Safety and immunogenicity of ChAd63-KH vaccine in post-kala-azar dermal leishmaniasis patients in Sudan.

Post-kala-azar dermal leishmaniasis (PKDL) is a chronic, stigmatizing skin condition occurring frequently after apparent clinical cure from visceral leishmaniasis. Given an urgent need for new treatments, we conducted a phase IIa safety and immunogenicity trial of ChAd63-KH vaccine in Sudanese patients with persistent PKDL. LEISH2a (ClinicalTrials.gov: NCT02894008) was an open-label three-phase clinical trial involving sixteen adult and eight adolescent patients with persistent PKDL (median duration, 30 months; range, 6-180 months). Patients received a single intramuscular vaccination of 1 × 1010 viral particles (v.p.; adults only) or 7.5 × 1010 v.p. (adults and adolescents), with primary (safety) and secondary (clinical response and immunogenicity) endpoints evaluated over 42-120 days follow-up. AmBisome was provided to patients with significant remaining disease at their last visit. ChAd63-KH vaccine showed minimal adverse reactions in PKDL patients and induced potent innate and cell-mediated immune responses measured by whole-blood transcriptomics and ELISpot. 7/23 patients (30.4%) monitored to study completion showed >90% clinical improvement, and 5/23 (21.7%) showed partial improvement. A logistic regression model applied to blood transcriptomic data identified immune modules predictive of patients with >90% clinical improvement. A randomized controlled trial to determine whether these clinical responses were vaccine-related and whether ChAd63-KH vaccine has clinical utility is underway.

Immunogenicity of HLA-DR1 and HLA-A2 peptides derived from Leishmania major Gp63 in golden hamsters.

AIMS: This study aimed to evaluate the toxicity and humoral and cellular immune response of three heterologous vaccines against Leishmania infantum, yet containing synthetic peptides from Leishmania major in the experimental model in hamsters. METHODS AND RESULTS: Through bioinformatics analyses, two Leishmania major Gp63 peptides were predicted and selected for vaccine formulations. Hamsters were divided into four groups, with each group receiving doses of three vaccine formulations containing HLA-DR1 or HLA-A2 peptides plus MontanideTM or both associated with the adjuvant. The animals received three vaccine doses and were evaluated for toxicity after each dose, in addition to being analysed for the production of antibodies and lymphoproliferation on day 211 after the last vaccine dose. Peptides predicted in association with oily adjuvant induced a humoral response and strong lymphoproliferation to Leishmania infantum antigen-specific stimulation.

Chitosan Contribution to Therapeutic and Vaccinal Approaches for the Control of Leishmaniasis.

The control of leishmaniases, a complex parasitic disease caused by the protozoan parasite Leishmania, requires continuous innovation at the therapeutic and vaccination levels. Chitosan is a biocompatible polymer administrable via different routes and possessing numerous qualities to be used in the antileishmanial strategies. This review presents recent progress in chitosan research for antileishmanial applications. First data on the mechanism of action of chitosan revealed an optimal in vitro intrinsic activity at acidic pH, high-molecular-weight chitosan being the most efficient form, with an uptake by pinocytosis and an accumulation in the parasitophorous vacuole of Leishmania-infected macrophages. In addition, the immunomodulatory effect of chitosan is an added value both for the treatment of leishmaniasis and the development of innovative vaccines. The advances in chitosan chemistry allows pharmacomodulation on amine groups opening various opportunities for new polymers of different size, and physico-chemical properties adapted to the chosen routes of administration. Different formulations have been studied in experimental leishmaniasis models to cure visceral and cutaneous leishmaniasis, and chitosan can act as a booster through drug combinations with classical drugs, such as amphotericin B. The various architectural possibilities given by chitosan chemistry and pharmaceutical technology pave the way for promising further developments.

The antibiotic resistance-free mammalian expression plasmid vector pPAL for development of third generation vaccines.

