . For this purpose, spleen cells from B10 . BR(H- the P. berghei CS protein based on 2k) mice immunized with WR4024/pADE171 R16, designed peptides from the sequence identified previously by Romero et al . (14) as WR4024/pADE171 RLF, WR4024, or recombinant vac- full-length a CTL target . Five different peptides from NH2 terminus, cinia (V-71) containing P. fakiparum CS gene were repeat region, and COOH terminus were used (Table 2) . stimulated in vitro for 6 d with CS-transfected L cells (H- either Salmonella- 2k) . cells for lytic activity Spleen cells from mice immunized with These were then assessed against P. berghei CS recombinant WR4024/pMGB2, S. typhimurium transfected or untransfected L cells. At an E/T ratio of 40 :1, were taken 4-10 wk after in three different sets ofexperiments, we observed 37 ± 2.2% carrier WR4024, or sporozoites immunization . After in vitro stimulation with peptide for specific killing ofCS gene-transfected cells by CTL induced by WR4024/pADE171 R16 compared with 20 ± 2.5% 6 d, the killing was measured by the "Cr release . DP015 a01wiUpeptic Table 3 . Protective Immunity Induced by S. typhimurium ®PB15a01wit estpwide (WR4024) Transformed with a Plasmid Expressing Full-Length P. berghei CS Gene (WR4024/pMGB2) Anti-CD8 No infected/ Percent Immunogen antibody no . challenged protection 20 6 1o WR40 - 29/31 WR4024 + 8/8 0 I M 1-6 - 10/22 55 0 WR4024/pMGB2 WN40244MG12 'YUZ WR4024/pMGB2 + 18/19 5 Figure 2 . Results of cytotoxic assays performed with P815 target cells - 30/30 0 None and CTL in the presence of synthetic peptides. Spleen cells from mice berghei recombinant vaccine None + 8/8 0 immunized with Salmonella-P . CS were stimu- lated in vitro for 6 d with 50 of peptide, and 6 d after in vitro stimula- Ag Irradiated Spz - 0/10 100 cells in the presence of tion cells were incubated with Cr-labeled P815 + 100 Irradiated Spz 0/10 51C . This experiment is rep- different peptides, and r release was measured resentative of four independent experiments . 1086 Salmonella Induces Protective Immunity against Malaria 50r duced CTL that caused 20 ± 4.5% lysis of untransfected cells. This nonspecific effect was induced by both recombinants . 40 and the control Salmonella vaccine, and contrasts to the 7 ± 1.5% lysis seen with recombinant vaccinia-immunized mice (Fig . 3) . After CD8 depletion, the lysis of untransfected cells by WR4024/pADE R16-induced CTL was reduced from 18 ± 2 4 ± .8 to 2% (p < 0 .05), and killing by WR4024/ pADE171 RLF-induced CTL was reduced from 16 ± 3 0 1 L i~~-k----IVi (7 to 7 ± 1.5% (p 0 .05) . These findings < suggest that the 0 5 10 20 0 5 10 20 40 majority of induced nonspecific cytotoxic T cells were CD8+ EFFECTOR/TARGET RATIO EFFECTOR/TARGET RATIO (Fig. 4) . Immunization of BALB/c mice with Salmonella vac- Figure Malaria Salmonella CS recombinants stimulate CS-specific CTL. 3 . cines followed by in vitro stimulation of spleen cells with In vitro stimulated spleen cells from mice immunized with Salmonella- peptides did not yield CTL reactive with non-peptide-pulsed incubated P. falciparum CS recombinants were with S'Cr-labeled CS- targets. This may be attributed to differences between in vitro transfected (A) or untransfected L cells (B) . Percent specific lysis was de- stimulation with termined . (0) Vaccinia recombinant ; transfected cells vs . in vitro stimulation with CS (A) R16 ; RLF ; (O) vector. (0) This experiment is representative of five independent experiments . peptides . To determine the epitope specificity of CTL induced by oral immunization with Salmonella-P. fakiparum CS constructs in B10 mice, .BR spleen cells from immunized mice were stimulated in vitro with CS-transfected killing of untransfected L cells (p < 0.05) . Likewise, at this L cells. After 6 d, untransfected L E/T spleen cells from animals cells were incubated with 10014g/ml ofpep- ratio, immunized with tide 371-390, single letter code DELDYENDIEKKICK- WR4024/pADE171 RLF killed 36 ± 3.1% ofCS gene-trans- MEKCSS, or peptide fected L cells compared with 4% killing 291-310 from the P.falciparum CS gene 20 ± of untrans- (12), and lysis was determined at an E/T of 40 :1 . Peptide fected L cells (p < 0.05) . These CTL responses were com- with amino acid parable with those obtained after immunization sequence 371-390 was recognized by CTL with the from mice immunized with the two recombinant recombinant vaccinia (Fig. 3) . CTLs from mice immunized Salmonella vaccines or the recombinant with the Salmonella carrier WR4024 gave 20 ± 4.5% 51Cr vaccinia (Fig . 5) . This same peptide release had previously been identified as the target of CTL induced by sporozoite or recombinant vaccinia-CS To determine if the cytotoxic activity in the spleen cell cul- immunization (12) . tures due L cells incubated with peptide 291-310 were not lysed by was to CD8+ T cells, in vitro depletions were'per- immune formed . Removal of CD8+ T cells from the cultures on the CTL above the levels seen with target cells alone (data not shown) . day of the CTL assay was confirmed by MF (data not shown) . WR4024/pADE171 R16-induced CS-specific CTL, when treated with anti-CD8 mAb and complement, yielded only Discussion 5 ± 2.8% specific lysis of CS-specific target cells at an E/T ratio of 40 :1 compared with specific lysis of 37 ± 2.2% (p We have demonstrated that oral immunization of mice with < 0.05), when the cells were treated with complement alone attenuated S. typhimurium recombinants expressing full-length (Fig. 4) . WR4024/pADE171 RLF induced CTL with similar P . berghei CS gene or full-length or repeatless P. fakiparum activity (12 ± 3 .5 vs. 36 ± 3 .1 ; p < 0 .05) . This demon- CS gene induce specific CD8+ CTL . We also provide evi- strated that the CS-specific cytotoxicity was mediated by dence that these CS-specific CTL are responsible for the pro- CD8+ cells . Reductions in specific cytotoxicity by depletion tection induced against P. berghei sporozoites after immuni- of CD8+ T cells similar were for spleen cells from animals zation ofBALB/c mice with Salmonella recombinant vaccines immunized with each of the three vaccines (Fig . 4) . Antibody-dependent cellular cytotoxicity has been shown Immunization of B10.BR mice with Salmonella also in- after immunization of humans with S. tpphi mutant strain Figure 4. Effect of CD8 depletion on CS-specific CTL . In vitro stimulated cells from the miceimmunized with Salmonella-P. falciparum CS recombinants were in- cubated with anti-CD8 mAb (19/178) followed by com- plement . Washed cells were then incubated with 5,000 (B) L 5'Cr-labeled transfected (A) or untransfected cells at various E/T ratios. The percent specific lysis of target cells by complement-treated (/) or CD8-depleted (®) CTL was determined . A representative experiment at the 40 :1 ratio is presented . 