Immunization with Persistent Attenuated lpg2 Leishmania major Parasites Requires Adjuvant To Provide Protective Immunity in C57BL/6 Mice
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感染与免疫杂志 2006年第1期
Department of Pathobiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110
ABSTRACT
Leishmania major parasites lacking the GDP-mannose transporter, termed lpg2 parasites, fail to induce disease in mice but persist long-term. We previously found that lpg2 organisms protect BALB/c mice from virulent L. major challenge. In contrast, we report here that lpg2 parasites induce protective immunity in C57BL/6 mice only when administered with CpG-containing oligodeoxynucleotides, indicating that parasite persistence alone is not sufficient to maintain protective immunity to L. major.
TEXT
Following infection with Leishmania major, mice that have achieved a clinical cure harbor persistent parasites and maintain a life-long immunity to reinfection (1, 3, 11). Persistent live parasites are required for the maintenance and/or renewal of anti-Leishmania effector memory cells that produce gamma interferon (IFN-), mediate a delayed-type hypersensitivity (DTH) response, and induce rapid protection against virulent challenge (13). Although they are required for durable immunity, persistent virulent parasites may cause disease recrudescence in immunocompromised individuals. Therefore, the generation of live, attenuated parasites that persist without causing disease would be a reasonable approach to generating a vaccine for leishmaniasis. BALB/c mice are extremely susceptible to L. major in part due to the development of a Th2 response (8). To determine if lpg2 parasites can protect mice that do not develop a dominant Th2 response, we tested the protective potential of lpg2 mutants in C57BL/6 mice. C57BL/6 mice develop a Th1 response following L. major infection and heal over 2 to 3 months. Following resolution of a primary infection, these mice exhibit substantial protective immunity to reinfection (7). In this report we show that although lpg2 parasites are maintained long-term in C57BL/6 mice, they provide minimal protection to virulent challenge, although when they were delivered with CpG oligodeoxynucleotides (ODNs), significant protection was observed.
C57BL/6 mice (6 to 8 weeks old; Jackson Laboratories, Bar Harbor, ME) were infected subcutaneously with 5 x 106 stationary-phase L. major substrain LV39 clone 5 (MRHO/SU/59/P; wild type [WT]) or the lpg2 mutant (9). The course of infection and parasite titers in the infected feet were then assessed. As expected, C57BL/6 mice infected with WT parasites developed small lesions that started to resolve spontaneously around week 8. In contrast, and consistent with our previous reports (9, 10), no lesions were observed in lpg2 mutant-infected C57BL/6 mice (Fig. 1A). The lack of disease in lpg2 mutant-infected mice was not due to their destruction, as comparable numbers of parasites could be reisolated from the footpads of WT or lpg2 mutant-infected animals by the limiting dilution assay at 10 weeks postinfection (Fig. 1B).
We then compared the immune responses in C57BL/6 mice following infection with WT or lpg2 mutant parasites. At various time points after infection (3 days, 4 weeks, and 10 weeks), single-cell suspensions from draining lymph nodes (LNs) were cultured in the presence of leishmanial freeze-thawed antigen (the equivalent of 107 parasites/ml), and the supernatants were collected and assayed for IFN- production. At 3 days postinfection, comparable levels of IFN- were detected after in vitro restimulation of cells from mice infected with WT and lpg2 parasites (Fig. 1C), indicating that the ability of lpg2 mutants to induce an early Th1 response in C57BL/6 mice was equivalent to that of WT parasites. However, this response rapidly waned; the levels of IFN- produced by cells from mice infected with lpg2 parasites significantly decreased at 4 weeks and were almost undetectable at 10 weeks postinfection. In contrast, IFN- production by cells from WT-infected mice increased over time (Fig. 1C). This lack of IFN- production by cells from lpg2 mutant-infected mice was not due to a shift to a Th2 cytokine response, as we also were unable to detect any measurable production of IL-4 or IL-10 by cells from lpg2 mutant-infected mice (data not shown). Taken together, these data indicate that the ability of lpg2 mutants to continuously stimulate an effector T-cell response is dramatically impaired.
