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Infectious Disease Research Institute,1 Corixa Corporation, Seattle, Washington 981042
Received 13 February 2002/ Returned for modification 2 May 2002/ Accepted 13 May 2002
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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No defined vaccine against leishmaniasis has been found, and killed or crude antigenic preparations of Leishmania promastigotes, so-called "first-generation vaccines," have been found to induce variable levels of protection in humans (26). Although chemotherapies against leishmaniasis do exist, they are few, are not affordable by those who need them, require daily injections for weeks, and are associated with side effects (29). In addition, drug resistance is becoming an increasing problem. Control of the vector, Phlebotomus sand flies, is not feasible in certain foci (forests, open desert areas), and where it is possible, the infrastructure and sustainability cost is prohibitively high. Vaccination through controlled infection with viable parasites or "leishmanization," which has been practiced in the Middle East (28), is being discontinued as a result of undesirable side effects and clinical complications (13, 14). Thus, systematic vaccine development may be the best control measure against leishmaniasis (11, 38).
Presumably, successful prophylactic immunization against at least one of the four main forms of leishmaniasis, cutaneous leishmaniasis (CL), should be amenable to vaccine-induced immunity given that this disease can be self-limiting and may be followed by resistance to reinfection. In recent years, several leishmanial antigens have been identified and evaluated as vaccine candidates in murine models of CL or visceral leishmaniasis (VL), with various degrees of protection (1, 5, 8, 9, 16, 17, 21, 24, 27, 30, 32, 33, 39, 42, 43, 48, 51). Thus, there is ample evidence that leishmanial antigens protect against disease in the murine model of the human disease.
Over the last decade, there has been considerable progress in understanding the immune responses involved in conferring protective immunity against leishmaniasis. In the murine model of leishmaniasis, interleukin-12 (IL-12)-driven Th1-type immune responses with gamma interferon (IFN-
), IL-2, and tumor necrosis factor beta production are associated with protection and self-cure while the Th2 phenotype with IL-4, IL-5, IL-10, IL-13, and transforming growth factor beta production is associated with susceptibility to disease (2, 12, 18, 19, 22, 23, 34, 35, 36). In the human disease, there is evidence that mixed Th cytokine profiles are present, while healing and protection against reinfection are associated with dominant Th1 and/or CD8+ T cells. These findings suggest that it is the balance between cytokines that activate or suppress activation of macrophages harboring Leishmania parasites that determines the outcome of the infection. Thus, treatments or antigen-adjuvant formulations that can alter the type of T-helper response may change the course of disease progression.
In previous studies, we identified and characterized three T-cell antigens, Leishmania elongation initiation factor (LeIF), Leishmania major stress-inducible protein 1 (LmSTI1), and thiol-specific antioxidant (TSA), that were found to be conserved among various Leishmania species and that elicit primarily a Th1-type immune response in murine or human cells (31, 40, 41, 49, 50). We have shown that two of these antigens, recombinant LmSTI1 (rLmSTI1) and recombinant TSA (rTSA), individually or when combined and delivered in the presence of recombinant IL-12 (rIL-12) or as a DNA-based vaccine, elicit protection against CL in the murine and nonhuman primate models (4, 25). The third antigen, recombinant LeIF (rLeIF), has been found to provide partial therapeutic protection against leishmaniasis and has adjuvant properties (41), including elicitation of IL-12. IL-12 is a potent inducer of IFN- (37, 47), maintains memory/effector Th1 cells (44), and is required for primary immunity to leishmaniasis (20, 45).
A vaccine suitable for use in humans and consisting of a single protein would be less costly to manufacture and has the advantage of a practical production process and more straightforward quality control testing than a vaccine consisting of several recombinant proteins. For this reason, a polyprotein composed of these three priority candidate antigens fused in tandem was made that is referred to as Leish-111f (Y. A. Skeiky, R. Coler, J. R. Webb, D. Carter, A. Campos-Neto, and S. G. Reed, 2nd Int. Congr. Leishmania Leishmaniasis [WorldLeishII], abstr. 34, 2001). Recombinant Leish-111f (rLeish-111f) adjuvanted with rIL-12 has been evaluated in the mouse model of Leishmania major and shown to be protective (Skeiky et al., 2nd Int. Congr. Leishmania Leishmaniasis). Because IL-12 has limitations as an adjuvant for human use, we have tested our polyprotein antigen in the presence of various adjuvants that may be more suitable for human use.
