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Background/objectives: Considering the large number of candidates in vaccine-testing studies against different pathogens and the amount of time spent in the preclinical and clinical trials, there is a pressing need to develop an improved in vivo system to quickly screen vaccine candidates. The model of a polyester-polyurethane sponge implant provides a rapid analysis of the specific stimulus-response, allowing the study of a compartmentalized microenvironment. The sponge implant's defined measurements were standardized as a compartment to assess the immune response triggered by the vaccinal antigen. The LBSap vaccine (composed of Leishmania braziliensis antigens associated with saponin adjuvant) was used in the sponge model to assess the antigen-specific immunological biomarker, including memory generation after initial contact with the antigen., Methods: Mice strains (Swiss, BALB/c, and C57BL/6) were previously immunized using LBSap vaccine, followed by an antigenic booster performed inside the sponge implant. The sponge implants were assessed after 72 h, and the immune response pattern was analyzed according to leukocyte immunophenotyping and cytokine production., Results: After LBSap vaccination, the innate immune response of the antigenic booster in the sponge implants demonstrated higher levels in the Ly+ neutrophils and CD11c+ dendritic cells with reduced numbers of F4/80+ macrophages. Moreover, the adaptive immune response in Swiss mice demonstrated a high CD3+CD4+ T-cell frequency, consisting of an effector memory component, in addition to a cytoxicity response (CD3+CD8+ T cells), displaying the central memory biomarker. The major cell surface biomarker in the BALB/c mice strain was related to CD3+CD4+ effector memory, while the increased CD3+CD8+ effector memory was highlighted in C57/BL6. The cytokine profile was more inflammatory in Swiss mice, with the highest levels of IL-6, TNF, IFN-g, and IL-17, while the same cytokine was observed in in C57BL/6 yet modulated by enhanced IL-10 levels. Similar to Swiss mice, BALB/c mice triggered an inflammatory environment after the antigenic booster in the sponge implant with the increased levels in the ILL-6, TNF, and IFN-g., Conclusions: The findings emphasized the impact of genetic background on the populations engaged in immune responses, suggesting that this model can be utilized to enhance and track both innate and adaptive immune responses in vaccine candidates. Consequently, these results may inform the selection of the most suitable experimental model for biomolecule testing, taking into account how the unique characteristics of each mouse strain affect the immune response dynamics.

The attenuated yellow fever (YF) vaccine is one of the most successful vaccines ever developed. After a single dose administration YF vaccine can induce balanced Th1/Th2 immune responses and long-lasting neutralizing antibodies. These attributes endorsed it as a model of how to properly stimulate the innate response to target protective immune responses. Despite their longstanding success, attenuated YF vaccines can cause rare fatal adverse events and are contraindicated for persons with immunosuppression, egg allergy and age < 6 months and >60 years. These drawbacks have encouraged the development of a non-live vaccine. The aim of the present study is to characterize and compare the immunological profile of two adjuvant formulations of an inactivated YF 17DD vaccine candidate. Inactivated YF vaccine formulations based on alum (Al(OH)3) or squalene (AddaVax®) were investigated by immunization of C57BL/6 mice in 3-dose or 2-dose schedules, respectively, and compared with a single dose of attenuated YF virus 17DD. Sera were analyzed by ELISA and Plaque Reduction Neutralization Test (PRNT) for detection of total IgG and neutralizing antibodies against YF virus. In addition, splenocytes were collected to evaluate cellular responses by ELISpot. Both inactivated formulations were able to induce high titers of IgG against YF, although neutralizing antibodies levels were borderline on pre-challenge samples. Analysis of IgG subtypes revealed a predominance of IgG2a associated with improved neutralizing capacity in animals immunized with the attenuated YF vaccine, and a predominance of IgG1 in groups immunized with experimental non-live formulations (alum and AddaVax®). After intracerebral (IC) challenge, attenuated and inactivated vaccine formulations showed an increase in neutralizing antibodies. The AddaVax®-based inactivated vaccine and the attenuated vaccine achieved 100% protection, and alum-based equivalent formulation achieved 70% protection.

Background: Coronavirus disease 2019 (COVID-19) can progress to severe pneumonia with respiratory failure and is aggravated by the deregulation of the immune system causing an excessive inflammation including the cytokine storm., Methods: In this study, we report that severe acutely infected patients have high levels of both type-1 and type-2 cytokines., Results: Our results show abnormal cytokine levels upon T-cell stimulation, in a nonpolarized profile. Furthermore, our findings indicate that this hyperactive cytokine response is associated with a significantly increased frequency of late-differentiated T cells with particular phenotype of effector exhausted/senescent CD28-CD57+ cells. Of note, we demonstrated for the first time an increased frequency of CD3+CD4+CD28-CD57+ T cells with expression of programmed death 1, one of the hallmarks of T-cell exhaustion., Conclusions: These findings reveal that COVID-19 is associated with acute immunodeficiency, especially within the CD4+ T-cell compartment, and points to possible mechanisms of loss of clonal repertoire and susceptibility to viral relapse and reinfection events., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The first formal description of the microbicidal activity of extracellular traps (ETs) containing DNA occurred in neutrophils in 2004. Since then, ETs have been identified in different populations of cells involved in both innate and adaptive immune responses. Much of the knowledge has been obtained from in vitro or ex vivo studies; however, in vivo evaluations in experimental models and human biological materials have corroborated some of the results obtained. Two types of ETs have been described-suicidal and vital ETs, with or without the death of the producer cell. The studies showed that the same cell type may have more than one ETs formation mechanism and that different cells may have similar ETs formation mechanisms. ETs can act by controlling or promoting the mechanisms involved in the development and evolution of various infectious and non-infectious diseases, such as autoimmune, cardiovascular, thrombotic, and neoplastic diseases, among others. This review discusses the presence of ETs in neutrophils, macrophages, mast cells, eosinophils, basophils, plasmacytoid dendritic cells, and recent evidence of the presence of ETs in B lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes. Moreover, due to recently collected information, the effect of ETs on COVID-19 is also discussed.