To determine whether lpg2 parasites can confer protection in C57BL/6 mice, after 10 weeks of infection with lpg2 or WT parasites, these animals, or nave controls, were challenged in their contralateral footpads with WT L. major parasites. Parasite loads were then compared between nave and infected animals 5 weeks after challenge. As shown in Fig. 2A, WT-infected mice had significantly lower parasite loads than nave (unvaccinated) animals, which harbored >104 parasites in their lesions. Surprisingly—and unlike BALB/c mice—C57BL/6 mice vaccinated with lpg2 parasites had lower parasite loads, but not significantly so, than nave controls. Furthermore, C57BL/6 mice previously infected with lpg2 parasites for 10 weeks mounted a very weak DTH response, comparable to that exhibited by nave animals, while healed WT mice exhibited a robust DTH response typical of a classical anti-Leishmania immunity (Fig. 2B).
The inability of lpg2 parasites to confer significant protection to C57BL/6 mice could be related to the minimal immune response seen prior to challenge (Fig. 2C), although in lpg2 mutant-infected BALB/c mice—which were protected from challenge with WT parasites—a similarly weak immune response was observed (10). Nevertheless, we tested whether enhancing the immune response with adjuvants would promote increased protective immunity. CpG ODNs, potent inducers of a Th1 response, have been widely used as effective adjuvants for diseases requiring Th1 immune responses, such as leishmaniasis (5, 6, 12). We therefore asked whether coinjection of lpg2 parasites with CpG ODNs would enhance the lpg2 mutant-induced protection. C57BL/6 mice were immunized with lpg2 parasites in combination with a single dose (50 μg/mouse) of immunostimulatory CpG ODNs (sequence 1826 [5'-TCC ATG ACG TTC CTG ACG TT-3']) or non-CpG ODNs (sequence 1982 [5'-TCC AGG ACT TCT CTC AGG TT-3'] used as a control) (Coley Pharmaceutical Group, Inc., Wellesley, MA). When challenged with WT parasites, lpg2 mutant- plus CpG ODN-vaccinated mice showed a hundredfold reduction in parasite burdens compared to those in nave unvaccinated animals (Fig. 2A). Interestingly, the protective immunity induced by inclusion of CpG ODNs with the lpg2 parasites was not associated with a strong effector T-cell response at the time of challenge. Thus, lpg2 mutant- plus CpG ODN-vaccinated-mice did not mount a significantly stronger DTH response than mice receiving lpg2 parasites alone (Fig. 2B). Moreover, lymph node cells taken from lpg2 mutant- plus CpG ODN-vaccinated-mice prior to challenge did not produce significantly more IFN- than lpg2 mutant-infected mice (Fig. 2C). Taken together, these data indicate that mice vaccinated with lpg2 parasites in combination with a single dose of CpG ODNs are protected against WT parasites in the absence of a strong immune response.
A striking feature of infection with lpg2 parasites in BALB/c mice was the rapid waning of the effector immune response, in spite of the long-term maintenance of protection (10). We saw a similar pattern in C57BL/6 mice infected with lpg2 parasites, even when CpG ODNs were included in the vaccine. Thus, these results argue that the minimal immune response seen in lpg2 mutant-infected C57BL/6 mice at 10 weeks of infection is not likely to be the reason that these animals are not strongly protected. Studies are under way to define the mechanism involved in the protective immunity associated with lpg2 parasites. However, whatever the mechanism, it is clear from the data reported in this study and our previous work using lpg2 parasites in BALB/c mice that the presence of DTH and large numbers of IFN--producing T cells prior to challenge are not required for protective immunity. Additional studies with more-sensitive assays will be required to determine if any effector T cells are present prior to challenge.
A substantial body of literature indicates that parasite persistence is associated with optimal immunity to L. major (3, 11, 13), which suggests that the best vaccine for leishmaniasis may require live, attenuated parasites. Spth et al. found that lpg2 parasites persist in BALB/c mice without disease (9), and we reported that lpg2 mutant-infected BALB/c mice are resistant to challenge with virulent L. major (10). Here we show that while C57BL/6 mice infected with lpg2 parasites alone are not protected against challenge with virulent L. major, mice vaccinated with CpG ODNs as an adjuvant with lpg2 parasites, exhibited significant protection. Furthermore, mice receiving this live, attenuated vaccine were resistant to rechallenge for at least 10 weeks after the immunization. These results are similar to those obtained when mice were immunized with virulent parasites in conjunction with CpG ODNs, but they have the advantage of the use of an attenuated organism (4). Taken together with our previous study of BALB/c mice (10), these results indicate that attenuated parasites are able to induce long-term immunity to leishmaniasis. However, the ability of lpg2 parasites to provide protection in BALB/c mice (10), but not in C57BL/6 mice, was a surprise, although we cannot rule out the possibility that different doses of parasites, or additional injections of the parasites, might give better protection. Nevertheless, while previous studies have demonstrated that the presence of WT L. major parasites in C57BL/6 mice is associated with immunity to reinfection (1, 2, 11), our data unexpectedly indicate that the presence of persistent parasites alone is not sufficient to maintain protective immunity. Thus, future studies using lpg2 parasites may help to identify the essential characteristics associated with persistent infection that promote the maintenance of protective immunity.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grant AI-059396 from the National Institute of Allergy and Infectious Disease.