1087 . Aggarwal et al 11), induction of CTL in mice against P. falciparum CS by sporozoites (13), and the protective role of CD8+ T cells in immunity to Toxoplasma gondii (36) . There is also evidence that protection against intracellular pathogens such as L . mono- cytogenes, M. tuberculosis, M. leprae, and others may in part be mediated through CD8+ T cells (37) . Our findings directly prove that an intracellular bacteria such as S. typhimu- rium can induce specific CD8+ CTL and suggest that the antigen processing and presentation is probably not very different from that which occurs with vaccinia or sporozoites, since the recognized peptide and quantitative data are very similar. Our finding that Salmonella-P. berghei CS recombinant-in- duced protection is mediated through specific CD8+ cyto- toxic T cells further demonstrates the importance of CTL Figure 5 . Identification of the CTL epitope on theP. fakiparum CS pro- in immunity against malaria . This is consistent with the re- tein . Spleen cells from Salmonella-P. fakiparum recombinant-immunized cent finding that CD8+ CTL clones directed against P. ber- mice were stimulated in vitro with CS-transfected L cells . S'Cr-labeled target cells were incubated with peptide and the in vitro stimulated spleen ghei CS by themselves are able to provide complete protec- cells at an E/T ratio of40 :1 (N) ; or without peptide (®), and S1Cr re- tion (14) . These CTL clones were induced by immunization lease was measured . This experiment is representative of three indepen- with radiation-attenuated sporozoites. The CTL we induced dent experiments . with Salmonella CS recombinants are directed against the same peptide in the CS sequence as the CTL clones defined by Romero et al. (14) . Not all of the CTL clones found by Romero were able to protect . Our study demonstrates that Ty21a (28) or auxotrophic S. typhi 541Ty(Vi+) or 543Ty(Vi - ) at least some of the CTL induced by Salmonella-P. berghei (29) . The effector cell has been identified as a nonadherent CS recombinants are protective. To increase the number of T3 + , T8 - , T4+ lymphocyte (28) . After immunization, protective CTL induced by oral immunization with Salmonella delayed-type hypersensitivity responses to Salmonella carbo- CS recombinants, we are inserting malarial genes into the hydrate antigens (30) and recombinant expressed proteins have bacterial chromosome in an attempt to stabilize and increase been demonstrated (15, 31) . Macrophage activation directly the expression of malaria gene products . also We are utilizing or through T cell-mediated mechanisms has also been seen Salmonella auxotrophs as carrier strains, which are safe but after immunization or infection (32, 33) . To our knowledge, more invasive than the attenuated S. typhimurium WR4024 induction of protective class I-restricted antigen-specific we utilized in this study. by CD8+ CTL immunization or infection with Salmonella We were able to achieve CTL that gave similar levels of has not, however, been previously reported . killing by immunization with Salmonella-P. fakiparum CS After oral ingestion, attenuated are Salmonella vaccines soon recombinant vaccines that expressed the entire gene CS or translocated from the intestinal lumen to an intracellular lo- a gene from which the central repeat region was deleted . Both cation, primarily inside macrophages (30) . Attenuated Sal- of these recombinant constructs produced the malaria CS an- monella carrier strains, such as S. typhimurium WR4024 used tigens as fusion proteins . The complete CS molecule is there- in this study and the aro A mutant of dublin SL1438 used S. fore not required for the in vivo induction of CD8+ CTL by us in other studies (34), have limited ability to multiply by Salmonella, and modification of the original geneby addi- but for periods of within macrophages do survive variable tion also does not interfere . Presentation of antigens in as- time . The intracellular expression ofrecombinant malaria CS sociation with class I molecules is generally dependent on responsible for presenta- antigens is probably processing and intracellular enzymatic cleavage to produce a peptide frag- tion in association with class I antigen with the subsequent ment that can bind to the class I molecule. Our findings sug- induction CTL . Bacterial enzymes may play some role in of gest a possible method for determining the minimal struc- degradation of the recombinant expressed protein before pro- ture from any given molecule that is necessary for such a process cessing by intracellular enzymes, although we have no direct to occur. This could be done by cloning deletion fragments evidence for this possibility. The presence of Salmonella in- as fusion proteins into Salmonella and looking for the ability side macrophages may also activate these macrophages and of these Salmonella recombinants to induce CTL . important as induce the release of cytokines thought to be We were able to determine the Salmonella-P. berghei CS differentiation factors for induction of CTL (35) . recombinant-induced protection by depletion of T CD8+ recently, thought that viruses were Until it was generally cells . This contrasts with our inability to eliminate protec- the only pathogens capable of presenting endogenous antigens tion by this technique in BALB/c mice immunized with on the surface of infected cells in association with class I mol- radiation-attenuated This result may due sporozoites. be to the ecules with subsequent induction of CD8+ CTL . This involvement of antibody, effector CD4+ T cells, other im- view was challenged by the evidence for a protective role of mune cell types or other sporozoite or preerythrocytic an- CD8+ CTL induced by sporozoites in murine malaria (10, tigens in BALB/c immunity to P. berghei . Schofield al . (11) et Salmonella Induces Protective Immunity against Malaria were able to eliminate sporozoite-induced protection in A/J 40) . This inherent safety plus their sensitivity to antibiotics mice by depletion of CD8+ T cells . This shows that the is important for immunization of human populations that im- may to HIV mechanism of radiation in attenuated sporozoite-induced have been exposed munity is dependent on the mouse haplotype . Although we Our findings that recombinant Salmonella constructs pro- were not able to eliminate sporozoite-induced protection in vide some protection and induce CTL effector mechanisms BALB/c mice, Weiss et al . (10) were able to accomplish this, similar to those induced by irradiated and live sporozoites provide a basis for further on the development of oral but the parasite was P. yoelii. work A vaccine for malaria that overcomes the sequence varia- vaccines against malaria . The CTL epitope, which has been differences due genetic identified in the of the human malaria parasite, tion in the CS CTL site as well as to CS protein restriction (13) will likely contain several different versions P . fakiparum, has only been shown to be recognized by primed well other mouse peptide also of the CS CTL site recognized by humans as as lymphocytes . Whether this sequence is sporozoite and liver stage antigens important for CTL rec- recognized by human CTL is currently unknown. Immuni- be constructed in zation of humans with irradiation-attenuated sporozoites or ognition . Such multivalent vaccines can Salmonella because of the size of the bacterial genome and possibly Salmonella recombinants may help answer this ques- the its manipulation . Because new Salmonella tion . These findings suggest that live attenuated Salmonella flexibility of vaccine strains designed for human use are auxotrophs (38), recombinants may also be useful in the study of other dis- CTL mediated immunity may important which can only replicate in the body to a limited extent, they eases where be . have been safe in studies of immunocompromised hosts (39, We wish to thank Ms . Hazel Sidberry for excellent technical assistance, Dr. Carolyn Deal for synthesis of for P. peptide 242-253, Ms. Lynett Smith providing the berghei CS peptides, Dr. Bernard Moss for the vaccinia recombinant, and Drs. Louis Baron, and Louis Miller for advice and helpful suggestions . Address correspondence to Jerald Sadog, Walter Reed Army Institute of Research, Washington DC 20307. Received for publication IS March 1990 and in revised form 8 June 1990 . References 1 . Miller, L.H ., R .J . Howard, R. Carter, M .F. Good, V Nus- effect of immunization of T and B cell deficient mice. J. Im- senzweig, and R.S . Nussenzweig . 