Vol. 58, No. 10
INFECTION AND IMMUNITY, OCt. 1990, p. 3425-3429
0019-9567/90/103425-05$02.00/0 Copyright X) 1990, American Society for Microbiology
Immunization of Mice against Plasmodium vinckei with Combination of Attenuated Salmonella typhimurium and Malarial Antigen
a
SANJAI KUMAR,' JED GORDEN,' JOANNE L. FLYNN,2 JAY A. BERZOFSKY,3 AND LOUIS H. MILLER'* Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases,' and Vaccine Development Section, Metabolsim Branch, National Cancer Institute,3 Bethesda, Maryland 20892, and Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 920372 Received 25 April 1990/Accepted 3 August 1990
Infection with the blood stage of the malaria parasite Plasmodium vinckei is uniformly lethal in mice. We found that immunization of BALB/c mice with a combination of killed P. vinckei antigens and an attenuated (aroA) Salmonella typhimurium strain induces high levels of protection against challenge with live P. vinckei. This is especially significant because, in our previous studies, immunization of mice with killed P. vinckei antigens and adjuvants such as Bordetella pertussis, complete Freund adjuvant, and saponin failed to induce protective immunity. Immunization with attenuated S. typhimurium alone did not provide any nonspecific immunity. In vivo depletion of CD4+ T cells in the mice immunized with attenuated S. typhimurium and P. vinckei antigens caused the loss of their immunity. Expression of this immunity required the presence of a spleen. These results support our previous hypothesis that a blood stage malaria vaccine may need both induction of CD4+ T cells specific for the parasite and modification of the spleen with a vaccine vehicle. Therefore, attenuated Salmonella strains such as the one used in this study, when expressing recombinant malarial antigens, might fulfill this requirement.
Immunization with crude preparations containing many malarial proteins requires adjuvants to induce protective immunity to asexual erythrocytic infections. Purified proteins and peptides tested to date also require adjuvants, and there is no reason to believe that vaccine candidates of the future will not require them. Adjuvants improve the T-cell response to antigens (3), but they may also have other as yet undefined roles in protective immunity. Even with adjuvants, it is difficult to immunize animals against certain malarias. For example, virulent Plasmodium knowlesi in rhesus monkeys requires three injections of Freund complete adjuvant with parasite antigens to induce protection (10). Previous attempts in our laboratory to immunize mice against universally fatal Plasmodium vinckei infections have been unsuccessful. These included injection of parasite antigens with various adjuvants such as Bordetella pertussis, saponin, and Freund complete adjuvant (9). We also immunized mice with a combination of B. pertussis and P. vinckei antigens crudely fractionated by fast protein liquid chromatography; again, no mice were protected (S. Kumar, unpublished results). Immunity to P. vinckei is CD4+ T-cell dependent on the basis of the findings that CD4 depletion eliminates immunity and that transfer of immune CD4+ T cells to CD4-depleted animals restores their immunity (9). Although CD4+ T cells are necessary for immunity, they are not sufficient, as the immune CD4+ T cells do not transfer protection to nonimmune mice. One possibility is that the non-T-cell factors reside in the spleen. The present study was undertaken to determine whether we could induce protection with an agent that modifies the spleen. Specifically, we infected animals with a live attenuated strain of Salmonella typhimurium (SL3235BV; J. L. *
Flynn, W. R. Weiss, K. A. Norris, H. S. Seifert, S. Kumar, and M. So, Mol. Microbiol., in press) and determined their resistance to challenge with P. vinckei. The S. typhimurium strain used was a derivative of the avirulent aroA mutant SL3235, and immunization of mice with this strain results in protection against a virulent S. typhimurium challenge (6, 8; Flynn et al., in press). The animals were protected if they received both attenuated S. typhimurium and crude P. vinckei antigens. The immunity was CD4+ T-cell dependent and required an intact spleen. MATERIALS AND METHODS Parasites. P. vinckei (ATCC 3009) was a generous gift from W. P. Weidanz, Hahnemann University, Philadelphia, Pennsylvania. The parasites were maintained by weekly intraperitoneal injection of 106 parasitized erythrocytes (PRBCs). The parasites were also maintained in a stable cryopreserved state in liquid nitrogen. The parasites were free of pathogens, including those of lactate dehydrogenase-elevating virus.
