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.

Recent Advances in Vaccines Against Leishmania Based on Patent Applications.

BACKGROUND: Leishmaniasis is caused by parasites of the genus Leishmania, and represents a group of chronic diseases with an epidemiological and clinical diversity. The disease is endemic in tropical regions, being found in 98 countries, affecting around 12 million people, with an estimated increase of 1.5 million per year. METHODS: The present review aims to analyze recent and most important patents regarding development of vaccines to improve immunization against leishmaniasis. For this purpose, the Web of Science - Derwent Innovations Index was consulted. There is also a short description of the licensed vaccines already on the market for commercialization, and a critical opinion on future developments. RESULTS: The data herein presented comprises national and international filings, thus considering the patent's country of origin, and can be used an indicator of a country's technological development regarding a specific field. Several types of vaccines against Leishmania were studied. The main classes comprise: vaccines using live cells (virulent or attenuated); dead cells; containing recombinant protein; using DNA of the parasite. United States (74 patents) leads the ranking of patent applications for vaccines against Leishmania, followed by Brazil (36 patents), which is an endemic region of leishmaniasis with 20,000 human cases of cutaneous leishmaniasis and over 3,000 cases of visceral form. CONCLUSION: This review showed that there is still a lot of space for development regarding the creation of a feasible, effective vaccine against leishmaniasis. The scientific community appears to be taking steps in the right direction, though.

Th-1 biased immunomodulation and synergistic antileishmanial activity of stable cationic lipid-polymer hybrid nanoparticle: biodistribution and toxicity assessment of encapsulated amphotericin B.

To address issues related to Amphotericin B (AmpB) clinical applications, we developed macrophage targeted cationic stearylamine lipid-polymer hybrid nanoparticles (LPNPs) with complementary characteristics of both polymeric nanoparticles and liposomes, for enhancement of therapeutic efficacy and diminishing toxic effect of encapsulated AmpB. The LPNPs (size 198.3 ± 3.52 nm, PDI 0.135 ± 0.03, zeta potential +31.6 ± 1.91 mV) provide core-shell type structure which has the ability to encapsulate amphiphilic AmpB in higher amount (Encapsulation efficiency 96.1 ± 2.01%), sustain drug release and stabilize formulation tremendously. Attenuated erythrocytes and J774A.1 toxicity of LPNPs demonstrated safe applicability for parenteral administration. Elevated macrophage uptake of LPNPs, rapid plasma clearance and higher drug allocation in macrophage abundant liver and spleen illustrated admirable antileishmanial efficacy of AmpB-LPNPs in vitro (IC50, 0.16 ± 0.04 µg AmpB/ml) and in vivo (89.41 ± 3.58% parasite inhibition) against visceral leishmaniasis models. Augmentation in antileishmanial activity due to Th-1 biased immune-alteration mediated by drug-free LPNPs which elevated microbicidal mediators of macrophages. Moreover, minimal distribution to kidney tissues and low level of nephrotoxicity markers (creatinine and BUN) demonstrated the safety profile of AmpB-LPNPs. Conclusively, reliable safety and macrophage directed therapeutic performance of AmpB-LPNPs suggest it as promising alternative to commercial AmpB-formulations for the eradication of intra-macrophage diseases.

Produção e avaliação de antígenos recombinantes candidatos a componente de uma avaliação contra leishmaniose visceral canina / Production and testing of candidates recombinant antigen component of an assessment against canine visceral leishmaniasis

