RESUMO
Deficiency in memory formation and increased immunosenescence are pivotal features of Trypanosoma cruzi infection proposed to play a role in parasite persistence and disease development. The vaccination protocol that consists in a prime with plasmid DNA followed by the boost with a deficient recombinant human adenovirus type 5, both carrying the ASP2 gene of T. cruzi, is a powerful strategy to elicit effector memory CD8+ T-cells against this parasite. In virus infections, the inhibition of mTOR, a kinase involved in several biological processes, improves the response of memory CD8+ T-cells. Therefore, our aim was to assess the role of rapamycin, the pharmacological inhibitor of mTOR, in CD8+ T response against T. cruzi induced by heterologous prime-boost vaccine. For this purpose, C57BL/6 or A/Sn mice were immunized and daily treated with rapamycin for 34 days. CD8+ T-cells response was evaluated by immunophenotyping, intracellular staining, ELISpot assay and in vivo cytotoxicity. In comparison with vehicle-injection, rapamycin administration during immunization enhanced the frequency of ASP2-specific CD8+ T-cells and the percentage of the polyfunctional population, which degranulated (CD107a+) and secreted both interferon gamma (IFNγ) and tumor necrosis factor (TNF). The beneficial effects were long-lasting and could be detected 95 days after priming. Moreover, the effects were detected in mice immunized with ten-fold lower doses of plasmid/adenovirus. Additionally, the highly susceptible to T. cruzi infection A/Sn mice, when immunized with low vaccine doses, treated with rapamycin, and challenged with trypomastigote forms of the Y strain showed a survival rate of 100%, compared with 42% in vehicle-injected group. Trying to shed light on the biological mechanisms involved in these beneficial effects on CD8+ T-cells by mTOR inhibition after immunization, we showed that in vivo proliferation was higher after rapamycin treatment compared with vehicle-injected group. Taken together, our data provide a new approach to vaccine development against intracellular parasites, placing the mTOR inhibitor rapamycin as an adjuvant to improve effective CD8+ T-cell response.
Assuntos
Vacinas Protozoárias , Trypanosoma cruzi , Animais , Linfócitos T CD8-Positivos , Camundongos , Camundongos Endogâmicos C57BL , Sirolimo/farmacologia , VacinaçãoRESUMO
Vaccine development against Plasmodium vivax malaria lags behind that for Plasmodium falciparum. To narrow this gap, we administered recombinant antigens based on P. vivax circumsporozoite protein (CSP) to mice. We expressed in Pichia pastoris two chimeric proteins by merging the three central repeat regions of different CSP alleles (VK210, VK247, and P. vivax-like). The first construct (yPvCSP-AllFL) contained the fused repeat regions flanked by N- and C-terminal regions. The second construct (yPvCSP-AllCT) contained the fused repeat regions and the C-terminal domain, plus RI region. Mice were vaccinated with three doses of yPvCSP in adjuvants Poly (I:C) or Montanide ISA720. We also used replication-defective adenovirus vectors expressing CSP of human serotype 5 (AdHu5) and chimpanzee serotype 68 (AdC68) for priming mice which were subsequently boosted twice with yPvCSP proteins in Poly (I:C) adjuvant. Regardless of the regime used, immunized mice generated high IgG titres specific to all CSP alleles. After challenge with P. berghei ANKA transgenic parasites expressing Pb/PvVK210 or Pb/PvVK247 sporozoites, significant time delays for parasitemia were observed in all vaccinated mice. These vaccine formulations should be clinically tried for their potential as protective universal vaccine against P. vivax malaria.