Background: Sporotrichosis occurs through contact with contaminated soil and plant. However, the incidence of sporotrichosis as a zoonotic epidemic has increased, particularly in Rio de Janeiro. Aim: In this work, we decided to evaluate some T-cell phenotypes involved in the immune response. Materials & methods: We used flow cytometry to quantify TCD4 + and TCD8 + and Treg from immunocompetent and immunosuppressed mice infected with Sporothrix species with different levels of virulence and pathogenicity. Results: It was demonstrated the predominance of TCD4 + over the TCD8 + cells in both groups, inoculated with all the species, and percentages of Treg observed in infected immunocompetent mice. Conclusion: This regulatory phenotype can be associated with a protective immunity in the initial periods of infection.

Leishmania ( V .) braziliensis is the etiological agent of Cutaneous (CL) and Mucocutaneous leishmaniasis (ML) in the New World. CL can be more benign but ML can be severe and disfiguring. Immunity to these diseases include hypersensitivity, an enhanced inflammatory response with strong IFN-γ and TNF-α secretion. Additionally, the production of IL-10 which down modulates the immune response is reduced. The Nucleoside hydrolase (NH36) of Leishmania ( L .) donovani is the main antigen of the Leishmune veterinary vaccine and its F3 domain induces a CD4 + T cell-mediated protection against L . ( L .) infantum chagasi infection. Prevention of L . ( L .) amazonensis infection requires in contrast an additional CD8 + T cell mediated response induced by the F1 domain. Consequently, the F1F3 recombinant chimera, which contains both domains cloned in tandem, optimized the vaccine efficacy against L . ( L .) amazonensis mouse infection. We compared the efficacies of NH36, F1, F3, and the FIF3 chimera against L . ( V .) braziliensis mouse infection. The F1F3 chimera increased the NH36 specific IgA and response before and after infection and the IgG and IgG3 levels after challenge. It also induced a 49% stronger intradermal response to leishmanial antigen (IDR) than NH36 that was positively correlated to the levels of IFN-γ and TNF-α, IgG, IgG2a, IgG2b, and IgG3 anti-NH36 antibodies. However, stronger Th1 responses with elevated IFN-γ / IL-10 and TNF-α / IL-10 ratios were promoted by the F3 and F1 vaccines and detected in infected controls while the F1F3 chimera promoted the highest IL-10 secretion, which reduced the pathological Th1 response, and characterized the induction of a mixed and/or T-cell regulatory response. We identified the epitopes responsible for these immune responses. The F3 vaccine induced the earliest immunity and after challenge, the F1F3 chimera promoted the highest CD4 + and CD8 + cytokine-secreting T cell responses, and the predominant frequencies of multifunctional CD4 + and CD8 + IL-2 + TNF-α + IFN-γ + T cells. Also as observed against L . ( L .) amazonensis infection, the F1F3 chimera showed the strongest reduction of the ear lesions sizes induced by L . ( V .) braziliensis . Our results confirm the potential use of the F1F3 chimera in a multi-species cross-protective vaccine against L . ( V .) braziliensis .

Plasmodium vivax Merozoite Surface Protein-9 (PvMSP-9) is a malaria vaccine candidate naturally immunogenic in humans and able to induce high antibody titers in animals when delivered as a recombinant protein. Recently, we identified the sequence EAAPENAEPVHENA (PvMSP9 E795-A808 ) as the main linear B-cell epitope in naturally exposed individuals. However, the potential of PvMSP9 E795-A808 as an immunogen in experimental animal models remained unexplored. Here we assess the immunogenicity of PvMSP9 E795-A808 using synthetic peptides. The peptides tested in BALB/c mice include two repeats of the sequence EAAPENAEPVHENA tested alone (peptide RII), or linked to an autologous (PvMSP9 peptide pL; pLRII) or heterologous (p2 tetanus toxin universal T cell epitope; TTRII) T cell epitope. Immune responses were evaluated by ELISA, FLUOROSPOT, and indirect immunofluorescence. We show that all of the peptide constructs tested were immunogenic eliciting specific IgG antibodies at different levels, with a prevalence of IgG1 and IgG2. Animals immunized with synthetic peptides containing T cell epitopes (pLRII or TTRII) had more efficient antibody responses that resulted in higher antibody titers able to recognize the native protein by immunofluorescence. Relevantly, the frequency of IFN-γ secreting SFC elicited by immunization with TTRII synthetic peptide was comparable to that reported to the PvMSP9-Nt recombinant protein. Taken together, our study indicates that PvMSP9 E795-A808 is highly immunogenic in mice and further studies to evaluate its value as promising vaccine target are warranted. Moreover, our study supports the critical role of CD4 T cell epitopes to enhance humoral responses induced by subunit based vaccines., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

[This corrects the article DOI: 10.3389/fimmu.2018.01690.].