Uma vacina efetiva contra a leishmaniose visceral (LV) canina pode contribuir para o controle da doença no homem. Visando o desenvolvimento de uma vacina contra LV canina, antígenos recombinantes de L. infantum foram selecionados em nosso laboratório pelo uso de uma mistura de soros de seres humanos ou cães naturalmente infectados pela L. infantum. Alguns destes antígenos foram testados em diversos protocolos de imunização, incluindo uso de diferentes adjuvantes, em camundongos ou cães. A imunização de camundongos ou cães com um dos antígenos recombinantes (rLci2B) usado isoladamente ou em associação com saponina induziu resposta imune Th2 ou Th1/Th2, respectivamente, não protetoras contra a infecção experimental. Com a determinação da sequência deduzida de aminoácidos notou-se que a maioria dos antígenos selecionados apresenta um segmento com sequência de aminoácidos única (domínio não repetitivo) e segmentos com sequência de aminoácidos com motivos repetitivos (domínios repetitivos).Possivelmente a incapacidade dos antígerecombinantes de induzir uma resposta imune predominantemente Th1, protetora contra a LV, seria por conta da presença de domínios repetitivos, que favorecem a apresentação antigênica por linfócitos B e, consequentemente, estimulam uma resposta imune Th2. Para avaliar o direcionamento da resposta imune pelos dois tipos de domínio, novas construções de DNA foram concebidas de modo a codificar apenas domínio(s) não repetitivo(s) ou domínio(s) não repetitivo(s) e domínios repetitivos.OBJETIVOS: Produzir quatro proteínas recombinantes com domínios não repetitivos (rLci2-NT-CT, rLci3-NT-CT, rLci10-NT e rLci12-NT-CT) e avaliar a capacidade desses polipeptídios de induzir resposta imune celular in vitro em cães assintomáticos inoculados por via dérmica com L. infantum. MATERIAIS E MÉTODOS: Foram realizadas: a)a subclonagem de construções de DNA (Lci3-NT-CT, Lci10-NT e Lci12-NT-CT) em um plasmídeo apropriado para expressão em Escherichia coli, b) a determinação de condições apropriadas para produção das proteínas recombinantes (rLci2-NT-5R-CT, rLci2-NT-CT, rLci3-NT-2R-CT, rLci3-NT-CT, rLci10-NT-2R e rLci10-NT) c) a purificação das proteínas recombinantes por cromatografia de afinidade e d)avaliação da capacidade dos polipeptídios de induzir estimulação de células mononucleares sangue periférico (PBMC) de cães assintomáticos inoculados por via dérmica com L.infantum. RESULTADOS:Três (rLci2-NT-CT, rLci2-NT-5R-CT, rLci3-NT-CT, rLci3-NT-2R-CT, rLci10-NT e rLci10-NT-2R) dos quatro pares de polipeptídios recombinantes foram expressos, produzidos e purificados. Três antígenos recombinantes (rLci2-NT-5R-CT,rLci2-NT-CT e rLci3-NT-2R-CT) promoveram a linfoproliferação in vitro utilizando PBMC de cães assintomáticos inoculados por via dérmica com L. infantum CONCLUSÕES: Três das seis proteínas produzidas induziram a linfoproliferação, sendo a maior linfoproliferação encontrada para PBMC estimulado com a proteína sem domínios repetitivos (rLci2-NT-CT). Avaliações adicionais são necessárias para comprovar a utilidade destas moléculas em formulação de vacina contra leishmaniose visceral canina.