Present address: Department of Immunology, University of Manitoba, Winnipeg, MB, Canada, R3E 0W3.
REFERENCES
1. Aebischer, T., S. F. Moody, and E. Handman. 1993. Persistence of virulent Leishmania major in murine cutaneous leishmaniasis: a possible hazard for the host. Infect. Immun. 61:220-226.
2. Belkaid, Y., K. F. Hoffmann, S. Mendez, S. Kamhawi, M. C. Udey, T. A. Wynn, and D. L. Sacks. 2001. The role of interleukin (IL)-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti-IL-10 receptor antibody for sterile cure. J. Exp. Med. 194:1497-1506.
3. Belkaid, Y., C. A. Piccirillo, S. Mendez, E. M. Shevach, and D. L. Sacks. 2002. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420:502-507.
4. Mendez, S., K. Tabbara, Y. Belkaid, S. Bertholet, D. Verthelyi, D. Klinman, R. A. Seder, and D. L. Sacks. 2003. Coinjection with CpG-containing immunostimulatory oligodeoxynucleotides reduces the pathogenicity of a live vaccine against cutaneous leishmaniasis but maintains its potency and durability. Infect. Immun. 71:5121-5129.
5. Rhee, E. G., S. Mendez, J. A. Shah, C. Y. Wu, J. R. Kirman, T. N. Turon, D. F. Davey, H. Davis, D. M. Klinman, R. N. Coler, D. L. Sacks, and R. A. Seder. 2002. Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against Leishmania major infection. J. Exp. Med. 195:1565-1573.
6. Roman, M., E. Martin-Orozco, J. S. Goodman, M. D. Nguyen, Y. Sato, A. Ronaghy, R. S. Kornbluth, D. D. Richman, D. A. Carson, and E. Raz. 1997. Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants. Nat. Med. 3:849-854.
7. Sacks, D., and N. Noben-Trauth. 2002. The immunology of susceptibility and resistance to Leishmania major in mice. Nat. Rev. Immunol. 2:845-858.
8. Scott, P., P. Natovitz, R. L. Coffman, E. Pearce, and A. Sher. 1988. Immunoregulation of cutaneous leishmaniasis. T cell lines that transfer protective immunity or exacerbation belong to different T helper subsets and respond to distinct parasite antigens. J. Exp. Med. 168:1675-1684.
9. Spth, G. F., L. F. Lye, H. Segawa, D. L. Sacks, S. J. Turco, and S. M. Beverley. 2003. Persistence without pathology in phosphoglycan-deficient Leishmania major. Science 301:1241-1243.
10. Uzonna, J. E., G. F. Spath, S. M. Beverley, and P. Scott. 2004. Vaccination with phosphoglycan-deficient Leishmania major protects highly susceptible mice from virulent challenge without inducing a strong Th1 response. J. Immunol. 172:3793-3797.
11. Uzonna, J. E., G. Wei, D. Yurkowski, and P. Bretscher. 2001. Immune elimination of Leishmania major in mice: implications for immune memory, vaccination, and reactivation disease. J. Immunol. 167:6967-6974.
12. Walker, P. S., T. Scharton-Kersten, A. M. Krieg, L. Love-Homan, E. D. Rowton, M. C. Udey, and J. C. Vogel. 1999. Immunostimulatory oligodeoxynucleotides promote protective immunity and provide systemic therapy for leishmaniasis via IL-12- and IFN-gamma-dependent mechanisms. Proc. Natl. Acad. Sci. USA 96:6970-6975.
