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Previous Article | Next Article 
Infection and Immunity, September 2003, p. 5121-5129, Vol. 71, No. 9
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.9.5121-5129.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Khaled Tabbara,1 Yasmine Belkaid,1 Sylvie Bertholet,1 Daniela Verthelyi,2 Dennis Klinman,3 Robert A. Seder,4 and David L. Sacks1*
Laboratory of Parasitic Diseases,1 Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health,4 Division of Therapeutic Proteins,2 Section of Retroviral Immunology, Cellular Immunology Section, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 208923
Received 13 March 2003/ Returned for modification 5 June 2003/ Accepted 19 June 2003
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast to the experience with killed vaccines, live vaccination, or leishmanization as it is called, involving the inoculation of virulent organisms in the arm to protect against the development of severe or multiple lesions, especially on the face, provides virtually complete and lifelong protection. Large-scale leishmanization trials were carried out in the former Soviet Union (21), Israel (14), and Iran (33) with highly successful results. However, leishmanization has had a number of problems, most notably an unacceptable frequency of large ulcerating lesions that are slow to heal or, in rare cases, nonhealing (30). Thus, while it remains the only vaccine with proven efficacy in humans, its widespread use has been discontinued in each of these regions where leishmaniasis is endemic. Leishmanization might again be adopted as a primary intervention strategy were its pathological consequences made more predictably benign, as for instance by using live, attenuated parasites. An alternative approach might be to use a live, nonattenuated vaccine containing an appropriate adjuvant that promotes a more rapid onset of antileishmanial immunity and more rapid healing of cutaneous lesions.
The ability of CpG-containing immunostimulatory oligodeoxynucleotides (CpG ODNs) to induce both innate and adaptive cellular immune responses has made them a prospective prophylactic and therapeutic vaccine adjuvant for diseases requiring cellular immunity. CpG ODNs have been shown to stimulate macrophages and dendritic cells to synthesize several cytokines, including IL-12, IL-18, tumor necrosis factor alpha, alpha interferon (IFN-
), IFN-ß, and IFN-
, to upregulate costimulatory molecules such as CD40 and major histocompatibility complex class II and to enhance the ability of dendritic cells to present soluble protein to class I-restricted T cells (reviewed in references 24 and 26). The use of CpG ODNs as an effective adjuvant in whole-cell killed and subunit vaccines against L. major (37, 41, 48) and as immunotherapy to promote resistance in L. major-infected BALB/c mice has already been demonstrated (47, 52). CpG ODNs have not, however, been tested in the context of live vaccination against L. major. In addition to the use of CpG ODNs as adjuvant, the inclusion of heat-killed Leishmania in a live vaccine might help to prevent the development of active lesions, or promote their more rapid healing, in a manner similar to its use in conjunction with BCG as immunotherapy for cutaneous leishmaniasis (11).
Following intradermal inoculation of L. major, C57BL/6 (B/6) mice develop a cutaneous lesion that resolves spontaneously after 8 to 10 weeks. Self-cure is associated with a low-level, persistent infection and with the development of long-lasting immunity to reinfection. L. major infection in B/6 mice, therefore, seems to be an appropriate model to study the pathology and immunity associated with leishmanization in humans. In the present paper, we investigated the potential of CpG ODNs, delivered with or without killed parasites at the site of live vaccination with L. major in B/6 mice, to enhance primary immunity and thereby moderate the pathology associated with leishmanization without compromising the potency or durability of protection that live vaccination normally confers.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Live vaccination protocol. L. major clone V1 (MHOM/IL/80/Friedlin) promastigotes were grown at 26°C in medium 199 supplemented with 20% Hi-FCE (HyClone, Logan, Utah), 100 U of penicillin/ml, 100 µg of streptomycin/ml, 2 mM L-glutamine, 40 mM HEPES, 0.1 mM adenine (in 50 mM HEPES), 5 mg of hemin (in 50% triethanolamine)/ml, and 1 mg of 6-biotin (M199/S)/ml. Infective-stage promastigotes (metacyclics) of L. major were isolated from stationary cultures (4 to 5 days old) by negative selection of infective forms using peanut agglutinin (Vector Laboratories Inc, Burlingame, Calif.). Mice were infected in the ear dermis by using a 27.5-gauge needle in a volume of 10 µl. Mice were injected in both ears or in only the right ear with 104 L. major metacyclic promastigotes alone or with a combination of two CpG ODNs (50 µg), freeze-thaw lysates of autoclaved L. major promastigotes bound to aluminum hydroxide (ALM; 50 µg) prepared as previously described (19), or the combination of both. CpG ODNs were synthesized at the Center for Biologics Evaluation and Research Core Facility (Bethesda, Md.). The sequences of the immunostimulatory ODNs (5' to 3') were TCAACGTTGA and GCTAGACGTTAGCGT, with each containing a phosphorothioate backbone, as previously described (25).