DNA vaccines require a vector to replicate genes and express encoding antigens. Antibiotic resistance genes are often used as selection markers, which must not be released to the environment upon final product commercialization. For this reason, generation of antibiotic resistance-free vectors is imperative. The pPAL vector contains the cytomegalovirus enhancer and promoter for expression in mammalian cells and the E. coli fabI chromosomal gene as a selectable marker. The fabI gene encodes the enoyl-ACP reductase (FabI). The bacteriostatic compound triclosan is an inhibitor of this enzyme. Therefore, the selection of positive clones depends on the enzyme:inhibitor molar ratio. According to western blot analysis, the pPAL vector is functional for expression of the Leishmania infantum (Kinetoplastid: Trypanosomatidae) gene encoding for the protein kinase C receptor analog (LACK/p36) in the HEK293T human cell line transfected with pPAL-LACK. The fabI gene sequence contains a 210 bp CpG island, suggesting a potential role as an adjuvant of the antibiotic resistance-free pPAL vector. In fact, Th1 response induction levels against canine leishmaniasis only using pPAL-LACK was shown to be as strong as in previous strategies using a recombinant vaccinia virus in combination with standard mammalian expression plasmid vectors. In summary, the pPAL plasmid contains the essential elements for manipulation and expression of any cloned DNA sequence in prokaryotic and mammalian cells using an E. coli endogenous gene as a selectable marker, which also provides a long CpG island. This element enhances Th1 immune response against L. infantum infection in dogs using the gene encoding for the LACK antigen. Therefore, this antibiotic resistance-free plasmid is a vaccine vector actively participating in protection against canine leishmaniasis and may be potentially tested as a vaccine vector with other antigens against different pathogens.

Antigenicity, immunogenicity and protective efficacy of a conserved Leishmania hypothetical protein against visceral leishmaniasis.

In this study, a Leishmania hypothetical protein, LiHyS, was evaluated regarding its antigenicity, immunogenicity and protective efficacy against visceral leishmaniasis (VL). Regarding antigenicity, immunoblottings and an enzyme-linked immunosorbent assay using human and canine sera showed high sensitivity and specificity values for the recombinant protein (rLiHyS) in the diagnosis of VL. When evaluating the immunogenicity of LiHyS, which is possibly located in the parasite's flagellar pocket, proliferative assays using peripheral blood mononuclear cells from healthy subjects or VL patients showed a high proliferative index in both individuals, when compared to the results obtained using rA2 or unstimulated cultures. Later, rLiHyS/saponin was inoculated in BALB/c mice, which were then challenged with Leishmania infantum promastigotes. The vaccine induced an interferon-γ, interleukin (IL)-12 and granulocyte-macrophage colony-stimulating factor production, which was maintained after infection and which was associated with high nitrite and IgG2a antibody levels, as well as low IL-4 and IL-10 production. Significant reductions in the parasite load in liver, spleen, bone marrow and draining lymph nodes were found in these animals. In this context, the present study shows that the rLiHyS has the capacity to be evaluated as a diagnostic marker or vaccine candidate against VL.

Molecular, biochemical characterization and assessment of immunogenic potential of cofactor-independent phosphoglycerate mutase against Leishmania donovani: a step towards exploring novel vaccine candidate.

Despite immense efforts, vaccine against visceral leishmaniasis has yet not been developed. Earlier our proteomic study revealed a novel protein, cofactor-independent phoshoglycerate mutase (LdiPGAM), an important enzyme in glucose metabolism, in T helper cells type 1 (Th1) stimulatory region of soluble Leishmania donovani antigen. In this study, LdiPGAM was biochemically and molecularly characterized and evaluated for its immunogenicity and prophylactic efficacy against L. donovani. Immunogenicity of recombinant LdiPGAM (rLdiPGAM) was initially assessed in naïve hamsters immunized with it by analysing mRNA expression of inducible nitric oxide (NO) synthase (iNOS) and other Th1/T helper cells type 2 cytokines, which revealed an upregulation of Th1 cytokines along with iNOS. Immunogenicity of rLdiPGAM was further evaluated in lymphocytes of treated Leishmania-infected hamsters and peripheral blood mononuclear cells of Leishmania patients in clinical remission by various parameters, viz. lymphoproliferation assay and NO production (hamsters and patients) and levels of various cytokines (patients). rLdiPGAM induced remarkable Lymphoproliferative response and NO production in treated Leishmania-infected hamsters as well as in patients and increase in interferon gamma (IFN-γ), interleukin-12 (IL-12p40) responses in Leishmania patients in clinical remission. Vaccination with rLdiPGAM exerted considerable prophylactic efficacy (73%) supported by increase in mRNA expression of iNOS, IFN-γ and IL-12p40 with decrease in transforming growth factor beta and interleukin-10. Above results indicate the importance of rLdiPGAM protein as a potential vaccine candidate against visceral leishmaniasis.