Bacteria. S. typhimurium SL3235BV was used for all immunizations. This strain is a modification of the aroA strain SL3235 (6) and has a stable mini-TnJO insertion encoding kanamycin resistance in the chromosome (Flynn et al., in press). This system uses a defective transposable element which randomly inserts into the bacterial chromosome to express foreign DNA sequences in S. typhimurium. The insertion in the chromosome does not noticeably affect the phenotype or the 50% lethal dose of the parental strain. The bacteria were grown in Luria broth in the presence of 50 ,ug of kanamycin per ml. The culture was initiated by inoculating bacteria into 5 ml of medium. The culture was allowed to grow at 37°C overnight with shaking, and the next morning the culture was diluted 1:50 in 50 ml of medium for 2 h at 37°C. This procedure provided bacteria growing in the log phase. The bacteria were centrifuged at 3,000 x g for 10
Corresponding author. 3425
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KUMAR ET AL.
min at 4°C and then washed once with phosphate-buffered saline (PBS), pH 7.2. The approximate bacterial counts were estimated by optical density (A6.; 8 x 108 bacteria per optical density unit). Finally, the bacteria were resuspended in PBS, and a 0.1-ml volume was given to each mouse. The accurate bacterial counts were determined by plating different dilutions of bacteria on Luria broth agar plates containing 50 ,ug of kanamycin per ml. The plates were incubated at 37°C overnight, and individual colonies were counted. Animals. Six- to eight-week-old female BALB/c and athymic nulnu mice were obtained from the National Institutes of Health Frederick Cancer Research Facility (Frederick, Md.) and were housed in the National Institutes of Health Animal
Facility. Antibodies. Anti-CD4 antibodies were produced in ascitic fluid in pristane-primed athymic nude mice or BALB/c mnice treated with cortisone and then irradiated at 500 rads (12) before injection of hybridoma cells designated GK1.5 (rat
TABLE 1. Survival of BALB/c mice vaccinated with attenuated S. typhimurium and P. vinckei antigens after challenge with live P. vinckei Peak parasitemia
No. of survivors after challengec
Immunization'
No. of mice
5 x 106 S. typhimurium and P. vinckei 5 x 106 S. typhimurium 1 X 106 S. typhimurium and P. vinckei 1 X 106 S. typhimurium 1 x 105 S. typhimurium and P. vinckei 1 x 105 S. typhimurium
4
22.0 ± 4.8
4
4 3
77.0 ± 3.5 12.0 ± 6.69
0 3
2
83.0 ± 3.54
0
4
35.5 ± 14.06
3
4
90.25 ± 2.16
0
a Each mouse received two i.p. injections at 3-week intervals. b Parasitemia is the percentage of erythrocytes that were infected and is expressed as the mean + the standard error of the mean. c Challenged by the i.v. route with 104 P. vinckei PRBCs 3 weeks after the second immunization.
immunoglobulin G2b) (4). Immunoglobulin fractions from the ascitic fluid were purified by 50% ammonium sulfate
precipitation followed by dialysis against PBS and then against 0.15 M NaCl. The immunoglobulin concentrations were determined by optical density at 280 nm (0.7 mg of protein per optical density unit). In vivo depletion of CD4' T cells. For in vivo depletion of CD4+ T cells, mice were injected with 1 mg of anti-CD4 monoclonal antibody every day for 10 days, followed by three doses every week during the experiment. The depletion of CD4+ T cells was confirmed by microfluorometry. The CD4-depleted mice were challenged with 104 P. vinckei PRBCs intravenously (i.v.) 10 days after the initiation of anti-CD4 monoclonal antibody treatment. Flow microfluorometry. Lymphocytes were phenotyped for surface marker by single-color indirect fluorescence by using an EPICS profile analyzer (Coulter Electronics, Inc., Hialeah, Fla.). A single cell suspension of 106 spleen cells from antibody-treated or control mice was incubated for 20 min at 4°C with a 10-pd solution of anti-CD4 monoclonal antibody in PBS containing 1% bovine serum albumin and 0.1% NaN3. The bound antibodies were visualized by incubation with 10 [lI of appropriately titrated mouse-absorbed goat anti-rat immunoglobulin G-fluorescein isothiocyanate (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.) for 20 min at 4°C. Splenectomy. The mice were splenectomized while anesthetized with the inhalation anesthetic