The Use of Specific Serological Biomarkers to Detect CaniLeish Vaccination in Dogs

Canine leishmaniosis (CanL) prevention in the Mediterranean basin is considered essential to stop human zoonotic visceral leishmaniasis. In this context, vaccination of dogs is expected to have a significant impact in disease control. CaniLeish® (Virbac Animal Health) is one of a few CanL vaccines that are at this moment licensed in Europe. This vaccine contains purified excreted-secreted proteins of Leishmania having several antigens/immunogens with potential to influence serological response. Therefore, it is important to know if CaniLeish vaccination increased the diagnostic challenges associated with conventional serology, limiting the value of some antigens. To address this 20 dogs from a cohort of 35 healthy dogs that were vaccinated, maintained indoor for 1 month and then returned to their natural domiciles for 2 years. After this period, they were re-called to evaluate their clinical/parasitological condition and assess the evolution of seroreactivity against different antigens: soluble promastigote Leishmania antigens (SPLA), recombinant protein Leishmania infantum cytosolic peroxiredoxin, recombinant protein K39 (rK39), recombinant protein K28 and recombinant kinesin degenerated derived repeat using ELISA. Two years after vaccination all vaccinated non-infected animals were seropositive for SPLA. For the other antigens the serological profile was indistinguishable from non-infected animals. Moreover, vaccinated animals presented a characteristic relative serological profile, with higher normalized serological response to SPLA than rK39. This fact enabled to distinguish with sensitivity 92.3% and specificity 95.4%, vaccinated non-infected dogs from infected and non-infected dogs. Ultimately, relative serological profile enabled the detection of healthy vaccinated animals enabling more accurate serological surveys.This work was financed by: FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project Institute for Research and Innovation in Health Sciences (POCI-01-0145-FEDER-007274) and project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). EC was supported by a research contract funded via the VII PN I+D+I 2013-2016 programme and FEDER Funds (RICET RD12/0018/0003)S

Identification of a Highly Antigenic Linear B Cell Epitope within Plasmodium vivax Apical Membrane Antigen 1 (AMA-1)

Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290–307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies

Induction of immunogenicity by live attenuated Leishmania donovani centrin deleted parasites in dogs

AbstractZoonotic visceral leishmaniasis, caused by the intracellular protozoan parasite Leishmania infantum, is a neglected tropical disease that is often fatal when untreated. Dogs are considered the main reservoir of L. infantum in zoonotic VL as the presence of infected dogs may increase the risk for human infection. Canine visceral leishmaniasis (CVL) is a major veterinary and public health problem in Southern Europe, Middle East and South America. Control of animal reservoirs relies on elimination of seropositive dogs in endemic areas. However, treatment of infected dogs is not considered a favorable approach as this can lead to emergence of drug resistance since the same drugs are used to treat human infections. Therefore, vaccination against CVL remains the best alternative in control of the animal reservoirs. In this study, we present data on the immunogenicity profile of a live attenuated parasite LdCen−/− in a canine infection model and compared it to that of Leishmune®, a commercially available recombinant vaccine. The immunogenicity of the LdCen−/− parasites was evaluated by antibody secretion, production of intracytoplasmic and secreted cytokines, activation and proliferation of T cells. Vaccination with LdCen−/− resulted in high immunogenicity as revealed by the higher IgGTotal, IgG1, and IgG2 production and higher lymphoproliferative response. Further, LdCen−/− vaccinated dogs showed higher frequencies of activated CD4+ and CD8+ T cells, IFN-γ production by CD8+ T cells, increased secretion of TNF-α and IL-12/IL-23p40 and decreased secretion of IL-4. These results contribute to the understanding of immunogenicity elicited by live attenuated L. donovani parasites and, consequently, to the development of effective vaccines against visceral leishmaniasis

Phenotypic profiling of CD8+ T cells during Plasmodium vivax blood-stage infection