An effective vaccine against visceral leishmaniasis (VL) dog can help to control the disease in man. Aiming at development of a vaccine against canine VL, recombinant antigens of L. infantum were selected in our laboratory by using a mixture of sera from humans or dogs naturally infected with L. infantum. Some of these antigens were tested in different immunization protocols, including use of different adjuvants in mice or dogs. The immunization of mice or dogs with a recombinant antigens (rLci2B) used alone or in combination with saponin induced Th2 response or Th1 / Th2, respectively, did not protective against experimental infection. With the determination of the deduced amino acid sequence it was noted that most of the antigens selected segment has a unique amino acid sequence (non-repetitive domain) and segments of amino acid sequence with repetitive motifs (repetitive domains). Possibly the inability of recombinant antigens to induce an immune response predominantly Th1 protective against LV would be due to the presence of repetitive domains that promote antigen presentation by B cells and thus stimulate an immune response Th2. To assess the direction of the immune response by two types of domain, new DNA constructs were designed to encode only the domain (s) not repetitive (s) or domain (s) not repetitive (s) and repetitive fields. MATERIALS AND METHODS: Were performed: a) subcloning DNA constructs (rLci3-NT-CT, rLci10-NT and rLci12-NT-CT) into a suitable plasmid for expression in Escherichia coli, b) determining the appropriate conditions for the production of proteins recombinant (rLci2NT-5R-CT, rLci2-NT-CT, rLci3-NT-2R-CT, rLci3-NT-CT, rLci10-NT-2R and rLci10-NT) c) purification of recombinant proteins by chromatography affinity d) evaluating the ability of polypeptides rLci2-NT-CT, rLci3-NT-CT and rLci10-NT to induce stimulation of peripheral blood mononuclear cells (PBMC) from healthy dogs inoculated dermal with L. infantum...

Rapid detection of human and canine visceral leishmaniasis: assessment of a latex agglutination test based on the A2 antigen from amastigote forms of Leishmania infantum.

The diagnosis of visceral leishmaniasis (VL) in humans and animal reservoir hosts is difficult, particularly in rural areas where the disease is endemic and laboratory facilities are limited. This study aimed to develop a latex agglutination test (LAT) for the rapid detection of anti-Leishmania antibodies against the A2 antigen derived from the amastigote form as well as those against crude antigens derived from the promastigote form of an Iranian strain of Leishmania (Leishmania) infantum. The A2 antigen (42-100 kDa) was prepared from the amastigote form of L. infantum, purified with electroelution and compared with the crude antigen from the promastigote form of L. infantum. Both antigens showed appropriate intensity reactions, were selected using dot blotting of positive and negative pooled sera and used to sensitize 0.9-µm latex beads. The tests were carried out on sera from 43 symptomatic, human patients with VL confirmed by parasitological examination and direct agglutination test (DAT), 30 healthy controls and 32 patients with other infections but without VL. Canine sera were collected from 63 domestic dogs with VL confirmed using parasitological examinations and DAT and 31 healthy dogs from areas non-endemic for VL. Compared with the controls, human sera from DAT-confirmed patients yielded a sensitivity of 88.4% (95% CI, 82.1-94.5%) and specificity of 93.5% (95% CI, 87.0-99.7%) on A2-LAT (amastigote) when 1:3200 was used as the cut-off titre. A good degree of agreement was found between A2-LAT and DAT (0.914). LAT required 3-5 min to complete, versus the 12-18 h needed for DAT. Compared with the controls, A2-LAT of canine sera from DAT-confirmed cases yielded a sensitivity of 95.2% (95% CI, 95.0-95.4%) and specificity of 100% (95% CI 100%) when 1:320 was used as the cut-off titre. A good degree of agreement was found between A2-LAT and DAT (0.968). Similarly, the sensitivity and specificity of Pro.-LAT (promastigote) was calculated to be 88.4% and 91.9%, respectively for human sera and 96.8% and 90.3%, respectively for canine sera. No statistically significant differences were observed between A2-LAT and Pro.-LAT for the detection of human and canine L. infantum infections. In conclusion, A2-LAT and Pro.-LAT could be used in parallel to screen for L. infantum infections in humans and dogs in areas endemic for VL in Iran.

Dynamics of the antibodies in cohorts of cured cases of visceral leishmaniasis: its implication on the validity of serological test, value in prognosis and in post therapeutic assessment.

