Adjuvant properties of IFN-γ and GM-CSF in the scFv6.C4 DNA vaccine against CEA-expressing tumors.

Tumor-associated carcinoembryonic antigen (CEA) is a natural target for vaccines against colorectal cancers. Our previous experience with a DNA vaccine with scFv6.C4, a CEA surrogate, showed a CEA-specific immune response with 40% of tumor-free mice after challenge with B16F10-CEA and 47% with MC38-CEA cells. These percentages increased to 63% after using FrC as an adjuvant. To further enhance the vaccine efficacy, we tested GM-CSF and IFNγ as adjuvants. C57BL/6J-CEA2682 mice were immunized 4 times with uP-PS/scFv6.C4, uP-PS/scFv6.C4 + uP-IFNγ, or uP-PS/scFv6.C4 + uP-GMCSF. After one week, the mice were challenged with MC38-CEA, and tumor growth was monitored over 100 days. Immunization with scFv6.C4 and scFv6.C4 + GM-CSF resulted in a gradual increase in the anti-CEA antibody titer, while scFv6.C4 + IFNγ immunization led to a rapid and sustained increase in the titer. The addition of IFNγ also induced higher CD4 + and CD8 + responses. When challenged, almost 80% of the scFv6.C4 + IFNγ-vaccinated mice did not develop tumors, while the others had a significant tumor growth delay. The probability of being tumor-free was 2700% higher using scFv6.C4 + IFNγ than scFv6.C4. The addition of GM-CSF had no additional effect on tumor protection. DNA immunization with scFv6.C4 + IFNγ, but not GM-CSF, increased the antitumor effect via readily sustained specific humoral and cytotoxic responses to CEA.

Correction: CXCR3 chemokine receptor contributes to specific CD8+ T cell activation by pDC during infection with intracellular pathogens.

[This corrects the article DOI: 10.1371/journal.pntd.0008414.].

STING Signaling Drives Production of Innate Cytokines, Generation of CD8+ T Cells and Enhanced Protection Against Trypanosoma cruzi Infection.

A variety of signaling pathways are involved in the induction of innate cytokines and CD8+ T cells, which are major players in protection against acute Trypanosoma cruzi infection. Previous data have demonstrated that a TBK-1/IRF3-dependent signaling pathway promotes IFN-ß production in response to Trypanosoma cruzi, but the role for STING, a main interactor of these proteins, remained to be addressed. Here, we demonstrated that STING signaling is required for production of IFN-ß, IL-6, and IL-12 in response to Trypanosoma cruzi infection and that STING absence negatively impacts activation of IRF-dependent pathways in response to the parasite. We reported no significant activation of IRF-dependent pathways and cytokine expression in RAW264.7 macrophages in response to heat-killed trypomastigotes. In addition, we showed that STING is essential for T. cruzi DNA-mediated induction of IFN-ß, IL-6, and IL-12 gene expression in RAW264.7 macrophages. We demonstrated that STING-knockout mice have significantly higher parasitemia from days 5 to 8 of infection and higher heart parasitism at day 13 after infection. Although we observed similar heart inflammatory infiltrates at day 13 after infection, IFN-ß, IL-12, CXCL9, IFN-γ, and perforin gene expression were lower in the absence of STING. We also showed an inverse correlation between parasite DNA and the expression of CXCL9, IFN-γ, and perforin genes in the hearts of infected animals at day 13 after infection. Finally, we reported that STING signaling is required for splenic IFN-ß and IL-6 expression early after infection and that STING deficiency results in lower numbers of splenic parasite-specific IFN-γ and IFN-γ/perforin-producing CD8+ T cells, indicating a pivotal role for STING signaling in immunity to Trypanosoma cruzi.

CXCR3 chemokine receptor contributes to specific CD8+ T cell activation by pDC during infection with intracellular pathogens.