1989 . Research towards munol. 118 :1322 . malaria vaccines . Science (Wash . DC). 234 :1349 . 9 . Egan, J .E .,J .L . Weber, WR . Ballou, M.R . Hollingdale, WR. 2 . Clyde, D.F., V McCarthy, R.M . Miller, and WE . Woodward . Majarian, D.M . Gordon, WL . Maloy, S .L . Hoffman, R.A. 1975 . Immunization of man against falciparum and vivax Wirtz, I . Schneider, G.R. Woollett, J .F. Young, and WT malaria by use of attenuated sporozoites . Am .J. Trop Med. Hyg. Hockmeyer. 1987 . Efficacy of murine malaria sporozoite vac- 24:397. cines : implication for human vaccine development . Science 3 . Nussenzweig, V, and R .S. Nussenzweig . 1986 . Development (Wash . DC). 236 :453 . vaccine Trop of a sporozoitb malaria . Am .J. Med. Hyg. 35 :678 . 10. Weiss, WR ., M . Sedegah, R .L . Beaudoin, L.H . Miller, and 4 . Nussenzweig, V, and R .S . Nussenzweig. 1989. Rationale for M .F. Good. 1988. CD8 + T cells (cytotoxic/suppressor) are re- the development of an engineered sporozoite malaria vaccine. quired for protection in mice immunized with malaria sporo- Adv. Immunol. 45:283. zoites . Proc Nad. Acad. Sci. USA . 85 :573 . 5 . Potocnjak, P., N . Yoshida, R .S. Nussenzweig, and V Nus- 11 . Schofield, L ., R. Villaquiran, A. Ferreira, H . Schellekens, R.S . senzweig. 1980. Monovalent fragments (Fab) of monoclonal Nussenzweig, and V Nussenzweig. 1987 . Interferon, CD8+ antibodies to sporozoite surface antigen protect mice T a (Pb44) cells and antibodies are required for immunity to malaria against malaria infection . J. Exp Med. 151 :1504 . sporozoites . Nature (Lond.). 330:664 . 6 . Hollingdale, M.R., F. Zavala, R .S. Nussenzweig, and V Nus- 12 . Hoffman, S .L ., D . Isenbarger, G.W. Long, M . Sedegah, A . senzweig. 1982 . Antibodies to the protective antigen of Plas- Szarfman, L . Waters, M.R . Hollingdale,P .H .V. Meide, D.S . modium berghei sporozoites prevent entry into culture cells. J. Finbloom, . vaccine andWR . Ballou . 1989 Sporozoite induces Immunol. 128 :1929 . genetically restricted T cell elimination of malaria from hepa- 7 . Charoenvit, Y, M .F. Leef, L .F. Yuan, M . Sedegah, and R.L . tocytes . Science (Wash. DC). 244 :1078 . Beaudoin . 1987 Characterization ofP. yoelii monoclonal anti- 13 . Kumar, S., L.H . Miller, I .A . Quakyi, D.B. Keister, R.A . bodies directed against stage specific sporozoite antigen . Infect. Houghten, WL . Maloy, B. Moss, J.A . Berzofsky, and M .F. Immun . 55 :604 Good . 1988 . Cytotoxic T cell specific for the circumsporozoite 8 . Chen, D.H ., R.E . Tigelaar, and F.I . Weinbaum . 1977 . Immu- protein of Plasmodium falciparum . Nature (Lond.). 334 :258 . nity to sporozoite-induced malaria infection in mice. I. The 14 . Romero, P., J.L . Maryanski, G. Corradin, R.S . Nussenzweig, 1089 Aggarwal et al . V. Nussenzweig, and F. Zavala . 1989 . Cloned cytotoxic T cells D. Wraith, andA.J . McMichael. 1986 . The epitopes of influenza recognize an epitope in the circumsporozoite protein and pro- nucleoprotein recognized by cytotoxic T lymphocytes can be tect against malaria . Nature (Lond.). 341:323 . defined with short synthetic peptides . Cell. 44 :959 . 15 . Sadoff, J.C ., W.R Ballou, L.S . . Baron, W.R . Majarian, R.N . 28 . Tagliabue, A., L . Nencioni, A. Caffarena, L . Villa, D. Boraschi, Brey, WT Hockmeyer, J.F . Young, S .J . Cryz, J . Ou, G.H. C. Cazzola, and S . Cavalieri . 1985 . Cellular immunity against Lowell, and J.D . Chulay. Salmonell typhimurium 1988 . a vac- Salmonella typhi live after oral vaccine. Clin . Exp Immunol. cine expressing circumsporozoite protein protects against 62:242 . malaria Science . (Wash . DC). 240:336 . 29 . Levine, M.M ., D. Herrington, J.R . Murphy, J.G . Morris, G. 16 . Hone, D., S . Atteridge, L.V. denBosch, andJ . Hackett . 1988 . Losonsky, B. Tall, A.A . Lindberg, S. Svenson, S . Baqar, and A chromosomal integration system for stabilization of heter- M.F. Edwards. 1987 . Safety, infectivity, immunogenicity, and ologous gene in Salmonella based vaccine strains. Micro& Patholog. in vivo stability oftwo attenuated auxotrophic mutant strains 5:407 . of Salmonella typhi, 541Ty and 542Ty, as live oral vaccines in 17 . Weber, J.L ., andWT Hockmeyer. 1985 . Structure of the cir- humans. 1986 . J. Clin . Invest. 79 :888 . cumsporozoite protein gene in 18 strains of Plasmodium fal- 30 . Lindberg, A.A ., and . J.A Robertson . 1983 . Salmonella typhimu- ciparum . Mol. Biochem . Parasitol . 15 :305 . rium infection in calves : Cell-mediated and humoral immune 18 . Gross, M., Sweet, R.W. G. Sathe, S . Yokoyama, O. Fasano, reactions before and after challenge with live virulent bacteria M. Goldfarb,M. Wigler, andM. Rosenberg . 1985 . Purification in calves given live or inactivated vaccines. Infect. Immun . 41 :751 . and characterization ofhuman H-ras protein expressed in Esch- 31 . Brown, A., C.E . Hormaeche, R. Demarco de Hormaeche, M. erichia coli . Mol. Cell. Biol . 5:1015 . Winther, G. Dougan, . Maskell, andB.A .D . Stocker. 1987 . DJ 19 . Birnboim, H. Doly. 1979 . C., and J . A rapid alkaline extrac- An attenuated araA Salmonella typhimurium vaccine elicits hu- tion procedure for screening of recombinant plasmid . DNA . moral and cellular immunity to cloned J3-galactosidase in mice Nucleic Acids Res. 7:1513 . J. Infect. Dis. 155:86 . 20 . Furness, G., and D. Rowley. 1956 . Transduction of virulence 32 . Collins, F.M . 1974 . Vaccines and cell-mediated immunity. Bac- within the species Salmonella typhimurium . J. Gen . Microbiol. teriol. Rev . . 38 :371 15 :140 . 33 . Schafer, R., C.A . Nary, andT.K . Eisenstein . 1988 . Induction 21 . Furness, G. 1958 . Interactionbetween Salmonella typhimurium of activated macrophages in C3H/Hej mice by avirulent and phagocytic cells in culture. J. Infect. Dis . 103 :272 . Salmonella . . Immunol. 140:1638 . 22 . Ou, J ., D.J . Kopecko, and L.S . Baron . 1986 . Genetic transfor- 34 . Sadoff, J.C., W.R . Ballou, L.S. Baron, J . Ou, W.R . Majarian, mation with large plasmids in Escherichia coli . In Recent Ad- and R. Brey. 1988 . Attenuated oral Salmonella vaccines ex- vances in Chemotherapy. Joji Ishigami, editor. University of pressing circumsporozoite antigen protect against malaria . In Tokyo Press, Tokyo . 383-384 . Technological Advances in Vaccine Development . Alan R. Liss, 23 . Cohen, S.N ., A.C .Y. Chang, and L . Hsu . 1972 . Non- Inc . ., New York . 