13. Zaph, C., J. Uzonna, S. M. Beverley, and P. Scott. 2004. Central memory T cells mediate long-term immunity to Leishmania major in the absence of persistent parasites. Nat. Med. 10:1104-1110.(Chahnaz Kebaer, Jude E. U)
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110
ABSTRACT
Leishmania major parasites lacking the GDP-mannose transporter, termed lpg2 parasites, fail to induce disease in mice but persist long-term. We previously found that lpg2 organisms protect BALB/c mice from virulent L. major challenge. In contrast, we report here that lpg2 parasites induce protective immunity in C57BL/6 mice only when administered with CpG-containing oligodeoxynucleotides, indicating that parasite persistence alone is not sufficient to maintain protective immunity to L. major.
TEXT
Following infection with Leishmania major, mice that have achieved a clinical cure harbor persistent parasites and maintain a life-long immunity to reinfection (1, 3, 11). Persistent live parasites are required for the maintenance and/or renewal of anti-Leishmania effector memory cells that produce gamma interferon (IFN-), mediate a delayed-type hypersensitivity (DTH) response, and induce rapid protection against virulent challenge (13). Although they are required for durable immunity, persistent virulent parasites may cause disease recrudescence in immunocompromised individuals. Therefore, the generation of live, attenuated parasites that persist without causing disease would be a reasonable approach to generating a vaccine for leishmaniasis. BALB/c mice are extremely susceptible to L. major in part due to the development of a Th2 response (8). To determine if lpg2 parasites can protect mice that do not develop a dominant Th2 response, we tested the protective potential of lpg2 mutants in C57BL/6 mice. C57BL/6 mice develop a Th1 response following L. major infection and heal over 2 to 3 months. Following resolution of a primary infection, these mice exhibit substantial protective immunity to reinfection (7). In this report we show that although lpg2 parasites are maintained long-term in C57BL/6 mice, they provide minimal protection to virulent challenge, although when they were delivered with CpG oligodeoxynucleotides (ODNs), significant protection was observed.
C57BL/6 mice (6 to 8 weeks old; Jackson Laboratories, Bar Harbor, ME) were infected subcutaneously with 5 x 106 stationary-phase L. major substrain LV39 clone 5 (MRHO/SU/59/P; wild type [WT]) or the lpg2 mutant (9). The course of infection and parasite titers in the infected feet were then assessed. As expected, C57BL/6 mice infected with WT parasites developed small lesions that started to resolve spontaneously around week 8. In contrast, and consistent with our previous reports (9, 10), no lesions were observed in lpg2 mutant-infected C57BL/6 mice (Fig. 1A). The lack of disease in lpg2 mutant-infected mice was not due to their destruction, as comparable numbers of parasites could be reisolated from the footpads of WT or lpg2 mutant-infected animals by the limiting dilution assay at 10 weeks postinfection (Fig. 1B).
We then compared the immune responses in C57BL/6 mice following infection with WT or lpg2 mutant parasites. At various time points after infection (3 days, 4 weeks, and 10 weeks), single-cell suspensions from draining lymph nodes (LNs) were cultured in the presence of leishmanial freeze-thawed antigen (the equivalent of 107 parasites/ml), and the supernatants were collected and assayed for IFN- production. At 3 days postinfection, comparable levels of IFN- were detected after in vitro restimulation of cells from mice infected with WT and lpg2 parasites (Fig. 1C), indicating that the ability of lpg2 mutants to induce an early Th1 response in C57BL/6 mice was equivalent to that of WT parasites. However, this response rapidly waned; the levels of IFN- produced by cells from mice infected with lpg2 parasites significantly decreased at 4 weeks and were almost undetectable at 10 weeks postinfection. In contrast, IFN- production by cells from WT-infected mice increased over time (Fig. 1C). This lack of IFN- production by cells from lpg2 mutant-infected mice was not due to a shift to a Th2 cytokine response, as we also were unable to detect any measurable production of IL-4 or IL-10 by cells from lpg2 mutant-infected mice (data not shown). Taken together, these data indicate that the ability of lpg2 mutants to continuously stimulate an effector T-cell response is dramatically impaired.
