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.

Identification and characterization of Mycobacterium tuberculosis antigens in urine of patients with active pulmonary tuberculosis: an innovative and alternative approach of antigen discovery of useful microbial molecules.

Despite the clear need to control tuberculosis, the diagnosis and prevention of this serious disease are poorly developed and have remained fundamentally unchanged for more than 50 years. Here, we introduce an innovative approach to directly identify Mycobacterium tuberculosis antigens produced in vivo in humans with tuberculosis. We combined reversed phase high performance liquid chromatography and mass spectrometry and categorize four distinct M. tuberculosis proteins produced presumably in lung lesions and excreted in the urine of patients with pulmonary tuberculosis. The genes (MT_1721, MT_1694, MT_2462 and MT_3444) coding for these proteins were cloned and the recombinant molecules were produced in Escherichia coli. The proteins were recognized by immunoglobulin G antibodies from tuberculosis patients but not from non-diseased subjects. In addition, the recombinant proteins were recognized strongly by peripheral blood mononuclear cells from healthy purified protein derivative of tuberculin-positive individuals and to a lesser extent from patients with tuberculosis. These molecules are the only proteins reported to date that are derived directly from bodily fluids of tuberculosis patients, therefore are interesting candidate antigens for the development of vaccine and/or antigen detection assay for accurate diagnosis of active tuberculosis.

Resistance to Paracoccidioides brasiliensis infection is linked to a preferential Th1 immune response, whereas susceptibility is associated with absence of IFN-gamma production.

The secretion of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, IL-5, and IL-10 by antigen-stimulated lymph node cells, eosinophil maturation, and the antibody isotypes produced were examined during intraperitoneal infection of susceptible (B10.A) and resistant (A/Sn) mice with Paracoccidioides brasiliensis. Lymph node cells from resistant mice produced early and sustained levels of IFN-gamma and IL-2, whereas susceptible animals secreted low to undetectable amounts of these type 1 cytokines. Both mouse strains presented late and transient production of IL-4, whereas IL-10 was produced constantly throughout the course of disease. Resistant animals produced increasing levels of IL-5 in the chronic phase of the infection (from the eighth week on), whereas susceptible mice showed two peaks of IL-5 production, at the first and twelfth weeks after infection. Only the susceptible strain presented medullary and splenic eosinophilia concomitant with the raised IL-5 production. In resistant mice, the levels of IgG2a antibodies were significantly higher than those observed in susceptible mice, which preferentially secreted IgG2b and IgA isotypes. Taken together, these results demonstrate that a sustained production of IFN-gamma and IL-2 and a predominant secretion of IgG2a antibodies are associated with resistance to P. brasiliensis. In contrast, the production of low levels of IFN-gamma, early secretion of high levels of IL-5 and IL-10, eosinophilia, and a preferential secretion of IgG2b and IgA isotypes characterize the progressive disease in susceptible animals.

Cytokines produced by susceptible and resistant mice in the course of Paracoccidioides brasiliensis infection.

Paracoccidioidomycosis (PCM) is the most prevalent deep mycosis in Latin America and presents a wide spectrum of clinical manifestations. We established a genetically controlled murine model of PCM, where A/Sn mice develop an infection which mimics the benign disease (immune responses which favor cellular immunity) and B10.A animals present the progressive disseminated form of PCM (preferential activation of B cells and impairment of cellular immune responses). To understand the immunoregulatory phenomena associated with resistance and susceptibility in experimental PCM, A/Sn and B10.A mice were studied regarding antigen-elicited secretion of monokines (TNF-alpha and TGF-beta) and type-1 (IL-2 and IFN-gamma) and type-2 (IL-4,5,10) cytokines. Total lymph node cells from resistant mice infected i.p. with P. brasiliensis produced early and sustained levels of IFN-gamma and IL-2; type-2 cytokines (IL-4 and IL-5) started to appear 8 weeks after infection. In contrast, susceptible mice produced low levels of IFN-gamma concomitant with significant levels of IL-5 and IL-10 early in the infection. In the chronic phase of the disease, susceptible animals presented a transitory secretion of IL-2, and IL-4. In the pulmonary infection IL-4, IL-5 and IL-10 were preferentially detected in the lung cells washings of susceptible animals. After in vitro challenge with fungal antigens, normal peritoneal macrophages from B10.A mice secreted high levels of TGF-beta and low levels of TNF-alpha. In contrast, macrophages from A/Sn animals released high levels of TNF-alpha associated with a small production of TGF-beta. The in vivo depletion of IFN-gamma not only abrogated the resistance of A/Sn mice but also diminished the relative resistance of B10.A animals. The in vivo depletion of IL-4 did not alter the disease outcome, whereas administration of rIL-12 significantly enhanced resistance in susceptible animals. Taken together, these results suggest that an early secretion of high levels of TNF-alpha and IFN-gamma followed by a sustained secretion of IL-2 and IFN-gamma plays a dominant role in the resistance mechanisms to P. brasiliensis infection. In contrast, an early and ephemeral secretion of low levels of TNF-alpha and IFN-gamma associated with production of IL-5, IL-10 and TGF-beta characterizes the progressive disease of susceptible animals.

