Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease

We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79 percent in attenuated parasite-vaccinated monkeys, versus 75 percent in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-g) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application

Leishmanial antigens in the diagnosis of active lesions and ancient scars of American tegumentary leishmaniasis patients.

Cutaneous biopsies (n = 94) obtained from 88 patients with American tegumentary leishmaniasis were studied by conventional and immunohistochemical techniques. Specimens were distributed as active lesions of cutaneous leishmaniasis (n = 53) (Group I), cicatricial lesions of cutaneous leishmaniasis (n = 35) (Group II) and suggestive scars of healed mucosal leishmaniasis patients (n = 6) (Group III). In addition, active cutaneous lesions of other etiology (n = 24) (Group C1) and cutaneous scars not related to leishmaniasis (n = 10) (Group C2) were also included in the protocol. Amastigotes in Group I biopsies were detected by routine histopathological exam (30.2%), imprint (28.2%), culture (43.4%), immunofluorescence (41.4%) and immunoperoxidase (58.5%) techniques; and by the five methods together (79.3%). In Group II, 5.7% of cultures were positive. Leishmanial antigen was also seen in the cytoplasm of macrophages and giant cells (cellular pattern), vessel walls (vascular pattern) and dermal nerves (neural pattern). Positive reaction was detected in 49 (92.5%), 20 (57%) and 4 (67%) biopsies of Groups I, II and III, respectively. Antigen persistency in cicatricial tissue may be related to immunoprotection or, on the contrary, to the development of late lesions. We suggest that the cellular, vascular and neural patterns could be applied in the immunodiagnosis of active and cicatricial lesions in which leishmaniasis is suspected.

Detection of early apoptosis and cell death in T CD4+ and CD8+ cells from lesions of patients with localized cutaneous leishmaniasis.

Human localized cutaneous leishmaniasis (LCL), induced by Leishmania braziliensis, ranges from a clinically mild, self-healing disease with localized cutaneous lesions to severe forms which can present secondary metastatic lesions. The T cell-mediated immune response is extremely important to define the outcome of the disease; however, the underlying mechanisms involved are not fully understood. A flow cytometric analysis of incorporation of 7-amino actinomycin D and CD4+ or CD8+ T cell surface phenotyping was used to determine whether different frequencies of early apoptosis or accidental cell death occur at different stages of LCL lesions. When all cells obtained from a biopsy sample were analyzed, larger numbers of early apoptotic and dead cells were observed in lesions from patients with active disease (mean = 39.5 +/- 2.7%) as compared with lesions undergoing spontaneous healing (mean = 17.8 +/- 2.2%). Cells displaying normal viability patterns obtained from active LCL lesions showed higher numbers of early apoptotic events among CD8+ than among CD4+ T cells (mean = 28.5 +/- 3.8 and 15.3 +/- 3.0%, respectively). The higher frequency of cell death events in CD8+ T cells from patients with LCL may be associated with an active form of the disease. In addition, low frequencies of early apoptotic events among the CD8+ T cells were observed in two patients with self-healing lesions. Although the number of patients in the latter group was small, it is possible to speculate that, during the immune response, differences in apoptotic events in CD4+ and CD8+ T cell subsets could be responsible for controlling the CD4/CD8 ratio, thus leading to healing or maintenance of disease.

Detection of early apoptosis and cell death in T CD4+ and CD8+ cells from lesions of patients with localized cutaneous leishmaniasis

Human localized cutaneous leishmaniasis (LCL), induced by Leishmania braziliensis, ranges from a clinically mild, self-healing disease with localized cutaneous lesions to severe forms which can present secondary metastatic lesions. The T cell-mediated immune response is extremely important to define the outcome of the disease; however, the underlying mechanisms involved are not fully understood. A flow cytometric analysis of incorporation of 7-amino actinomycin D and CD4+ or CD8+ T cell surface phenotyping was used to determine whether different frequencies of early apoptosis or accidental cell death occur at different stages of LCL lesions. When all cells obtained from a biopsy sample were analyzed, larger numbers of early apoptotic and dead cells were observed in lesions from patients with active disease (mean = 39.5 + or - 2.7 per cent) as compared with lesions undergoing spontaneous healing (mean = 17.8 + or - 2.2 per cent). Cells displaying normal viability patterns obtained from active LCL lesions showed higher numbers of early apoptotic events among CD8+ than among CD4+ T cells (mean = 28.5 + or - 3.8 and 15.3 + or - 3.0 per cent, respectively). The higher frequency of cell death events in CD8+ T cells from patients with LCL may be associated with an active form of the disease. In addition, low frequencies of early apoptotic events among the CD8+ T cells were observed in two patients with self-healing lesions. Although the number of patients in the latter group was small, it is possible to speculate that, during the immune response, differences in apoptotic events in CD4+ and CD8+ T cell subsets could be responsible for controlling the CD4/CD8 ratio, thus leading to healing or maintenance of disease.