In Brazil, canine visceral leishmaniasis (CVL) is caused by Leishmania infantum, presenting a broad spectrum of clinical manifestations. Dogs are the main parasite reservoir in urban areas and canine cases precede human infection. Currently, A2 protein based Leish-Tec® vaccine is the only vaccine commercially available against CVL in Brazil. Considering that the main screening and confirmatory tests of canine infection are serological, it is possible that the antibody response elicited after vaccination interfere with diagnosis, leading to the inability to distinguish between vaccinated and infected animals. In order to identify the specific B-cell response induced after vaccination, A2 protein sequence was screened for main linear B-cell epitopes using in silico prediction (Bepipred) and immunological confirmation by ELISA. Three amino acid sequences were described as potential B-cell epitopes (SV11-SAEPHKAAVDV, PP16-PQSVGPLSVGPQSVGP, and VQ34-VGPLSVGPQSVGPLSVGPLSVGPQAVGPLSVGPQ). Specific IgG ELISAs were performed in sera of 12 immunized dogs living in non-endemic areas, followed for up to 1 year after immunization. The results were compared with those obtained in a group of 10 symptomatic and 10 asymptomatic CVL dogs. All predicted epitopes were confirmed as linear B-cell epitopes broadly recognized by sera from studied dogs. Total IgG ELISAs demonstrated distinct patterns of response between peptides in the immunized and CVL groups. VQ34 peptide was recognized by the majority of sera from vaccinated and symptomatic dogs, and increases after vaccination. PP16 induced low levels of specific IgG that increased 1 year after immunization. Interestingly, a low frequency of reactivity was found against SV11 in naturally infected dogs (symptomatic and asymptomatic), while 83.3% of vaccinated dogs presented positive responses 1 year after immunization. The two animals in the vaccinated group that did not respond to SV11 1 year after immunization presented positive serology both 30 days and 6 months after immunization. In summary, we identified three main linear B-cell epitopes in A2 based vaccine. Moreover, the humoral response against SV11 presented marked differences between infected and Leish-Tec vaccinated dogs, and should be further investigated, in large trials, to confirm its potential as a serological marker able to distinguish between infected and vaccinated dogs.

Introduction: A proliferation-inducing ligand (APRIL) and B cell activation factor (BAFF) are known to play a significant role in the pathogenesis of several diseases, including BAFF in malaria. The aim of this study was to investigate whether APRIL and BAFF plasma concentrations could be part of inflammatory responses associated with P. vivax and P. falciparum malaria in patients from the Brazilian Amazon., Methods: Blood samples were obtained from P. vivax and P. falciparum malaria patients (n = 52) resident in Porto Velho before and 15 days after the beginning of treatment and from uninfected individuals (n = 12). We investigated APRIL and BAFF circulating levels and their association with parasitaemia, WBC counts, and cytokine/chemokine plasma levels. The expression levels of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) on PBMC from a subset of 5 P. vivax-infected patients were analyzed by flow cytometry., Results: APRIL plasma levels were transiently increased during acute P. vivax and P. falciparum infections whereas BAFF levels were only increased during acute P. falciparum malaria. Although P. vivax and P. falciparum malaria patients have similar cytokine profiles during infection, in P. vivax acute phase malaria, APRIL but not BAFF levels correlated positively with IL-1, IL-2, IL-4, IL-6, and IL-13 levels. We did not find any association between P. vivax parasitaemia and APRIL levels, while an inverse correlation was found between P. falciparum parasitaemia and APRIL levels. The percentage of TACI positive CD4+ and CD8+ T cells were increased in the acute phase P. vivax malaria., Conclusion: These findings suggest that the APRIL and BAFF inductions reflect different host strategies for controlling infection with each malaria species., (© 2018 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.)

Physical contact between dendritic cells (DCs) and T cell lymphocytes is necessary to trigger the immune cell response. CCL19 and CCL21 chemokines bind to the CCR7 receptor of mature DCs, and of T cells and regulate DCs migration to the white pulp (wp) of the spleen, where they encounter lymphocytes. In visceral leishmaniasis (VL), cellular immunosuppression is mediated by impaired DC migration due to the decreased chemokine secretion by endothelium and to the reduced DCs CCR7 expression. The Leishmania (L.) donovani nucleoside hydrolase NH36 and its C-terminal domain, the F3 peptide are prominent antigens in the generation of preventive immunity to VL. We assessed whether these vaccines could prevent the migrating defect of DCs by restoring the expression of CCR7 receptors. C57Bl6 mice were vaccinated with NH36 and F3 and challenged with L. (L.) infantum chagasi . The F3 vaccine induced a 100% of survival and a long-lasting immune protection with an earlier CD4 + Th1 response, with secretion of higher IFN-γ and TNF-α/IL-10 ratios, and higher frequencies of CD4 + T cells secreting IL-2 + , TNF-α + , or IFN-γ + , or a combination of two or the three cytokines (IL-2 + TNF-α + IFN-γ + ). The CD8 + T cell response was promoted earlier by the NH36-vaccine, and later by the F3-vaccine. Maximal number of F3-primed DCs migrated in vitro in response to CCL19 and showed a high expression of CCR7 receptors (26.06%). Anti-CCR7 antibody treatment inhibited DCs migration in vitro (90%) and increased parasite load in vivo . When transferred into 28-day-infected mice, only 8% of DCs from infected, 59% of DCs from NH36-vaccinated, and 84% of DCs from F3-vaccinated mice migrated to the wp. Consequently, immunotherapy of infected mice with F3-primed DCs only, promoted increases in corporal weight and reductions of spleen and liver parasite loads and relative weights. Our findings indicate that vaccination with F3-vaccine preserves the maturation, migration properties and CCR7 expression of DCs, which are essential processes for the generation of cell-mediated immunity. The F3 vaccine is more potent in reversing the migration defect that occurs in VL and, therefore, more efficient in immunotherapy of VL.