Cytokines produced by suscesptible and resistant mice in the course of Paracoccidioides brasiliensis infection

Paracoccidioidomycosis (PCM) is the most prevalent deep mycosis in Latin America and presents a wide spectrum of clinical manifestations. We established a genetically controlled murine model of PCM, where A/Sn mice develop an infection which mimics the benign disease (immune responses which favor cellular immunity) and B10.A animals present the progressive disseminated form of PCM (preferential activation of B cells and impairment of cellular immune responses). To understand the immunoregulatory phenomena associated with resistance and susceptibility in experimental PCM, A/Sn and B10.A mice were studied regarding antigen-elicited secretion of monokines (TNF-alpha and TGF-beta) and type-1 (IL-2 and IFN-gamma) and type-2 (IL-4,5,10) cytokines. Total lymph node cells from resistant mice infected ip with P. brasiliensis produced early and sustained levels of IFN-gamma and IL-2; type-2 cytokines (IL-4 and IL-05) started to appear 8 weeks after infection. In contrast, susceptible mice produced low levels of IFN-gamma concomitant with significant levels of IL-5 and IL-10 early in the infection. In the chronic phase of the disease, susceptible animals presented a transitory secretion of IL-2, and IL-4. In the pulmonary infection IL-4, IL-5 and IL-10 were preferentially detected in the lung cells washings of susceptible animals. After in vitro challenge with fungal antigens, normal peritoneal macrophages from B10.A mice secreted high levels of TGF-beta and low levels of TNF-alpha. In contrast, macrophages from A/Sn animals released high levels of TNF-alpha associated with a small production of TGF-beta. The in vivo depletion of IFN-gamma not only abrogated the resistance of A/Sn mice but also diminished the relative resistance of B10.B animals. The in vivo depletion of IL-4 did not alter the disease outcome, whereas administration of rIL-12 significantly enhanced resistance in susceptible animals. Taken together, these results suggest that an early secretion of high levels of TNF-alpha and IFN-gamma followed by a sustained secretion of IL-2 and IFN-gamma plays a dominant role in the resistance mechanisms to P. brasiliensis infection. In contrast, an early and ephemeral secretion of low levels of TNF-alpha and IFN-gamma associated with production of IL-5 IL-10 and TGF-beta characterizes the progressive disease of susceptible animals.

Protective role of gamma interferon in experimental pulmonary paracoccidioidomycosis.

We have developed a murine model of pulmonary infection by Paracoccidioides brasiliensis in which resistance was associated with immunological activities governed by gamma interferon (IFN-gamma). To better characterize this model, we measured type 1 and type 2 cytokines in the lungs and investigated the effect of endogenous IFN-gamma depletion by monoclonal antibodies in the course of infection of susceptible (B10.A) and resistant (A/Sn) mice. At weeks 4 and 8 after infection, lungs from susceptible animals presented levels of IFN-gamma, interleukin-4 (IL-4), IL-5, and IL-10 higher than those in resistant mice. In both mouse strains, neutralization of endogenous IFN-gamma induced exacerbation of the pulmonary infection, earlier fungal dissemination to the liver and spleen, impairment of the specific cellular immune response resulting in significantly lower delayed-type hypersensitivity reactions, and increased levels of immunoglobulin G1 (IgG1)- and IgG2b-specific antibodies. Histopathological analysis demonstrated that depletion of IFN-gamma changes the focal granulomatous lesions found in the lungs of B10.A and A/Sn mice into coalescent granulomata which destroy the pulmonary architecture. These results suggest that irrespective of the mouse strain, IFN-gamma plays a protective role and that this cytokine is one major mediator of resistance against P. brasiliensis infection in mice.

Type 1 and type 2 cytokines: from basic science to fungal infections.

At the present time, it is clear that Th1 responses afford protection against the fungi; however, the development, maintenance and function of the protective immune responses are complex mechanisms and are influenced by multiple factors. The route of infection has been shown to affect initial cytokine production and, consequently, the induction of protective Th1 responses. The ability of different isolates of the same fungal agent to induce and sustain a protective response has also been emphasized. Protective immune responses have been shown to vary in genetically different mouse strains after infection. In addition, these protective responses, such as cellular influx and cytokine production, also vary within the same animal depending on the tissue infected. The functional dominance of certain cytokines over others in influencing development and maintenance of protective responses has been discussed. Certain cytokines may act differently in hosts lacking important components of their innate or immune repertoire. It is evident from these presentations that a more comprehensive understanding of the protective mechanisms against different fungal agents is emerging. However, there is still much to learn before cytokine modulatory therapy can be used effectively without risk in the human host.