Atypical mucocutaneous leishmaniasis caused by Leishmania braziliensis in an acquired immunodeficiency syndrome patient: T-cell responses and remission of lesions associated with antigen immunotherapy.

An atypical case of acquired immunodeficiency syndrome-associated mucocutaneous lesions due to Leishmania braziliensis is described. Many vacuolated macrophages laden with amastigote forms of the parasite were found in the lesions. Leishmanin skin test and serology for leishmaniasis were both negative. The patient was resistant to therapy with conventional drugs (antimonial and amphotericin B). Interestingly, remission of lesions was achieved after an alternative combined therapy of antimonial associated with immunotherapy (whole promastigote antigens). Peripheral blood mononuclear cells were separated and stimulated in vitro with Leishmania antigens to test the lymphoproliferative responses (LPR). Before the combined immunochemotherapy, the LPR to leishmanial antigens was negligible (stimulation index - SI=1.4). After the first course of combined therapy it became positive (SI=4.17). The antigen responding cells were predominantly T-cells (47.5%) most of them with CD8+ phenotype (33%). Very low CD4+ cells (2.2%) percentages were detected. The increased T-cell responsiveness to leishmanial antigens after combined therapy was accompanied by interferon-g (IFN-g) production as observed in the cell culture supernatants. In this patient, healing of the leishmaniasis lesions was associated with the induction of a specific T-cell immune response, characterized by the production of IFN-g and the predominance of the CD8+ phenotype among the Leishmania-reactive T-cells.

Cutaneous scars in American tegumentary leishmaniasis patients: a site of Leishmania (Viannia) braziliensis persistence and viability eleven years after antimonial therapy and clinical cure.

Two former patients treated for the cutaneous form of American tegumentary leishmaniasis were reviewed eight and 11 years, respectively, following clinical cure. We were able to isolate Leishmania parasites in a culture of material from the two scar biopsies, and in one of them the parasite was characterized as Leishmania (Viannia) braziliensis. In both cases, the histopathology revealed discreet hyperceratosis and a slight infiltrate of mononuclear cells surrounding and on the walls of the surface and deep dermal vessels. No amastigotes were seen on immunohistochemical or histopathologic examination. The Montenegro skin test result and the in vitro lymphoproliferative response to Leishmania antigen were positive, but no specific IgG and IgM antibodies were detected. Otorhinolaryngologic examination showed no macroscopic alteration in the mucosae. These findings are important for the evaluation and criteria of post-treatment cure.

Immunologic patterns associated with cure in human American cutaneous leishmaniasis

Patients with American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-gamma) and interleukin 4 (IL-4) produced were also determined in the culture supernatants. Two different patterns of Lb-induced T cell responses were observed: a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-gamma and IL-4) during the active disease, and b) similar proportions of responding CD4+ and CD8+ cells, and type 1 cytokine production (presence of INF-gamma and very low IL-4) at the end of therapy (healed lesions). This last pattern is probably associated with a beneficial T cell response.

T-cell responsiveness of American cutaneous leishmaniasis patients to purified Leishmania pifanoi amastigote antigens and Leishmania braziliensis promastigote antigens: immunologic patterns associated with cure.

Patients suffering from American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) or by three proteins (A-2/P-2, P-4, and P-8) derived from Leishmania pifanoi amastigotes were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-gamma), interleukin 2 (IL-2) and interleukin 4 (IL-4) produced were also determined. Results show two different patterns of Lb-induced T cell responses: (a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-gamma, IL-2, and IL-4) during the active disease, (b) similar proportions of responding CD4+ and CD8+ cells and type 1 cytokine production (presence of IFN-gamma and IL-2 and very low IL-4) at the end of therapy (healed lesions). Thus, this last pattern is probably associated with a beneficial T cell response. The A-2/P-2 amastigote cysteine proteinase provided only marginal (s.i. approximately or = 2.5) T cell stimulation in 25% of patients studied; in contrast, the L. pifanoi P-4 and P-8 amastigote antigens induced significant stimulation (s.i. approximately or = 5) in approximately 50% of the patients. In comparison to Lb-stimulated cultures, lower proliferative responses of T lymphocytes to P-4 or P-8 were observed. However, the P-4- or P-8-stimulated cultures had similar percentages of reactive CD4+ and CD8+ cells, as well as type 1 cytokines (presence of IFN-gamma and IL-2, and low levels or absence of IL-4) in the supernatants both before and at the end of therapy. The consistent induction of apparently beneficial T cell responses by the P-4 and P-8 amastigote glycoproteins points to the possibility that these molecules be considered as candidates for future defined vaccines against leishmaniasis.