Purpureocillium lilacinum is a filamentous, hyaline fungus considered an emerging pathogen in humans. The aim of our study was to evaluate the outcome of hyalohyphomycosis in C57BL/6 murine models inoculated with two clinical P. lilacinum isolates (S1 and S2). Each isolate was inoculated in mice randomly distributed in immunocompetent (CPT) and immunosuppressed (SPS) groups. Mice were evaluated at day 7, 21, and 45 after inoculation for histopathological analysis, recovery of fungal cells, and immunological studies. Histological analysis showed scarce conidia-like structures in lung tissue from CPT mice and a lot of fungal cells in SPS mice inoculated with S2 compared to mice inoculated with S1. The maximum recovery of fungal cells was seen in CPT mice inoculated with both isolates at day 7, but with mean significantly higher in those inoculated with S2 isolate. Phenotypical characterization of T cells showed TCD8 + lymphocytes predominance over TCD4 + in immunosuppressed mice infected and control groups. We also observed higher percentages of the central and effector memory/effector phenotype in CPT mice infected with S2 strain, especially in TCD8 + in the initial period of infection. Regulatory T cells showed higher percentages in immunosuppressed, predominantly after the acute phase. Our results showed that the P. lilacinum is a fungus capable to cause damages in competent and immunosuppressed experimental hosts. Furthermore, S2 isolate seems to cause more damage to the experimental host and it was possible to identify different cellular subsets involved in the mice immune response.

The Leishmania (Leishmania) donovani nucleoside hydrolase NH36 is the main antigen of the Leishmune ® vaccine and one of the promising candidates for vaccination against visceral leishmaniasis. The antigenicity of the N-terminal (F1), the central (F2), or the C-terminal recombinant domain (F3) of NH36 was evaluated using peripheral blood mononuclear cells (PBMC) from individuals infected with L. (L.) infantum from an endemic area of visceral leishmaniasis of Spain. Both NH36 and F1 domains significantly increased the PBMC proliferation stimulation index of cured patients and infected asymptomatic individuals compared to healthy controls. Moreover, F1 induced a 19% higher proliferative response than NH36 in asymptomatic exposed subjects. In addition, in patients cured from visceral leishmaniasis, proliferation in response to NH36 and F1 was accompanied by a significant increase of IFN-γ and TNF-α secretion, which was 42-43% higher, in response to F1 than to NH36. The interleukin 17 (IL-17) secretion was stronger in asymptomatic subjects, in response to F1, as well as in cured cutaneous leishmaniasis after NH36 stimulation. While no IL-10 secretion was determined by F1, a granzyme B increase was detected in supernatants from cured patients after stimulation with either NH36 or F1. These data demonstrate that F1 is the domain of NH36 that induces a recall cellular response in individuals with acquired resistance to the infection by L. (L.) infantum . In addition, F1 and NH36 discriminated the IgG3 humoral response in patients with active visceral leishmaniasis due to L. (L.) donovani (Ethiopia) and L. (L.) infantum (Spain) from that of endemic and non-endemic area controls. NH36 showed higher reactivity with sera from L. (L.) donovani -infected individuals, indicating species specificity. We conclude that the F1 domain, previously characterized as an inducer of the Th1 and Th17 responses in cured/exposed patients infected with L. (L.) infantum chagasi , may also be involved in the generation of a protective response against L. (L.) infantum and represents a potential vaccine candidate for the control of human leishmaniasis alone, or in combination with other HLA epitopes/antigens.