methoxyflurane (Pitman Moore, Washington Crossing, N.J.). The mice were splenectomized 2 days before challenge infection. Parasite antigens. The asexual-stage P. vinckei antigens were prepared by the procedure described by Playfair et al. (11), with slight modifications. Briefly, the PRBCs were lysed with 0.01% saponin for 30 min at room temperature. After being washed twice in PBS, the parasites were killed by treatment with 0.06% Formalin in PBS for 15 min at room temperature. The parasites were further washed twice in PBS and then lysed by freeze-thawing. The final parasite preparations were resuspended in PBS at 1010 parasites per ml, and a 0.1-ml volume was given to each mouse. This concentration was based on the number of PRBCs before saponin lysis. Immunizations. The mice were immunized with a combination of different numbers of live attenuated S. typhimurium organisms and P. vinckei antigens from 109 P. vinckei PRBCs. The immunizations were performed by using the following routes: intraperitoneal (i.p.), oral (p.o.), or subcu-
taneous (s.c.). Naive BALB/c mice were given either one or two doses of the vaccine. The second immunization was
given 3 weeks after the primary immunization. All the vaccinated mice and controls in the experiment were challenged 6 weeks after the primary immunization with 104 P. vinckei PRBCs inoculated i.v. The blood smears were prepared from tail vein blood, and the parasitemia was enumerated by counting about 1,000 erythrocytes on Giemsastained slides. RESULTS Intraperitoneal immunization. Compared to mice infected with the universally lethal P. vinckei, mice that were immunized i.p. with attenuated S. typhimurium and P. vinckei antigens survived the infection. The data were as follows. In two preliminary experiments, mice that were immunized twice with 5 x 106, 1 x 106, or 5 x 105 attenuated S. typhimurium and P. vinckei antigens survived infection after reaching peak parasitemias of 12 to 36% (expressed as the percentage of erythrocytes that were infected), whereas attenuated S. typhimurium alone gave no protection (Table 1). In a third experiment, 10 mice were immunized twice with 106 attenuated S. typhimurium and P. vinckei antigens i.p. After i.v. infection with 104 P. vinckei PRBCs, mice reached a mean peak parasitemia of 4% on day 8 and then cleared the infection on day 10 (Fig. 1). Immunization of mice with 106 attenuated S. typhimurium alone did not protect the mice. Of the five mice in the group, three died on day 10 and two died on day 11. In the same experiment, five mice that received a single immunization (rather than two immunizations as described above) with 106 attenuated S. typhimurium and P. vinckei antigens survived infection (Fig. 1), although they reached a significantly higher peak parasitemia than after two immunizations (P < 0.01). The control mice, which received two immunizations with P. vinckei antigens alone, were not protected. Of the five mice in the group, two died on day 10 and three died on day 11. Role of the spleen in immunity. To determine the role of the spleen in immunity induced by immunization with attenuated S. typhimurium and P. vinckei antigens, five BALB/c mice that had received two immunizations were splenectomized 2 days before challenge infection. Following infection, the splenectomized mice lost their immunity and died 11 days after infection; however, the course of parasitemia in
IMMUNIZATION OF MICE AGAINST P. VINCKEI
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. 6
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9
10
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DAYS AFTER INFECTION
FIG. 1. Immunization of BALB/c mice with S. typhimurium and P. vinckei antigens by the i.p. route. Mice received either two immunizations (i) (10 mice in this group) or a single immunization (i) (S mice in this group) with S. typhimurium and P. vinckei antigens. Control mice, five per group, were given two immunizations (O) or a single immunization (O) with S. typhimurium alone. Five mice received two immunizations each with P. vinckei antigen alone (A). Six weeks after primary immunization, mice were challenged i.v. with 104 PRBCs. Each datum point represents the mean parasitemia, and each vertical bar represents the standard error of the mean.