Submitted by Repositório Arca ([email protected]) on 2019-04-24T17:38:50Z No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Approved for entry into archive by Janaína Nascimento ([email protected]) on 2019-08-13T14:21:37Z (GMT) No. of bitstreams: 2 ve_ Hojo-Souza_Natália_etal_INI_2015.pdf: 1172050 bytes, checksum: b1948378bfee8669ad90694b3aa2cb60 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-08-13T14:21:37Z (GMT). No. of bitstreams: 2 ve_ Hojo-Souza_Natália_etal_INI_2015.pdf: 1172050 bytes, checksum: b1948378bfee8669ad90694b3aa2cb60 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Centro de Pesquisa em Medicina Tropical. Porto Velho, RO, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Centro de Pesquisa em Medicina Tropical. Porto Velho, RO, Brasil.Fundação Oswaldo Cruz. Instituto de Pesquisa Clínica Evandro Chagas. Rio de Janeiro. RJ, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Parasitologia. Belo Horizonte, MG, Brasil.Background: For a long time, the role of CD8+ T cells in blood-stage malaria was not considered important because erythrocytes do not express major histocompatibility complex (MHC) class I proteins. While recent evidences suggest that CD8+ T cells may play an important role during the erythrocytic phase of infection by eliminating parasites, CD8+ T cells might also contribute to modulate the host response through production of regulatory cytokines. Thus, the role of CD8+ T cells during blood-stage malaria is unclear. Here, we report the phenotypic profiling of CD8+ T cells subsets from patients with uncomplicated symptomatic P. vivax malaria. Methods: Blood samples were collected from 20 Plasmodium vivax-infected individuals and 12 healthy individuals. Immunophenotyping was conducted by flow cytometry. Plasma levels of IFN-γ, TNF-α and IL-10 were determined by ELISA/CBA. Unpaired t-test or Mann–Whitney test was used depending on the data distribution. Results: P. vivax-infected subjects had lower percentages and absolute numbers of CD8+ CD45RA+ and CD8+ CD45RO+ T cells when compared to uninfected individuals (p ≤ 0.0002). A significantly lower absolute number of circulating CD8+ CD45+ CCR7+ cells (p = 0.002) was observed in P. vivax-infected individuals indicating that infection reduces the number of central memory T cells. Cytokine expression was significantly reduced in the naïve T cells from infected individuals compared with negative controls, as shown by lower numbers of IFN-γ + (p = 0.001), TNF-α+ (p < 0.0001) and IL-10+ (p < 0.0001) CD8+ T cells. Despite the reduction in the number of CD8+ memory T cells producing IFN-γ (p < 0.0001), P. vivax-infected individuals demonstrated a significant increase in memory CD8+ TNF-α+ (p = 0.016) and CD8+ IL-10+ (p = 0.004) cells. Positive correlations were observed between absolute numbers of CD8+ IL-10+ and numbers of CD8+ IFN-γ + (p < 0.001) and CD8+ TNF-α+ T cells (p ≤ 0.0001). Finally, an increase in the plasma levels of TNF-α (p = 0.017) and IL-10 (p = 0.006) and a decrease in the IFN-γ plasma level (p <0.0001) were observed in the P. vivax-infected individuals. Conclusions: P. vivax infection reduces the numbers of different subsets of CD8+ T cells, particularly the memory cells, during blood-stage of infection and enhances the number of CD8+ memory T cells expressing IL-10, which positively correlates with the number of cells expressing TNF-α and IFN-γ

IgG Induced by Vaccination With Ascaris suum Extracts Is Protective Against Infection

Human ascariasis has a global and cosmopolitan distribution, and has been characterized as the most prevalent neglected tropical disease worldwide. The development of a preventive vaccine is highly desirable to complement current measures required for this parasitic infection control and to reduce chronic childhood morbidities. In the present study, we describe the mechanism of protection elicited by a preventive vaccine against ascariasis. Vaccine efficacy was evaluated after immunization with three different Ascaris suum antigen extracts formulated with monophosphoryl lipid A (MPLA) as an adjuvant: crude extract of adult worm (ExAD); crude extract of adult worm cuticle (CUT); and crude extract of infective larvae (L3) (ExL3). Immunogenicity elicited by immunization was assessed by measuring antibody responses, cytokine production, and influx of tissue inflammatory cells. Vaccine efficacy was evaluated by measuring the reductions in the numbers of larvae in the lungs of immunized BALB/c mice that were challenged with A. suum eggs. Moreover, lung physiology and functionality were tested by spirometry to determine clinical efficacy. Finally, the role of host antibody mediated protection was determined by passive transfer of serum from immunized mice. Significant reductions in the total number of migrating larvae were observed in mice immunized with ExL3 61% (p &lt; 0.001), CUT 59% (p &lt; 0.001), and ExAD 51% (p &lt; 0.01) antigens in comparison with non-immunized mice. For the Ascaris antigen-specific IgG antibody levels, a significant and progressive increase was observed with each round of immunization, in association with a marked increase of IgG1 and IgG3 subclasses. Moreover, a significant increase in concentration of IL-5 and IL-10 (pre-challenge) in the blood and IL-10 in the lung tissue (post-challenge) was induced by CUT immunization. Finally, ExL3 and CUT-immunized mice showed a marked improvement in lung pathology and tissue fibrosis as well as reduced pulmonary dysfunction induced by Ascaris challenge, when compared to non-immunized mice. Moreover, the passive transfer of specific IgG antibodies from ExL3, CUT, and ExAD elicited a protective response in naïve mice, with significant reductions in parasite burdens in lungs of 65, 64, and 64%, respectively. Taken together, these studies indicated that IgG antibodies contribute to protective immunity