Paracoccidioides brasiliensis infection in nude mice: studies with isolates differing in virulence and definition of their T cell-dependent and T cell-independent components.

Athymic and euthymic BALB/c mice infected with highly (Pb18) or slightly (Pb265) virulent Paracoccidioides brasiliensis isolates were compared regarding mortality, presence of viable yeasts, specific immunoglobulin M (IgM) and IgG titers, and the antigen recognition patterns of these antibodies. Isolate Pb18 caused a more severe disease in athymic mice, as supported by higher number of infected organs and shorter survival times. These animals, however, were resistant to Pb265 infection. High titers of antibodies were found only in euthymic mice, seven weeks after Pb18 infection. At this time, euthymic animals presented IgG antibodies to numerous protein bands that were not detected at four weeks postinfection or after Pb265 inoculation. In contrast, antibodies from athymic mice always reacted with few antigen bands. Although the majority of P. brasiliensis antigens are T cell-dependent, the immunodominant gp43 and also the 41.5- and 27.5-kD antigens are here, for the first time, characterized as T cell-independent antigens of P. brasiliensis.

Effect of macrophage blockade on the resistance of inbred mice to Paracoccidioides brasiliensis infection.

The effect of macrophage blockade on the natural resistance and on the adaptative immune response of susceptible (B10.D2/oSn) and resistant (A/Sn) mice to Paracoccidioides brasiliensis infection was investigated. B10.D2/oSn and A/Sn mice previously injected with colloidal carbon were infected ip with yeast cells to determine the 50% lethal dose, and to evaluate the anatomy and histopathology, macrophage activation, antibody production and DTH reactions. Macrophage blockade rendered both resistant and susceptible mice considerably more susceptible to infection, as evidenced by increased mortality and many disseminated lesions. P. brasiliensis infection and/or carbon treatment increased the ability of macrophages from resistant mice to spread up to 25 days after treatment. In susceptible mice the enhanced spreading capacity induced by carbon treatment was impaired at all assayed periods except at 1 week after infection. Macrophage blockade enhanced DTH reactions in resistant mice, but did not alter these reactions in susceptible mice, which remained anergic. To the contrary, macrophage blockade enhanced specific antibody production by susceptible mice, but did not affect the low levels produced by resistant mice. The effect of macrophage blockade confirms the natural tendency of resistant animals to mount DTH reactions in the course of the disease and the preferential antibody response developed by susceptible mice after P. brasiliensis infection. On the whole, macrophage functions appear to play a fundamental role in the natural and acquired resistance mechanisms to P. brasiliensis infection.

Delayed-type hypersensitivity response in an isogenic murine model of paracoccidioidomycosis.

The specific delayed-type hypersensitivity (DTH) response was evaluated in resistant (A/SN) and susceptible (B10.A) mice intraperitoneally infected with yeasts from a virulent (Pb18) or from a non-virulent (Pb265) Paracoccidioides brasiliensis isolates. Both strains of mice were footpad challenged with homologous antigens. Pb18 infected A/SN mice developed an evident and persistent DTH response late in the course of the disease (90th day on) whereas B10.A animals mounted a discrete and ephemeral DTH response at the 14th day post-infection. A/SN mice infected with Pb265 developed cellular immune responses whereas B10.A mice were almost always anergic. Histological analysis of the footpads of infected mice at 48 hours after challenge showed a mixed infiltrate consisting of predominantly mononuclear cells. Previous infection of resistant and susceptible mice with Pb18 did not alter their DTH responses against heterologous unrelated antigens (sheep red blood cells and dinitrofluorobenzene) indicating that the observed cellular anergy was antigen-specific. When fungal related antigens (candidin and histoplasmin) were tested in resistant mice, absence of cross-reactivity was noted. Thus, specific DTH responses against P. brasiliensis depend on both the host's genetically determined resistance and the virulence of the fungal isolate.

The source of the growth-promoting factor(s) affects the plating efficiency of Paracoccidioides brasiliensis.

We studied the influence of the growth factor (GF) source, concentration and production time on the plating efficiency of Paracoccidioides brasiliensis yeast cells. The highest plating efficiencies were achieved when the GF was derived from a fast growing P. brasiliensis isolate which was not homologous to the plated samples.

Alterations in the pathogenicity of one Paracoccidioides brasiliensis isolate do not correlative with its in vitro growth.

