Navegando por Autor "Ostolin, Thais Lopes Valentim Di Paschoale"

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Avaliação da dose-resposta e da associação de adjuvantes na potência de vacinas quiméricas contra leishmaniose visceral : estudo de fase I em camundongos BALB/c.
(2020) Ostolin, Thais Lopes Valentim Di Paschoale; Reis, Alexandre Barbosa; Brito, Rory Cristiane Fortes de; Reis, Alexandre Barbosa; Brito, Rory Cristiane Fortes de; Fujiwara, Ricardo Toshio; Borges, William de Castro
No Brasil, a leishmaniose visceral canina (LVC) encontra-se em expansão sendo um sério problema de saúde pública. A alta prevalência de cães infectados reforça a necessidade de uma vacina para ser empregada em campanhas de vacinação de forma profilática. Nesse trabalho foram avaliadas duas quimeras desenhadas a partir do mapeamento de epítopos de células T pertencentes a proteínas de Leishmania infantum descritas na literatura como candidatas vacinais. O objetivo do estudo foi avaliar a dose-resposta e o efeito da associação de adjuvantes na eficácia de vacinas quiméricas contra a leishmaniose visceral em camundongos BALB/c. Os animais foram divididos em 14 grupos Salina, Saponina e Monofosforil lipídio A (SM) e quimeras A e B, nas doses de 5, 10 e 20 g, associados ou não aos adjuvantes. Esses foram imunizados com 3 doses, com intervalo de 15 dias entre as doses, e desafiados com 1x107 promastigotas de L. infantum. Foi avaliada a atividade proliferativa de linfócitos T totais (CD3+ ) e suas subpopulações (CD3+CD4+ e CD3+CD8+ ) e a produção de citocinas intracitoplasmáticas (IL-2, IFN-, TNF-, IL-4 e IL-10) após estímulo com antígeno solúvel de L. infantum (ASLi) por citometria de fluxo. Além disso, foi determinada a produção de óxido nítrico (NO) em sobrenadante de cultura de esplenócitos estimulados com ASLi e produção de imunoglobulinas murinas anti-Leishmania (IgG total, IgG1 e IgG2) no soro dos camundongos por ELISA. A quantificação da carga parasitária foi feita no baço por PCR em tempo real (qPCR). Ambas quimeras nas três doses avaliadas, apresentaram aumento da proliferação de linfócitos T e suas subpopulações quando comparadas ao grupo Salina. Quando associadas aos adjuvantes, o mesmo comportamento foi observado sendo o aumento em comparação aos grupos Salina e SM. Observou-se aumento de células T CD4+ e CD8+ produtoras de IFN-, TNF- e IL-2, enquanto houve redução de IL-4 e IL-10, tanto em A quanto em B nas três doses. Cabe ressaltar, que em B na dose de 5 g associada a SM houve aumento na produção de IFN- e IL-2 e redução de IL-4 e IL-10 pelos linfócitos TCD4+ em comparação ao grupo Salina. Os resultados da reatividade anti-Leishmania, mostraram que as proteínas quiméricas A e B aumentaram a produção de IgG total e IgG2 nas três doses avaliadas. Quando associadas a SM, apenas a proteína quimérica A, mostrou-se reativa. Em relação ao subclasse IgG1, houve aumento nas doses de 5 g e 10 g. E para IgG2 houve diferença significativa das doses de 5 e 10 g com a dose de 20 g. Quando quantificamos a carga parasitária foi observado que as proteínas quiméricas sozinhas não foram capazes de reduzir a carga no baço dos animais. Em contrapartida, quando foram associadas ao sistema SM, houve redução da carga parasitária no baço em todas as doses avaliadas para ambas quimeras. Observou-se aumento na produção de NO em todos grupos que foram imunizados com as quimeras associadas ao sistema SM. Os resultados obtidos foram promissores, demonstrando que mesmo em doses reduzidas as quimeras foram capazes de desencadear uma resposta imunológica satisfatória. Os resultados mostram que as duas quimeras sozinhas e ou associadas ao sistema de adjuvantes foram imunogênicas mesmo em doses reduzidas. Além disso, esse estudo contribuirá para o desenvolvimento de novas vacinas contra a LV.