In the present report, we show that Leish-111f has relatively low inherent immunogenicity and requires a strong adjuvant to prime a specific immune response. We evaluated and compared the immunogenic and protective efficacies of formulating Leish-111f with either the naturally derived disaccharide adjuvant of Salmonella minnesota monophosphoryl lipid A (MPL) with squalene (MPL-SE) or a synthetic monosaccharide analog of MPL, termed Ribi 529, with SE (Ribi 529-SE). MPL has been used as an adjuvant in several human clinical trials of safety and immunogenicity, including vaccines for malaria (10), hepatitis B (46), genital herpes (G. Leroux-Roels, E. Moreau, B. Verhasselt, S. Biernaux, V. Brulein, M. Francotte, P. Pala, M. Slaopui, and P. Vandepapeliere, Program Abstr. 33rd Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1209, 1993), and allergy desensitization. These studies all found that MPL was well tolerated and there was no evidence of systemic toxicity. In combination with MPL-SE, Leish-111f induces a very potent immune response that protects mice against CL when they are challenged 3 or 12 weeks postimmunization. Our data therefore emphasize the importance of adjuvant for the screening of candidate antigens for Leishmania vaccines and demonstrate that Leish-111f shows great promise for inclusion in a future leishmaniasis vaccine.
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MATERIALS AND METHODS |
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Leishmanial antigens. rTSA, LmSTI1, LeIF, and Leish-111f were produced as described previously (40, 49, 50; Skeiky et al., 2nd Int. Congr. Leishmania Leishmaniasis). Briefly, Leish-111f protein was produced in recombinant Escherichia coli BL21(DE3)/pLysS (Novagen, Madison, Wis.). Stationary cultures of bacteria expressing Leish-111f were grown overnight with kanamycin (30 µg/ml) and chloramphenicol (34 µg/ml) and then diluted in fresh medium and induced with isopropyl-ß-D-thiogalactopyranoside (IPTG) for 3 h. The cells were harvested and lysed in the presence of Complete Protease Inhibitor Tablets (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and 10 mM Tris (pH 8.0) with a French pressure cell at 16,000 lb/in2. Inclusion bodies were harvested by centrifugation, washed once with 10 mM Tris (pH 8.0)-0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), and then solubilized in 8 M urea-30 mM ethanolamine (pH 10.0) (buffer A). Solubilization was allowed to continue overnight with agitation, and the solubilized Leish-111f protein was loaded onto a column containing High Q Strong Anion Exchange resin (Bio-Rad, Hercules, Calif.). Fractionation occurred by elution with a gradient from buffer A to buffer A containing 1 M NaCl (50% in 70 min and then 100% in 10 min). The fractions containing the protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were bound to nickel-nitrilotriacetic acid resin (Qiagen Inc., Valencia, Calif.) overnight at 4°C. The recombinant-bound-resin was equilibrated at room temperature, washed with 10 mM Tris (pH 8.0)-8 M urea-0.5% deoxycholic acid-10 mM imidazole-250 mM NaH2PO4, and eluted with 8 M urea-10 mM Tris (pH 8.0)-300 mM imidazole. The eluted protein was dialyzed against excess 10 mM Tris (pH 8.0) containing 1 mM reduced glutathione and stored at -80°C for further analysis. The lipopolysaccharide content of each antigen preparation was measured by a Limulus amoebocyte lysate test (BioWhittaker, Walkersville, Md.) and shown to be below 10 endotoxin units (EU)/mg of protein.