Development of immunoprotection against visceral leishmaniasis (VL) focused on the identification of antigens capable of inducing a Th1 immune response. Alternatively, antigens targeting the CD8 and T-regulatory responses are also relevant in VL pathogenesis and worthy of being included in a preventive human vaccine. We assessed in active and cured patients and VL asymptomatic subjects the clinical signs and cytokine responses to the Leishmania donovani nucleoside hydrolase NH36 antigen and its N-(F1), central (F2) and C-terminal (F3) domains. As markers of VL resistance, the F2 induced the highest levels of IFN-γ, IL-1β, and TNF-α and, together with F1, the strongest secretion of IL-17, IL-6, and IL-10 in DTH + and cured subjects. F2 also promoted the highest frequencies of CD3 + CD4 + IL-2 + TNF-α - IFN-γ - , CD3 + CD4 + IL-2 + TNF-α + IFN-γ - , CD3 + CD4 + IL-2 + TNF-α - IFN-γ + , and CD3 + CD4 + IL-2 + TNF-α + IFN-γ + T cells in cured and asymptomatic subjects. Consistent with this, the IFN-γ increase was correlated with decreased spleen ( R  = -0.428, P  = 0.05) and liver sizes ( R  = -0.428, P  = 0.05) and with increased hematocrit counts ( R  = 0.532, P  = 0.015) in response to F1 domain, and with increased hematocrit ( R  = 0.512, P 0.02) and hemoglobin counts ( R  = 0.434, P  = 0.05) in response to F2. Additionally, IL-17 increases were associated with decreased spleen and liver sizes in response to F1 ( R  = -0.595, P  = 0.005) and F2 ( R  = -0.462, P  = 0.04). Conversely, F1 and F3 increased the CD3 + CD8 + IL-2 + TNF-α - IFN-γ - , CD3 + CD8 + IL-2 + TNF-α + IFN-γ - , and CD3 + CD8 + IL-2 + TNF-α + IFN-γ + T cell frequencies of VL patients correlated with increased spleen and liver sizes and decreased hemoglobin and hematocrit values. Therefore, cure and acquired resistance to VL correlate with the CD4 + -Th1 and Th-17 T-cell responses to F2 and F1 domains. Clinical VL outcomes, by contrast, correlate with CD8 + T-cell responses against F3 and F1, potentially involved in control of the early infection. The in silico -predicted NH36 epitopes are conserved and bind to many HL-DR and HLA and B allotypes. No human vaccine against Leishmania is available thus far. In this investigation, we identified the NH36 domains and epitopes that induce CD4 + and CD8 + T cell responses, which could be used to potentiate a human universal T-epitope vaccine against leishmaniasis.

Malaria and Cutaneous Leishmaniasis (CL) are co-endemic throughout large regions in tropical countries and co-infection may impact the evolution of host-parasite interactions. In the present study, we evaluate Malaria/Leishmaniasis disease outcome, Th1/Th2 cytokine levels and the CD4 and CD8 T-cell profiles in a co-infection murine model (BALB/c) of Plasmodium yoelii 17XNL (Py) and Leishmania amazonensis (La) or L. braziliensis (Lb). Malaria parasitaemia was assessed through blood strains stained with Giemsa. Leishmania lesions were monitored with a digital caliper and parasite loads determined by limiting-dilution assay. Serum levels of IFN-γ, TNF, IL-2, IL-4, IL-6, IL-10, and IL-17 were determined using multiplexed bead assay and expression of CD3, CD4, and CD8 T-cells markers were determined by Flow Cytometry in the thymus, spleens and lymph nodes. Parasitaemia in Lb+Py co-infected group was lower than in Py single-infected group, suggesting a protective effect of Lb co-infection in Malaria progression. In contrast, La+Py co-infection increased parasitaemia, patent infection and induced mortality in non-lethal Malaria infection. Regarding Leishmaniasis, Lb+Py co-infected group presented smaller lesions and less ulceration than Lb single-infected animals. In contrast, La+Py co-infected group presented only a transitory delay on the development of lesions when compared to La single-infected mice. Decreased levels of IFN-γ, TNF, IL-6, and IL-10 were observed in the serum of co-infected groups, demonstrating a modulation of Malaria immune response by Leishmania co-infections. We observed an intense thymic atrophy in Py single-infected and co-infected groups, which recovered earlier in co-infected animals. The CD4 and CD8 T cell profiles in thymus, spleens and lymph nodes did not differ between Py single and co-infected groups, except for a decrease in CD4(+)CD8(+) T cells which also increased faster in co-infected mice. Our results suggest that Py and Leishmania co-infection may change disease outcome. Interestingly Malaria outcome can be altered according to the Leishmania specie involved. Alternatively Malaria infection reduced the severity or delayed the onset of leishmanial lesions. These alterations in Malaria and CL development seem to be closely related with changes in the immune response as demonstrated by alteration in serum cytokine levels and thymus/spleens T cell phenotypes dynamics during infection.

There have been exhaustive efforts to develop an efficient vaccine against leishmaniasis. Factors like host and parasite genetic characteristics, virulence, epidemiological scenarios, and, mainly, diverse immune responses triggered by Leishmania species make the achievement of this aim a complex task. It is already clear that the induction of a Th1, pro-inflammatory response, is important in the protection against Leishmania infection. However, many questions must still be answered to fully understand Leishmania immunopathology, especially regarding Leishmania-specific Th1 response induction, regulation, and persistence. A large number of Leishmania antigens able to induce pro-inflammatory response have been selected so far, but none of them demonstrated efficiency in protection assays. A possible explanation is that CD4 T cells display marked heterogeneity at a single-cell level especially regarding the production of Th1-defining cytokines and multifunctionality. It has been established in the literature that Th1 cells undergo a differentiation process, which can generate cells with diverse phenotypes and survival capabilities. Despite that, only a few studies evaluate this heterogenic response and the amount of multifunctional CD4 T cells induced by Leishmania vaccine candidates, missing what can be a crucial point in defining a correlate of protection after vaccination. Moreover, most of the knowledge involving the development of cutaneous leishmaniasis (CL) vaccines comes from the mouse model of infection with Leishmania major, which cannot be fully applied to New World Leishmaniasis. For this reason, the immune response triggered by infection with New World Leishmania species, as well as vaccine candidates, need further studies. In this review, we will reinforce the importance of evaluating the quality of immune response against Leishmania, using a multiparametric analysis in order to understand better this complex host-parasite interaction, discussing the differences in the responses triggered by different New World Leishmania species, as well as the impact on the development of an effective vaccine against CL.