Cell immune response in mice skin stimulated with different adjuvants by intradermal route.
(2022) Mathias, Fernando Augusto Siqueira; Cardoso, Jamille Mirelle de Oliveira; Reis, Levi Eduardo Soares; Souza, Juliana Vitoriano de; Moreira, Nádia das Dores; Ostolin, Thais Lopes Valentim Di Paschoale; Brito, Rory Cristiane Fortes de; Soares, Rodrigo Dian de Oliveira Aguiar; Vieira, Paula Melo de Abreu; Carneiro, Cláudia Martins; Roatt, Bruno Mendes; Reis, Alexandre Barbosa
Adjuvants act in the innate immunity and, when combined to vaccine antigens, can produce a more intense response, improving the antigen presentation, directing the immune system, excellent for new vaccine formulations. This study evaluated the use of the intradermal route and the immune response triggered by a single dose of the adjuvants Aluminum Hydroxide (Al(OH)3 ), Montanide Pet Gel A (MPGA), Glucopyranosyl Lipid A Stable Emulsion (GLA-SE), and Resiquimod (R-848) in the mice skin. As control mice received sterile saline. MPGA and GLA-SE led to cell recruitment when compared with control group, with intense presence of neutrophils in first 12 hours, replaced by macrophages after 168 hours. R-848 and Al(OH)3 showed similar cell recruitment profiles. Regarding cytokine production, groups that received MPGA and GLA-SE produced high levels of IL-6, TNF-α, and IFN-γ. R-848 and Al(OH)3 groups displayed similar profile of cytokine production only at the first hour. Our results suggest that the intradermal route is efficient inducing immune system activation and GLA-SE was promising adjuvants for a type 1 immune response vaccine.
A chimeric vaccine combined with adjuvant system induces immunogenicity and protection against visceral leishmaniasis in BALB/c mice.
(2021) Ostolin, Thais Lopes Valentim Di Paschoale; Gusmão, Miriã Rodrigues; Mathias, Fernando Augusto Siqueira; Cardoso, Jamille Mirelle de Oliveira; Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Ruiz, Jeronimo Conceição; Resende, Daniela de Melo; Brito, Rory Cristiane Fortes de; Reis, Alexandre Barbosa
In Brazil, canine visceral leishmaniasis is an important public health problem due to its alarming growth. The high prevalence of infected dogs reinforces the need for a vaccine for use in prophylactic vaccination campaigns. In the present study, we evaluate the immunogenicity and protection of the best dose of Chimera A selected through the screening of cytokines production important in disease. BALB/c mice were vaccinated subcutaneously with three doses and challenged intravenously with 1 107 L. infantum promastigotes. Spleen samples were collected to assess the intracellular cytokine profile production, T cell proliferation and parasite load. At first, three different doses of Chimera A (5 lg, 10 lg and 20 lg) were evaluated through the production of IFN-c and IL-10 cytokines. Since the dose of 20 lg showed the best results, it was chosen to continue the study. Secondarily, Chimera A at dose of 20 lg was formulated with Saponin plus Monophosphoryl lipid A. Vaccination with Chimera A alone and formulated with SM adjuvant system was able to increase the percentage of the proliferation of specific T lymphocytes and stimulated a Th1 response with increased levels of IFN-c, TNF-a and IL-2, and decreased of IL-4 and IL-10. The vaccine efficacy through real-time PCR demonstrated a reduction in the splenic parasite load in animals that received Chimera A formulated with the SM adjuvant system (92%). Additionally, we observed increased levels of nitric oxide in stimulated-culture supernatants. The Chimera A formulated with the SM adjuvant system was potentially immunogenic, being able to induce immunoprotective mechanisms and reduce parasite load. Therefore, the use of T-cell multi-epitope vaccine is promising against visceral leishmaniasis.
Chimeric vaccines designed by immunoinformatics-activated polyfunctional and memory T cells that trigger protection against experimental visceral leishmaniasis.