splenectomized mice was slower than in naive mice (Fig. 2). Immunized control mice that did not undergo splenectomy controlled their infection after developing low-level parasitemia. Effect of CD4+ T-cell depletion on immunity. The role of CD4+ T cells in immunity induced by immunization with attenuated S. typhimurium and P. vinckei antigens was studied by in vivo depletion of CD4+ T cells with anti-CD4 monoclonal antibodies. In vivo depletion of CD4+ T cells was confirmed by microfluorometry. The number of CD4+ T cells in CD4-depleted mice was
INFECTION AND IMMUNITY, OCt. 1990, p. 3425-3429
0019-9567/90/103425-05$02.00/0 Copyright X) 1990, American Society for Microbiology
Immunization of Mice against Plasmodium vinckei with Combination of Attenuated Salmonella typhimurium and Malarial Antigen
a
SANJAI KUMAR,' JED GORDEN,' JOANNE L. FLYNN,2 JAY A. BERZOFSKY,3 AND LOUIS H. MILLER'* Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases,' and Vaccine Development Section, Metabolsim Branch, National Cancer Institute,3 Bethesda, Maryland 20892, and Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 920372 Received 25 April 1990/Accepted 3 August 1990
Infection with the blood stage of the malaria parasite Plasmodium vinckei is uniformly lethal in mice. We found that immunization of BALB/c mice with a combination of killed P. vinckei antigens and an attenuated (aroA) Salmonella typhimurium strain induces high levels of protection against challenge with live P. vinckei. This is especially significant because, in our previous studies, immunization of mice with killed P. vinckei antigens and adjuvants such as Bordetella pertussis, complete Freund adjuvant, and saponin failed to induce protective immunity. Immunization with attenuated S. typhimurium alone did not provide any nonspecific immunity. In vivo depletion of CD4+ T cells in the mice immunized with attenuated S. typhimurium and P. vinckei antigens caused the loss of their immunity. Expression of this immunity required the presence of a spleen. These results support our previous hypothesis that a blood stage malaria vaccine may need both induction of CD4+ T cells specific for the parasite and modification of the spleen with a vaccine vehicle. Therefore, attenuated Salmonella strains such as the one used in this study, when expressing recombinant malarial antigens, might fulfill this requirement.
Immunization with crude preparations containing many malarial proteins requires adjuvants to induce protective immunity to asexual erythrocytic infections. Purified proteins and peptides tested to date also require adjuvants, and there is no reason to believe that vaccine candidates of the future will not require them. Adjuvants improve the T-cell response to antigens (3), but they may also have other as yet undefined roles in protective immunity. Even with adjuvants, it is difficult to immunize animals against certain malarias. For example, virulent Plasmodium knowlesi in rhesus monkeys requires three injections of Freund complete adjuvant with parasite antigens to induce protection (10). Previous attempts in our laboratory to immunize mice against universally fatal Plasmodium vinckei infections have been unsuccessful. These included injection of parasite antigens with various adjuvants such as Bordetella pertussis, saponin, and Freund complete adjuvant (9). We also immunized mice with a combination of B. pertussis and P. vinckei antigens crudely fractionated by fast protein liquid chromatography; again, no mice were protected (S. Kumar, unpublished results). Immunity to P. vinckei is CD4+ T-cell dependent on the basis of the findings that CD4 depletion eliminates immunity and that transfer of immune CD4+ T cells to CD4-depleted animals restores their immunity (9). Although CD4+ T cells are necessary for immunity, they are not sufficient, as the immune CD4+ T cells do not transfer protection to nonimmune mice. One possibility is that the non-T-cell factors reside in the spleen. The present study was undertaken to determine whether we could induce protection with an agent that modifies the spleen. Specifically, we infected animals with a live attenuated strain of Salmonella typhimurium (SL3235BV; J. L. *
Flynn, W. R. Weiss, K. A. Norris, H. S. Seifert, S. Kumar, and M. So, Mol. Microbiol., in press) and determined their resistance to challenge with P. vinckei. The S. typhimurium strain used was a derivative of the avirulent aroA mutant SL3235, and immunization of mice with this strain results in protection against a virulent S. typhimurium challenge (6, 8; Flynn et al., in press). The animals were protected if they received both attenuated S. typhimurium and crude P. vinckei antigens. The immunity was CD4+ T-cell dependent and required an intact spleen. MATERIALS AND METHODS Parasites. P. vinckei (ATCC 3009) was a generous gift from W. P. Weidanz, Hahnemann University, Philadelphia, Pennsylvania. The parasites were maintained by weekly intraperitoneal injection of 106 parasitized erythrocytes (PRBCs). The parasites were also maintained in a stable cryopreserved state in liquid nitrogen. The parasites were free of pathogens, including those of lactate dehydrogenase-elevating virus.