Chemokine receptor type 3 (CXCR3) plays an important role in CD8+ T cells migration during intracellular infections, such as Trypanosoma cruzi. In addition to chemotaxis, CXCR3 receptor has been described as important to the interaction between antigen-presenting cells and effector cells. We hypothesized that CXCR3 is fundamental to T. cruzi-specific CD8+ T cell activation, migration and effector function. Anti-CXCR3 neutralizing antibody administration to acutely T. cruzi-infected mice decreased the number of specific CD8+ T cells in the spleen, and those cells had impaired in activation and cytokine production but unaltered proliferative response. In addition, anti-CXCR3-treated mice showed decreased frequency of CD8+ T cells in the heart and numbers of plasmacytoid dendritic cells in spleen and lymph node. As CD8+ T cells interacted with plasmacytoid dendritic cells during infection by T. cruzi, we suggest that anti-CXCR3 treatment lowers the quantity of plasmacytoid dendritic cells, which may contribute to impair the prime of CD8+ T cells. Understanding which molecules and mechanisms guide CD8+ T cell activation and migration might be a key to vaccine development against Chagas disease as those cells play an important role in T. cruzi infection control.

CXCR3 chemokine receptor guides Trypanosoma cruzi-specific T-cells triggered by DNA/adenovirus ASP2 vaccine to heart tissue after challenge.

CD8+ T lymphocytes play an important role in controlling infections by intracellular pathogens. Chemokines and their receptors are crucial for the migration of CD8+ T-lymphocytes, which are the main IFNγ producers and cytotoxic effectors cells. Although the participation of chemokine ligands and receptors has been largely explored in viral infection, much less is known in infection by Trypanosoma cruzi, the causative agent of Chagas disease. After T. cruzi infection, CXCR3 chemokine receptor is highly expressed on the surface of CD8+ T-lymphocytes. Here, we hypothesized that CXCR3 is a key molecule for migration of parasite-specific CD8+ T-cells towards infected tissues, where they may play their effector activities. Using a model of induction of resistance to highly susceptible A/Sn mice using an ASP2-carrying DNA/adenovirus prime-boost strategy, we showed that CXCR3 expression was upregulated on CD8+ T-cells, which selectively migrated towards its ligands CXCL9 and CXCL10. Anti-CXCR3 administration reversed the vaccine-induced resistance to T. cruzi infection in a way associated with hampered cytotoxic activity and increased proapoptotic markers on the H2KK-restricted TEWETGQI-specific CD8+ T-cells. Furthermore, CXCR3 receptor critically guided TEWETGQI-specific effector CD8+ T-cells to the infected heart tissue that express CXCL9 and CXCL10. Overall, our study pointed CXCR3 and its ligands as key molecules to drive T. cruzi-specific effector CD8+ T-cells into the infected heart tissue. The unveiling of the process driving cell migration and colonization of infected tissues by pathogen-specific effector T-cells is a crucial requirement to the development of vaccine strategies.

scFv6.C4 DNA vaccine with fragment C of Tetanus toxin increases protective immunity against CEA-expressing tumor.

The carcinoembryonic antigen (CEA) is the main tumor-associated antigen of colorectal cancers. Previously, we developed a DNA vaccine using scFv6.C4, a CEA surrogate, against CEA-expressing tumors; 40% of the vaccinated mice were tumor-free after tumor challenge. In order to enhance vaccine efficacy, fragment C of Tetanus Toxin (FrC) was tested as adjuvant. C57BL/6J-CEA2682 mice were electroporated intramuscularly 4 times with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4, challenged by s.c. injection of 1 × 105 MC38-CEA cells, and tumor growth was monitored over 100 days. The humoral and cellular immune responses were assessed by ELISA, immunocytochemistry, in-vitro lymphocyte proliferation, and CTL cytotoxicity assays. Immunization with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4 induced similar anti-CEA antibody titers. However, immunocytochemistry analysis showed stronger staining with uP-PS/scFv6.C4-FrC-immunized mice sera. When challenged with MC38-CEA cells, 63% of the FrC-vaccinated mice did not develop tumors, half of the rest had a significant tumor growth delay, and the probability of being free of tumors was on average 40% higher than that of scFv6.C4-immunized mice. Addition of the adjuvant led to higher CD4+ and CD8+ proliferative responses and strong CD8+ CTL response against MC38-CEA cells. DNA immunization with scFv6.C4 and FrC increased antitumor effect via induction of high and specific humoral and cellular immune responses to CEA.

Therapeutical effects of vaccine from Trypanosoma cruzi amastigote surface protein 2 by simultaneous inoculation with live parasites.

