Investigation of the antigenicity and protective efficacy of Leishmania promastigote membrane antigens in search of potential diagnostic and vaccine candidates against visceral leishmaniasis.

BACKGROUND: Visceral leishmaniasis (VL), is a parasitic disease that causes serious medical consequences if treatment is delayed. Despite a decline in the number of VL cases in the Indian subcontinent, the commencement of the disease in newer areas continues to be a major concern. Although serological diagnosis mainly by immunochromatographic tests has been found to be effective, a test of cure in different phases of treatment is still desired. Even though a good prophylactic response has been obtained in murine models by a number of vaccine candidates, few have been proposed for human use. METHODS: In this study, nine antigenic components (31, 34, 36, 45, 51, 63, 72, 91 and 97 kDa) of Leishmania promastigote membrane antigens (LAg), were electroeluted and evaluated through ELISA to diagnose and distinguish active VL from one month cured and six months post-treatment patients. Further, to investigate the immunogenicity of electroeluted proteins, human PBMCs of cured VL patients were stimulated with 31, 34, 51, 63, 72 and 91 kDa proteins. RESULTS: We found that 34 and 51 kDa proteins show 100% sensitivity and specificity with healthy controls and other diseases. After six months post-treatment, antibodies to 72 and 91 kDa antigens show a significant decline to almost normal levels. This suggests that 34 and 51 kDa proteins are efficient in diagnosis, whereas 72 and 91 kDa proteins may be used to monitor treatment outcome. In another assay, 51 and 63 kDa proteins demonstrated maximum ability to upregulate IFN-γ and IL-12 with minimum induction of IL-10 and TGF-ß. The results indicating that 51 and 63 kDa proteins could be strong candidates for human immunization against VL. In contrast, 34 and 91 kDa proteins demonstrated a reverse profile and may not be a good vaccine candidate. CONCLUSIONS: The preliminary data obtained in this study proposes the potential of some of the antigens in Leishmania diagnosis and for test of cure. Additionally, some antigens demonstrated good immunoprophylactic cytokine production through T cell-mediated immune response, suggesting future vaccine candidates for VL. However, further studies are necessary to explore these antigens in diagnosis and to access the long-term immune response.

Liposomal Elongation Factor-1α Triggers Effector CD4 and CD8 T Cells for Induction of Long-Lasting Protective Immunity against Visceral Leishmaniasis.

Despite advances, identification and formulation of safe and effective vaccine for long-lasting protection against leishmaniasis is still inadequate. In this study, we have identified a novel antigen, leishmanial elongation factor-1α (EF1-α), as an immunodominant component of solubilized leishmanial membrane antigens that reacts with visceral leishmaniasis (VL) sera and induces cellular proliferative and cytokine response in PBMCs of cured VL subjects. Leishmanial EF1-α is a 50 kDa antigen that plays a crucial role in pathogen survival by regulating oxidative burst in the host phagocytes. Previously, immunodominant truncated forms of EF1-α from different species of Leishmania have been reported. Formulation of the L. donovani 36 kDa truncated as well as the cloned recombinant EF1-α in cationic liposomes induce strong resistance to parasitic burden in liver and spleen of BALB/c mice through induction of DTH and a IL-10 and TGF-ß suppressed mixed Th1/Th2 cytokine responses. Multiparametric analysis of splenocytes for generation of antigen-specific IFN-γ, IL2, and TNF-α producing lymphocytes indicate that cationic liposome facilitates expansion of both CD4+ as well as CD8+ memory and effector T cells. Liposomal EF1-α is a novel and potent vaccine formulation against VL that imparts long-term protective responses. Moreover, the flexibility of this formulation opens up the scope to combine additional adjuvants and epitope selected antigens for use in other disease forms also.

IL-4 contributes to failure, and colludes with IL-10 to exacerbate Leishmania donovani infection following administration of a subcutaneous leishmanial antigen vaccine.

BACKGROUND: Visceral leishmaniasis caused by the protozoan parasite Leishmania donovani complex is a potentially fatal disease if left untreated. Few treatment options exist and are toxic, costly and ineffective against resistant strains. Thus a safe and efficacious vaccine to combat this disease is needed. Previously, we reported that intraperitoneal administration of leishmanial antigens (LAg) entrapped in liposomes conferred protection to BALB/c mice against L. donovani challenge infection. However, this vaccine failed to protect mice when administered subcutaneously. We therefore evaluated whether formulation of LAg in combination with two commonly used human-compatible adjuvants, alum and saponin, could improve the protective efficacy of subcutaneously administered LAg, to a level comparable to that of the intraperitoneal liposomal vaccination. RESULTS: Vaccine formulations of LAg with alum or saponin failed to reduce parasite burden in the liver, and alum + LAg immunized mice also failed to reduce parasite burden in the spleen. Interestingly, saponin + LAg vaccination actually resulted in an increased L. donovani parasitic load in the spleen following L. donovani challenge, suggesting this regimen exacerbates the infection. In contrast, mice immunized intraperitoneally with Lip + LAg demonstrated significant protection in both liver and spleen, as expected. Mechanistically, we found that failure of alum + LAg to protect mice was associated with elevated levels of IL-4, whereas both IL-4 and IL-10 levels were increased in saponin + LAg immunized mice. This outcome served to exacerbate L. donovani infection in the saponin + LAg group, despite a concurrent increase in proinflammatory IFN-γ production. On the contrary, protection against L. donovani challenge in Lip + LAg immunized mice was associated with elevated levels of IFN-γ in conjunction with low levels of IL-4 and IL-10 production. CONCLUSIONS: These findings indicate that elevated levels of IL-4 may contribute to LAg vaccine failure, whereas combined elevation of IL-4 together with IL-10 exacerbated the disease as observed in saponin + LAg immunized mice. In contrast, a robust IFN-γ response, in the absence of IL-4 and IL-10 production, was associated with protective immunity following administration of the Lip + LAg vaccine. Together these findings suggest that optimization of antigen/adjuvant formulations to minimize IL-4 and IL-10 induction may be helpful in the development of high efficacy vaccines targeting Leishmania.