To determine whether lpg2 parasites can confer protection in C57BL/6 mice, after 10 weeks of infection with lpg2 or WT parasites, these animals, or nave controls, were challenged in their contralateral footpads with WT L. major parasites. Parasite loads were then compared between nave and infected animals 5 weeks after challenge. As shown in Fig. 2A, WT-infected mice had significantly lower parasite loads than nave (unvaccinated) animals, which harbored >104 parasites in their lesions. Surprisingly—and unlike BALB/c mice—C57BL/6 mice vaccinated with lpg2 parasites had lower parasite loads, but not significantly so, than nave controls. Furthermore, C57BL/6 mice previously infected with lpg2 parasites for 10 weeks mounted a very weak DTH response, comparable to that exhibited by nave animals, while healed WT mice exhibited a robust DTH response typical of a classical anti-Leishmania immunity (Fig. 2B).
The inability of lpg2 parasites to confer significant protection to C57BL/6 mice could be related to the minimal immune response seen prior to challenge (Fig. 2C), although in lpg2 mutant-infected BALB/c mice—which were protected from challenge with WT parasites—a similarly weak immune response was observed (10). Nevertheless, we tested whether enhancing the immune response with adjuvants would promote increased protective immunity. CpG ODNs, potent inducers of a Th1 response, have been widely used as effective adjuvants for diseases requiring Th1 immune responses, such as leishmaniasis (5, 6, 12). We therefore asked whether coinjection of lpg2 parasites with CpG ODNs would enhance the lpg2 mutant-induced protection. C57BL/6 mice were immunized with lpg2 parasites in combination with a single dose (50 μg/mouse) of immunostimulatory CpG ODNs (sequence 1826 [5'-TCC ATG ACG TTC CTG ACG TT-3']) or non-CpG ODNs (sequence 1982 [5'-TCC AGG ACT TCT CTC AGG TT-3'] used as a control) (Coley Pharmaceutical Group, Inc., Wellesley, MA). When challenged with WT parasites, lpg2 mutant- plus CpG ODN-vaccinated mice showed a hundredfold reduction in parasite burdens compared to those in nave unvaccinated animals (Fig. 2A). Interestingly, the protective immunity induced by inclusion of CpG ODNs with the lpg2 parasites was not associated with a strong effector T-cell response at the time of challenge. Thus, lpg2 mutant- plus CpG ODN-vaccinated-mice did not mount a significantly stronger DTH response than mice receiving lpg2 parasites alone (Fig. 2B). Moreover, lymph node cells taken from lpg2 mutant- plus CpG ODN-vaccinated-mice prior to challenge did not produce significantly more IFN- than lpg2 mutant-infected mice (Fig. 2C). Taken together, these data indicate that mice vaccinated with lpg2 parasites in combination with a single dose of CpG ODNs are protected against WT parasites in the absence of a strong immune response.
A striking feature of infection with lpg2 parasites in BALB/c mice was the rapid waning of the effector immune response, in spite of the long-term maintenance of protection (10). We saw a similar pattern in C57BL/6 mice infected with lpg2 parasites, even when CpG ODNs were included in the vaccine. Thus, these results argue that the minimal immune response seen in lpg2 mutant-infected C57BL/6 mice at 10 weeks of infection is not likely to be the reason that these animals are not strongly protected. Studies are under way to define the mechanism involved in the protective immunity associated with lpg2 parasites. However, whatever the mechanism, it is clear from the data reported in this study and our previous work using lpg2 parasites in BALB/c mice that the presence of DTH and large numbers of IFN--producing T cells prior to challenge are not required for protective immunity. Additional studies with more-sensitive assays will be required to determine if any effector T cells are present prior to challenge.
A substantial body of literature indicates that parasite persistence is associated with optimal immunity to L. major (3, 11, 13), which suggests that the best vaccine for leishmaniasis may require live, attenuated parasites. Spth et al. found that lpg2 parasites persist in BALB/c mice without disease (9), and we reported that lpg2 mutant-infected BALB/c mice are resistant to challenge with virulent L. major (10). Here we show that while C57BL/6 mice infected with lpg2 parasites alone are not protected against challenge with virulent L. major, mice vaccinated with CpG ODNs as an adjuvant with lpg2 parasites, exhibited significant protection. Furthermore, mice receiving this live, attenuated vaccine were resistant to rechallenge for at least 10 weeks after the immunization. These results are similar to those obtained when mice were immunized with virulent parasites in conjunction with CpG ODNs, but they have the advantage of the use of an attenuated organism (4). Taken together with our previous study of BALB/c mice (10), these results indicate that attenuated parasites are able to induce long-term immunity to leishmaniasis. However, the ability of lpg2 parasites to provide protection in BALB/c mice (10), but not in C57BL/6 mice, was a surprise, although we cannot rule out the possibility that different doses of parasites, or additional injections of the parasites, might give better protection. Nevertheless, while previous studies have demonstrated that the presence of WT L. major parasites in C57BL/6 mice is associated with immunity to reinfection (1, 2, 11), our data unexpectedly indicate that the presence of persistent parasites alone is not sufficient to maintain protective immunity. Thus, future studies using lpg2 parasites may help to identify the essential characteristics associated with persistent infection that promote the maintenance of protective immunity.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grant AI-059396 from the National Institute of Allergy and Infectious Disease.