Tumor necrosis factor-alpha in human american tegumentary leishmaniasis.

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine produced by activated macrophages and other cells. In order to verify whether the serum levels of TNF-alpha in American tegumentary leishmaniasis patients are associated with the process of cure or aggravation of the disease, 41 patients were studied: 26 cases of cutaneous leishmaniasis (CL) and 15 of mucocutaneous leishmaniasis (MCL). During active disease the serum levels of TNF-alpha of MCL patients were significantly higher than those of CL patients and control subjects (healthy individuals and cutaneous lesions from other etiologies). The MCL patients had serum titers of TNF-alpha significantly lower at the end of antimonial therapy than before therapy. After a six-month follow-up, the MCL patients had serum levels of TNF-alpha similar to those observed at the end of the therapy as well as to those of CL patients and control subjects. No significant variation in the serum levels of TNF-alpha was observed in CL patients throughout the study period (before, at the end of therapy and after a six-month follow-up). The possible relationship between the high TNF-alpha serum levels and severity of the disease is discussed.

Leishmania amazonensis: the Asian rhesus macaques (Macaca mulatta) as an experimental model for study of cutaneous leishmaniasis.

As a means of assessing the usefulness of the Rhesus macaque (Macaca mulatta) as a nonhuman primate model for studying cutaneous leishmaniasis, monkeys were infected with Leishmania amazonensis. Variation in the level of susceptibility was found; however, animals inoculated with 10(8) promastigotes provided consistent results as indicated by an earlier onset and/or larger size of lesions. Three monkeys, which had recovered from skin lesions, were challenge-infected using the same parasite strain/dose; although these animals remained susceptible to homologous infection, lesion size was smaller and healed faster than in the initial infection. The immunologic features during infection were assessed. Levels of IgM and IgG antibodies to promastigote antigens rose during active infection and then declined; immunoblot analyses indicated that numerous leishmanial antigens (predominately >30 kDa) were recognized. Delayed type hypersensitivity (DTH) responses and proliferative responses (PBL) developed during active infection and/or rechallenge. Circulating peripheral T cell subpopulations varied throughout the course of infection. Initially (6-8 weeks p.i.), CD4+ T cells appear to predominate; subsequently (15-21 weeks p.i.), an increase in CD8+ T cells was observed. Pathologic analyses indicated that lesions contained amastigotes with a mononuclear infiltrate of macrophages, lymphocytes, and plasma cells, and formation of tuberculoid-type granulomas. As the progression and resolution of leishmanial infection in rhesus macaques are very similar to those observed in humans, this primate model could be employed for elucidating the mechanisms of protective immunity in cutaneous leishmaniasis.

An experimental model of the production of metastases in murine cutaneous leishmaniasis.

An experimental investigation into the influence of artificially induced trauma in the production of leishmanial metastatic lesions and into the possible role played by Leishmania-reactive T cell populations in the metastatic process was carried out. Trauma was induced by incising a small cut into the shaved rump of Leishmania amazonensis-infected BALB/c mice. Ten days after the trauma, mice were killed to quantify the parasite load in the traumatic lesion or in the equivalent area in nontraumatized mice, by limiting dilution analysis. Results demonstrated that metastatic lesions occurred earlier in traumatized animals and that parasites could be detected sooner in traumatic lesions than in equivalent areas in nontraumatized mice. When lymph node cells from L. amazonensis antigen-immunized BALB/c mice were adoptively transferred intravenously to L. amazonensis-infected syngeneic mice, the parasite load in the metastatic lesions was greater in the animals that received La Ag-reactive T cells than in the controls. When CD4(+)- or CD8(+)-depleted T cell populations from La Ag-immunized mice were adoptively transferred to infected traumatized or nontraumatized animals, we observed that the metastatic lesions in CD4(+)-inoculated animals had a greater number of parasites than the lesions in mice from all other groups. Thus, a new and reliable mouse model for studying the mechanisms involved in leishmanial metastasis is described.

Cellular and humoral immune responses of a patient with American cutaneous leishmaniasis and AIDS.

