RESUMO
Integrins mediate the lymphocyte migration into an infected tissue, and these cells are essential for controlling the multiplication of many intracellular parasites such as Trypanosoma cruzi, the causative agent of Chagas disease. Here, we explore LFA-1 and VLA-4 roles in the migration of specific CD8+ T cells generated by heterologous prime-boost immunization during experimental infection with T. cruzi. To this end, vaccinated mice were treated with monoclonal anti-LFA-1 and/or anti-VLA-4 to block these molecules. After anti-LFA-1, but not anti-VLA-4 treatment, all vaccinated mice displayed increased blood and tissue parasitemia, and quickly succumbed to infection. In addition, there was an accumulation of specific CD8+ T cells in the spleen and lymph nodes and a decrease in the number of those cells, especially in the heart, suggesting that LFA-1 is important for the output of specific CD8+ T cells from secondary lymphoid organs into infected organs such as the heart. The treatment did not alter CD8+ T cell effector functions such as the production of pro-inflammatory cytokines and granzyme B, and maintained the proliferative capacity after treatment. However, the specific CD8+ T cell direct cytotoxicity was impaired after LFA-1 blockade. Also, these cells expressed higher levels of Fas/CD95 on the surface, suggesting that they are susceptible to programmed cell death by the extrinsic pathway. We conclude that LFA-1 plays an important role in the migration of specific CD8+ T cells and in the direct cytotoxicity of these cells.
RESUMO
BACKGROUND: Polyparasitism is a common condition in humans but its impact on the host immune system and clinical diseases is still poorly understood. There are few studies of the prevalence and the effect of malaria-intestinal parasite co-infections in the immune response to malaria vaccine candidates. The present study determines whether the presence of malaria and intestinal parasites co-infection is associated with impaired IgG responses to Plasmodium vivax AMA-1 and MSP-119 in a rural population of the Brazilian Amazon. METHODS: A cross-sectional survey was performed in a rural area of Rondonia State and 279 individuals were included in the present study. At recruitment, whole blood was collected and Plasmodium and intestinal parasites were detected by microscopy and molecular tests. Blood cell count and haemoglobin were also tested and antibody response specific to P. vivax AMA-1 and MSP-119 was measured in plasma by ELISA. The participants were grouped according to their infection status: singly infected with Plasmodium (M); co-infected with Plasmodium and intestinal parasites (CI); singly infected with intestinal parasites (IP) and negative (N) for both malaria and intestinal parasites. RESULTS: The prevalence of intestinal parasites was significantly higher in individuals with malaria and protozoan infections were more prevalent. IgG antibodies to PvAMA-1 and/or PvMSP-119 were detected in 74 % of the population. The prevalence of specific IgG was similar for both proteins in all four groups and among the groups the lowest prevalence was in IP group. The cytophilic sub-classes IgG1 and IgG3 were predominant in all groups for PvAMA-1 and IgG1, IgG3 and IgG4 for PvMSP-119. In the case of non-cytophilic antibodies to PvAMA-1, IgG2 was significantly higher in IP and N group when compared to M and CI while IgG4 was higher in IP group. CONCLUSIONS: The presence of intestinal parasites, mainly protozoans, in malaria co-infected individuals does not seem to alter the antibody immune responses to P. vivax AMA-1 and MSP-119. However, IgG response to both AMA1 and MSP1 were lower in individuals with intestinal parasites.