The major disadvantage of a Serological test like Direct Agglutination Test (DAT) for Visceral Leishmaniasis (also called Kala-azar) is its inability to distinguish between recent and past infection. The objective of our study was to look at rate of decline of antibodies in fully cured cases of Kala-azar and length of time it takes for DAT to become negative. Cohort Study involving completely treated Kala-azar cases from Government Hospital during one calendar year of study. Cases were selected on the basis of treatment cohorts 0, 3, 6, 9 & 12 mo after completion of treatment.. Phase I--The cases were traced and after obtaining the informed consent they were subjected to Direct Agglutination Test (DAT). Phase II--The five treatment cohorts, constituting 82 cured cases (average of 15 cured cases per each treatment cohort) were tested again with DAT three months after the first test. The titers of Phase-I and phase-II tests were analyzed for the dynamics of the antibodies for the period. Cutoff-Values of DAT below 1:800 are considered negative. Values of 1:800, 1:1200, 1:1600 and so on are considered positive. The mean titer [Geometric Mean Titer (GMT)] at the start of treatment was 1:1120, which showed steady decline up to six months, plummeting below the cutoff titer for the DAT (1:800) at the ninth month. Antibodies continue to linger for about one year in cured Kala-azar cases even after correct and complete treatment. Single DAT results may be misleading due to high false positivity up to one year after the cure. Paired test defined as two tests 3 mo apart on the same subject. Paired test is highly recommended for diagnosis and prognosis. DAT is still a very useful tool for diagnosis if used along with clinical correlation.

Comparative assessment of a DNA and protein Leishmania donovani gamma glutamyl cysteine synthetase vaccine to cross-protect against murine cutaneous leishmaniasis caused by L. major or L. mexicana infection.

Leishmaniasis is a major health problem and it is estimated that 12 million people are currently infected. A vaccine which could cross-protect people against different Leishmania spp. would facilitate control of this disease as more than one species of Leishmania may be present. In this study the ability of a DNA vaccine, using the full gene sequence for L. donovani gamma glutamyl cysteine synthetase (γGCS) incorporated in the pVAX vector (pVAXγGCS), and a protein vaccine, using the corresponding recombinant L. donovani γGCS protein (LdγGCS), to protect against L. major or L. mexicana infection was evaluated. DNA vaccination gave transient protection against L. major and no protection against L. mexicana despite significantly enhancing specific antibody titres in vaccinated infected mice compared to infected controls. Vaccination with the LdγGCS protected against both species but only if the protein was incorporated into non-ionic surfactant vesicles for L. mexicana. The results of this study indicate that a L. donovani γGCS vaccine could be used to vaccinate against more than one Leishmania species but only if the recombinant protein is used.

Visceral leishmaniasis: immunology and prospects for a vaccine.

Human visceral leishmaniasis (HVL) is the most severe clinical form of a spectrum of neglected tropical diseases caused by protozoan parasites of the genus Leishmania. Caused mainly by L. donovani and L. infantum/chagasi, HVL accounts for more than 50 000 deaths every year. Drug therapy is available but costly, and resistance against several drug classes has evolved. Here, we review our current understanding of the immunology of HVL and approaches to and the status of vaccine development against this disease.

Anti-Leishmania chagasi immunoglobulin G3 detected by flow cytometry for early cure assessment in American visceral leishmaniasis.