Present address: Department of Immunology, University of Manitoba, Winnipeg, MB, Canada, R3E 0W3.
REFERENCES
1. Aebischer, T., S. F. Moody, and E. Handman. 1993. Persistence of virulent Leishmania major in murine cutaneous leishmaniasis: a possible hazard for the host. Infect. Immun. 61:220-226.
2. Belkaid, Y., K. F. Hoffmann, S. Mendez, S. Kamhawi, M. C. Udey, T. A. Wynn, and D. L. Sacks. 2001. The role of interleukin (IL)-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti-IL-10 receptor antibody for sterile cure. J. Exp. Med. 194:1497-1506.
3. Belkaid, Y., C. A. Piccirillo, S. Mendez, E. M. Shevach, and D. L. Sacks. 2002. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 420:502-507.
4. Mendez, S., K. Tabbara, Y. Belkaid, S. Bertholet, D. Verthelyi, D. Klinman, R. A. Seder, and D. L. Sacks. 2003. Coinjection with CpG-containing immunostimulatory oligodeoxynucleotides reduces the pathogenicity of a live vaccine against cutaneous leishmaniasis but maintains its potency and durability. Infect. Immun. 71:5121-5129.
5. Rhee, E. G., S. Mendez, J. A. Shah, C. Y. Wu, J. R. Kirman, T. N. Turon, D. F. Davey, H. Davis, D. M. Klinman, R. N. Coler, D. L. Sacks, and R. A. Seder. 2002. Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against Leishmania major infection. J. Exp. Med. 195:1565-1573.
6. Roman, M., E. Martin-Orozco, J. S. Goodman, M. D. Nguyen, Y. Sato, A. Ronaghy, R. S. Kornbluth, D. D. Richman, D. A. Carson, and E. Raz. 1997. Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants. Nat. Med. 3:849-854.
7. Sacks, D., and N. Noben-Trauth. 2002. The immunology of susceptibility and resistance to Leishmania major in mice. Nat. Rev. Immunol. 2:845-858.
8. Scott, P., P. Natovitz, R. L. Coffman, E. Pearce, and A. Sher. 1988. Immunoregulation of cutaneous leishmaniasis. T cell lines that transfer protective immunity or exacerbation belong to different T helper subsets and respond to distinct parasite antigens. J. Exp. Med. 168:1675-1684.
9. Spth, G. F., L. F. Lye, H. Segawa, D. L. Sacks, S. J. Turco, and S. M. Beverley. 2003. Persistence without pathology in phosphoglycan-deficient Leishmania major. Science 301:1241-1243.
10. Uzonna, J. E., G. F. Spath, S. M. Beverley, and P. Scott. 2004. Vaccination with phosphoglycan-deficient Leishmania major protects highly susceptible mice from virulent challenge without inducing a strong Th1 response. J. Immunol. 172:3793-3797.
11. Uzonna, J. E., G. Wei, D. Yurkowski, and P. Bretscher. 2001. Immune elimination of Leishmania major in mice: implications for immune memory, vaccination, and reactivation disease. J. Immunol. 167:6967-6974.
12. Walker, P. S., T. Scharton-Kersten, A. M. Krieg, L. Love-Homan, E. D. Rowton, M. C. Udey, and J. C. Vogel. 1999. Immunostimulatory oligodeoxynucleotides promote protective immunity and provide systemic therapy for leishmaniasis via IL-12- and IFN-gamma-dependent mechanisms. Proc. Natl. Acad. Sci. USA 96:6970-6975.
13. Zaph, C., J. Uzonna, S. M. Beverley, and P. Scott. 2004. Central memory T cells mediate long-term immunity to Leishmania major in the absence of persistent parasites. Nat. Med. 10:1104-1110.(Chahnaz Kebaer, Jude E. U)