Comparison of BCG, MPL and cationic liposome adjuvant systems in leishmanial antigen vaccine formulations against murine visceral leishmaniasis.

BACKGROUND: The development of an effective vaccine against visceral leishmaniasis (VL) caused by Leishmania donovani is an essential aim for controlling the disease. Use of the right adjuvant is of fundamental importance in vaccine formulations for generation of effective cell-mediated immune response. Earlier we reported the protective efficacy of cationic liposome-associated L. donovani promastigote antigens (LAg) against experimental VL. The aim of the present study was to compare the effectiveness of two very promising adjuvants, Bacille Calmette-Guerin (BCG) and Monophosphoryl lipid A (MPL) plus trehalose dicorynomycolate (TDM) with cationic liposomes, in combination with LAg, to confer protection against murine VL. RESULTS: All the three formulations afforded significant protection against L. donovani in both the visceral organs, liver and spleen. Although comparable level of protection was observed in BCG+LAg and MPL-TDM+LAg immunized mice, highest level of protection was exhibited by the liposomal LAg immunized group. Significant increase in anti-LAg IgG levels were detected in both MPL-TDM+LAg and liposomal LAg immunized animals with higher levels of IgG2a than IgG1. But BCG+LAg failed to induce any antibody response. As an index of cell-mediated immunity DTH responses were measured and significant response was observed in mice vaccinated with all the three different formulations. However, highest responses were observed with liposomal vaccine immunization. Comparative evaluation of IFN-gamma and IL-4 responses in immunized mice revealed that MPL-TDM+LAg group produced the highest level of IFN-gamma but lowest IL-4 level, while BCG+LAg demonstrated generation of suboptimum levels of both IFN-gamma and IL-4 response. Elicitation of moderate levels of prechallenge IFN-gamma along with optimum IL-4 corresponds with successful vaccination with liposomal LAg. CONCLUSION: This comparative study reveals greater effectiveness of the liposomal vaccine for protection against progressive VL in BALB/c. Again, evaluation of the immune responses by vaccination emphasizes the need of stimulation of potent cellular immunity based on both Th1 and Th2 cell responses to confer protection against VL.

Identification of novel Leishmania donovani antigens that help define correlates of vaccine-mediated protection in visceral leishmaniasis.

Visceral leishmaniasis (VL), caused by the intracellular parasite Leishmania donovani is a major public health problem in the developing world. But there is no effective and safe vaccine approved for clinical use against any form of leishmaniasis. Through reactivity with kala-azar patient and cured sera, polypeptides ranging from 91 to 31-kDa from L. donovani promastigotes were previously identified as potential protective vaccine candidates. In this study four polypeptides 91(LD91), 72 (LD72), 51(LD51) and 31 (LD31)-kDa were purified using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by electroelution. We compared the vaccine efficacy of these antigens encapsulated in cationic liposomes in BALB/c mice against challenge infection with L. donovani. Our results demonstrated that liposomal LD31 (74%-77%) and LD51 (72%-75%) vaccination reduced parasite burden to the greatest degree followed by liposomal LD72 (65%-67%) and LD91 (46%-49%). Analysis of the cytokine responses in immunized mice revealed that all the vaccinated groups produced prechallenge interferon-gamma, interleukin-12 and interleukin-4. Interestingly, the degree of reduction in parasite load could be predicted by the magnitude of the cytokine responses which correlated inversely with the parasite burden both in liver and spleen. The 31, 51 and 72-kDa bands were identified as ATP synthase alpha chain, beta-tubulin and heat shock 70-related protein 1 precursor of L. major, respectively using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) mass spectrometry. These three leishmanial antigens have not been described before as successful vaccine candidates examined against in vivo VL model. Thus, these antigens can be potential components of future antileishmaniasis vaccines.

Vaccination route that induces transforming growth factor beta production fails to elicit protective immunity against Leishmania donovani infection.