Bacteria. S. typhimurium SL3235BV was used for all immunizations. This strain is a modification of the aroA strain SL3235 (6) and has a stable mini-TnJO insertion encoding kanamycin resistance in the chromosome (Flynn et al., in press). This system uses a defective transposable element which randomly inserts into the bacterial chromosome to express foreign DNA sequences in S. typhimurium. The insertion in the chromosome does not noticeably affect the phenotype or the 50% lethal dose of the parental strain. The bacteria were grown in Luria broth in the presence of 50 ,ug of kanamycin per ml. The culture was initiated by inoculating bacteria into 5 ml of medium. The culture was allowed to grow at 37°C overnight with shaking, and the next morning the culture was diluted 1:50 in 50 ml of medium for 2 h at 37°C. This procedure provided bacteria growing in the log phase. The bacteria were centrifuged at 3,000 x g for 10
Corresponding author. 3425
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KUMAR ET AL.
min at 4°C and then washed once with phosphate-buffered saline (PBS), pH 7.2. The approximate bacterial counts were estimated by optical density (A6.; 8 x 108 bacteria per optical density unit). Finally, the bacteria were resuspended in PBS, and a 0.1-ml volume was given to each mouse. The accurate bacterial counts were determined by plating different dilutions of bacteria on Luria broth agar plates containing 50 ,ug of kanamycin per ml. The plates were incubated at 37°C overnight, and individual colonies were counted. Animals. Six- to eight-week-old female BALB/c and athymic nulnu mice were obtained from the National Institutes of Health Frederick Cancer Research Facility (Frederick, Md.) and were housed in the National Institutes of Health Animal
Facility. Antibodies. Anti-CD4 antibodies were produced in ascitic fluid in pristane-primed athymic nude mice or BALB/c mnice treated with cortisone and then irradiated at 500 rads (12) before injection of hybridoma cells designated GK1.5 (rat
TABLE 1. Survival of BALB/c mice vaccinated with attenuated S. typhimurium and P. vinckei antigens after challenge with live P. vinckei Peak parasitemia
No. of survivors after challengec
Immunization'
No. of mice
5 x 106 S. typhimurium and P. vinckei 5 x 106 S. typhimurium 1 X 106 S. typhimurium and P. vinckei 1 X 106 S. typhimurium 1 x 105 S. typhimurium and P. vinckei 1 x 105 S. typhimurium
4
22.0 ± 4.8
4
4 3
77.0 ± 3.5 12.0 ± 6.69
0 3
2
83.0 ± 3.54
0
4
35.5 ± 14.06
3
4
90.25 ± 2.16
0
a Each mouse received two i.p. injections at 3-week intervals. b Parasitemia is the percentage of erythrocytes that were infected and is expressed as the mean + the standard error of the mean. c Challenged by the i.v. route with 104 P. vinckei PRBCs 3 weeks after the second immunization.
immunoglobulin G2b) (4). Immunoglobulin fractions from the ascitic fluid were purified by 50% ammonium sulfate
precipitation followed by dialysis against PBS and then against 0.15 M NaCl. The immunoglobulin concentrations were determined by optical density at 280 nm (0.7 mg of protein per optical density unit). In vivo depletion of CD4' T cells. For in vivo depletion of CD4+ T cells, mice were injected with 1 mg of anti-CD4 monoclonal antibody every day for 10 days, followed by three doses every week during the experiment. The depletion of CD4+ T cells was confirmed by microfluorometry. The CD4-depleted mice were challenged with 104 P. vinckei PRBCs intravenously (i.v.) 10 days after the initiation of anti-CD4 monoclonal antibody treatment. Flow microfluorometry. Lymphocytes were phenotyped for surface marker by single-color indirect fluorescence by using an EPICS profile analyzer (Coulter Electronics, Inc., Hialeah, Fla.). A single cell suspension of 106 spleen cells from antibody-treated or control mice was incubated for 20 min at 4°C with a 10-pd solution of anti-CD4 monoclonal antibody in PBS containing 1% bovine serum albumin and 0.1% NaN3. The bound antibodies were visualized by incubation with 10 [lI of appropriately titrated mouse-absorbed goat anti-rat immunoglobulin G-fluorescein isothiocyanate (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.) for 20 min at 4°C. Splenectomy. The mice were splenectomized while anesthetized with the inhalation anesthetic methoxyflurane (Pitman Moore, Washington Crossing, N.J.). The mice were splenectomized 2 days before challenge infection. Parasite antigens. The asexual-stage P. vinckei antigens were prepared by the procedure