Induction of CD4+CD25+FOXP3+ Regulatory T Cells during Human Hookworm Infection Modulates Antigen-Mediated Lymphocyte Proliferation

Hookworm infection is considered one of the most important poverty-promoting neglected tropical diseases, infecting 576 to 740 million people worldwide, especially in the tropics and subtropics. These blood-feeding nematodes have a remarkable ability to downmodulate the host immune response, protecting themselves from elimination and minimizing severe host pathology. While several mechanisms may be involved in the immunomodulation by parasitic infection, experimental evidences have pointed toward the possible involvement of regulatory T cells (Tregs) in downregulating effector T-cell responses upon chronic infection. However, the role of Tregs cells in human hookworm infection is still poorly understood and has not been addressed yet. In the current study we observed an augmentation of circulating CD4+CD25+FOXP3+ regulatory T cells in hookworm-infected individuals compared with healthy non-infected donors. We have also demonstrated that infected individuals present higher levels of circulating Treg cells expressing CTLA-4, GITR, IL-10, TGF-β and IL-17. Moreover, we showed that hookworm crude antigen stimulation reduces the number of CD4+CD25+FOXP3+ T regulatory cells co-expressing IL-17 in infected individuals. Finally, PBMCs from infected individuals pulsed with excreted/secreted products or hookworm crude antigens presented an impaired cellular proliferation, which was partially augmented by the depletion of Treg cells. Our results suggest that Treg cells may play an important role in hookworm-induced immunosuppression, contributing to the longevity of hookworm survival in infected people

Resposta imune celular e humoral de pacientes infectadospor Plasmodium vivax frente ao antígeno 1 de membrana apical(AMA-1)

Exportado OPUSMade available in DSpace on 2019-08-12T17:41:55Z (GMT). No. of bitstreams: 1 tese_final_corrigida.pdf: 8664269 bytes, checksum: c59c6f76a89e8d25866292dbcd7f83ee (MD5) Previous issue date: 18A malária é uma das doenças parasitárias de maior importância global e das principais causas de morbidade e mortalidade nas áreas tropicais e subtropicais do mundo. Apesar dos esforços para o controle da infecção em diferentes áreas endêmicas, a malária continua em expansão, e as medidas tradicionais de controle da transmissão são pouco eficazes. Portanto, como ferramenta adicional de controle, uma vacina efetiva contra essa doença parasitária se faz necessária. O entendimento dos mecanismos envolvidos na determinação da imunidade naturalmente adquirida é um importante pré-requisito para o estabelecimento de vacinas realmente eficazes. Entretanto, os mecanismos envolvidos na modulação da função da resposta celular durante a infecção ainda não estão totalmente esclarecidos. Na tentativa de compreender os possíveis mecanismos imunes envolvidos na infecção por P. vivax, descrevemos, na primeira parte do trabalho, a maturação de células dendríticas derivadas de monócitos de pacientes infectados por P. vivax. Foi observado que os indivíduos infectados apresentaram uma modulação da expressão da maioria dos marcadores de superfíce celular avaliados, verificada pela redução significativa da expressão de CD11c, CD1a, HLA-ABC, HLA-DR, CD80, CD86, CD40, CD64 e CD14 (P # 0,05). Além disso, foi observado que as células dendríticas desses pacientes, quando diferenciadas na presença do antígeno AMA-1, desencadearam um aumento da expressão das moléculas apresentadoras de antígeno CD1a e HLA-DR. Na segunda parte do trabalho, avaliamos a participação de células T reguladoras (Treg) no processo de infecção por P. vivax. Demonstramos que pacientes infectados apresentaram um aumento significativo do número de células Treg circulantes produtoras de citocinas anti-inflamatórias (IL-10 e TGF-$) e pró-inflamatórias (IFN-! e IL-17), correlacionando-se positivamente com a carga parasitária. A expressão das moléculas GITR e CTLA-4 em células Treg foi significativamente maior em indivíduos infectados, apresentando, também, uma associação positiva com a parasitemia. O efeito supressivo de células Treg na resposta proliferativa de linfócitos T de indivíduos infectados, estimuladas pelo antígeno Pv-AMA-1, um importante candidato vacinal, também foi experimentalmente evidenciado. Na terceira etapa do trabalho, demonstramos que a frequência de células Th17 está aumentada durante a infecção por P. vivax, e que o número absoluto dessas células está diretamente correlacionado com o número de células T CD4+ produtoras de IFN-!, IL-10 e TGF-$. Finalmente, avaliamos in silico a predição de possíveis epitopos de células B na proteína Pv-AMA- 1. Nossos resultados demonstraram que o DII da proteína se apresenta como a região de maior potencial antigênico, baseado nos parâmetros entropia e sequência de aminoácidos. O peptídeo sintético gerado a partir dessas análises apresentou reatividade sorológica semelhante ao observado para o ectodomínio e para o DII da proteína. Nossos resultados sugerem que a infecção por malária vivax apresenta diferentes mecanismos de modulação da resposta imune do hospedeiro que poderiam influenciar significativamente o curso da infecção malárica