The in vitro subcultivation of some microorganisms for long periods causes measurable loss of their pathogenicity, which can be reverted by reisolation from infected hosts. We compared the pathogenicity and the in vitro growth pattern of one P. brasiliensis isolate (Pb 18) in its yeast phase, using the following samples: 1) The original pathogenic Pb 18 (OP). 2) Pb 18 attenuated by continuous in vitro subcultivation (AT). 3) Pb 18 (AT) reisolated from susceptible B10.A mice (RS). 4) Pb 18 (AT) reisolated from resistant A/SN mice (RR). Pathogenicity was evaluated by anatomopathology and mortality of mice infected i.p. with 5 x 10(6) fungi. Median survival times of mice infected with OP ranged from 74 to 117 days during the first 51 months of subculturing; with more cycles of subculturing the median survival time increased, reaching 250 days at the 64th month. This indicated decreasing virulence of OP during this period of subculturing. Survival of mice infected with RS and RR was respectively 112 and 123 days, which is similar to the behavior of the OP variant. The in vitro growth curve profile of RR showed significantly higher numbers of total and viable yeasts than the other studied variant. These results show that: 1) Pb 18 isolate loses its pathogenicity by continuous subcultivation. This phenomenon is reverted by reisolation from mice, independently from their susceptibility to the fungus; 2) the in vitro growth patterns of Pb 18 do not correlate with alterations in pathogenicity but are influenced by the host's environment.

Growth curves, morphology and ultrastructure of ten Paracoccidioides brasiliensis isolates.

The yeast phase of ten P. brasiliensis isolates were studied to characterize their growth pattern, morphology and ultrastructure. Growth curves were determined after counts of total and viable fungi units (FU) during 20 days. Three growth patterns were observed: slow, reaching approximately 10-30 X 10(6) FU/tube (Pb 18, Pb 265 and PB 2); intermediate, reaching 60-150 X 10(6) FU/tube (IVIC Pb 9, IVIC Pb 267, Pb SN, Pb Vitor and Pb Campo Grande) and fast, reaching 180-370 X 10(6) FU/tube (Pb 2052 and Pb 192). The highest percentage of viable cells occurred on the 6th day of culture for Pb 192, Pb Campo Grande, Pb 2052 and IVIC Pb 9; on the 8th day for Pb Vitor, Pb SN, Pb 18 and IVIC Pb 267; on the 10th day for Pb 265 and on the 12th day of culture for Pb 2. Mean generation times varied from approximately 21.2 (Pb 2052) to 102.6 hours (Pb 265). The isolates showed similar morphology, except IVIC Pb 267 which did not present a typical yeast-phase at 35 degrees C and the two fast-growing isolates (Pb 2052 and Pb 192) that presented smaller cell sizes and less tendency to clump. The ultrastructure of the isolates was similar: the cell walls presented a width of 0.1 to 0.2 mu; the mitochondria presented few cristae and had equivalent patterns of distribution and morphology; the endoplasmic reticulum was scanty, presenting narrow cisternae; the vacuoles, empty or filled with electron-dense material, were numerous and two to five nuclei with pores were constantly observed.

Resistance to Paracoccidioides brasiliensis in mice is controlled by a single dominant autosomal gene.

In a previous report it was shown that there are resistant, susceptible, and intermediate strains of mice to intraperitoneal Paracoccidioides brasiliensis infection. In the present work, we investigated the type of inheritance and the number of genes that determine resistance to paracoccidioidomycosis. Parental and hybrid mice were inoculated intraperitoneally with 5 X 10(6) P. brasiliensis yeast cells, and mortality was scored daily. Analysis of susceptible and resistant parental strains and of F1, F2, and backcross mice showed that the resistance to P. brasiliensis seems to be controlled genetically by a single dominant gene, which we designated the Pbr locus. The mean survival times of susceptible F2 and backcross hybrids were very similar to that of the susceptible parent. Examination of the pathological changes observed in parental and F1 mice, 6 months after infection, showed that F1 offspring presented a similar number and distribution of lesions to those of the resistant strains. The Pbr gene is not linked to H-2, Hc, and albino genes. Furthermore, resistance to paracoccidioidomycosis is controlled by an autosomal gene.

In vivo and in vitro characteristics of six Paracoccidioides brasiliensis strains.

The yeast-like forms of six P. brasiliensis strains were characterized and compared using in vitro (growth curve determination) and in vivo (pathogenicity to sensitive inbred mice) criteria. Strains Pb 18 and Pb 265 which behaved similarly in vitro, showing low counts of fungi and long mean generation times, were respectively the most and the least pathogenic strains. Strains Pb 2052 and IVIC Pb 267, which grow abundantly in vitro were, respectively virulent and avirulent. Strains Pb SN and IVIC Pb 9 behaved similarly both in vitro and in vivo displaying an intermediate pattern of virulence and growing conditions.