Assuntos
Antígenos de Protozoários/genética , Imunoglobulina G/imunologia , Enteropatias Parasitárias/epidemiologia , Malária/epidemiologia , Proteínas de Membrana/genética , Proteína 1 de Superfície de Merozoito/genética , Proteínas de Protozoários/genética , Adulto , Antígenos de Protozoários/metabolismo , Brasil/epidemiologia , Coinfecção/epidemiologia , Coinfecção/imunologia , Coinfecção/parasitologia , Humanos , Enteropatias Parasitárias/imunologia , Enteropatias Parasitárias/parasitologia , Malária/imunologia , Malária/parasitologia , Proteínas de Membrana/metabolismo , Proteína 1 de Superfície de Merozoito/metabolismo , Plasmodium vivax/fisiologia , Prevalência , Proteínas de Protozoários/metabolismo , Adulto JovemRESUMO
Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is a prototypical neglected tropical disease. Specific immunity promotes acute phase survival. Nevertheless, one-third of CD patients develop chronic chagasic cardiomyopathy (CCC) associated with parasite persistence and immunological unbalance. Currently, the therapeutic management of patients only mitigates CCC symptoms. Therefore, a vaccine arises as an alternative to stimulate protective immunity and thereby prevent, delay progression and even reverse CCC. We examined this hypothesis by vaccinating mice with replication-defective human Type 5 recombinant adenoviruses (rAd) carrying sequences of amastigote surface protein-2 (rAdASP2) and trans-sialidase (rAdTS) T. cruzi antigens. For prophylactic vaccination, naïve C57BL/6 mice were immunized with rAdASP2+rAdTS (rAdVax) using a homologous prime/boost protocol before challenge with the Colombian strain. For therapeutic vaccination, rAdVax administration was initiated at 120 days post-infection (dpi), when mice were afflicted by CCC. Mice were analyzed for electrical abnormalities, immune response and cardiac parasitism and tissue damage. Prophylactic immunization with rAdVax induced antibodies and H-2Kb-restricted cytotoxic and interferon (IFN)γ-producing CD8+ T-cells, reduced acute heart parasitism and electrical abnormalities in the chronic phase. Therapeutic vaccination increased survival and reduced electrical abnormalities after the prime (analysis at 160 dpi) and the boost (analysis at 180 and 230 dpi). Post-therapy mice exhibited less heart injury and electrical abnormalities compared with pre-therapy mice. rAdVax therapeutic vaccination preserved specific IFNγ-mediated immunity but reduced the response to polyclonal stimuli (anti-CD3 plus anti-CD28), CD107a+ CD8+ T-cell frequency and plasma nitric oxide (NO) levels. Moreover, therapeutic rAdVax reshaped immunity in the heart tissue as reduced the number of perforin+ cells, preserved the number of IFNγ+ cells, increased the expression of IFNγ mRNA but reduced inducible NO synthase mRNA. Vaccine-based immunostimulation with rAd might offer a rational alternative for re-programming the immune response to preserve and, moreover, recover tissue injury in Chagas' heart disease.
Assuntos
Cardiomiopatia Chagásica/prevenção & controle , Doença de Chagas/imunologia , Doença de Chagas/terapia , Vacinas Protozoárias/uso terapêutico , Trypanosoma cruzi/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Doença Crônica , Feminino , Fenômenos do Sistema Imunitário , Camundongos , Camundongos Endogâmicos C57BL , Vacinação , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
The development of cancer immunotherapy has long been a challenge. Here, we report that prophylactic vaccination with a highly attenuated Trypanosoma cruzi strain expressing NY-ESO-1 (CL-14-NY-ESO-1) induces both effector memory and effector CD8(+) T lymphocytes that efficiently prevent tumor development. However, the therapeutic effect of such a vaccine is limited. We also demonstrate that blockade of Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) during vaccination enhances the frequency of NY-ESO-1-specific effector CD8(+) T cells producing IFN-γ and promotes lymphocyte migration to the tumor infiltrate. As a result, therapy with CL-14-NY-ESO-1 together with anti-CTLA-4 is highly effective in controlling the development of an established melanoma.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/imunologia , Vacinas Anticâncer/imunologia , Imunoterapia/métodos , Melanoma Experimental/terapia , Proteínas de Membrana/imunologia , Animais , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/genética , Linfócitos T CD8-Positivos/parasitologia , Antígeno CTLA-4/antagonistas & inibidores , Feminino , Humanos , Melanoma Experimental/imunologia , Melanoma Experimental/parasitologia , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologiaRESUMO
The circumsporozoite protein (CSP), the most abundant surface antigen of sporozoites, has been extensively studied in different expression platforms as a vaccine candidate. Clinical trials have shown the necessity of broad and highly avid humoral immune responses together with high numbers of CSP-specific TCD4+ and TCD8+ cells, especially those producing IFN-γ, to induce protection. To this aim, we designed two distinct recombinant immunogens based on previously-described antigenic fragments of Plasmodium vivax CSP (PvCSP) to be used as vaccine candidates. The first one is a virus-like particle (VLP) comprising the repeat region of PvCSP (B and TCD4+ epitopes) within the loop of the hepatitis B virus core antigen (HBcAgPvCSP). The second one is a PvCSP multi-epitope polypeptide, rPvCSP-ME, designed based on antigenic regions of PvCSP recognized by lymphocytes of individuals from endemic areas. Mice immunized with 2 doses of these proteins, administered individually or combined and formulated in Montanide ISA 720 adjuvant, were able to induce strong effector and memory humoral responses with IgG titers ranging from 10(4) to 10(5) and avidity indexes toward full-length PvCSP reaching up to 66%, even 3 months after the last immunization. Furthermore, balanced Th1/Th2 responses were generated, as determined by titers of IgG subclasses and further confirmed by ELISPOT analyses, which detected that these vaccination protocols were able to elicit long-term IFN-γ and IL-2-secreting memory T-cells. Overall, these results show that our vaccine candidates generate, in mice, immune responses against regions within PvCSP that have been associated with protection against malaria in humans.