197-204 chromosomal antibiotic resistance in bacteria : Genetic trans- 35 . Lafferty, K.J ., L. Anders, and S.J . Prowse . 1980 . Role of lym- Escherichia Natl. formation of coli by R-factorDNA. Proc Acad. phokine and antigen in the control of specific T cell response. Sci. USA . 69 :2110 . Immunol. Rev. 51 :279 . 24 . Sidberry, H., B. Kaufman, D.G . Wright, and J . Sadoff. 1985 . 36 . Suzuki, Y., and J . S . Remington . 1988 . Dual regulation of Immunoenzymatic analysis by monoclonal antibodies of bac- resistance against Toxoplasma gondii infection by Ly2' and terial lipopolysaccharides after transfer to nitrocellulose . J. Im- Ly2.1', L3T4+ T cells in mice. J. Immunol. 40 :2943 . munol. Methods. 76 :299 . 37 . Kauffman, S.H.E . 1988 . CD8' T lymphocytes in intracellular 25 . Hammerling, G.J., U. Hammerling, and L. Flaherty. 1979 . microbial infection . Immunol. Today. 9:1668 . Qat-4 and Qat-5, new murine T cell antigens governed by the 38 . Hoiseth, S.K ., and B.A.D. Stocker. 1981 . Aromatic-dependen t Tla region and identified by monoclonal antibodies. J. Exp. Salmonella typhimurium are non virulent and effective as live Med. 150:108 . oral vaccines. Nature (Loud.). 291:238 . 26 . Dialynas, D.P., Z.S. Quan, K.A . Wall, A. Pierres, J . Quintans, . 39 Hoiseth, S.K . 1983 . Aromatic deficient mutants as live M.R . Loken, M. Pierres, and F. Fitch . 1983 . Characterization salmonella vaccines . Ph.D. Thesis, Stanford University, Stan- of the murine T cell surface molecule, designated L3T4, ford, CA. identified by monoclonal antibody GK 1.5 : similarity ofL3T4 40 . Stocker, B.A.D., S.K . Hoiseth, and B.P. Smith . 1983 . Aromatic to the human Leu-3/T4 molecule . J. Immunol. 131:2445 . dependent Salmonella species as live vaccines in mice and calves. 27 . Townsend, A ., J . Rothbard, F.M . Gotch, G. Bahadur, .R.M Dev. Biol. Stand. 53 :47 . 1090 Salmonella Induces Protective Immunity against Malaria http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

Oral Salmonella: malaria circumsporozoite recombinants induce specific CD8+ cytotoxic T cells

The Journal of Experimental Medicine , Volume 172 (4) – Oct 1, 1990

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Abstract

Oral Salmonella : Malaria Circumsporozoite Recombinants Induce Specific CD8+ Cytotoxic T Cells By Anita Aggarwal," San .ai Kumar,$ Richard Jaff'e,* David Hone,§ Mitchell Gross,11 and Jerald Sadoff* From the 'Department of Bacterial Diseases, Walter Reed Army Institute of Researrh, Washington, DC 20307; the tLaboratory Parasitic of Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892; the §Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland 21201; and the IlDepartment of Molecular Genetics, Smith Kline and French Laboratories, Suvdeland, Pennsylvania 19406 Summary Oral immunization with an attenuated Salmonella typhimurium recombinant containing the full- length Plasmodium berghei circumsporozoite (CS) gene induces against protective immunity P. berghei sporozoite challenge in the absence of antibody. We found that this immunity was mediated through the induction of specific CD8+ T cells since in vivo elimination of CD8+ cells abrogated protection . In vitro studies revealed that this Salmonella-P. berghei CS recombinant induced class I-restricted CD8+ cytotoxic T cells are directed P that against the berghei CS peptide epitope spanning amino acids 242-253 . This is the same peptide that previously was identified as the target of cytotoxic T lymphocytes (CTL) induced by sporozoite immunization. Salmonella-P. fkiparum CS recombinants were constructed that contained either the full-length CS gene or a repeatless gene consisting of CS flanking sequences . Both of these vaccines were able to induce CD8+ CTL directed against P. fakiparum CS peptide 371-390, which is identical to the target of CTL induced by sporozoites and vaccinia CS recombinants. These results directly demonstrate the ability of an intracellular bacteria such as Salmonella to induce class I-restricted CD8+ CTL and illustrate the importance CTL of CD8 + in immunity to malaria . alaria occurs in of repeat hundreds millions ofpeople and kills region ofthe circumsporozoite protein in terms of the M one to two million children every year (reviewed in numbers of sporozoites that can be protected against (9) . reference 1) . A vaccine to prevent malaria needed is because Recent work has shown that in some mouse strains after control measures, including the use of antibiotics and insec- immunization with radiation-attenuated sporozoites, CD8+ ticides, have been ineffective in most parts the T cells of developing (cytotoxic/suppressors) are induced that are involved world . Under experimental conditions, humans have been in protection against malaria . Protection can be eliminated protected from Plasmodiumfkiparum malaria by by in depletion immuniza- vivo of CD8+ T cells (10, 11) . CTL from tion with the bites of hundreds of infected mosquitoes that P. berghei-immunized mice recognize the protein CS on the have been irradiated (2) . Animals can also immunized in surface infected be of hepatocytes and eliminate these cells from this manner or by the direct injection of radiation-attenuated the culture in a genetically restricted manner (12) . P.f kiparum sporozoites (3) . This protection is mediated in part sporozoites by anti- have been shown to induce circumsporozoite pro- bodies, some of which are directed against the repeat region tein-specific CD8+ CTL (13), and the epitope these CTL of the circumsporozoite (CS)l which are protein, covers the directed against has been mapped to amino acid position sporozoite surface (4-7) . Early animal experiments, however, 370-391 . More recently, cloned cytotoxic T cell lines directed also demonstrated that 1A-suppressed mice, which lack B cells against P. berghei CS protein (position 249-260) have been and circulating Igs, could be immunized with radiation- shown to passively transfer protection against challenge (14) . attenuated sporozoites, indicating that T cells alone are We have previously reported that an orally administered sufficient for sporozoite-induced immunity in mice (8) . This live attenuated Salmonella recombinant vaccine, which ex- cellular immunity is more potent than antibody against the pressed the full-length P. berghei circumsporozoite antigen, protected mice against malaria (15) . Because the protection was achieved in the absence of antibody, we postulated that cellular immune mechanisms were involved. We proposed ' Abbreviations used in this paper: CS, circumsporozoite; MF, microfluoro- metry ; RLF, repeatless fragment . that intracellular targeting of the Salmonella recombinant or- 1083 The Journal of Experimental Medicine " Volume 172 October 1990 1083-1090 ganisms led to expression of CS antigen on the cell surface Immunization Protocol. BALB/c (H2d) or B10.BR (H2t) female mice, 5-6 in association with class I MHC molecules wk old, were immunized with Salmonella-P. berghei or with the subse- Salmonella-P. falciparum CS recombinants, respectively. Mice were quent induction of specific CTL . To test this hypothesis, we immunized orally with three doses of 10' Salmonella CS recom- constructed Salmonella typhimurium-P berghei CS and