A Non-pathogenic Recombinant Leishmania Expressing Lipophosphoglycan 3 Against Experimental Infection with Leishmania infantum.

Visceral leishmaniasis (VL) is caused by Leishmania infantum in the Mediterranean basin and affects primarily children and immunosuppressed individuals. Various strategies of vaccination have so far been examined by either protein or DNA without achievable complete protection against the disease. The live non-pathogenic lizard parasite, Leishmania tarentolae, expressing elected Leishmania antigens has recently provided a promising new approach as a safe and effective live vaccine candidate to prevent leishmaniasis. Here, we evaluated the immunoprotective potential of a live recombinant L. tarentolae expressing Lipophosphoglycan 3 (LPG3) antigen against L. infantum infection in BALB/c mice. Results indicated that the administration of live recombinant Leishmania produced a significant high level of IFN-γ accompanied by reduced levels of IL-10 as compared to wild-type parasites as live vaccine control, thus suggesting the induction of a Th1-type immune response in a mouse model of visceral leishmaniasis. Analysis of the IgG antibody response also showed high levels of IgG2a relative to IgG1 in sera of mice immunized with recombinant Leishmania parasites. However, immune responses elicited by this live vaccine conferred partial protection against infectious challenge. Therefore, further studies are required to increase its protective efficacy.

Cross-protective efficacy from a immunogen firstly identified in Leishmania infantum against tegumentary leishmaniasis.

Experimental vaccine candidates have been evaluated to prevent leishmaniasis, but no commercial vaccine has been proved to be effective against more than one parasite species. LiHyT is a Leishmania-specific protein that was firstly identified as protective against Leishmania infantum. In this study, LiHyT was evaluated as a vaccine to against two Leishmania species causing tegumentary leishmaniasis (TL): Leishmania major and Leishmania braziliensis. BALB/c mice were immunized with rLiHyT plus saponin and lately challenged with promastigotes of the two parasite species. The immune response generated was evaluated before and 10 weeks after infection, as well as the parasite burden at this time after infection. The vaccination induced a Th1 response, which was characterized by the production of IFN-γ, IL-12 and GM-CSF, as well as by high levels of IgG2a antibodies, after in vitro stimulation using both the protein and parasite extracts. After challenge, vaccinated mice showed significant reductions in their infected footpads, as well as in the parasite burden in the tissue and organs evaluated, when compared to the control groups. The anti-Leishmania Th1 response was maintained after infection, being the IFN-γ production based mainly on CD4(+) T cells. We described one conserved Leishmania-specific protein that could compose a pan-Leishmania vaccine.

Studies on cocktails of 31-kDa, 36-kDa and 51-kDa antigens of Leishmania donovani along with saponin against murine visceral leishmaniasis.

A substantial number of antigens of Leishmania donovani have been described in the past. However, identifying candidate antigens is not enough. Appropriate antigen delivery to induce the right type of immune response against leishmaniasis (i.e. induction of a strong antigen-specific Th1 type of immune response) is another crucial component of an effective vaccine. Therefore, 'cocktail' vaccines are proposed based on the assumption that such cocktails will show enhanced efficacy. Studies have been carried out on LD31 and LD51 polypeptides from L. donovani promastigotes, which have proven to be potential vaccine candidates. This study was designed to check the protective efficacy of various cocktails of low molecular weight antigens alone and along with saponin as adjuvant. Mice were sacrificed on different post-challenge days for evaluation of parasite load and other immunological parameters. Protective efficacy of different vaccine formulations was revealed by significant decline in parasite burden and increased DTH Delayed Type Hypersenstivity responses. The antibody response was of IgG type with elevated IgG2a and decreased production of IgG1, whereas cytokine levels pointed towards the generation of protective Th1 type of immune response. Among all vaccine formulations, cocktail of 31+51+saponin was found to be highly immunogenic and imparted maximum protection.

Live attenuated Leishmania donovani p27 gene knockout parasites are nonpathogenic and elicit long-term protective immunity in BALB/c mice.