Plasmodium vivax: efeito de antígenos recombinantes candidatos à vacina (AMA-1 e MSP1-19) na resposta imune inata.

Exportado OPUSMade available in DSpace on 2019-08-14T07:13:10Z (GMT). No. of bitstreams: 1 disserta__o_lilian_lacerda_bueno.pdf: 2351417 bytes, checksum: a2a844114edc94bac7f5dc7ffde66099 (MD5) Previous issue date: 15A malária é uma das principais causas de morbidade e mortalidade nas áreas tropicais e subtropicais do mundo. Uma vez que a disseminação da malária tende a aumentar, uma vacina efetiva contra essa doença parasitária é necessária. Dois principais candidatos à vacina contra a fase eritrocítica do parasito são o antígeno 1 de membrana apical (AMA-1) e o fragmento de 19 kDa da proteína de superfície de merozoíto (MSP-119). A maioria dos estudos clínicos utilizando esses antígenos apresenta maior enfoque na identificação de proteínas que desencadeiam uma resposta protetora do que necessariamente no entendimento de seus mecanismos efetores. Além disso, devido a natureza do processo de imunização, os estudos sobre a resposta imune de candidatos à vacina são voltados principalmente para a imunidade adaptativa. Neste estudo investigamos o efeito dos candidatos a vacina Pv-AMA-1 e Pv-MSP-119 na resposta imune de doadores nunca expostos à infecção por malária. Células dendríticas derivadas de monócitos, maturadas na presença de Pv-AMA-1, apresentaram alteração de fenótipo celular, como evidenciado pelo aumento da expressão de CD80 e CD64 e redução da expressão de CD11c, CD86 e CD40. Além disso, Pv-AMA-1 induziu um aumento da produção de MIP-1/CCL3 e queda nos níveis de TARC/CCL17 tanto em células dendríticas quanto em células mononucleares do sangue periférico (PBMCs). Finalmente, uma resposta pró-inflamatória significativa foi observada em culturas de PBMCs estimuladas por Pv-AMA-1. Esses resultados sugerem que o antígeno recombinante Pv-AMA-1 pode atuar diretamente na resposta imune inata contribuindo para a eliminação do parasito

Direct effect of Plasmodium vivax recombinant vaccine candidates AMA-1 and MSP-1(19) on the innate immune response

The recombinant apical membrane antigen 1 (AMA-1) and 19-kDa fragment of merozoite surface protein (MSP-1(19)) are the lead candidates for inclusion in a vaccine against blood stages of malaria due to encouraging protective studies in humans and animals. Despite the importance of an efficacious malaria vaccine, vaccine-related research has focused on identifying antigens that result in protective immunity rather than determining the nature of anti-malarial immune effector mechanisms. Moreover, emphasis has been placed on adaptive rather than innate immune responses. In this study, we investigated the effect of Plasmodium vivax vaccine candidates Pv-AMA-1 and Pv-MSP-1(19) on the immune response of malaria-naive donors. Maturation of dendritic cells is altered by Pv-AMA-1 but not Pv-MSP-1(19), as observed by differential expression of cell surface markers. In addition, Pv-AMA-1 induced an increased production of MIP-1 alpha/CCL3 and decreased production of TARC/CCL17 levels in both dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs). Finally, a significant pro-inflammatory response was elicited by Pv-AMA-1-stimulated PBMCs. These results suggest that the recombinant vaccine candidate Pv-AMA-1 may play a direct role on innate immune response and might be involved in parasite destruction. (C) 2007 Elsevier Ltd. All rights reserved