We have previously reported a novel flow cytometric based methodology to access the reactivity of seric anti-live (FC-ALPA) and fixed (FC-AFPA) L. chagasi IgG antibodies applicable for cure assessment after specific therapy of VL. Both, FC-ALPA-IgG and FC-AFPA-IgG are promising targets to be used for early cure assessment. However, our finding suggested that further refinements were still required to improve the performance of FC-AFPA IgG for early cure assessment in VL. In the present investigation, we have established and evaluated the performance of FC-AFPA-IgG1/IgG2/IgG3/IgG4 aiming to increase the performance index of the previously reported for FC-AFPA-IgG. The data was expressed as percentage of fluorescent positive parasites after incubation of pre-fixed L. chagasi promastigotes with the test sera samples and addition of second-step FITC-labeled anti-human IgG subclasses conjugates. The analysis of anti-L. chagasi IgG reactivity in polled sera samples from VL patients demonstrated that, before the etiological treatment, the IgG subclass profile was characterized by IgG1>IgG3 with the absence of IgG2 and IgG4 at the specific sera dilution tested. Following the establishment of specific PPFP cut-off-edges to segregate negative and positive results (PPFP of 50% for FC-AFPA-IgG1 and PPFP of 40% for FC-AFPA-IgG3), the analysis of IgG1 and IgG3 reactivity demonstrated good performance for early cure assessment in VL. The analysis of individual samples indicated that despite at 2 mAT, most treated VL patients (81%) still displayed positive results in FC-AFPA-IGg1 analysis, an increased fraction of treated patients (76%) presented negative in FC-AFPA-IgG1 analysis at 6 mAT. Interestingly, the data from FC-AFPA-IgG3 demonstrated an outstanding performance of this method to early cure assessment in VL with increased frequency of treated patients displaying negative results at 2 mAT (90.5%) as well as at 6 mAT (95.2%). The analysis of likelihood ratio (LR) further confirmed the remarkable performance of FC-AFPA-IgG3 as an early complementary biomarker useful to monitor the post-therapeutic cure in human VL.

Anti-fixed Leishmania chagasi promastigotes IgG antibodies detected by flow cytometry (FC-AFPA-IgG) as a tool for serodiagnosis and for post-therapeutic cure assessment in American visceral leishmaniasis.

Visceral leishmaniasis (VL) is a systemic infection, caused by an intracellular protozoan parasite belonging to the Leishmania donovani complex. The diagnosis of VL is complex because most clinical features are shared with other commonly occurring febrile hepatosplenic diseases that can be endemic along with VL. A number of serological devices are available but still require improvement mainly due to residual post-therapeutic serology and the cross-reactivity with other Trypanosomatidae protozooses. This study intended to describe and evaluate the performance of an indirect immunofluorescence assay referred as flow cytometry anti-fixed Leishmania chagasi promastigote IgG antibodies (FC-AFPA-IgG) for serodiagnosis of VL and assessment of post-therapeutic cure. The sera reactivity is reported as the percentage of positive fluorescent parasite (PPFP) along the titration curve. The analysis of sera titration curve indicated the sera dilution 1/32,000 and the PPFP=25% as the cut-off to segregate positive and negative results. Using these parameters, the FC-AFPA-IgG displayed outstanding sensitivity and specificity for diagnosis and post-therapeutic cure assessment purposes. The inter-test reproducibility of FC-AFPA-IgG was also verified, considering two independent Analysts and validated the results obtained by FC-AFPA-IgG. Moreover, the comparison between FC-AFPA-IgG and the conventional serologic test (ELISA) showed that besides the statistically analogous results with strong positive correlation the FC-AFPA-IgG displayed higher performance indexes. Further analysis demonstrated that while cross-reactivity was observed in 8% of samples tested by ELISA, no cross-reactivity was detected by FC-AFPA-IgG. Together, the findings presented in this study showed the potential of FC-AFPA-IgG in both diagnosis and post-therapeutic cure assessment of VL.

Drug regimens for visceral leishmaniasis in Mediterranean countries.

Until the early 1990s, pentavalent antimony was the only documented first-line drug employed for the treatment of zoonotic visceral leishmaniasis (VL) in the Mediterranean, with reported cure rates exceeding 95% in immunocompetent patients. The emergence of antimony resistance in other endemic settings and the increase in drug options have stimulated re-evaluation of the current therapeutic approaches and outcomes in Mediterranean countries. A scientific consortium ('LeishMed' network) collected updated information from collaborating clinical health centres of 11 endemic countries of Southern Europe, Northern Africa and the Middle East. In contrast with the previous situation, VL is now treated differently in the region, basically through three approaches: (1) In Northern Africa and in part of the Middle East, pentavalent antimony is still the mainstay for therapy, with no alternative drug options for treating relapses; (2) In some European countries and Israel, both pentavalent antimony and lipid-associated amphotericin B (AmB) formulations are used as first-line drugs, although in different patients' categories; (3) In other countries of Europe, mainly liposomal AmB is employed. Importantly, cure rates exhibited by different drugs, including antimonials in areas where they are still in routine use, are similarly high (>/=95%) in immunocompetent patients. Our findings show that antimony resistance is not an emerging problem in the Mediterranean. A country's wealth affects the treatment choice, which represents a balance between drug efficacy, toxicity and cost, and costs associated with patient's care.