Introduction: Purpureocillium lilacinum is emerging as a causal agent of hyalohyphomycosis that is refractory to antifungal drugs; however, the pathogenic mechanisms underlying P. lilacinum infection are not understood. In this study, we investigated the interaction of P. lilacinum conidia with human macrophages and dendritic cells in vitro., Methods: Spores of a P. lilacinum clinical isolate were obtained by chill-heat shock. Mononuclear cells were isolated from eight healthy individuals. Monocytes were separated by cold aggregation and differentiated into macrophages by incubation for 7 to 10 days at 37°C or into dendritic cells by the addition of the cytokines human granulocyte-macrophage colony stimulating factor and interleukin-4. Conidial suspension was added to the human cells at 1:1, 2:1, and 5:1 (conidia:cells) ratios for 1h, 6h, and 24h, and the infection was evaluated by Giemsa staining and light microscopy., Results: After 1h interaction, P. lilacinum conidia were internalized by human cells and after 6h contact, some conidia became inflated. After 24h interaction, the conidia produced germ tubes and hyphae, leading to the disruption of macrophage and dendritic cell membranes. The infection rate analyzed after 6h incubation of P. lilacinum conidia with cells at 2:1 and 1:1 ratios was 76.5% and 25.5%, respectively, for macrophages and 54.3% and 19.5%, respectively, for cultured dendritic cells., Conclusions: P. lilacinum conidia are capable of infecting and destroying both macrophages and dendritic cells, clearly demonstrating the ability of this pathogenic fungus to invade human phagocytic cells.

Purpureocillium lilacinum is an emerging pathogenic fungus that can cause different clinical manifestations ranging from cutaneous and sub-cutaneous infections to severe oculomycosis. In this study, using both conventional indirect immunofluorescence and non-conventional flow cytometry approaches, IgG antibodies were readily detected in both C57BL/6 immunocompetent and immunosuppressed mice after i.v. infection with P. lilacinum. The humoral immune response was specific, since virtually no antibodies were detected in the serum of control mice. Flow cytometry assays also showed both quantitative and qualitative differences in total IgG and its isotypes in sera of immunocompetent and immunosupressed infected mice. Although a good starting point, it is clear that the effectiveness of serological assays for P. lilacinum hyalohyphomycosis identification in clinical studies still requires further standardization. Upon further validation in humans, these techniques have the potential to be suitable to detect P. lilacinum infection in patients, thereby avoiding current laborious and time-consuming culture techniques., (© 2013.)

Leishmaniasis is a group of important parasitic diseases affecting millions worldwide. To understand more clearly the quality of T helper type 1 (Th1) response stimulated after Leishmania infection, we applied a multiparametric flow cytometry protocol to evaluate multifunctional T cells induced by crude antigen extracts obtained from promastigotes of Leishmania braziliensis (LbAg) and Leishmania amazonensis (LaAg) in peripheral blood mononuclear cells from healed cutaneous leishmaniasis patients. Although no significant difference was detected in the percentage of total interferon (IFN)-γ-producing CD4(+) T cells induced by both antigens, multiparametric flow cytometry analysis revealed clear differences in the quality of Th1 responses. LbAg induced an important proportion of multifunctional CD4(+) T cells (28% of the total Th1 response evaluated), whereas LaAg induced predominantly single-positive cells (68%), and 57% of those were IFN-γ single-positives. Multifunctional CD4(+) T cells showed the highest mean fluorescence intensity (MFI) for the three Th1 cytokines assessed and MFIs for IFN-γ and interleukin-2 from those cells stimulated with LbAg were significantly higher than those obtained after LaAg stimulation. These major differences observed in the generation of multifunctional CD4(+) T cells suggest that the quality of the Th1 response induced by L. amazonensis antigens can be involved in the mechanisms responsible for the high susceptibility observed in L. amazonensis-infected individuals. Ultimately, our results call attention to the importance of studying a Th1 response regarding its quality, not just its magnitude, and indicate that this kind of evaluation might help understanding of the complex and diverse immunopathogenesis of American tegumentary leishmaniasis., (© 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.)

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during meta-cyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.

CD4+ T cells have a crucial role in mediating protection against a variety of pathogens through production of specific cytokines. However, substantial heterogeneity in CD4+ T-cell cytokine responses has limited the ability to define an immune correlate of protection after vaccination. Here, using multiparameter flow cytometry to assess the immune responses after immunization, we show that the degree of protection against Leishmania major infection in mice is predicted by the frequency of CD4+ T cells simultaneously producing interferon-gamma, interleukin-2 and tumor necrosis factor. Notably, multifunctional effector cells generated by all vaccines tested are unique in their capacity to produce high amounts of interferon-gamma. These data show that the quality of a CD4+ T-cell cytokine response can be a crucial determinant in whether a vaccine is protective, and may provide a new and useful prospective immune correlate of protection for vaccines based on T-helper type 1 (TH1) cells.