S. typhi- binants on alternate days . Salmonella were cultured from liver murium-P . falciparum CS recombinants, immunized mice, and homogenates for up to wk . tested for CS-specific CTL. We now report that orally ad- Challenge. wk after primary immunization with Salmonella- ministered Salmonella-CS recombinants induce CS-specific P. berghei CS recombinants, mice were injected intravenously with CTL . In vivo depletion experiments demonstrate that these 1,000 NK65 P . berghei sporozoites . Thin blood films were made CS-specific CTL are responsible for the protection against every day beginning 5 d after challenge, Giemsa stained, and scanned P berghei . for parasites in 50 oil immersion fields . Mice were considered pro- tected ifno parasites were detected by day 21 after challenge. Serum was collected from individual mice, and anti-CS antibody was de- Materials and Methods tected using an ELISA (15) . Depletion ofT Lymphocytes. Groups of BALB/c mice immunized Plasmids . pADE171 (16) was kindly provided by Dr. David either withWR 4024/pMGB2 or WR4024, or unimmunized mice, Hone, University ofMaryland (Baltimore, MD), and pMGB2 (15), were depleted of CD8' T cells or CD4 ` T plasmid csp, cells by the method AROR-16 and plasmid AR58 repeatless/NS (Gross, of Weiss 4 et al. (10) . Briefly, wk after the immunization, mice M., D.R . Sylvester, G. Sathe, andT Theisen, manuscript submitted were injected intraperitoneally with ofanti-CD8 antibody for publication) were from the Department of Molecular Genetics, 300,ug anti-Lyt-2 .2 hybridoma clone 19/178 (mouse IgG 2a) (25), or anti- Smith Kline Laboratories (Swedeland, PA) . CD4 antibody, anti-L3T4 clone GK 1.5 (rat IgG 2b (26) for four Bacteria. S typhimurium r - S . LB 5010 m*, typhimurium WR4024 successive days . These antibodies were purchased from Bioproducts trp R- were the generous gifts of Dr. Louis Baryon, Walter Reed for Science, Inc. (Madison, WI), and were kindly purified by . Army Institute Dr of Research (Washington, DC) . Walter Weiss, Naval Medical Research Institute, Rockville, MD. Construction ofSalmonella-P. berghei CS Recombinants . The full- 3 d after the last depletion dose, spleen cells from antibody-treated length CS gene of berghei strain was P . NK65 cloned in the Stul mice were stained with FITC-conjugated goat anti-mouse IgG2a restriction site of the PL-based plasmid expression vector, pMG27 or goat anti-rat IgG2b (Kierkegaard & Perry Laboratories, Inc ., NSterm, as described elsewhere in detail Escherichia coli, (17, 18) . In Gaithersburg, MD). Depletion was quantitated microfluorom- by pMGB2 expresses full-length CS protein with six additional amino etry (MF) . Mice were challenged with 1,000 NK65 P berghei acids at its NH2 terminus (Met, Pro, Trp, Arg, Lys) Plasmid Asp, . sporozoites intravenously the fourth day . on of depletion These pMGB2 was purified (19) and transformed into Salmonella typhimu- mice received additional injections of 300 of anti-CD8 or anti- rium WR 4024 trp R- . This strain is a derivative of WR4017, Ftg CD4 antibody every third day for 21 d after challenge . A control which was previously known as strain M206, an avirulent strain group of mice received the same dose of a mouse IgG2a on the with impaired ability to multiply within macrophages (20, 21) . same schedule. Ampicillin-resistant colonies were examined for the expression of for CTL Assay P. berghei Experiments. TheCTL assay was per- P . berghei CS antigen . formed as described by Townsend et al. (27) . Spleen cells from of . CS . Construction Salmonella-P fakiparum Recombinants R16 WR4024/pMGB2, WR4024, or sporozoite-immunized BALB/c CSP, containing repeats of the CS repeat region fused 16 to the mice (haplotype H-2d) were stimulated in vitro in the presence of full-length CS gene ofP .falciparum, strain 7G8, and NS 181 repeat- various concentrations of the five different P . berghei CS peptides less fragment (RLF), containing the first 81 amino acids of influenza listed in Table 2 . Cells were harvested after 6 d in culture, counted, NS 1 protein fused to the CS gene containing no repeat sequences and incubated for 6 h at E/T ratios from 2:1 to 50 :1 with 5,000 (RLF), were digested by Bg1II and Sall from plasmids AR 13R-16 5'Cr-labeled P815 (haplotype H-2d) or EL4 (haplotype H-2b) target csp and AR 58 repeatless/NS, respectively (Gross, M., D.R . Syl- cells in the presence of each individual peptide. Controls included vester, G. Sathe, andT Theisen, manuscript submitted for publi- incubation with 5'Cr-labeled cells the absence of peptide. After in cation) . The gel-purified inserts R16 CSP and NS 181 RLF were 6 h of incubation at 37 °C, the plates were centrifuged, and 100 blunt-end the site plasmid pADE171(16), which ligated into PstI of ul of supernatant was removed to measure chromium release . Per- carries the his OGD region ofS. typhimurium, resulting in plasmids cent lysis determined (experimental - was as 100x cpm medium containing the malaria gene flanked by S. typhimurium his OGD control cpm)/(detergent-released cpm - medium control cpm) . sequences (Fig . 1, Table 1) . Plasmids pADE171 R16 and pADE171 CTL Assays for P. fakiparum Experiments The methods used RLF were purified and used to transform strain LB 5010, which for cytotoxicity assays and to identify peptide target epitope(s) in is an r-m* S. typhimurium strain . Spectinomycin-resistant colonies P. fakiparum CS protein were essentially the same as described by were for the expression of CS gene. examined Kumar et al . (13) . Spleen cells (5 x 106 ) from B10.BR (H-2t) mice Plasmid pMGB2, pADE171 R16, and pADE171 RLF were trans- immunized orally with WR4024/pADE171 R16, WR4024/ formed into S. typhimurium WR4024 trp R- by a modification pADE171 RLF, WR4024, or recombinant vaccinia (V-71) were in- (22) of standard methods (23) Transformants WR4024/pMGB2 cubated with 2 x 10 5 CS-transfected L cells or with 2 x 105 un- expressing P . berghei CS were identified by mAb 3.28 1 (24) . transfected L cells. Cells were harvested after 6 d in culture, counted, Salmonella transformants WR4024/pADE171 R16 and WR4024/ and incubated for 6 h at various E/T ratios with 5,000 "Cr- pADE171 RLF were examined for the expression of P . fakiparum cells . labeled CS-transfected L cells or labeled untransfected L Per- using 49 .41) 9.1 and rabbit antirepeatless anti- CS antigen mAb cent specific lysis was determined as described above. body 579.15, respectively . mAb 49.4D 9.1 (IgG) was made against To identify the CTL epitope, spleen cells from immunized mice the full-length CS gene by Dr. K. Esser, Walter Reed Army Insti- were in L cells for d . stimulated vitro with CS-transfected 6 Puta- . raised against the repeat- tute of Research Polyclonal rabbit sera CS tive CTL were then incubated with untransfected L cells at an E/T less molecule NS 1 81RLF 9 was a kind gift of Dr. D.M . Gordon ratio of 40 :1 for 6 h in the presence of various concentrations of (Walter Reed Army Institute of Research) . 