The aim of this study was to evaluate the efficacy of vaccine using replication-deficient human recombinant Type 5 replication-defective adenoviruses (AdHu5) carrying sequences of the amastigote surface protein 2 (ASP2) (AdASP2) in mice infected with the Trypanosoma cruzi ( T cruzi) Y strain. A total of 16 A/Sn mice female were distributed into four groups, as follows (n = 4 per group): Group 1 - Control Group (CTRL); Group 2 - Infected Group (TC): animals were infected by subcutaneous route with 150 bloodstream trypomastigotes of T cruzi Y strain; Group 3 - Immunized Group (AdASP-2): animals were immunized by intramuscular injection (im) route with 50 µL of AdSP-2 (2 × 10 8 plaque forming units [pfu]/cam) at day 0; Group 4-Immunized and Infected Group (AdASP-2+TC): animals were immunized by im route with 50 µL of ASP-2 (2 × 10 8 pfu/cam) and infected by T cruzi at the same day (day 0). It was observed a significant decrease of nests in the group that was immunized with AdASP-2 and infected on the same day. Tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS) gene expressions showed a significant increase in the AdASP-2+TC group when compared to TC group, but it was noted that Cyclooxygenase-2 (Cox-2) was increased in TC group when compared to AdASP-2+TC group. Increase of matrix metalloproteinases-2 (MMP-2) and decrease of MMP-9 immunoexpression in the AdASP-2+TC group was noticed as well. Oxidative DNA damage was present in myocardium for AdASP-2+TC group as a result of 8-hydroxydeoxyguanosine immunoexpression. Taken together, our results highlighted an increased oxidative stress, MMP-2 activity and inflammatory host response promoted by AdASP-2 against T cruzi infection.

Therapeutic effects of vaccine derived from amastigote surface protein-2 (ASP-2) against Chagas disease in mouse liver.

This study investigated the efficacy of the vaccine in liver of mice infected with the Trypanosoma cruzi (T. cruzi) and immunized with AdASP-2. For this purpose, histopathological analysis and gene expression of COX-2, TNF-alpha, TNFR, iNOS, cytochrome C, caspase-3, TLR4, IL-6 and IL10 were evaluated. The following groups were used in this study: Group 1 - Control Group (CTRL) animals received AdßGal vehicle; Group 2 - Infected Group (TC) animals were infected with T. cruzi; Group 3 - Immunized Group (AdASP-2): animals were immunized by AdASP-2 vaccine; Group 4 - Immunized and Infected Group (AdASP-2+TC) animals were infected with T. cruzi and immunized by AdSP-2 vaccine. A significant decrease of amastigote nests was noticed in the group of animals that were immunized with AdASP-2 and infected on the same day. COX-2 and TNF-alpha gene expressions increased in TC group, whereas TNF-alpha decreased in the TC+AdASP-2 group. TNFR expression was high in AdASP-2+TC group. iNOS expression was high for all experimental groups whereas cytochrome C decreased for all experimental groups. Caspase 3 increased in TC and TC+AdASP-2 groups. The gene expression of TLR4 and IL-10 showed an increase in AdASP-2+TC group. Finally, hepatic fibrosis was noticed to TC and AdASP-2 + TC groups. Taken together, our results demonstrated that vaccination with AdASP-2 was effective against the acute phase of experimental Chagas disease as a result of a more powerful and rapid immune response closely related to expression of some inflammatory genes, such as iNOS, TNF-alpha, TLR 4, and IL-10.

Qualidade higienicossanitária de hortaliças cruas servidas em restaurante institucional da Baixada Santista, SP / Hygienicosanitary quality of raw vegetables served in institutional restaurant of Baixada Santista, SP

A qualidade higienicossanitária de verduras consumidas cruas constitui fator importante para a prevenção das doenças de origem alimentar. O objetivo dessa pesquisa foi analisar a qualidade higienicossanitária de hortaliças cruas servidas em um restaurante da Baixada Santista. Para isso, foram realizadas 30 coletas de amostras, num período de 4 meses e analisada a qualidade microbiológica e a presença de parasitas. Para avaliação de Boas Práticas em serviço de alimentação, foi aplicado um checklist e realizado o treinamento de colaboradores. Nas análises microbiológicas constatou-se que 53,3% das amostras foram consideradas impróprias para consumo para o parâmetro de coliformes a 35ºC e termotolerantes e também foi constatada ausência total de Salmonella segundo a RDC nº 12 de 02/01/2001. Quanto às análises parasitológicas, 10% das amostras apresentaram resultados positivos. A partir da aplicação do checklist, os dados coletados demonstraram falta de qualidade sanitária no preparo das saladas, inadequação quanto à higiene pessoal e ambiental, e inadequação das edificações e instalações em 51,83% do checklist aplicado. O treinamento em Boas Práticas de Manipulação deve ser executado periodicamente com todos os colaboradores. Conclui-se que a falta de qualidade higienicossanitária no preparo das saladas e a inadequação das boas práticas observadas, podem comprometer a qualidade da refeição e a saúde do consumidor.