Leishmaniasis causes significant morbidity and mortality worldwide, and no vaccines against this disease are available. Previously, we had shown that the amastigote-specific protein p27 (Ldp27) is a component of an active cytochrome c oxidase complex in Leishmania donovani and that upon deletion of its gene the parasite had reduced virulence in vivo. In this study, we have shown that Ldp27(-/-) parasites do not survive beyond 20 wk in BALB/c mice and hence are safe as an immunogen. Upon virulent challenge, mice 12 wk postimmunization showed significantly lower parasite burden in the liver and spleen. When mice were challenged 20 wk postimmunization, a significant reduction in parasite burden was still noted, suggesting long-term protection by Ldp27(-/-) immunization. Immunization with Ldp27(-/-) induced both pro- and anti-inflammatory cytokine responses and activated splenocytes for enhanced leishmanicidal activity in association with NO production. Protection in both short- and long-term immunized mice after challenge with the wild-type parasite correlated with the stimulation of multifunctional Th1-type CD4 and CD8 T cells. Adoptive transfer of T cells from long-term immunized mice conferred protection against virulent challenge in naive recipient mice, suggesting involvement of memory T cell response in protection against Leishmania infection. Immunization of mice with Ldp27(-/-)also demonstrated cross-protection against Leishmania major and Leishmania braziliensis infection. Our data show that genetically modified live attenuated Ldp27(-/-) parasites are safe, induce protective immunity even in the absence of parasites, and can provide protection against homologous and heterologous Leishmania species.

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.

The protective immune response produced in dogs after primary vaccination with the LiESP/QA-21 vaccine (CaniLeish®) remains effective against an experimental challenge one year later.

Control of canine leishmaniasis is an important objective for the benefit of dogs living in or visiting endemic areas and for public health because of the zoonotic nature of this disease. Resistance or susceptibility to developing canine leishmaniasis after exposure to Leishmania infantum is primarily determined by the ability of the immune system to develop an appropriate Th1-dominated specific response to the parasite. For this reason there is a need for effective canine vaccines that can decrease the number of dogs developing progressive infections. In this study, we followed the impact of the LiESP/QA-21 canine vaccine (composed of excreted-secreted proteins of L. infantum and the QA-21 saponin adjuvant), recently launched commercially in Europe, on selected humoral and cellular immune parameters following an infectious intravenous challenge with L. infantum promastigotes administered one year after the primary vaccine course. We also followed parasitological parameters to determine the parasitological status of the challenged dogs. In contrast to controls, vaccinated dogs retained significantly stronger cell-mediated immune responses against the parasite despite a virulent challenge and had significantly lower mean parasite burdens at the end of the study, associated with a lower probability of developing active infections. These results confirm that the immune responses generated by vaccination with LiESP/QA-21 are still effective against an intravenous challenge one year after the primary vaccine course.

Immunoliposomes containing Soluble Leishmania Antigens (SLA) as a novel antigen delivery system in murine model of leishmaniasis.

Development of new generation of vaccines against leishmaniasis requires adjuvants to elicit the type and intensity of immune response needed for protection. The coupling of target-specific antibodies to the liposomal surface to create immunoliposomes has appeared as a promising way in achieving a liposome active targeting. In this study, immunoliposomes were prepared by grafting non-immune mouse IgG onto the liposomal surface. The influence of active targeted immunoliposomes on the type and intensity of generated immune response against Leishmania was then investigated and compared with that of liposomes and control groups which received either SLA or HEPES buffer alone. All formulations contained SLA and were used to immunize the mice in the left hind footpad three times in 3-week intervals. Evaluation of lesion development and parasite burden in the foot and spleen after challenge with Leishmania major, evaluation of Th1 cytokine (IFN-γ), and titration of IgG isotypes were carried out to assess the type of generated immune response and the extent of protection. The results indicated that liposomes might be effective adjuvant systems to induce protection against L. major challenge in BALB/c mice, but stronger cell mediated immune responses were induced when immunoliposomes were utilized. Thus, immune modulation using immunoliposomes might be a practical approach to improve the immunization against L. major.

Revolutionizing Leishmaniasis Treatment with Cutting Edge Drug Delivery Systems and Nanovaccines: An Updated Review.