1084 Salmonella Induces Protective Immunity against Malaria peptide 371-390 (DELDYENDIEKKICKMEKCSS) . Peptide res- nization provided protection in 55% of these animals, whereas idue 291-310 was used as control. Killing was measured in 100 all of the unimmunized and 94% of the mice receiving the pl of supernatant as described above. WR4024 carrier strain were infected (Table 3) . No antibody To demonstrate that CS-specific CTL were CD8+ cells, CS- in the serum of individual mice could be demonstrated against specific CTL were incubated with anti-CD8 19/178) mAb (clone recombinant CS protein using ELISA techniques (data not (25) followed by complement . Washed cells were then stained with shown) . Mice immunized intravenously with 50,000 irradi- FITC-conjugated goat anti-mouse IgG2a, and depletion was quan- ated sporozoites were fully protected . titated by microfluorimetry. Cells were incubated with 5,000 s'Cr- To determine the role of CD8+ T cells in protection, labeled transfected L cells or untransfected L cells for 6 h at various mice were depleted of CD8' T cells by intraperitoneal in- E/T ratios . The percent lysis of target cells bycomplement-treated or CD8-depleted CTL was determined. jections of anti-CD8 mAb 4 wk after immunization . This procedure resulted in removal of >98% of the CD8+ T cells from the spleens of these animals (data not shown) . The deple- Results tion was confirmed by surface phenotyping of CD8+ T cells For construction ofSalmonella-P. berghei CS recombinants, from the spleen by single-color indirect fluorescent staining expression plasmid pMGB2 containing the full-length P. ber- and monitored by microfluorometry. Elimination of CD8+ ghei CS gene was transformed into S. typhimurium WR4024, T cells abrogated the protection after immunization with the an avirulent strain . Ampicillin-resistant transformants were Salmonella recombinant vaccine WR4024/pMGB2 . Protec- examined for the expression of the CS protein . One of the tion was reduced from 55% to 5% (Table 3) . CD8+ transformants, P WR4024/pMGB2, expressing berghei CS cell-depleted control mice were all infected after challenge. protein, was selected for further studies (Table 1) . Mice depleted of T CD4 cells did not lose their immunity For construction . of Salmonella-P falciparum CS recom- (data not shown) . These results indicate that CD8+ T cells binants, plasmid pADE171 R16 containing the full-length are the mediators of protection induced by Salmonella recom- P. falciparum CS gene and plasmid pADE171 RLF containing binant vaccines . However, in contrast to immunity induced the flanking regions without the repeats were constructed by Salmonella recombinant vaccines, immunity achieved by (Fig. 1) . The plasmids were Typhimurium transformed into S. immunization with radiation-attenuated sporozoites could WR4024, and spectinomycin-resistant transformants were not after be eliminated even complete depletion of CD8+ examined for expression of CS antigens . of these Two trans- T cells (Table 3) . formants were picked for further studies (Table 1) . To further characterize the CD8+ T cells induced by Protection studies were performed by immunization of Salmonella-P. berghei CS recombinant immunization, in vitro BALB/c mice with Salmonella-P. berghei CS recombinants . T cytotoxic cell assays were performed. For these assays, we Mice were immunized orally with WR4024/pMGB2 or used the methods ofTownsend et al. (27), where target cells WR4024 and were intravenously challenged 5 wk later with are made by incubation of histocompatible cells with short 1,000 P . berghei infectious . This (NK65) sporozoites immu- synthetic peptides that associate with class I molecules . We Table 1 . Salmonella-CS Recombinant Vaccines Immunogen Plasmid Gene Species WR4024/pMGB2 pMGB2 Full-length CS P. berghei WR4024/pADE171 R16 pADE171 Full-length CS P. falciparum WR4024/pADE171 RLF pADE171 Flanking regions P. falciparum WR4024 None None Carrier Table 2 . Plasmodium berghei CS Peptides Sequence Region Residue ADAPEGKKNEKKNEKIERNN NHz terminus 69-78 (DPPPPNPN)3 Repeat 102-124 NDDSYIPSAEKI COON terminus 242-253 SYIPSAEKILEFVKQQIRDSITEEWSQ COOH terminus 245-270 CFVKQIRDSITEEWSQCNVTCG COOH terminus 256-276 1085 Aggarwal et al . Bglll BamH[ The spleen cells from mice immunized with WR4024/ pMGB2 caused 24%D specific lysis of P815 (H-2d) R11 ANCHOR target cells APL I RIB RI (NANP)37(NVDP)4 R-16CSP incubatedwith peptide 242-253 at a concentration of50 NAg/ml and an E/T ratio of 40 :1 (Fig . 2) . Spleen cells from WR4024/ pMGB2-immunized mice had no Cytolytic activity without 99111 BaMH1 SalVmol in vitro peptide stimulation . In vitro stimulated cells did not show activity if the target cells were not incubated with pep- NS181 R1 R11 APL tide cells 242-253 . Spleen from unimmunized mice or mice NS1 81RLF immunized with the carrier Salmonella WR4024 and stimu- lated with the peptide 242-253 had cytolytic activity, <4%D ECORI Hn0111 ECORI indicating that the CTL arose after immunization and were Aalil Pa11 (E 0 not vitro due to in peptide stimulation. Specific Cytolytic ac- pADE171 tivity of34%D was measured with peptide 242-253-stimulated spleen cells from mice immunized with radiation-attenuated sporozoites. None of the other four peptides stimulated spleen cells from mice immunized either with Salmonella recombinants or Psll/SaII radiation-attenuated sporozoites . These peptides also failed to generate P815 cell targets. There was no specific lysis of P815 cells after incubation with any of the five peptides at RECOMBINANT the concentration used, demonstrating that they were not directly toxic to cells . The CTL lytic activity generated by Construction of WR4024/phDE171 R16 and WR4024/ Figure 1 . WR4024/pMGB2 immunization was genetically restricted . pADE171 RLF. The plasmid containing R16 full-length or NSI repeatless peptide After stimulation of spleen cells with 242-253, no gene P . fakiparum was digested with Bg1II and Sall . The gel-purified CS of specific lytic activity was observed when EL4(H-26) cells in- which insert was blunt-end ligated into the Pstl site of plasmid phDE171, cubated with the same peptide were used as targets carries his OGD region of Salmonella, resulting in the plasmid containing CS gene flanked by Salmonella his OGD . We asked whether Salmonella recombinant vaccines could also induce CTL responses to the human malaria parasite Pfalciparum . For this purpose, spleen cells from B10 . BR(H- the P. berghei CS protein based on 2k) mice immunized with WR4024/pADE171 R16, designed peptides from the sequence identified previously by Romero et al . (14) as WR4024/pADE171 RLF, WR4024, or recombinant vac- full-length a CTL target . Five different peptides from NH2 terminus, cinia (V-71) containing P. fakiparum CS gene were repeat region, and COOH terminus were used (Table 2) . stimulated in vitro for 6 d with CS-transfected L cells (H- either Salmonella- 2k) . cells for lytic activity Spleen cells from mice immunized with These were then assessed against P. berghei CS recombinant WR4024/pMGB2, S. typhimurium transfected or untransfected L cells. At an E/T ratio of 40 :1, were taken 4-10 wk after in three different sets ofexperiments, we observed 37 ± 2.2% carrier WR4024, or sporozoites immunization . After in vitro stimulation with peptide for specific killing ofCS gene-transfected cells by CTL induced by WR4024/pADE171 R16 compared with 20 ± 2.5% 6 d, the killing was measured by the "Cr release . DP015 a01wiUpeptic Table 3 . Protective Immunity Induced by S. typhimurium ®PB15a01wit estpwide (WR4024) Transformed with a Plasmid Expressing Full-Length P. berghei CS Gene (WR4024/pMGB2) Anti-CD8 No infected/ Percent Immunogen antibody no . challenged protection 20 6 1o WR40 - 29/31 WR4024 + 8/8 0 I M 1-6 - 10/22 55 0 WR4024/pMGB2 WN40244MG12 'YUZ WR4024/pMGB2 + 18/19 5 Figure 2 . Results of cytotoxic assays performed with P815 target cells - 30/30 0 None and CTL in the presence of synthetic peptides. Spleen cells from mice berghei recombinant vaccine None + 8/8 0 immunized with Salmonella-P . CS were stimu- lated in vitro for 6 d with 50 of peptide, and 6 d after in vitro stimula- Ag Irradiated Spz - 0/10 100 cells in the presence of tion cells were incubated with Cr-labeled P815 + 100 Irradiated Spz 0/10 51C . This experiment is rep- different peptides, and r release was measured resentative of four independent experiments . 