Immunization and determination of parasite burden after challenge. Groups of five mice were immunized with 10 µg of the Leish-111f polyprotein containing TSA, LmSTI1, and LeIF alone or with 5, 10, or 20 µg of either MPL-SE or Ribi 529-SE (Corixa Corporation, Seattle, Wash.) in a volume of 0.1 ml. Control groups received either adjuvant alone or saline. Three subcutaneous (s.c.) injections were given in the right footpad and at the base of the tail at 3-week intervals. Mice were infected 3 or 12 weeks after completion of the immunization protocol. As a challenge, 2 x 105 or 4 x 105 L. major WHOM/IL/80/Friedlin metacyclic promastigotes or 1 x 106 L. amazonensis promastigotes were suspended in 25 µl of saline and injected s.c. into the left hind footpad. The progress of infection was followed by measuring footpad swelling or thickness with a metric caliper (Mitutoyo Measuring Instruments, Aurora, Ill.). Grading of lesion size was done by subtracting the thickness of the uninfected contralateral footpad from that of the infected one. Animals were euthanized when they were unable to walk or when the lesions became necrotic.
ELISA for anti-Leish-111f IgG. Serum samples were taken from all animals, and antigen-specific enzyme-linked immunosorbent assays (ELISAs) were performed for the identification of specific anti-rLeish-111f, anti-rTSA, anti-rLmSTI1, and anti-rLeIF IgG1 and IgG2a. Briefly, 96-well microtiter plates (Costar) were coated (100 µl/well) with each recombinant antigen at 2 µg/ml in phosphate-buffered saline (PBS) and incubated for 4 h at 37°C. Plates were washed and blocked overnight at 4°C with 200 µl of 10% fetal calf serum (FCS) in PBS-Tween per well. Serum samples were diluted to 1:100 with PBS-Tween-10% FCS and applied to plates in twofold serial dilutions. Plates were incubated at 37°C for 4 h. Plates were washed, horseradish peroxidase-conjugated goat anti-mouse IgG1 or IgG2a (Southern Biotechnology Associates Ltd., Birmingham, Ala.) was added at a 1:2,000 dilution, and the mixture was incubated for 2 h at 37°C. Detection was done with the substrate 3,3',5,5'-tetramethylbenzidine (Kirkegaard & Perry, Gaithersburg, Md.). Optical density at 450 nm was determined by using 570 nm as the reference wavelength.
Culture of spleen cells.
Proliferative and cytokine responses were measured by preparing single-cell spleen suspensions by Lympholyte-M density gradient centrifugation (CedarLane Labs, Hornby, Ontario, Canada). Splenic mononuclear cells were resuspended in complete medium (RPMI 1640 medium supplemented with 10% FCS, 50 µg of gentamicin per ml, 2 mM L-glutamine, and 5 x 10-5 M ß-mercaptoethanol) and plated at 2 x 106/ml in 96-well flat-bottom plates (Costar). The spleen mononuclear cells and lymph node cells were cultured in the presence of anti-IL-4R antibody (Immunex Corporation, Seattle, Wash.) at 1 µg/ml to increase the sensitivity of the IL-4 measurement. The spleen cells were stimulated in vitro at 37°C in 5% CO2 with Leish-111f, rTSA, rLmSTI1, or rLeIF at 10 and 2 µg/ml; soluble leishmania antigen (SLA; 10 µg/ml); or medium alone. Concanavalin A, at a concentration of 1 µg/ml, was used in all experiments as a positive control for cell viability. Supernatants were collected after 48 and 72 h of culture and tested by ELISA for IL-4 and IFN- elicitation as previously described (7). Alternatively, after 5 days, the cultures were pulsed with 1 µCi of [3H]thymidine for 18 h, harvested, and counted in a gas scintillation counter.
Infection of antigen-presenting cells (APCs) with L. major. Macrophages were obtained from bone marrow precursor cells eluted from the femurs of BALB/c mice. Bone marrow cells were washed once in Hanks balanced salt solution and plated at 2 x 105/ml in six-well plates (Costar) in Dulbecco MEM containing 10% FCS, 1 mM sodium pyruvate, 2 mM L-glutamine, and 10 ng of recombinant granulocyte-macrophage colony-stimulating factor (Immunex Corporation) per ml. The medium was changed on days 2 and 4 of culture. On day 5 of culture, adherent cells (macrophages) were washed twice with cold PBS, harvested on ice, and infected with metacyclic L. major promastigotes.