Leishmaniasis, one of the most overlooked tropical diseases, is a life-threatening illness caused by the parasite Leishmania donovani that is prevalent in underdeveloped nations. Over 350 million individuals in more than 90 different nations worldwide are at risk of contracting the disease, which has a current fatality rate of 50 000 mortalities each year. The administration of liposomal Amp B, pentavalent antimonials, and miltefosine are still considered integral components of the chemotherapy regimen. Antileishmanial medications fail to treat leishmaniasis because of their numerous drawbacks. These include inadequate effectiveness, toxicity, undesired side effects, drug resistance, treatment duration, and cost. Consequently, there is a need to overcome the limitations of conventional therapeutics. Nanotechnology has demonstrated promising outcomes in addressing these issues because of its small size and distinctive characteristics, such as enhanced bioavailability, lower toxicity, biodegradability, and targeted drug delivery. This review is an effort to highlight the recent progress in various nanodrug delivery systems (nDDSs) over the past five years for treating leishmaniasis. Although the preclinical outcomes of nDDSs have shown promising treatment for leishmaniasis, further research is needed for their clinical translation. Advancement in three primary priority domains─molecular diagnostics, clinical investigation, and knowledge dissemination and standardization─is imperative to propel the leishmaniasis field toward translational outcomes.

Vaccine efficacy induced by virus-like particles containing Leishmania donovani surface glycoprotein GP63.

Leishmania donovani surface glycoprotein 63 (GP63) is a major virulence factor involved in parasite escape and immune evasion. In this study, we generated virus-like particles (VLPs) expressing L. donovani GP63 using the baculovirus expression system. Mice were intramuscularly immunized with GP63-VLPs and challenged with L. donovani promastigotes. GP63-VLP immunization elicited higher levels of L. donovani antigen-specific serum antibodies and enhanced splenic B cell, germinal center B cell, CD4+, and CD8+ T cell responses compared to unimmunized controls. GP63-VLPs inhibited the influx of pro-inflammatory cytokines IFN-γ and IL-6 in the livers, as well as thwarting the development of splenomegaly in immunized mice. Upon L. donovani challenge infection, a drastic reduction in splenic parasite burden was observed in VLP-immunized mice. These results indicate that GP63-VLPs immunization conferred protection against L. donovani challenge infection by inducing humoral and cellular immunity in mice.

SIV infection of rhesus macaques results in dysfunctional T- and B-cell responses to neo and recall Leishmania major vaccination.

HIV infection is characterized by immune system dysregulation, including depletion of CD4+ T cells, immune activation, and abnormal B- and T-cell responses. However, the immunologic mechanisms underlying lymphocytic dysfunctionality and whether it is restricted to immune responses against neo antigens, recall antigens, or both is unclear. Here, we immunized SIV-infected and uninfected rhesus macaques to induce immune responses against neo and recall antigens using a Leishmania major polyprotein (MML) vaccine given with poly-ICLC adjuvant. We found that vaccinated SIVuninfected animals induced high frequencies of polyfunctional MML-specific CD4+ T cells. However, in SIV-infected animals, CD4+ T-cell functionality decreased after both neo (P = .0025) and recall (P = .0080) MML vaccination. Furthermore, after SIV infection, the frequency of MML-specific antibody-secreting classic memory B cells was decreased compared with vaccinated, SIV-uninfected animals. Specifically, antibody-secreting classic memory B cells that produced IgA in response to either neo (P = .0221) or recall (P = .0356) MML vaccinations were decreased. Furthermore, we found that T-follicular helper cells, which are essential for priming B cells, are preferentially infected with SIV. These data indicate that SIV infection results in dysfunctional T-cell responses to neo and recall vaccinations, and direct SIV infection of T-follicular helper cells, both of which probably contribute to deficient B-cell responses and, presumably, susceptibility to certain opportunistic infections.

Cationic solid-lipid nanoparticles are as efficient as electroporation in DNA vaccination against visceral leishmaniasis in mice.

The use of an appropriate delivery system has recently emerged as a promising approach for the development of effective vaccination against visceral leishmaniasis (VL). Here, we compare two vaccine delivery systems, namely electroporation and cationic solid-lipid nanoparticle (cSLN) formulation, to administer a DNA vaccine harbouring the L. donovani A2 antigen along with L. infantum cysteine proteinases [CPA and CPB without its unusual C-terminal extension (CPB(-CTE) )] and evaluate their potential against L. infantum challenge. Prime-boost administration of the pcDNA-A2-CPA-CPB(-CTE) delivered by either electroporation or cSLN formulation protects BALB/c mice against L. infantum challenge and that protective immunity is associated with high levels of IFN-γ and lower levels of IL-10 production, leading to a strong Th1 immune response. At all time points, the ratio of IFN-γ: IL-10 induced upon restimulation with rA2-rCPA-rCPB and F/T antigens was significantly higher in vaccinated animals. Moreover, Th2-efficient protection was elicited through a high humoral immune response. Nitric oxide production, parasite burden and histopathological analysis were also in concordance with other findings. Overall, these data indicate that similar to the electroporation delivery system, cSLNs as a nanoscale vehicle of Leishmania antigens could improve immune response, hence indicating the promise of these strategies against visceral leishmaniasis.