Assuntos
Vacinas Antimaláricas/imunologia , Malária Vivax/imunologia , Malária Vivax/prevenção & controle , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/imunologia , Adenoviridae/genética , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Afinidade de Anticorpos/imunologia , Modelos Animais de Doenças , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/química , Imunização , Imunogenicidade da Vacina , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Vacinas Antimaláricas/genética , Malária Vivax/mortalidade , Camundongos , Plasmodium vivax/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genéticaRESUMO
Secretory granules released by cytotoxic T lymphocytes (CTLs) are powerful weapons against intracellular microbes and tumor cells. Despite significant progress, there is still limited information on the molecular mechanisms implicated in target-driven degranulation, effector cell survival and composition and structure of the lytic granules. Here, using a proteomic approach we identified a panel of putative cytotoxic granule proteins, including some already known granule constituents and novel proteins that contribute to regulate the CTL lytic machinery. Particularly, we identified galectin-1 (Gal1), an endogenous immune regulatory lectin, as an integral component of the secretory granule machinery and unveil the unexpected function of this lectin in regulating CTL killing activity. Mechanistic studies revealed the ability of Gal1 to control the non-secretory lytic pathway by influencing Fas-Fas ligand interactions. This study offers new insights on the composition of the cytotoxic granule machinery, highlighting the dynamic cross talk between secretory and non-secretory pathways in controlling CTL lytic function.
Assuntos
Degranulação Celular/imunologia , Citotoxicidade Imunológica , Proteína Ligante Fas/genética , Galectina 1/genética , Linfócitos T Citotóxicos/imunologia , Receptor fas/genética , Animais , Proliferação de Células , Proteína Ligante Fas/imunologia , Galectina 1/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteômica , Vesículas Secretórias/química , Vesículas Secretórias/imunologia , Vesículas Secretórias/metabolismo , Transdução de Sinais , Linfócitos T Citotóxicos/citologia , Receptor fas/imunologiaRESUMO
Plasmodium vivax is the most common species that cause malaria outside of the African continent. The development of an efficacious vaccine would contribute greatly to control malaria. Recently, using bacterial and adenoviral recombinant proteins based on the P. vivax circumsporozoite protein (CSP), we demonstrated the possibility of eliciting strong antibody-mediated immune responses to each of the three allelic forms of P. vivax CSP (PvCSP). In the present study, recombinant proteins representing the PvCSP alleles (VK210, VK247, and P. vivax-like), as well as a hybrid polypeptide, named PvCSP-All epitopes, were generated. This hybrid containing the conserved C-terminal of the PvCSP and the three variant repeat domains in tandem were successfully produced in the yeast Pichia pastoris. After purification and biochemical characterization, they were used for the experimental immunization of C57BL/6 mice in a vaccine formulation containing the adjuvant Poly(I:C). Immunization with a recombinant protein expressing all three different allelic forms in fusion elicited high IgG antibody titers reacting with all three different allelic variants of PvCSP. The antibodies targeted both the C-terminal and repeat domains of PvCSP and recognized the native protein on the surface of P. vivax sporozoites. More importantly, mice that received the vaccine formulation were protected after challenge with chimeric Plasmodium berghei sporozoites expressing CSP repeats of P. vivax sporozoites (Pb/PvVK210). Our results suggest that it is possible to elicit protective immunity against one of the most common PvCSP alleles using soluble recombinant proteins expressed by P. pastoris. These recombinant proteins are promising candidates for clinical trials aiming to develop a multiallele vaccine against P. vivax malaria.
RESUMO
Plasmodium vivax is the most widely distributed malaria species and the most prevalent species of malaria in America and Asia. Vaccine development against P. vivax is considered a priority in the global program for the eradication of malaria. Earlier studies have characterized the Apical Membrane Antigen 1 (AMA-1) ectodomain and the C-terminal region (19kDa) of the Merozoite Surface Protein 1 (MSP-1) of P. vivax as immunodominant antigens. Based on this characterization, we designed a chimeric recombinant protein containing both merozoite immunodominant domains (PvAMA166-MSP119). The recombinant PvAMA166-MSP119 was successfully expressed in Pichia pastoris and used to immunize two different mouse strains (BALB/c and C57BL/6) in the presence of the Poly (I:C) as an adjuvant. Immunization with the chimeric protein induced high antibody titers against both proteins in both strains of mice as detected by ELISA. Antisera also recognized the native proteins expressed on the merozoites of mature P. vivax schizonts. Moreover, this antigen was able to induce IFN-gamma-secreting cells in C57BL/6 mice. These findings indicate that this novel yeast recombinant protein containing PvAMA166 and PvMSP119 is advantageous, because of improved antibody titers and cellular immune response. Therefore, this formulation should be further developed for pre-clinical trials in non-human primates as a potential candidate for a P. vivax vaccine.
Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Proteínas de Membrana/imunologia , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologia , Proteínas Recombinantes de Fusão/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/genética , Proteínas de Membrana/genética , Proteína 1 de Superfície de Merozoito/genética , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pichia/genética , Poli I-C/administração & dosagem , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
In vivo antigen targeting to dendritic cells (DCs) has been used as a way to improve immune responses. Targeting is accomplished with the use of monoclonal antibodies (mAbs) to receptors present on the DC surface fused with the antigen of interest. An anti-DEC205 mAb has been successfully used to target antigens to the DEC205+CD8α+ DC subset. The administration of low doses of the hybrid mAb together with DC maturation stimuli is able to activate specific T cells and induce production of high antibody titres for a number of different antigens. However, it is still not known if this approach would work with any fused protein. Here we genetically fused the αDEC205 mAb with two fragments (42-kDa and 19-kDa) derived from the ~200 kDa Plasmodium vivax merozoite surface protein 1 (MSP1), known as MSP142 and MSP119, respectively. The administration of two doses of αDEC-MSP142, but not of αDEC-MSP119 mAb, together with an adjuvant to two mouse strains induced high anti-MSP119 antibody titres that were dependent on CD4+ T cells elicited by peptides present in the MSP133 sequence, indicating that the presence of T cell epitopes in antigens targeted to DEC205+ DCs increases antibody responses.
Assuntos
Formação de Anticorpos/fisiologia , Células Dendríticas/imunologia , Epitopos de Linfócito T/imunologia , Lectinas Tipo C/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos CD4/deficiência , Antígenos CD4/genética , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/metabolismo , Feminino , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Interferon gama/metabolismo , Interleucina-2/metabolismo , Proteína 1 de Superfície de Merozoito/química , Proteína 1 de Superfície de Merozoito/genética , Proteína 1 de Superfície de Merozoito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Baço/citologia , Baço/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Babesia bovis is a tick-transmitted protozoan hemoparasite and the causative agent of bovine babesiosis, a potential risk to more than 500 million cattle worldwide. The vaccines currently available are based on attenuated parasites, which are difficult to produce, and are only recommended for use in bovines under one year of age. When used in older animals, these vaccines may cause life-threatening clinical symptoms and eventually death. The development of a multi-subunit recombinant vaccine against B. bovis would be attractive from an economic standpoint and, most importantly, could be recommended for animals of any age. In the present study, recombinant ectodomains of MSA-2a1, MSA-2b and MSA-2c antigens were expressed in Pichia pastoris yeast as secreted soluble peptides. RESULTS: The antigens were purified to homogeneity, and biochemically and immunologically characterized. A vaccine formulation was obtained by emulsifying a mixture of the three peptides with the adjuvant Montanide ISA 720, which elicited high IgG antibody titers against each of the above antigens. IgG antibodies generated against each MSA-antigen recognized merozoites and significantly inhibited the invasion of bovine erythrocytes. Cellular immune responses were also detected, which were characterized by splenic and lymph node CD4+ T cells producing IFN-γ and TNF-α upon stimulation with the antigens MSA-2a1 or MSA-2c. CONCLUSIONS: These data strongly suggest the high protective potential of the presented formulation, and we propose that it could be tested in vaccination trials of bovines challenged with B. bovis.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Antígenos de Superfície/imunologia , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Proteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Antígenos de Protozoários/genética , Antígenos de Superfície/genética , Bovinos , Manitol/administração & dosagem , Manitol/análogos & derivados , Proteínas de Membrana/genética , Ácidos Oleicos/administração & dosagem , Proteínas de Protozoários/genética , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/genética , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Although CD4+ Foxp3+ T cells are largely described in the regulation of CD4+ T cell responses, their role in the suppression of CD8+ T cell priming is much less clear. Because the induction of CD8+ T cells during experimental infection with Trypanosoma cruzi is remarkably delayed and suboptimal, we raised the hypothesis that this protozoan parasite