The hygienic-sanitary quality of ready-to-eat foods is important to the prevention of food-borne diseases. The objective of this research was to evaluate the hygienic-sanitary quality of raw vegetables served at Baixada Santista. For this, 30 samples of raw salads were sampled, in a period of four months and were submitted to microbiological and parasitologycal analysis. For the evaluation of Good Practices in food service, a checklist was applied and training of handlers. In the microbiological analysis, 53,3% of the samples were considered improper for consumption for the coliform parameter at 35ºC and thermotolerant and was also found absence of Salmonella according to RDC nº12 of 01/02/2001. Regarding the parasitological analysis, ten percent of samples had parasites. From the application of the checklist, the data show lack of sanitary quality without preparation of salads, inadequate personal and environmental hygiene, and inadequate buildings and facilities in 51,83% of the checklists applied. Training in Good Handling Practices should be performed periodically with all handlers. It is concluded that the lack of hygienic-sanitary quality without preparation of the salads and an inadequacy of the observed good practices can compromise a quality of the meal and a health of the consumer.

LFA-1 Mediates Cytotoxicity and Tissue Migration of Specific CD8+ T Cells after Heterologous Prime-Boost Vaccination against Trypanosoma cruzi Infection.

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.

Vacinação genética utilizando genes de Trypanosoma cruzi em linhagem de camundongos altamente suscetível à infecção / Genetic vaccination using genes of Trypanosoma cruzi in a strain of mice highly susceptible to infection

Diversos trabalhos demonstraram que camundongos BALB/c ou C57BL/6 imunizados com plasmídios que contém genes do Trypanosoma cruzi desenvolveram resposta imune específica e imunidade protetora contra infecção parasitária letal. Nossos resultados anteriores mostraram que camundongos A/Sn altamente suscetíveis a infecção pelo T. cruzi, vacinados com DNA plasmidial contendo o gene que codifica a trans-sialidase apresentaram uma redução no pico da parasitemia após a infecção experimental. Entretanto, após o desafio com as formas tripomastigotas sangüíneas da cepa Y, estes animais foram incapazes de sobreviver. 0 objetivo inicial desta tese foi tentar aumentar a resposta imune do tipo 1 em camundongos A/Sn utilizando protocolos de imunização combinados com DNA plasmidal e proteína recombinante. Para tal, camundongos A/Sn foram imunizados com: i) o plasmídio p154/13 que contém o gene que codifica a trans-sialidase (TS) de T cruzi, ii) com a proteína recombinante TS em alum, iii) simultaneamente com o plasmídio p154/13 e com a proteína recombinante TS, ou iv) seqüencialmente com o plasmídio p154/13 seguido da proteína recombinante TS. Após a análise da resposta imune humoral e celular, observamos que camundongos A/Sn imunizados com o plasmídio p154/13 desenvolveram uma resposta imune predominante do tipo 1. Em contraste, a imunização com a proteína recombinante TS acoplada em alum gerou uma típica resposta imune do tipo 2. A administração simultânea do plasmídio p154/13 e proteína recombinante TS também induziu uma resposta imune predominante do tipo 2. Porém, a imunização seqüencial denominada "DNA-priming protein-boosting", que consistiu de duas doses iniciais do plasmídio p154/13 seguida de injeções de reforço com a proteína recombinante TS, melhorou significativamente a resposta imune específica do tipo 1, determinado pela drástica redução da relação das subclasses de imunoglobulina IgG1/IgG2a no soro dos camundongos e pelo aumento da produção in vitro de IFN- por células T CD4 dos camundongos imunizados. Nossas observações confirmaram e estenderam dados prévios que mostraram que o protocolo "DNA-priming protein-boosting" pode ser uma estratégia geral para aumentar resposta imune do tipo 1 das vacinas de MA. Após a infecção com T cruzi, nenhuma melhoria significativa da imunidade protetora foi observada nestes camundongos quando comparados aos camundongos que só receberam o DNA plasmidial...(au)