DNA and killed vaccination protocols. For DNA vaccination, mice were injected in the right ear with 10 µg of total DNA of a mixture of plasmid DNAs encoding the antigens LACK, LmSTI1, and TSA suspended in sterile phosphate-buffered saline as previously described (28). Some mice were vaccinated with a mixture of ALM plus CpG ODNs by using the same dose employed in the live vaccination protocol. Vaccinated mice were given booster injections 2 weeks later using the same regimen.
L. major challenge. Protection against L. major challenge was evaluated in mice immunized in the right ear only. Fifteen weeks or 6 months after vaccination, 500 L. major metacyclic promastigotes in a volume of 10 µl were inoculated intradermally into the opposite (left) ear by using a 27.5-gauge needle. The evolution of the lesions was monitored by measuring the diameter of the induration with a direct-reading vernier caliper (Thomas, Swedesboro, N.J.).
Parasite quantitation. Parasite loads in the ears were determined as previously described (5). Briefly, the ventral and dorsal sheets of the infected ears were separated, deposited dermal-side down in Dulbecco's modified Eagle medium containing 100 U of penicillin/ml, 100 µg of streptomycin/ml, and Liberase CI enzyme blend (50 µg/ml; Boehringer Mannheim). Ears were incubated for 2 h at 37°C. The sheets were cut into small pieces and homogenized using a Teflon-coated microtissue grinder in a microcentrifuge tube containing 100 µl of M199/S. The tissue homogenates were filtered using a 70-µm-pore-size cell strainer (Falcon Products Inc., St. Louis, Mo.), serially diluted in a 96-well flat-bottom microtiter plate containing biphasic medium, prepared by using 50 µl of NNN medium containing 20% defibrinated rabbit blood, and overlaid with 100 µl of M199/S (5). The number of viable parasites in each ear was determined from the highest dilution at which promastigotes could be grown out after 7 days of incubation at 26°C. The number of parasites was also determined in the local draining lymph nodes (retromaxillar). The lymph nodes were recovered and mechanically dissociated using a pellet pestle and then serially diluted as described above.
In vivo recall response.
Mice were injected in both ears with a combination of living and killed antigens consisting of 106 metacyclic L. major promastigotes and 12.5 µg of soluble leishmanial antigen prepared from freeze-thawed (three times) stationary-phase L. major promastigotes. Forty-eight hours later, mice were sacrificed and cells from the ear dermis and local draining lymph nodes (three or four mice) were obtained. Single-cell suspensions from the ear dermis were obtained as described above. For the analysis of surface markers and intracytoplasmic staining for IFN-, cells were stimulated with L. major-infected bone marrow-derived murine dendritic cells (BMDDC) as a source of antigen for 16 h, at which time brefeldin A was added (10 µg/ml) (8). The cells were cultured for an additional 6 h and then fixed in 4% paraformaldehyde. Prior to staining, cells were incubated with an anti-Fc III/II (Pharmingen) receptor and 10% normal mouse serum in phosphate-buffered saline containing 0.1% bovine serum albumin and 0.01% NaN3. Cells were permeabilized and stained for the surface marker CD3 (145-2 C11; fluorescein isothiocyanate labeled; Pharmingen), CD4, or CD8 (RM4-5 and 53-6.7; cychrome conjugated; Pharmingen) and for the cytokine IFN- conjugated to R-phycoerythrin (JE56-5H4; Pharmingen). Incubations were carried out for 30 min on ice. The isotype controls used were rat immunoglobulin G2b (IgG2b; A95-1; Pharmingen) and rat IgG2a (R35-95; Pharmingen). The frequency of CD4+ and CD8+ T cells was determined by gating on CD3+ cells. For each sample, at least 100,000 cells were analyzed. The data were collected and analyzed using CELLQuest software and a FACScalibur flow cytometer (Becton Dickinson, San Jose, Calif.).