Whole-cell and soluble extracts of Leishmania promastigotes have both been used as skin test antigens and have also been tested as vaccine candidates. However, the differences in antigenicity between soluble and particulate Leishmania fractions are not known. We evaluated in vitro responses of PBMC from 30 American tegumentary leishmaniasis (ATL) patients and seven noninfected donors to different antigen preparations from Leishmania promastigotes, namely Leishmania amazonensis and L. braziliensis whole-cell extracts, as well as soluble and particulate fractions of L. amazonensis. All Leishmania antigen preparations stimulated significantly higher proliferation and interferon (IFN)-gamma production (but not interleukin (IL)-10 production) in PBMC from the leishmaniasis patients than in cells from the control subjects. The L. braziliensis whole-cell extract stimulated significantly higher cell proliferation and IFN-gamma production than the L. amazonensis whole-cell extract in the group of patients but not in the control group. This result can be explained by the fact that the patients were infected with L. braziliensis. Again in the group of patients, the PBMC proliferative responses as well as the levels of IFN-gamma and IL-10 stimulated by L. amazonensis whole-cell extract were significantly greater than those elicited by the L. amazonensis soluble fraction but were not significantly different from those elicited by the L. amazonensis particulate fraction. We found a higher antigenicity of the particulate fraction as compared to the soluble fraction, what suggests that the antigens present in the particulate fraction account for most of the antigenicity of whole-cell Leishmania promastigote antigen extracts.

Herein we investigate the ability of live promastigotes and total lysate of Leishmania (Viannia) braziliensis, derived from parasites in the logarithmic (L-Lb) or stationary phase (S-Lb), to induce human mast cell line (HMC-1) activation. In comparison with medium-treated cells, a significant histamine release was observed in HMC-1 cultures stimulated with S-Lb. Lipophosphoglycan also induced histamine release by HMC-1 cells. In immunocytochemical assays, we found a marked staining for tryptase in medium-treated HMC-1 cells, however, stimulation with L-Lb or S-Lb caused a marked decrease in the color reaction as well as in the number of tryptase-positive cells. L-Lb and S-Lb induced an evident decrease in the intracellular expression of IL-4 but not IL-12. Live stationary promastigotes were able to induce high levels of IL-4 release in HMC-1 cultures. Furthermore, these cells released significant amounts of IL-12 when incubated with both types of live promastigotes. These results indicate that L. (V.) braziliensis promastigotes differ in their ability to induce direct human mast cells activation, according to the growth phase of the parasite. Furthermore, the release of pro-inflammatory mediators and cytokines could represent an important phenomenon that might favor the initial establishment of the infection.

A positive reaction to the leishmanin skin test (LST) indicates previous contact with Leishmania antigens and is a useful criterion for the diagnosis of cutaneous leishmaniasis. In leishmaniasis vaccine trials, selection of volunteers has always been based on skin testing. During 1999 we performed a randomized controlled study in order to evaluate the immunogenicity of the LST. Fifty-nine (29 male and 30 female) healthy volunteer undergraduate students from the Medical School of Volta Redonda, Rio de Janeiro State, Brazil, with no evidence of previous infection with Leishmania, were randomly assigned into 2 groups: 29 subjects received LST and 30 received a placebo (merthiolate-phosphate-buffered saline). All volunteers received LST 41 d after the first injection of LST or placebo. Blood samples were taken immediately before the applications of LST or placebo for the assessment of Leishmania antigen-induced proliferation and cytokine production in peripheral blood mononuclear cell cultures. A significant increase in proliferative responses to L. braziliensis (P < 0.005) and L. amazonensis (P = 0.01) antigens as well as in L. braziliensis antigen-induced interferon-gamma production (P < 0.01) followed the application of LST but not the administration of the placebo. A single LST application is therefore able to induce Leishmania-specific cell-mediated immune responses. This observation should be considered in human trials of candidate vaccines against leishmaniasis.

A primate model of leishmaniasis was developed with the objective of future vaccine testing. Lesion development and immunological parameters were studied upon primary and secondary infections. Seven Cebus apella were injected subcutaneously with 2 x 10(6) Leishmania (Leishmania) amazonensis promastigotes. Erythematous nodules appeared 19-29 days p.i., which disappeared 100 days p.i. Four months later, six of the monkeys were challenged with the same inoculum; three of them developed erythematous nodules after 7 days p.i., with ulcer formation in two of these subjects. The lesions were short-lived and all were cured 40 days post challenge. Anti-Leishmania IgG antibodies were detected and they increased after the challenge infection. Leishmania antigen-induced lymphoproliferation was found 1 month post-primary infection, which coincided with IFN-gamma production and lesion development. It decreased to control levels afterwards, but at the time of the challenge dose, it was significantly above the initial level. After the challenge infection, it first increased then decreased sharply at 40 days post-challenge, coinciding with the healing of the lesion. It increased again to a higher level at 60 days post-challenge. Leishmania (Leishmania) amazonensis-infection in C. apella did not induce complete protection against a secondary infection with a homologous parasite although specific antibody production and lymphoproliferation with IFN-gamma production were observed. This fact indicates that vaccine has to be better than infection in the induction of protective immunity, and raises a question on in vitro parameters that should be considered as a counterpart of expected protection induced by vaccine candidate. In addition, we conclude that this is a useful primate model for the evaluation of candidate vaccines.