A standardized cytological and immunochemical method for the analysis of fine-needle spleen aspirates: assessment of leukocyte population changes in canine visceral leishmaniosis.

A method for the evaluation of splenic cellularity using samples collected by fine-needle aspirative biopsy was standardized in this work. The procedure includes erythrocyte lysing, preparation of cytospin films and staining by histochemical and immunocytochemical techniques. The cellular profiles of spleen preparations were compared with those observed in peripheral blood samples subjected to the same procedure. Two groups were compared, one consisting of 14 healthy uninfected and the other of 15 polysymptomatic Leishmania chagasi/infantum-infected dogs, from an endemic area for visceral leishmaniosis. Cell populations were identified by conventional hematoxilin-eosin and Wright' stainings, and by immunocytochemistry using monoclonal antibodies against canine CD45RA and CD45RB, phagocytes and a pan-leukocyte antigen. Larger neutrophil (P < 0.0001) and monocyte/macrophage (P = 0.0036) relative counts and lower lymphocyte relative counts (P < 0.0001) were found in the spleen, and not in the blood, of the animals with leishmaniosis than in those of the healthy animals. The proportions of CD45RB+ cells were higher, and of CD45RA+ cells were lower, both in the spleen and in the blood of animals with leishmaniosis than in those of healthy dogs (P < 0.05). Additionally, hematoxilin-eosin-stained cytospins of spleen aspirates from Leishmania-infected animals permitted the easy visualization of amastigote forms inside phagocytes, under light microscopy.

Visceral leishmaniasis in the Syrian Arab Republic: early detection using rK39.

Leishmaniasis causes significant morbidity and mortality in areas where it is endemic. A seroprevalence survey was conducted in 2 endemic villages in Daraa, Syrian Arab Republic, where 80 out of 345 children (23.2%) tested positive for visceral leishmaniasis (VL) using rK39 dipstick test. Only 10 cases were symptomatic (12.5%), and 27.5% were positive by ELISA test. All the sera (N = 138) obtained from the control village were negative. Of the rK39 initially positive cases, 52 had seroconverted to negative 9 months later, 55 remained ELISA negative, and none developed the full-blown disease. Being faster and less expensive than other diagnostic tests, rK39 is a rapid, sensitive and specific diagnostic tool for symptomatic cases of VL in remote areas with poor accessibility to health services.

Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP.

The repetitive sequence of Leishmania major gene B protein (GBP) has previously been shown to be a useful tool in the diagnosis of cutaneous leishmaniasis (CL). Here, we have assessed enzyme-linked immunosorbent assays (ELISAs) using recombinant L. donovani GBP (rGBP) and a peptide sequence of L. donovani GBP (GBPP) in the diagnosis of L. donovani infections in Sudan. The sensitivity of the rGBP ELISA in diagnosing visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) was 92% and 93%, respectively. In contrast, the sensitivity of the GBPP ELISA was 55% for VL and 63% for PKDL. Plasma antibody reactivity of donors with VL and PKDL remained high for an extended period after the end of treatment. Antibody-reactivity to rGBP and GBPP was detected in 71% and 14% of plasma samples from CL patients, respectively. Plasma from healthy Sudanese donors living in an area endemic for malaria but free of leishmaniasis was negative in both assays.