BALB/c mice immunized intraperitoneally (i.p.) and intravenously (i.v.) with Leishmania donovani promastigote membrane antigens (LAg), either free or encapsulated in liposomes, were protected against challenge infection with L. donovani, whereas mice immunized by the subcutaneous (s.c.) and intramuscular routes were not protected. Protected mice showed strong parasite resistance in both the liver and spleen, along with enhanced immunoglobulin G2a and delayed-type hypersensitivity responses. Again, mice vaccinated through the i.p. and i.v. routes showed high levels of NO production after challenge infection. s.c. vaccination resulted in an increased capacity of the spleen cells to produce prechallenge transforming growth factor beta (TGF-beta) levels during the in vitro antigen recall response, whereas i.p. immunization induced production of prechallenge gamma interferon, interleukin-12 (IL-12), and IL-4 levels, with a Th1 bias. Exposure to antigen-stimulated splenocyte supernatants of i.p. but not s.c. immunized mice activated macrophages for in vitro parasite killing. As an enhanced level of TGF-beta was detected in supernatants from unprotected s.c. immunized mice, neutralization by anti-TGF-beta antibody enhanced in vitro macrophage killing activity. The suppressive role of this cytokine was evaluated in vivo by vaccination with liposomal LAg and anti-TGF-beta antibody. Upon parasite challenge, these animals showed significant protection in both the liver and spleen. Moreover, the addition of recombinant TGF-beta in splenocyte supernatants of i.p. immunized mice in vitro as well as in vivo inhibited the protective ability of the macrophages by the i.p. route. Thus, the induction of high prechallenge TGF-beta limits the efficacy of vaccination by routes that are nonprotective.

Recent developments in leishmaniasis vaccine delivery systems.

BACKGROUND: The observation that recovery from infection with Leishmania confers immunity to reinfection suggests that control of leishmaniasis by vaccination may be possible. New generation vaccines, particularly those based on recombinant proteins and DNA, are found to be less immunogenic. OBJECTIVE: There is an urgent need for the development of new and improved vaccine adjuvants. METHODS: Based on their principal mechanisms of action, adjuvants can be broadly separated into two classes: immunostimulatory adjuvants and vaccine delivery systems. Vaccine delivery systems can carry both antigen and adjuvant for effective delivery to the antigen-presenting cells (APCs). In this article, we review the adjuvants, the delivery systems and their combinations used in the search of an effective vaccine against leishmaniasis. CONCLUSION: Based on current knowledge, cationic liposomes appear to have better prospects as effective delivery systems for developing a vaccine for leishmaniasis.

Leishmanial antigens in liposomes promote protective immunity and provide immunotherapy against visceral leishmaniasis via polarized Th1 response.

Leishmaniasis affects 12 million people, and it is generally agreed that vaccination provides the best long-term strategy for its control. An ideal vaccine should be effective in both preventing and treating leishmaniasis. However, immunological correlates to predict vaccine efficacy and success of treatment in visceral leishmaniasis (VL) remain ill defined. Here, we correlated the vaccine efficacy of soluble leishmanial antigens (SLA) from Leishmania donovani promastigote membrane, entrapped in negative, neutral and positively charged liposomes with the elicited immune responses to predict vaccine success in experimental VL. Production of both IFN-gamma and IL-4 with a dominance of Th1 response following immunization was required for optimum success against L. donovani infection in BALB/c mice. The best vaccine formulation, SLA in positively charged liposomes, was then used for immunotherapy. This vaccine induced more than 90% elimination of parasites from both liver and spleen. The success of immunotherapy exhibited an immune modulation with surge in Th1 cytokines, IFN-gamma and IL-12 with extreme down regulation of disease promoting IL-4 and IL-10. These findings suggest that an immune modulation towards Th1 is effective for both successful vaccination and immunotherapy.

Immune responses in kala-azar.

Human infection with Leishmania results in diverse clinical and immunopathological situations. The capacity of the parasites to cause this wide range of disease manifestations depends upon their ability to evade the immune defense mechanisms by performing a well-tuned orchestra of hostparasite interactions inside the macrophages. While updated knowledge focus on the key role of cell-mediated immunity (CMI) in protection, the survival strategies of the parasites leads to the suppression of CMI which can further be aggravated by the co-infections with HIV, tuberculosis etc. The present review describes the immune mechanisms in human leishmaniasis with a special attention to visceral leishmaniasis or kala-azar, one of the most important epidemiological health problems in Indian subcontinent. Modulations of the both humoral and cell-mediated immune responses during asymptomatic infections, active disease and after successful chemotherapy are discussed. The components responsible for the regulation of the critical balance of Th1/Th2 type of responses are re-evaluated. Co-infection of HIV and visceral leishmaniasis and their interdependence has been addressed. Although the specific role of an elevated humoral response in kala-azar is yet to be established, attempts for its application in diagnosis, precisely for the development of field diagnostic techniques, are presented. Also discussed are attempts to utilize the immunogenic potentials of different leishmanial antigens in the development of anti-leishmanial vaccines.