This study was aimed at evaluating the immunogenicity of a vaccine composed of killed Leishmania amazonensis promastigotes using several different protocols in a randomized, double-blind and controlled trial design in order to select one of them for further efficacy trials. One hundred and fourteen leishmanin skin test (LST)-negative healthy volunteers were allocated into eight groups that received either two or three deep intramuscular injections of vaccine at doses of 180, 360 and 540 microg or similar injections of placebo. Cell-mediated immune responses were evaluated before and after vaccination by means of LST as well as proliferative responses and cytokine production in Leishmania antigen-stimulated peripheral blood mononuclear cell cultures. The majority of the subjects who actually received vaccine converted to positive LST (89.5%). On the other hand, none of the subjects who received placebo converted to positive LST. Proliferative responses and production of interferon-gamma and interleukin-2 were significantly higher after vaccination than before vaccination in all groups, including those that received placebo. The dose of 360 microg provided the highest LST conversion rate (100%), as well as the greatest increase in interferon-gamma and interleukin-2 production after vaccination.

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.

This study was designed to evaluate the immunogenicity of autoclaved and nonautoclaved preparations of a vaccine composed of whole antigens from killed promastigotes of Leishmania amazonensis. Leishmanin skin-test (LST)-negative volunteers were immunized with either autoclaved or nonautoclaved vaccine preparations (32 and 36 subjects, respectively) that had been maintained at 4 degrees C for one year before the onset of this trial. Immunological tests were performed two days before and 40 days after vaccination. The LST conversion rates induced by the autoclaved and nonautoclaved vaccines were significantly different: 59% and 83%, respectively. Leishmania antigen-stimulated proliferative responses of peripheral blood mononuclear cells (PBMC) were significantly higher after vaccination than before vaccination in both groups. The CD8+ subset was predominant over the CD4+ subset among the leishmania-reactive cells after vaccination in both groups. The production of IFN-gamma by the leishmania antigen-stimulated PBMC was significantly higher after vaccination than before vaccination in the group receiving the nonautoclaved vaccine but not in the autoclaved vaccine group. IL-2 was found both before and after vaccination with no differences between its levels in these time points in either group. IL-4 was not detected for either group during the study period.

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 (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.

Forty-three Brazilians were immunized against American tegumentary leishmaniasis using a vaccine made of whole antigens from killed promastigotes of five American dermotropic Leishmania strains. None of the immunized subjects had a positive reaction in the Montenegro skin test (leishmanin) before vaccination, and 74% developed positive reactions in the skin test after vaccination. The proliferative responses of peripheral blood mononuclear cells (PBMC) induced by antigens from dermotropic Leishmania species were significantly higher after vaccination than before vaccination. However, with antigens from L. chagasi (a causative agent of American visceral leishmaniasis), there was no significant difference between the proliferative responses obtained before and after vaccination. Interferon-gamma was detected in the supernatants of L. braziliensis antigen-stimulated PBMC cultures after vaccination (but not before vaccination). One year after vaccination, PBMC were obtained from eight of the immunized individuals and stimulated with L. braziliensis antigens in proliferative response assays. In all cases, the majority of the responding cells were CD8+ T cells, in contrast to the results of a group of patients with active lesions of tegumentary leishmaniasis, whose L. braziliensis-reactive cells were mainly of the CD4+ T cell phenotype.

Peripheral blood mononuclear cells (PBMC) from 13 American tegumentary leishmaniasis (ATL) patients and two healthy controls were tested in in vitro proliferative response assays with crude lipophosphoglycan (LPG), purified LPG, LPG-associated proteins (LPGAP) and synthetic LPGAP from L. donovani. Cells from another group of six ATL patients were similarly tested with LPGAP obtained from L. major. L. major LPGAP was more stimulatory than L. donovani LPGAP. Crude LPG from L. donovani was significantly more stimulatory than all the other L. donovani antigens tested, including L. donovani LPGAP. The present results indicate that the association with the glycolipid (LPG) may enhance the antigenicity of LPGAP for human T lymphocytes.

Ultrasoft X-rays are useful for mechanistic studies of ionizing radiation damage in living cells due to the localized nature of their energy depositions. To date radiobiology experiments in this energy region have relied on characteristic X-rays (mainly Alk and Ck) from X-ray tubes. However, limitations in the photon intensity and the available energies from X-ray tube sources prevent a definitive characterization of the relationship between photon energy and biological damage. Synchrotron radiation has the potential to avoid these limitations, since it produces X-rays with high intensity over a continuous spectrum. We have established a synchrotron-based system for radiation biology studies using the ES-0 exposure station of the Center for X-ray Lithography at the University of Wisconsin Synchrotron Radiation Center storage ring, Aladdin. A characterization of the system including spectral and intensity properties of the photon beam is presented. The first mammalian cell survival curve for synchrotron-produced ultrasoft X-rays was generated and is presented. Cell survival curves of C3H/10T 1/2 cells using synchrotron radiation of 1.48 keV agree with previous data using Alk X-rays (1.49 keV). An RBE of 1.47 +/- 0.30 at the 10% survival level was measured with reference to 250 kVp X-rays.