For cytokine measurement in culture supernatants, pooled cells from draining lymph nodes were resuspended in RPMI medium containing fetal bovine serum-penicillin-streptomycin at 6 x 106 cells/ml, and 0.1 ml was plated in U-bottom 96-well plates. Cells were incubated at 37°C in an atmosphere of 5% CO2 with uninfected or L. major-infected BMDDC. IFN- and IL-10 production in 48-h culture supernatants was quantitated by enzyme-linked immunosorbent assay.
Statistical analysis. Statistical tests were performed with SigmaStat (Jandel Software). Because most comparisons derived from data with nonhomogeneous variances, Kruskal-Wallis analysis of variance on ranks was performed and multiple comparisons were done by the Dunn method. Dual comparisons were made with the Mann-Whitney rank sum test. All data from parasite numbers were log transformed before statistical tests were conducted.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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following restimulation with infected BMDDC in vitro (Fig. 4A). The number of CD4+ IFN--producing cells at 4.5 weeks was fivefold higher than that in mice infected with parasites alone (156,400 versus 30,300). The resolution of the transient inflammation in the parasite- and CpG ODN-infected mice coincided with the decrease of CD4+ lymphocytes in the site, whereas these cells began to accumulate appreciably in the ears of mice infected with parasites alone by 6 weeks (>60,000 cells). The IL-10-producing CD4+ lymphocytes migrated to the infection site of mice receiving live parasites plus CpG ODNs with a similar kinetic as the IFN--producing CD4+ T cells, although their absolute numbers were less. Interestingly, the transient inflammation and parasite control were not associated with an increase in CD8+ IFN--producing T cells in the site, which (Fig. 4C) at 4.5 weeks remained low, at around 300 cells in both groups. The number of IFN--producing CD8+ T cells was in fact higher in the mice infected with parasites alone, reaching a peak at 6 weeks postinfection (approximately 2,500 cells).
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and IL-10 were also determined in supernatants of cells isolated from draining lymph nodes after in vitro restimulation using L. major-infected BMDDC (Fig. 5). By 2.5 weeks, significant IFN- secretion (3 to 4 ng/ml) was detected in both groups of mice, and despite the increased accumulation of cytokine-producing cells in the dermis of mice vaccinated with parasites plus CpG ODNs, the levels of cytokine produced by draining lymph node cells was not greater in these mice than in mice infected with parasites alone during early time points. IFN- production peaked sharply at week 6 in the mice infected with parasites alone, whereas it maintained roughly the same levels in mice infected with parasites plus CpG ODNs. IL-10 was detectable in both groups by 3.5 weeks and was higher at every time point thereafter in the CpG ODN-treated mice.
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Since the combination of ALM and CpG ODNs can itself induce long-lasting protection (37), it was decided to rigorously compare after 6 months the durability of protection and immune responses elicited by live vaccination using L. major and CpG ODNs plus ALM with that elicited by the nonlive vaccine containing CpG ODNs plus ALM alone. Comparison was also made with mice vaccinated 6 months previously with a cocktail vaccine containing DNA encoding the antigens LACK, LmST1, and TSA, which have previously been reported to provide long-lived protection against L. major (28). Mice immunized with live parasites with CpG ODNs plus ALM again showed significantly reduced dermal pathology during their primary infection compared with mice immunized with live parasites alone (P < 0.001) (Fig. 7A). These mice were completely protected against cutaneous leishmaniasis when challenged with L. major in the opposite ear after 6 months (Fig. 7B). The mice vaccinated with antigen DNA and CpG ODN plus ALM were also strongly protected, although some of the mice in each group developed a small induration. When the parasite loads in the ears were determined at 4 weeks (acute phase) and 20 weeks (chronic phase) postchallenge (Fig. 7C), the live, attenuated vaccine provided an approximately 1,000-fold reduction in parasite number at each time point compared to what was seen with the unvaccinated animals (P < 0.001). In contrast, the number of dermal parasites was not significantly reduced at either time point in mice immunized with antigen DNA, while the group vaccinated with CpG ODNs plus ALM showed a small but significant reduction (P < 0.05) in parasite burden only at the early time point.