Assuntos
Imunidade Celular , Imunidade Humoral , Vacinas Antimaláricas/imunologia , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Afinidade de Anticorpos , Epitopos/imunologia , Feminino , Imunoglobulina G/sangue , Memória Imunológica , Interferon gama/imunologia , Interleucina-2/imunologia , Malária/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium vivax , Proteínas Recombinantes/imunologia , Linfócitos T/imunologiaRESUMO
BACKGROUND: Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil. METHODS: The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-119) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay. RESULTS: The prevalence of activated CD4+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient's cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses. CONCLUSIONS: The results presented here shows that PvMSP-119 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.
Assuntos
Anticorpos Antiprotozoários/sangue , Doenças Endêmicas , Leucócitos Mononucleares/imunologia , Malária Vivax/epidemiologia , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium vivax/imunologia , Adolescente , Adulto , Idoso , Brasil/epidemiologia , Proliferação de Células , Criança , Feminino , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Interferon gama/metabolismo , Interleucina-10/metabolismo , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
In the present study we evaluated the protection raised by immunization with recombinant influenza viruses carrying sequences coding for polypeptides corresponding to medial and carboxi-terminal moieties of Trypanosoma cruzi ´s amastigote surface protein 2 (ASP2). Those viruses were used in sequential immunization with recombinant adenovirus (heterologous prime-boost immunization protocol) encoding the complete sequence of ASP2 (Ad-ASP2) in two mouse strains (C57BL/6 and C3H/He). The CD8 effector response elicited by this protocol was comparable to that observed in mice immunized twice with Ad-ASP2 and more robust than that observed in mice that were immunized once with Ad-ASP2. Whereas a single immunization with Ad-ASP2 sufficed to completely protect C57BL/6 mice, a higher survival rate was observed in C3H/He mice that were primed with recombinant influenza virus and boosted with Ad-ASP2 after being challenged with T. cruzi. Analyzing the phenotype of CD8+ T cells obtained from spleen of vaccinated C3H/He mice we observed that heterologous prime-boost immunization protocol elicited more CD8+ T cells specific for the immunodominant epitope as well as a higher number of CD8+ T cells producing TNF-α and IFN-γ and a higher mobilization of surface marker CD107a. Taken together, our results suggest that immunodominant subpopulations of CD8+ T elicited after immunization could be directly related to degree of protection achieved by different immunization protocols using different viral vectors. Overall, these results demonstrated the usefulness of recombinant influenza viruses in immunization protocols against Chagas Disease.
Assuntos
Doença de Chagas/prevenção & controle , Neuraminidase/imunologia , Vacinas Protozoárias/imunologia , Trypanosoma cruzi/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Especificidade de Anticorpos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Doença de Chagas/imunologia , Doença de Chagas/mortalidade , Doença de Chagas/parasitologia , Epitopos de Linfócito T/imunologia , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Camundongos , Orthomyxoviridae/genética , Orthomyxoviridae/imunologia , Fenótipo , Vacinas Protozoárias/genética , Baço/imunologia , Trypanosoma cruzi/genética , VacinaçãoRESUMO
Chagas' disease is a major public health problem affecting nearly 10 million in Latin America. Despite several experimental vaccines have shown to be immunogenic and protective in mouse models, there is not a current vaccine being licensed for humans or in clinical trial against T. cruzi infection. Towards this goal, we used the backbone of Yellow Fever (YF) 17D virus, one of the most effective and well-established human vaccines, to express an immunogenic fragment derived from T. cruzi Amastigote Surface Protein 2 (ASP-2). The cDNA sequence of an ASP-2 fragment was inserted between E and NS1 genes of YF 17D virus through the construction of a recombinant heterologous cassette. The replication ability and genetic stability of recombinant YF virus (YF17D/ENS1/Tc) was confirmed for at least six passages in Vero cells. Immunogenicity studies showed that YF17D/ENS1/Tc virus elicited neutralizing antibodies and gamma interferon (IFN-γ) producing-cells against the YF virus. Also, it was able to prime a CD8(+) T cell directed against the transgenic T. cruzi epitope (TEWETGQI) which expanded significantly as measured by T cell-specific production of IFN-γ before and after T. cruzi challenge. However, most important for the purposes of vaccine development was the fact that a more efficient protective response could be seen in mice challenged after vaccination with the YF viral formulation consisting of YF17D/ENS1/Tc and a YF17D recombinant virus expressing the TEWETGQI epitope at the NS2B-3 junction. The superior protective immunity observed might be due to an earlier priming of epitope-specific IFN-γ-producing T CD8(+) cells induced by vaccination with this viral formulation. Our results suggest that the use of viral formulations consisting of a mixture of recombinant YF 17D viruses may be a promising strategy to elicit protective immune responses against pathogens, in general.