described by Playfair et al. (11), with slight modifications. Briefly, the PRBCs were lysed with 0.01% saponin for 30 min at room temperature. After being washed twice in PBS, the parasites were killed by treatment with 0.06% Formalin in PBS for 15 min at room temperature. The parasites were further washed twice in PBS and then lysed by freeze-thawing. The final parasite preparations were resuspended in PBS at 1010 parasites per ml, and a 0.1-ml volume was given to each mouse. This concentration was based on the number of PRBCs before saponin lysis. Immunizations. The mice were immunized with a combination of different numbers of live attenuated S. typhimurium organisms and P. vinckei antigens from 109 P. vinckei PRBCs. The immunizations were performed by using the following routes: intraperitoneal (i.p.), oral (p.o.), or subcu-
taneous (s.c.). Naive BALB/c mice were given either one or two doses of the vaccine. The second immunization was
given 3 weeks after the primary immunization. All the vaccinated mice and controls in the experiment were challenged 6 weeks after the primary immunization with 104 P. vinckei PRBCs inoculated i.v. The blood smears were prepared from tail vein blood, and the parasitemia was enumerated by counting about 1,000 erythrocytes on Giemsastained slides. RESULTS Intraperitoneal immunization. Compared to mice infected with the universally lethal P. vinckei, mice that were immunized i.p. with attenuated S. typhimurium and P. vinckei antigens survived the infection. The data were as follows. In two preliminary experiments, mice that were immunized twice with 5 x 106, 1 x 106, or 5 x 105 attenuated S. typhimurium and P. vinckei antigens survived infection after reaching peak parasitemias of 12 to 36% (expressed as the percentage of erythrocytes that were infected), whereas attenuated S. typhimurium alone gave no protection (Table 1). In a third experiment, 10 mice were immunized twice with 106 attenuated S. typhimurium and P. vinckei antigens i.p. After i.v. infection with 104 P. vinckei PRBCs, mice reached a mean peak parasitemia of 4% on day 8 and then cleared the infection on day 10 (Fig. 1). Immunization of mice with 106 attenuated S. typhimurium alone did not protect the mice. Of the five mice in the group, three died on day 10 and two died on day 11. In the same experiment, five mice that received a single immunization (rather than two immunizations as described above) with 106 attenuated S. typhimurium and P. vinckei antigens survived infection (Fig. 1), although they reached a significantly higher peak parasitemia than after two immunizations (P < 0.01). The control mice, which received two immunizations with P. vinckei antigens alone, were not protected. Of the five mice in the group, two died on day 10 and three died on day 11. Role of the spleen in immunity. To determine the role of the spleen in immunity induced by immunization with attenuated S. typhimurium and P. vinckei antigens, five BALB/c mice that had received two immunizations were splenectomized 2 days before challenge infection. Following infection, the splenectomized mice lost their immunity and died 11 days after infection; however, the course of parasitemia in
IMMUNIZATION OF MICE AGAINST P. VINCKEI
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FIG. 1. Immunization of BALB/c mice with S. typhimurium and P. vinckei antigens by the i.p. route. Mice received either two immunizations (i) (10 mice in this group) or a single immunization (i) (S mice in this group) with S. typhimurium and P. vinckei antigens. Control mice, five per group, were given two immunizations (O) or a single immunization (O) with S. typhimurium alone. Five mice received two immunizations each with P. vinckei antigen alone (A). Six weeks after primary immunization, mice were challenged i.v. with 104 PRBCs. Each datum point represents the mean parasitemia, and each vertical bar represents the standard error of the mean.
splenectomized mice was slower than in naive mice (Fig. 2). Immunized control mice that did not undergo splenectomy controlled their infection after developing low-level parasitemia. Effect of CD4+ T-cell depletion on immunity. The role of CD4+ T cells in immunity induced by immunization with attenuated S. typhimurium and P. vinckei antigens was studied by in vivo depletion of CD4+ T cells with anti-CD4 monoclonal antibodies. In vivo depletion of CD4+ T cells was confirmed by microfluorometry. The number of CD4+ T cells in CD4-depleted mice was