The lymphocyte responsiveness to leishmanial antigens and its influence on the course of cutaneous leishmaniasis was studied in a patient with AIDS-associated American cutaneous leishmaniasis caused by Leishmania braziliensis. The patient had cutaneous disseminated erythematous papules or nodules and mucosal lesions as well as moniliasis and weight loss. The patient had a poor delayed-type hypersensitivity to leishmanial antigens, showing 3 mm of induration. The cellular immune responses were studied in vitro by lymphocyte proliferative assays induced by leishmanial antigens and concanavalin A. The T cell phenotypes were analysed by flow cytometry. The peripheral blood mononuclear cells before proliferation showed an inversion of the CD4/CD8 ratio (0.28:1). The lymphoproliferative responses to antigen and mitogen were very low (indices < 2.5). The blast-like cell phenotypes after antigen stimulation in culture were: CD3+ 44.8%, CD4+ 7.53% and CD8+ 17.45%. In AIDS patients the decrease in the pool of CD4+ cells, and consequent diminution of the CD4/CD8 ratio, produced by HIV infection provokes a generalized immune depression. The patient's disseminated clinical picture was probably related to the inability of his T cell-mediated immune responses to control the spread of Leishmania infection.

Flow cytometry in the study of the interaction between murine macrophages and the protozoan parasite Leishmania amazonensis.

A flow cytometry method was adapted to study interaction between murine macrophages and Leishmania amazonensis. Using this method it was possible to detect internalization of parasites through an increase in macrophage granularity (side scatter), with the latter indicating the presence of parasites inside parasitophorus vacuoles. A quenching technique was used to confirm the feasibility of the method and to distinguish between internalized and externally attached parasites. Experiments using fixed-labeled and killed-unlabeled parasites gave similar results, demonstrating that granularity was an adequate parameter in the study of parasite-macrophage interaction, when compared with labeling methods. Experiments that measured internalization using only the increase in macrophage granularity as an indicator showed that living L. amazonensis was internalized to a greater extent than killed-unlabeled parasites. This finding suggests that the parasite has an active role in the process of internalization. The 2 methods in combination, flow cytometry and labeling, can be used to study murine peritoneal cell-L. amazonensis interactions and to sort phagocytosing and nonphagocytosing subpopulations of macrophages for further studies.

Serological survey for canine cutaneous and visceral leishmaniasis in areas at risk for transmission in Rio de Janeiro where prophylatic measures had been adopted

A serological survery for canine visceral (VL) and American cutaneous leishmaniasis (ACL) has been carried out during 1984-1989, to assess the effects of the prophylactic measures adopted in areas where there was a risk of transmission of the diseases in Rio de Janeiro. A previous serologival survey (1982/83) had detected serum positive dogas as well as the human disease in these same areas. A total fo 22,828 dogs have been examined in this last survey, 7,807 of which came from Campo Grande (VL and ACL area), 4,110 from Jacarepaguá (ACL area), 4,l46 from Realengo, 3,879 from Bangu and 2,886 from Senador Camará, (three VL areas). The analysis of these results showed a notable reduction in the number of serum positve dogs, compared to those of the first survey was 12.7%, against 0.62% of the second; (b) in Jacarepaguá (ACL) it decreased from 8.6%) to l.8% (c) in Bangu, Realengo and Senador Camará (VL) the rate decreased from 4.3% to 0.38%. The results indicate that this decrease was due to the prophylactic measures adopted in those areas

Aspectos ecologicos da leishmaniose tegumentar americana: 8. avaliacao da atividade enzootica de Leishmania (Viannia) braziliensis, em ambiente florestal e peridomiciliar, regiao do vale do Ribeira, Estado de Sao Paulo, Brasil / Ecological aspects of American tegumentary leishmaniasis: 8. Evaluation of the enzootic activity of Leishmania (Vianna) braziliensis, in forest and peridomiciliary environments of the Ribeira Valey region, Sao Paulo State, Brazil

A evidencia da transmissao extraflorestal da leishmaniose cutaneo-mucosa na regiao do Vale do Ribeira ensejou o presente estudo epidemiologico prospectivo, visando avaliar a atividade enzootica de L. (V.) braziliensis. A pesquisa paratisologica da infeccao natural em pequenos mamiferos e populacao canina foi complementada com o teste de imunofluorescencia indireta (IFI) para caes e captura de flebotomineos em ambiente florestal e peridomiciliar. A positividade para o teste sorologico e exame parasitologico somente foi observada para caes residentes e com taxas de 5,6 e 2,4 por cento, respectivamente. Entre animais silvestres e sinantropicos capturados, destacam-se os pertencentes a Oryzomys (Oligoryzomys) e Rattus rattus, ambos assinalados em proporcoes equivalentes (29,3 por cento) em ambiente peridomiciliar. Foram capturados apenas 166 exemplares femininos de Lutzomyia intermedia, fato atribuido a borrifacao das habitacoes humanas e anexos com DDT. No contexto epidemiologico mais amplo, discute-se a fragilidade do ciclo extraflorestal da L. (V.) braziliensis; o papel do cao e de pequenos mamiferos, como fonte de infeccao domiciliar, alem de analisar o potencial deles na dispersao do parasita na area estudada