To infect APCs, parasites and 105 macrophages/well in a 48-well plate were centrifuged at 1,200 x g for 1 h at room temperature to give a multiplicity of infection of two to four parasites per cell. At the end of infection, noninternalized protozoa were separated from macrophages by washing with warm Hanks balanced salt solution. An aliquot of infected APCs was fixed for 30 min in 1% glutaraldehyde and stained with Giemsa. The level of infection was measured by counting 1,000 cells in two separate cultures. Infected APCs were then cultured in fresh medium at 37°C in 5% CO2 for 60 h before being used in T-cell stimulation assays.
Statistical methods. The efficacies of different vaccination formulations were compared by one-way analysis of variance of footpad swelling in millimeters. A P value of <0.05 was considered significant.
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RESULTS |
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Recombinant Leish-111f was formulated with MPL and Ribi 529 adjuvants in SE. These adjuvants have been proven to boost the potency of modern vaccines or antigens, including those used for various infectious diseases and allergy desensitization. MPL has demonstrated safety and efficacy in human clinical trials involving more than 30,000 doses. RC-529 is a synthetic monosaccharide product based on structure-function studies of the MPL adjuvant. Since it can be synthesized organically in large-scale, high-purity lots, Ribi 529 has the advantage of a facile production process and quality control testing. Ribi 529 also has been shown to have an excellent safety profile and enhances cellular and humoral immune responses, including cytotoxic T lymphocytes, IgG2a, and IgA. Extensive preclinical studies have shown that Ribi 529 compares favorably to MPL, and phase I clinical trails are ongoing.
All vaccines were given three times at 3-week intervals, and the antigen-specific immune responses primed by immunization with vaccine formulations or controls were investigated. The titers of antibodies against Leish-111f and its individual components were determined in sera harvested from immunized mice. The isotype profile of antibody responses depends on the cytokines produced by antigen-specific T cells. Production of IgG2a is dependent on IFN-, whereas IL-4 is associated with high levels of IgG1 (6). The relative production of immunoglobulin isotypes is thus a marker for the induction of Th1-like versus Th2-like immune responses. Anti-Leish111f IgG1 and IgG2a titers were measured in the sera taken from all mice receiving Leish-111f vaccines, regardless of the adjuvant or the dose of the adjuvant administered. The highest levels of IgG1 were observed when Leish-111f was formulated with 20 µg of Ribi 529-SE (IgG1 titer, 2.174 ± 0.087) versus 20 µg of MPL-SE (IgG1 titer, 1.587 ± 0.1164) (Fig. 1). In contrast, the highest levels of IgG2a were measured in mice receiving Leish-111f plus various concentrations of MPL-SE (anti-MPL-SE titer, 2.24 ± 0.158; anti-Ribi 529-SE titer, 1.409 ± 0.133) (Fig. 1A to C). These mice also all had both IgG1 and IgG2a responses to the individual components of Leish-111f, suggesting that an immune response to each component is mounted when the polyprotein fusion molecule is formulated with the MPL-SE and Ribi 529-SE adjuvants and used for vaccination. These results also suggest that no strong antigenic competition occurs among TSA, LmSTI1, and LeIF, at least when immune responses are measured after administration of three immunizing doses of the vaccine. Sera from animals receiving saline or different amounts (5, 10, or 20 µg) of adjuvant alone were essentially negative for Leish-111f-specific IgG1 and IgG2a.