actively induces the regulation of CD8+ T cell priming. Using an in vivo assay that eliminated multiple variables associated with antigen processing and dendritic cell activation, we found that injection of bone marrow-derived dendritic cells exposed to T. cruzi induced regulatory CD4+ Foxp3+ T cells that suppressed the priming of transgenic CD8+ T cells by peptide-loaded BMDC. This newly described suppressive effect on CD8+ T cell priming was independent of IL-10, but partially dependent on CTLA-4 and TGF-ß. Accordingly, depletion of Foxp3+ cells in mice infected with T. cruzi enhanced the response of epitope-specific CD8+ T cells. Altogether, our data uncover a mechanism by which T. cruzi suppresses CD8+ T cell responses, an event related to the establishment of chronic infections.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/imunologia , Células Dendríticas/imunologia , Evasão da Resposta Imune/imunologia , Linfócitos T Reguladores/imunologia , Animais , Feminino , Citometria de Fluxo , Humanos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Trypanosoma cruzi/imunologiaRESUMO
The ß1i, ß2i and ß5i immunoproteasome subunits have an important role in defining the repertoire of MHC class I-restricted epitopes. However, the impact of combined deficiency of the three immunoproteasome subunits in the development of protective immunity to intracellular pathogens has not been investigated. Here, we demonstrate that immunoproteasomes play a key role in host resistance and genetic vaccination-induced protection against the human pathogen Trypanosoma cruzi (the causative agent of Chagas disease), immunity to which is dependent on CD8+ T cells and IFN-γ (the classical immunoproteasome inducer). We observed that infection with T. cruzi triggers the transcription of immunoproteasome genes, both in mice and humans. Importantly, genetically vaccinated or T. cruzi-infected ß1i, ß2i and ß5i triple knockout (TKO) mice presented significantly lower frequencies and numbers of splenic CD8+ effector T cells (CD8+CD44highCD62Llow) specific for the previously characterized immunodominant (VNHRFTLV) H-2Kb-restricted T. cruzi epitope. Not only the quantity, but also the quality of parasite-specific CD8+ T cell responses was altered in TKO mice. Hence, the frequency of double-positive (IFN-γ+/TNF+) or single-positive (IFN-γ+) cells specific for the H-2Kb-restricted immunodominant as well as subdominant T. cruzi epitopes were higher in WT mice, whereas TNF single-positive cells prevailed among CD8+ T cells from TKO mice. Contrasting with their WT counterparts, TKO animals were also lethally susceptible to T. cruzi challenge, even after an otherwise protective vaccination with DNA and adenoviral vectors. We conclude that the immunoproteasome subunits are key determinants in host resistance to T. cruzi infection by influencing both the magnitude and quality of CD8+ T cell responses.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Vacinas Protozoárias/imunologia , Adolescente , Adulto , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Trypanosoma cruzi , Adulto JovemRESUMO
Malaria remains a world-threatening disease largely because of the lack of a long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane molecule with a chimeric cysteine-rich ectodomain homologous to regions of the Duffy binding-like erythrocyte binding protein and apical membrane antigen 1 (AMA1) antigens. Although MAEBL does not appear to be essential for the survival of blood-stage forms, ectodomains M1 and M2, homologous to AMA1, seem to be involved in parasite attachment to erythrocytes, especially M2. MAEBL is necessary for sporozoite infection of mosquito salivary glands and is expressed in liver stages. Here, the Plasmodium yoelii MAEBL-M2 domain was expressed in a prokaryotic vector. C57BL/6J mice were immunized with doses of P. yoelii recombinant protein rPyM2-MAEBL. High levels of antibodies, with balanced IgG1 and IgG2c subclasses, were achieved. rPyM2-MAEBL antisera were capable of recognizing the native antigen. Anti-MAEBL antibodies recognized different MAEBL fragments expressed in CHO cells, showing stronger IgM and IgG responses to the M2 domain and repeat region, respectively. After a challenge with P. yoelii YM (lethal strain)-infected erythrocytes (IE), up to 90% of the immunized animals survived and a reduction of parasitemia was observed. Moreover, splenocytes harvested from immunized animals proliferated in a dose-dependent manner in the presence of rPyM2-MAEBL. Protection was highly dependent on CD4(+), but not CD8(+), T cells toward Th1. rPyM2-MAEBL antisera were also able to significantly inhibit parasite development, as observed in ex vivo P. yoelii erythrocyte invasion assays. Collectively, these findings support the use of MAEBL as a vaccine candidate and open perspectives to understand the mechanisms involved in protection.