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-producing CD4+ and CD8+ T cells recruited to an intradermal challenge site 6 months after vaccination. Table 1 shows the total number of cells extracted from the ear dermis 48 h following injection of antigen in the nonvaccinated ear and the frequencies and total numbers of recruited CD4+ and CD8+ T cells able to produce IFN- following restimulation with infected BMDDC in vitro. Recruitment of T cells to the site of antigen challenge was negligible in naïve mice. By comparison, the number of recruited lymphocytes able to make IFN- was maintained at a high level in mice that had been vaccinated with live parasites and CpG ODNs plus ALM (4.7 x 105 and 1.8 x 105 per ear for CD4+ and CD8+ T cells, respectively). The number of CD4+ T cells able to produce IFN- was especially striking and was 10-fold greater than the number determined for either of the nonlive vaccines. The number of IFN--producing CD8+ T cells was significantly increased relative to unvaccinated controls in mice vaccinated with ALM plus CpG ODNs (1.1 x 105/ear). The recall response in mice vaccinated with antigen DNA was relatively poor for either T-cell subset, though still greater than the response in unvaccinated mice.
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DISCUSSION |
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CpG ODNs act on innate immunity and rapidly trigger immune activation, including NO production. Thus, the CpG ODN may have acted locally on skin macrophages to facilitate Leishmania clearance, as has been described recently (47). Alternatively, the earlier killing may have been due to the ability of the CpG ODN to promote Leishmania-specific immunity, including the recruitment of effector T cells to the site. Coadministration of live parasites and CpG ODNs provoked a transient inflammation in the skin that was dominated by an early accumulation of IFN--producing CD4+ lymphocytes. The mechanism by which CD4+ T cells specifically migrated to the dermis of CpG ODN-treated mice is under investigation. Stan et al. (42) have suggested that CpG ODNs can induce transfected myocytes to upregulate major histocompatibility complex class II molecules and chemokines with subsequent recruitment of inflammatory cells secreting IFN-. Takeshita et al. (44) showed that murine BALB/c macrophages upregulate the expression of chemokines such as MIP1B, MIP2, RANTES, JE/MCP-1, and IP-10 after exposure to CpG ODNs. These chemokines are associated with Th1 responses (34) and therefore contribute to preferential generation of Th1-driven responses after treatment with CpG ODNs. It is interesting that CD8+ T cells comprised a relatively minor component of the early response to L. major in the mice treated with CpG ODN and that their numbers in the site remained low relative to the mice infected with L. major alone. As it is known that both CD8+ and CD4+ T cells are necessary to efficiently kill the high number of parasites that accumulate during the normal "silent" phase of L. major expansion in the skin (8), these results suggest that the earlier activation and recruitment of effector CD4+ T cells to the site is sufficient to control the initial stage of parasite growth, resulting in a significantly reduced inflammatory response, including CD8+-T-cell recruitment, that would normally accompany the immune clearance of high antigen loads from the skin. It is interesting that compared with the mice immunized with L. major alone, the mice immunized with L. major plus CpG ODNs also had an earlier recruitment and/or expansion of CD4+ IL-10+ cells in the site and a higher level of IL-10 produced by draining lymph node cells at early time points following infection. Their numbers were still approximately fivefold less than the numbers of IFN--producing effector cells in the site, which is consistent with the rapid control of infection that was achieved. Nonetheless, it is likely that the IL-10 produced by these cells contributed to parasite persistence and to the moderation of tissue pathology associated with the early control of infection in the skin (4, 7). Whether or not these cells were CD4+ CD25+ regulatory T cells and were driven by CpG ODNs directly or as a homeostatic response to the inflammation elicited by the vaccine are important questions to be addressed in future studies.
The addition of ALM without CpG ODNs did not seem to substantially reduce the pathogenicity of the live vaccine, and its application with CpG ODNs produced only a slightly better outcome compared to that with CpG ODNs alone. Nonetheless, as there is evidence from a preliminary clinical study in Uzbekistan that killed promastigotes delivered with live vaccine reduced the size and duration of active lesions (12), and as there is by now extensive experience regarding the safety of ALM in human trials (3, 22, 39), there may be a rationale for pursuing clinical studies using CpG ODNs in conjunction with ALM for attenuation of live vaccine.