Elongation factor-2, a Th1 stimulatory protein of Leishmania donovani, generates strong IFN-γ and IL-12 response in cured Leishmania-infected patients/hamsters and protects hamsters against Leishmania challenge.

In visceral leishmaniasis, Th1 types of immune responses correlate with recovery from and resistance to disease, and resolution of infection results in lifelong immunity against the disease. Leishmanial Ags that elicit proliferative and cytokine responses in PBMCs from cured/exposed/Leishmania patients have been characterized through proteomic approaches, and elongation factor-2 is identified as one of the potent immunostimulatory proteins. In this study, we report the cloning and expression of Leishmania donovani elongation factor-2 protein (LelF-2) and its immunogenicity in PBMCs of cured/exposed Leishmania-infected patients and hamsters (Mesocricetus auratus). Leishmania-infected cured/exposed patients and hamsters exhibited significantly higher proliferative responses to recombinant Lelf-2 (rLelF-2) than those with L. donovani-infected hosts. The soluble L. donovani Ag stimulated PBMCs of cured/exposed and Leishmania patients to produce a mixed Thl/Th2-type cytokine profile, whereas rLelF-2 stimulated the production of IFN-γ, IL-12, and TNF-α but not IL-4 or IL-10. Further, rLelF-2 downregulated LPS-induced IL-10 as well as soluble L. donovani Ag-induced IL-4 production by Leishmania patient PBMCs. The immunogenicity of rLelF-2 was also checked in hamsters in which rLelF-2 generates strong IL-12- and IFN-γ-mediated Th1 immune response. This was further supported by a remarkable increase in IgG2 Ab level. We further demonstrated that rLelF-2 was able to provide considerable protection (∼65%) to hamsters against L. donovani challenge. The efficacy was supported by the increased inducible NO synthase mRNA transcript and Th1-type cytokines IFN-γ, IL-12, and TNF-α and downregulation of IL-4, IL-10, and TGF-ß. Hence, it is inferred that rLelF-2 elicits a Th1 type of immune response exclusively and confers considerable protection against experimental visceral leishmaniasis.

Vaccination with liposomal leishmanial antigens adjuvanted with monophosphoryl lipid-trehalose dicorynomycolate (MPL-TDM) confers long-term protection against visceral leishmaniasis through a human administrable route.

The development of a long-term protective subunit vaccine against visceral leishmaniasis depends on antigens and adjuvants that can induce an appropriate immune response. The immunization of leishmanial antigens alone shows limited efficacy in the absence of an appropriate adjuvant. Earlier we demonstrated sustained protection against Leishmania donovani with leishmanial antigens entrapped in cationic liposomes through an intraperitoneal route. However, this route is not applicable for human administration. Herein, we therefore evaluated the immune response and protection induced by liposomal soluble leishmanial antigen (SLA) formulated with monophosphoryl lipid-trehalose dicorynomycolate (MPL-TDM) through a subcutaneous route. Subcutaneous immunization of BALB/c mice with SLA entrapped in liposomes or with MPL-TDM elicited partial protection against experimental visceral leishmaniasis. In contrast, liposomal SLA adjuvanted with MPL-TDM induced significantly higher levels of protection in liver and spleen in BALB/c mice challenged 10 days post-vaccination. Protection conferred by this formulation was sustained up to 12 weeks of immunization, and infection was controlled for at least 4 months of the challenge, similar to liposomal SLA immunization administered intraperitoneally. An analysis of cellular immune responses of liposomal SLA + MPL-TDM immunized mice demonstrated the induction of IFN-γ and IgG2a antibody production not only 10 days or 12 weeks post-vaccination but also 4 months after the challenge infection and a down regulation of IL-4 production after infection. Moreover, long-term immunity elicited by this formulation was associated with IFN-γ production also by CD8⁺ T cells. Taken together, our results suggest that liposomal SLA + MPL-TDM represent a good vaccine formulation for the induction of durable protection against L. donovani through a human administrable route.

Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates.

Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500,000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients' urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1) and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.