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To further investigate the phenotype (Th1 versus Th2) of the immune response elicited by immunization of Leish-111f, suspensions of spleen cells from immunized mice were restimulated in vitro with Leish-111f, its components, or controls and then proliferative and cytokine levels were measured. Splenocytes harvested from mice immunized with Leish-111f formulated with the MPL-SE or Ribi 529-SE adjuvant proliferated and produced IFN- with little IL-4 when stimulated in vitro with SLA (10 µg/ml) or rLeish-111f, TSA, LmSTI1, or LeIF (10 and 2 µg/ml) (Fig. 2A and B). These results confirm that immunization with rLeish-111f plus the MPL-SE or Ribi 529-SE adjuvant induces an immunity that is polarized toward a Th1 phenotype.
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when cultured with SLA (16,200 pg of IFN-/ml) or with the purified antigen rLmSTI1 (6,600 pg of IFN-/ml), rTSA (12,500 pg of IFN-/ml), rLeIF (22,000 pg of IFN-/ml), or Leish-111f (6,100 pg of IFN-/ml) (Fig. 3A). Furthermore, in the presence of APCs infected at a multiplicity of infection of 2 to 4 and T-cell lines generated against Leish-111f or SLA, high levels of proliferation and IFN- were measured (Fig. 3B and C). Uninfected APCs cultured with Leish-111f-specific T-cell lines did not proliferate or produce IFN- (Fig. 3B and C).
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production than did nonprotected or nonhealing mice. Mice immunized with Leish-111f in combination with other adjuvants, such as alum or MPL adsorbed to alum, developed strong Th2-polarized antigen-specific immune responses and did not develop a protective immune response upon a challenge with L. major (data not shown). The most effective combination was Leish-111f formulated with 20 µg of MPL-SE, which afforded protection for at least 14 weeks (Fig. 4A).
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Mice immunized with both 2 and 10 µg of Leish-111f plus MPL-SE were protected against an L. major challenge for at least 10 weeks, at which point all of the mice in the saline and adjuvant control groups had to be euthanized. At 10 weeks postchallenge, the difference in the mean level of protection observed in mice receiving 2 µg (0.38 ± 0.55) versus 10 µg (0.74 ± 0.57) of Leish-111f plus MPL-SE was not statistically significant (Fig. 6A). This experiment has been repeated twice with similar results. One experiment was extended for as long as 35 weeks postinfection, at which time the mean difference in footpad swelling measured in groups of mice immunized with 2 µg (1.7 ± 1.72 mm) versus 10 µg (1.74 ± 1.53 mm) of Leish-111f still did not reach statistical significance (data not shown). This suggests that in BALB/c mice, immunizing doses of 2 and 10 µg of Leish-111f with MPL-SE perform equally as well at eliciting protection against an L. major challenge.
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Changing the challenge dose from 2 x 105 to 4 x 105 L. major metacyclic promastigotes affected the kinetics of footpad swelling but did not change the protective effect of rLeish-111f with MPL-SE (data not shown). With the larger challenge dose, a significant difference in lesion size could be discerned between the control and vaccinated groups from week 2 to 3 onward, whereas with the smaller challenge dose, at least 4 weeks was required before a significant difference between these groups of mice became observable. Additional experiments showed that similar protection results were obtained when mice were given two instead of three s.c. immunizations of the Leish-111f vaccine followed by a challenge with 2 x 105 L. major promastigotes or 8 x 103 L. major amastigotes (data not shown).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this report, we have shown that a vaccine composed of MPL-SE and a recombinant polyprotein containing three different Leishmania antigens fused in tandem induced effective protection against L. major and L. amazonensis challenges in two different strains of mice. MPL-SE at 20 µg was the most potent of the six different adjuvant formulations tested at eliciting protection against leishmaniasis in BALB/c mice. Whether the vaccine contained 2 or 10 µg of rLeish-111f or whether the challenge dose was increased from 2 x 105 to 4 x 105 parasites was not relevant with regard to the protective effect. Furthermore, this vaccine formulation conferred durable protection against leishmaniasis for up to 14 weeks. Of particular importance is the finding that susceptible BALB/c mice immunized with the Leish-111f vaccine were protected whether they were challenged 3 or 12 weeks postvaccination. Achievement of such long-lasting protection against leishmaniasis with a protein-based vaccine has not been reported previously.