1086 Salmonella Induces Protective Immunity against Malaria 50r duced CTL that caused 20 ± 4.5% lysis of untransfected cells. This nonspecific effect was induced by both recombinants . 40 and the control Salmonella vaccine, and contrasts to the 7 ± 1.5% lysis seen with recombinant vaccinia-immunized mice (Fig . 3) . After CD8 depletion, the lysis of untransfected cells by WR4024/pADE R16-induced CTL was reduced from 18 ± 2 4 ± .8 to 2% (p < 0 .05), and killing by WR4024/ pADE171 RLF-induced CTL was reduced from 16 ± 3 0 1 L i~~-k----IVi (7 to 7 ± 1.5% (p 0 .05) . These findings < suggest that the 0 5 10 20 0 5 10 20 40 majority of induced nonspecific cytotoxic T cells were CD8+ EFFECTOR/TARGET RATIO EFFECTOR/TARGET RATIO (Fig. 4) . Immunization of BALB/c mice with Salmonella vac- Figure Malaria Salmonella CS recombinants stimulate CS-specific CTL. 3 . cines followed by in vitro stimulation of spleen cells with In vitro stimulated spleen cells from mice immunized with Salmonella- peptides did not yield CTL reactive with non-peptide-pulsed incubated P. falciparum CS recombinants were with S'Cr-labeled CS- targets. This may be attributed to differences between in vitro transfected (A) or untransfected L cells (B) . Percent specific lysis was de- stimulation with termined . (0) Vaccinia recombinant ; transfected cells vs . in vitro stimulation with CS (A) R16 ; RLF ; (O) vector. (0) This experiment is representative of five independent experiments . peptides . To determine the epitope specificity of CTL induced by oral immunization with Salmonella-P. fakiparum CS constructs in B10 mice, .BR spleen cells from immunized mice were stimulated in vitro with CS-transfected killing of untransfected L cells (p < 0.05) . Likewise, at this L cells. After 6 d, untransfected L E/T spleen cells from animals cells were incubated with 10014g/ml ofpep- ratio, immunized with tide 371-390, single letter code DELDYENDIEKKICK- WR4024/pADE171 RLF killed 36 ± 3.1% ofCS gene-trans- MEKCSS, or peptide fected L cells compared with 4% killing 291-310 from the P.falciparum CS gene 20 ± of untrans- (12), and lysis was determined at an E/T of 40 :1 . Peptide fected L cells (p < 0.05) . These CTL responses were com- with amino acid parable with those obtained after immunization sequence 371-390 was recognized by CTL with the from mice immunized with the two recombinant recombinant vaccinia (Fig. 3) . CTLs from mice immunized Salmonella vaccines or the recombinant with the Salmonella carrier WR4024 gave 20 ± 4.5% 51Cr vaccinia (Fig . 5) . This same peptide release had previously been identified as the target of CTL induced by sporozoite or recombinant vaccinia-CS To determine if the cytotoxic activity in the spleen cell cul- immunization (12) . tures due L cells incubated with peptide 291-310 were not lysed by was to CD8+ T cells, in vitro depletions were'per- immune formed . Removal of CD8+ T cells from the cultures on the CTL above the levels seen with target cells alone (data not shown) . day of the CTL assay was confirmed by MF (data not shown) . WR4024/pADE171 R16-induced CS-specific CTL, when treated with anti-CD8 mAb and complement, yielded only Discussion 5 ± 2.8% specific lysis of CS-specific target cells at an E/T ratio of 40 :1 compared with specific lysis of 37 ± 2.2% (p We have demonstrated that oral immunization of mice with < 0.05), when the cells were treated with complement alone attenuated S. typhimurium recombinants expressing full-length (Fig. 4) . WR4024/pADE171 RLF induced CTL with similar P . berghei CS gene or full-length or repeatless P. fakiparum activity (12 ± 3 .5 vs. 36 ± 3 .1 ; p < 0 .05) . This demon- CS gene induce specific CD8+ CTL . We also provide evi- strated that the CS-specific cytotoxicity was mediated by dence that these CS-specific CTL are responsible for the pro- CD8+ cells . Reductions in specific cytotoxicity by depletion tection induced against P. berghei sporozoites after immuni- of CD8+ T cells similar were for spleen cells from animals zation ofBALB/c mice with Salmonella recombinant vaccines immunized with each of the three vaccines (Fig . 4) . Antibody-dependent cellular cytotoxicity has been shown Immunization of B10.BR mice with Salmonella also in- after immunization of humans with S. tpphi mutant strain Figure 4. Effect of CD8 depletion on CS-specific CTL . In vitro stimulated cells from the miceimmunized with Salmonella-P. falciparum CS recombinants were in- cubated with anti-CD8 mAb (19/178) followed by com- plement . Washed cells were then incubated with 5,000 (B) L 5'Cr-labeled transfected (A) or untransfected cells at various E/T ratios. The percent specific lysis of target cells by complement-treated (/) or CD8-depleted (®) CTL was determined . A representative experiment at the 40 :1 ratio is presented . 1087 . Aggarwal et al 11), induction of CTL in mice against P. falciparum CS by sporozoites (13), and the protective role of CD8+ T cells in immunity to Toxoplasma gondii (36) . There is also evidence that protection against intracellular pathogens such as L . mono- cytogenes, M. tuberculosis, M. leprae, and others may in part be mediated through CD8+ T cells (37) . Our findings directly prove that an intracellular bacteria such as S. typhimu- rium can induce specific CD8+ CTL and suggest that the antigen processing and presentation is probably not very different from that which occurs with vaccinia or sporozoites, since the recognized peptide and quantitative data are very similar. Our finding that Salmonella-P. berghei CS recombinant-in- duced protection is mediated through specific CD8+ cyto- toxic T cells further demonstrates the importance of CTL Figure 5 . Identification of the CTL epitope on theP. fakiparum CS pro- in immunity against malaria . This is consistent with the re- tein . Spleen cells from Salmonella-P. fakiparum recombinant-immunized cent finding that CD8+ CTL clones directed against P. ber- mice were stimulated in vitro with CS-transfected L cells . S'Cr-labeled target cells were incubated with peptide and the in vitro stimulated spleen ghei CS by themselves are able to provide complete protec- cells at an E/T ratio of40 :1 (N) ; or without peptide (®), and S1Cr re- tion (14) . These CTL clones were induced by immunization lease was measured . This experiment is representative of three indepen- with radiation-attenuated sporozoites. The CTL we induced dent experiments . with Salmonella CS recombinants are directed against the same peptide in the CS sequence as the CTL clones defined by Romero et al. (14) . Not all of the CTL clones found by Romero were able to protect . Our study demonstrates that Ty21a (28) or auxotrophic S. typhi 541Ty(Vi+) or 543Ty(Vi - ) at least some of the CTL induced by Salmonella-P. berghei (29) . The effector cell has been identified as a nonadherent CS recombinants are protective. To increase the number of T3 + , T8 - , T4+ lymphocyte (28) . After immunization, protective CTL induced by oral immunization with Salmonella delayed-type hypersensitivity responses to Salmonella carbo- CS recombinants, we are inserting malarial genes into the hydrate antigens (30) and recombinant expressed proteins have bacterial chromosome in an attempt to stabilize and increase been demonstrated (15, 31) . Macrophage activation directly the expression of malaria gene products . also We are utilizing or through T cell-mediated mechanisms has also been seen Salmonella auxotrophs as carrier strains, which are safe but after immunization or infection (32, 33) . To our knowledge, more invasive than the attenuated S. typhimurium WR4024 induction of protective class I-restricted antigen-specific we utilized in this study. by CD8+ CTL immunization or infection with Salmonella We were able to achieve CTL that gave similar levels of has not, however, been previously reported . killing by immunization with Salmonella-P. fakiparum CS After oral ingestion, attenuated are Salmonella vaccines soon recombinant vaccines that expressed the entire gene CS or translocated from the intestinal lumen to an intracellular lo- a gene from which the central repeat region was deleted . Both cation, primarily inside macrophages (30) . Attenuated Sal- of these recombinant constructs produced the malaria CS an- monella carrier strains, such as S. typhimurium WR4024 used tigens as fusion proteins . The complete CS molecule is there- in this study and the aro A mutant of dublin SL1438 used S. fore not required for the in vivo induction of CD8+ CTL by us in other studies (34), have limited ability to multiply by Salmonella, and modification of the original geneby addi- but for periods of within macrophages do survive variable tion also does not interfere . Presentation of antigens in as- time . The intracellular expression ofrecombinant malaria CS sociation with class I molecules is generally dependent on responsible for presenta- antigens is probably processing and intracellular enzymatic cleavage to produce a peptide frag- tion in association with class I antigen with the subsequent ment that can bind to the class I molecule. Our findings sug- induction CTL . Bacterial enzymes may play some role in of gest a possible method for determining the minimal struc- degradation of the recombinant expressed protein before pro- ture from any given molecule that is necessary for such a process cessing by intracellular enzymes, although we have no direct to occur. This could be done by cloning deletion fragments evidence for this possibility. The presence of Salmonella in- as fusion proteins into Salmonella and looking for the ability side macrophages may also activate these macrophages and of these Salmonella recombinants to induce CTL . important as induce the release of cytokines thought to be We were able to determine the Salmonella-P. berghei CS differentiation factors for induction of CTL (35) . recombinant-induced protection by depletion of T CD8+ recently, thought that viruses were Until it was generally cells . This contrasts with our inability to eliminate protec- the only pathogens capable of presenting endogenous antigens tion by this technique in BALB/c mice immunized with on the surface of infected cells in association with class I mol- radiation-attenuated This result may due sporozoites. be to the ecules with subsequent induction of CD8+ CTL . This involvement of antibody, effector CD4+ T cells, other im- view was challenged by the evidence for a protective role of mune cell types or other sporozoite or preerythrocytic an- CD8+ CTL induced by sporozoites in murine malaria (10, tigens in BALB/c immunity to P. berghei . Schofield al . (11) et Salmonella Induces Protective Immunity against Malaria were able to eliminate sporozoite-induced protection in A/J 40) . This inherent safety plus their sensitivity to antibiotics mice by depletion of CD8+ T cells . This shows that the is important for immunization of human populations that im- may to HIV mechanism of radiation in attenuated sporozoite-induced have been exposed munity is dependent on the mouse haplotype . Although we Our findings that recombinant Salmonella constructs pro- were not able to eliminate sporozoite-induced protection in vide some protection and induce CTL effector mechanisms BALB/c mice, Weiss et al . (10) were able to accomplish this, similar to those induced by irradiated and live sporozoites provide a basis for further on the development of oral but the parasite was P. yoelii. work A vaccine for malaria that overcomes the sequence varia- vaccines against malaria . The CTL epitope, which has been differences due genetic identified in the of the human malaria parasite, tion in the CS CTL site as well as to CS protein restriction (13) will likely contain several different versions P . fakiparum, has only been shown to be recognized by primed well other mouse peptide also of the CS CTL site recognized by humans as as lymphocytes . Whether this sequence is sporozoite and liver stage antigens important for CTL rec- recognized by human CTL is currently unknown. Immuni- be constructed in zation of humans with irradiation-attenuated sporozoites or ognition . Such multivalent vaccines can Salmonella because of the size of the bacterial genome and possibly Salmonella recombinants may help answer this ques- the its manipulation . Because new Salmonella tion . These findings suggest that live attenuated Salmonella flexibility of vaccine strains designed for human use are auxotrophs (38), recombinants may also be useful in the study of other dis- CTL mediated immunity may important which can only replicate in the body to a limited extent, they eases where be . have been safe in studies of immunocompromised hosts (39, We wish to thank Ms . Hazel Sidberry for excellent technical assistance, Dr. Carolyn Deal for synthesis of for P. peptide 242-253, Ms. Lynett Smith providing the berghei CS peptides, Dr. Bernard Moss for the vaccinia recombinant, and Drs. Louis Baron, and Louis Miller for advice and helpful suggestions . Address correspondence to Jerald Sadog, Walter Reed Army Institute of Research, Washington DC 20307. Received for publication IS March 1990 and in revised form 8 June 1990 . References 1 . Miller, L.H ., R .J . Howard, R. Carter, M .F. Good, V Nus- effect of immunization of T and B cell deficient mice. J. Im- senzweig, and R.S . 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Journal

The Journal of Experimental MedicinePubmed Central

Published: Oct 1, 1990

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