Quantitative study of Leishmania braziliensis braziliensis reactive T cells in peripheral blood and in the lesions of patients with American mucocutaneous leishmaniasis.

A limiting dilution analysis (LDA) was utilized to estimate the frequency of L. braziliensis braziliensis reactive T cells (Lbb-T cells) in peripheral blood and in the lesions of patients with mild localized cutaneous leishmaniasis (LCL) or with severe mucosal leishmaniasis (MCL). The frequencies of Lbb-T cells in peripheral blood varied from 1:107300 to 1:3587 and were not significantly different in MCL and LCL patients. However, a significant difference was encountered (P less than 0.02) between the T cells frequencies in cutaneous (1:748 to 1:45) and mucosal lesions (1:152 to 1:13). A positive correlation was also observed between these frequencies and the magnitude of delayed-type hypersensitivity (DTH) (P less than 0.01) and the presence of fibrinoid necrosis and granulomatous reaction in the site of the lesions (P less than 0.05). The lack of correlation between the severity of disease (MCL or LCL) and the frequency of Lbb-T cells in peripheral blood gave no indications towards understanding the physiopathology of severe or mild disease. However, the correlation between high T cell frequencies in the site of the lesions, the magnitude of DTH, the fibrinoid necrosis and the severity of the disease (MCL lesions) points to the possibility that the presence of a strong T cell dependent cellular immune response in the site of the lesions may have a deleterious effect. However, a local well modulated T cell immune response might provide healing of the lesions.

Quantification of Leishmania-specific T cells in human American cutaneous leishmaniasis (Leishmania braziliensis braziliensis) by limiting dilution analysis.

Limiting dilution analysis was used to estimate the frequency of Leishmania-specific T cells from the peripheral blood of 18 human cases of American Cutaneous Leishmaniasis (ACL). Sixteen patients had localized cutaneous leishmaniasis (LCL) and two were recovered LCL patients. In 10 patients with active disease and in two with healed lesions the Leishmania-specific T cell frequencies ranged from 1/10(5) to 1/10(3). In six patients no proliferation was detected after 21 days of cell culture. This finding points to very low precursor frequencies in the peripheral blood of these patients. A significant correlation was found between the two groups with low or high Leishmania-specific T cell frequencies and the lymphoproliferative responses to leishmanial antigens. The majority of the blast-like Leishmania-specific T cells showed a helper/inducer (CD4) phenotype.

Human american cutaneous leishmaniasis (Leishmania b. braziliensis) in Brazil: lymphoproliferative responses and influence of therapy.

The host defence to Leishmania parasites is believed to depend on cell-mediated immune responses. Three groups of inhabitants from an endemic area in Rio de Janeiro were studied: Group I consisted of 28 patients with cutaneous lesions, Group II of 28 healthy persons (without ulcers) but with positive Montenegro skin tests (MST) and Group III of 29 healthy persons with negative MST. The peripheral blood lymphocyte proliferative responses induced by leishmanial-antigens (Leishmania b. braziliensis lymphoproliferative response) as well as by Concanavalin A (Con A-lymphoproliferative response), both measured by 3H-thymidine incorporation were tested in each group. The results showed that: The Leishmania b. braziliensis lymphoproliferative response (L.b.b.-LPR) in healthy persons with positive MST (Group II) was higher than in patients prior to therapy (Group I); A significantly higher L.b.b.-LPR was found in patients and healthy persons with positive MST as compared to Group III (negative MST); The L.b.b.-LPR of Group I (patients) increased during antimonial therapy--this might possibly be related to the destruction of parasites; The levels of L.b.b.-LPR after therapy became similar to the ones before therapy; All individuals from the three groups had a positive Con A-lymphoproliferative response (Con A-LPR); All patients who had a histopathological picture of granulomatous reaction also had a positive L.b.b.-LPR; A poor response to antimonial therapy observed in six patients was associated with a low L.b.b.-LPR.