Effective vaccination against Leishmania infection depends on the generation of antigen-specific memory T cells that, upon exposure to infecting parasites, rapidly expand as effector Th1 T cells for production of IFN-. In turn, IFN--mediated activation of infected macrophages should lead to killing of intracellular parasites if the recall response is rapid and of sufficient magnitude to overcome the adverse effects of the Leishmania infection on macrophage function. Maintenance of adequate vaccine-induced memory T cells for long-term protective immunity against leishmaniasis is another important consideration. It has been argued that the maintenance of memory T cells and lasting protective immunity against leishmaniasis depends on sustained IL-12 production and periodic antigen reexposure, either through natural exposure to Leishmania parasites, persistence of subclinical parasites, or repeated vaccine delivery. Thus, an important consideration in leishmaniasis vaccine design is delivery in a form that is likely to persist, such as DNA or formulation with bacterium-derived or slow-release adjuvants such as Corynebacterium parvum or alum, respectively. Remarkably, we found that the immune protection conferred by the Leish-111f-plus-MPL-SE vaccine was systemic and maintained for more than 3 months after infection. These data stand in contrast to previous reports that durable protection against leishmaniasis was not achieved with protein-based vaccines when the challenge was delayed for 12 weeks after primary immunization (25) unless IL-12 DNA was administered concurrently (15) or unless the mice were given supplemental (every 2 weeks) treatment with the recombinant protein and/or rIL-12 (44).
Several lines of evidence indicate that the protection induced by our multicomponent vaccine was due not to a nonspecific immunostimulatory effect of the adjuvants used but rather to immune responses induced by both the sequences specific to our recombinant polyprotein antigen and the MPL-SE adjuvant. The levels of spontaneous release of IFN- by spleen cells from vaccinated and control mice were similar. Control mice showed no evidence of antigen-specific cellular or humoral immune responses, reduction in lesion size, or reduction in parasite burden (data not shown). The finding that Leish-111f is not highly immunogenic or protective in the absence of adjuvant suggests that this molecule does not have sufficient inherent adjuvant activity in vivo. Furthermore, the antigen-specific antibody isotype profile shifts significantly to higher levels of IgG2a on addition of MPL-SE to rLeish-111f. Thus, the protective ability of Leish-111f is adjuvant dependent.
MPL is thought to activate monocytes-macrophages and stimulate the release of several cytokines, including IL-12, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor. Through the action of these cytokines, it is thought that T cells and APCs are recruited to local lymphoid organs, where cellular interactions occur that result in induction of strong Th1-type cellular responses characterized by increased production of IFN- and IL-2, as well as production of IgG2a. We are currently investigating the various cell types and associated cytokines that are recruited by the Leish-111f vaccine and that are involved in conferring durable protection against leishmaniasis.
BALB/c mice are remarkably susceptible to L. major and develop infections relating to the induction of an intense Th2 response. Thus, it is significant that the Leish-111f vaccine is capable of eliciting robust protection against leishmaniasis in this animal model of the disease. It has been reported that IL-12 is the key endogenous initiator of the cell-mediated immune response that underlies defense against L. major (47). Correlating with long-term protection, we have found that mice immunized with Leish-111f plus MPL-SE have persistent memory cells that are capable of antigen-specific restimulation for at least 6 months.
In theory, linking these three antigens in tandem increases the number of epitopes available for inducing Th1-type immune responses and protective immunity in a heterogeneous human population with diverse major histocompatibility complex types. For subunit vaccine development, induction of protective immunity will likely require responses against different epitopes, probably from several antigens. Durable immunity is likely to require the delivery of the vaccine in such a way that there is sustained antigen release or antigen persistence, to mimic, as it were, the persistent antigenic stimulation of subclinical or posthealing infection. Our Leish-111f formulation satisfies these requirements for vaccination against leishmaniasis. For these reasons, we are currently testing the Leish-111f vaccine in a nonhuman primate model and plan to initiate phase I human safety and immunogenicity trials in the near future.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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