Previous studies have shown that maintenance of acquired immunity in resistant mice is due to the persistence of low numbers of parasites in the inoculation site and draining lymph node after challenge and that complete elimination of parasites results in loss of immunity (7, 46). Thus, it is to be emphasized that despite the early containment of parasite growth and the absence of dermal lesions, the attenuated live vaccines still established a latent infection in which a small number of parasites persisted in the dermis and local draining lymph nodes. When long-term protection conferred by the live parasites plus CpG ODNs and ALM was carefully compared with that conferred by two powerful nonlive vaccines that have been described previously, i.e., the same CpG plus ALM used alone (37) or a cocktail antigen DNA vaccine (29), we found that the protection achieved by the nonlive vaccines, while still substantial, began to wane by 6 months, as revealed by development of small lesions and only slightly reduced parasite burdens compared to unvaccinated controls. By comparison, live vaccination with CpG ODNs and ALM provided complete protection against rechallenge infection. The live vaccine prevented the development of dermal lesions and significantly reduced dermal parasite numbers while maintaining greater numbers of antigen-specific CD4+ and CD8+ T cells that were tissue seeking and able to produce IFN-. It is likely that the difference between live and nonlive vaccination protocols in terms of the durability of protection would become even more pronounced as the interval between vaccination and challenge is prolonged.
The more conventional approach to the development of a nonpathogenic, live L. major vaccine has involved the use of parasites attenuated by irradiation, serial culture, or targeted gene deletion. Partial protection of BALB/c mice could be induced by inoculation of high doses of nonpathogenic clones or irradiated parasites (20, 23) or parasites bearing auxotrophic gene deletions (45). In these studies, however, only short-term protection was evaluated and was achieved only if a high-dose inoculum was administered by the intraperitoneal or intravenous route. Since in the BALB/c model short-term protection can also be achieved by intravenous, but not by subcutaneous, immunization using killed parasites (20), it seems unlikely in this study that the protection was dependent on parasite viability and infection. In the study by Rivier et al. (38) in which radioattenuated parasites were shown to protect CBA mice following subcutaneous inoculation, the protection required viable parasites. Those authors also demonstrated that a small number of parasites remained viable in the inoculation site at the time of the challenge and could be propagated in culture, suggesting that the protection may have been dependent on subclinical infection established by the few parasites that were not inactivated by irradiation. Again, the long-term persistence of the parasites or the durability of protection was not further addressed. The only other studies, so far as we are aware, to show that subcutaneous inoculation of live parasites can confer protection without producing a lesion involved the use of low doses (100 to 1,000) of nonattenuated promastigotes to establish subclinical infection (9, 27, 35). In a follow-up study by Uzonna et al. (46), the relationship between parasite persistence and immunity was established when it was shown that transfer of immune cells from subclinical mice could protect naïve BALB/c mice against L. major challenge and could completely clear the parasite, leaving the mice susceptible to a rechallenge infection. Thus, there is accumulating evidence suggesting that the key to long-term immunity is parasite persistence and there is no clear indication that the live, attenuated parasites used to date retain this ability.
Our approach to vaccine attenuation has been to employ live, virulent organisms inoculated with an appropriate adjuvant in order to induce an immune response similar to natural infection, only more rapid and robust so as to promote earlier control of parasite growth and healing of cutaneous lesions. Modifications of this same approach might include using the ALM plus CpG ODNs prophylactically to protect against the pathogenic effects of the live vaccine delivered within weeks or months of the killed vaccine or therapeutically to promote healing of vaccine lesions as they appear. The inoculation of CpG ODNs with or without ALM at the time and site of live vaccination seems to be the most practical approach to improving the safety while maintaining the immunogenicity of a highly effective vaccine that has already been widely applied.
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FOOTNOTES |
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Present address: Department of Microbiology and Tropical Medicine, George Washington University, Washington, DC 20052.
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REFERENCES |
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) or in combination with either 50 µg of ALM (
), 50 µg of CpG ODNs (
), or 50 µg of ALM and 50 µg of CpG ODNs (•). Data shown represent the mean induration ± standard deviation (SD) for 10 mice per group. Asterisks indicate statistically significant differences compared with results for mice vaccinated with parasites alone or with parasites plus ALM (P < 0.05).
) or in combination with 50 µg of ALM (
), or 50 µg each of ALM and CpG ODNs (X). Results are expressed as the mean induration ± SD for six mice per group.