(2020) Brito, Rory Cristiane Fortes de; Ruiz, Jeronimo Conceição; Cardoso, Jamille Mirelle de Oliveira; Ostolin, Thais Lopes Valentim Di Paschoale; Reis, Levi Eduardo Soares; Mathias, Fernando Augusto Siqueira; Soares, Rodrigo Dian de Oliveira Aguiar; Roatt, Bruno Mendes; Oliveira, Rodrigo Corrêa de; Resende, Daniela de Melo; Reis, Alexandre Barbosa
Many vaccine candidates against visceral leishmaniasis (VL) have been proposed; however, to date, none of them have been efficacious for the human or canine disease. On this basis, the design of leishmaniasis vaccines has been constantly changing, and the use of approaches to select specific epitopes seems to be crucial in this scenario. The ability to predict T cell-specific epitopes makes immunoinformatics an even more necessary approach, as in VL an efficient immune response against the parasite is triggered by T lymphocytes in response to Leishmania spp. immunogenic antigens. Moreover, the success of vaccines depends on the capacity to generate long-lasting memory and polyfunctional cells that are able to eliminate the parasite. In this sense, our study used a combination of different approaches to develop potential chimera candidate vaccines against VL. The first point was to identify the most immunogenic epitopes of Leishmania infantum proteins and construct chimeras composed of Major histocompatibility complex (MHC) class I and II epitopes. For this, we used immunoinformatics features. Following this, we validated these chimeras in a murine model in a thorough memory study and multifunctionality of T cells that contribute to a better elucidation of the immunological protective mechanisms of polyepitope vaccines (chimera A and B) using multicolor flow cytometry. Our results showed that in silico-designed chimeras can elicit polyfunctional T cells producing T helper (Th)1 cytokines, a strong immune response against Leishmania antigen, and the generation of central and effector memory T cells in the spleen cells of vaccinated animals that was able to reduce the parasite burden in this organ. These findings contribute two potential candidate vaccines against VL that can be used in further studies, and help in this complex field of vaccine development against this challenging parasite.
Comparative evaluation of meglumine antimoniate encapsulated in a mixture of conventional and PEGylated liposomes and immunotherapy using an anti-canine IL-10 receptor-blocking monoclonal antibody on canine visceral leishmaniasis.
(2022) Cardoso, Jamille Mirelle de Oliveira; Brito, Rory Cristiane Fortes de; Mathias, Fernando Augusto Siqueira; Reis, Levi Eduardo Soares; Vieira, João Filipe Pereira; Ostolin, Thais Lopes Valentim Di Paschoale; Andrade, Hélida Monteiro de; Ramos, Guilherme Santos; Frezard, Frederic; Soares, Rodrigo Dian de Oliveira Aguiar; Roatt, Bruno Mendes; Reis, Alexandre Barbosa
This study compared the therapeutic potential of the chemotherapy using meglumine antimoniate encapsulated in a mixture of conventional and PEGylated liposomes (Nano Sbv ) and immunotherapy with anti-canine IL-10 receptor-blocking monoclonal antibody (Anti IL-10R) on canine visceral leishmaniasis (CVL). Twenty mongrel dogs naturally infected by L. infantum, displaying clinical signs of visceral leishmaniasis were randomly divided in two groups. In the first one, nine dogs received six intravenous doses of a mixture of conventional and PEGylated liposomes containing meglumine antimoniate at 6.5 mg Sb/kg/dose. In the second one, eleven dogs received two intramuscular doses of 4 mg of anti-canine IL-10 receptor-blocking monoclonal antibody. The animals were evaluated before (T0) and 30, 90, and 180 days after treatments. Our major results demonstrated that both treatments were able to maintain hematological and biochemical parameters, increase circulating T lymphocytes subpopulations, increase the IFN-γ producing T-CD4 lymphocytes, restore the lymphoproliferative capacity and improve the clinical status. However, although these improvements were observed in the initial post-treatment times, they did not maintain until the end of the experimental follow-up. We believe that the use of booster doses or the association of chemotherapy and immunotherapy (immunochemotherapy) is promising to improve the effectiveness of treating CVL for improving the clinical signs and possibly reducing the parasite burden in dogs infected with Leishmania infantum.
Immunoprophylaxis using polypeptide chimera vaccines plus adjuvant system promote Th1 response controlling the spleen parasitism in hamster model of visceral leishmaniasis.
(2022) Gusmão, Miriã Rodrigues; Ostolin, Thais Lopes Valentim Di Paschoale; Carvalho, Lívia Mendes; Costa, Ana Flávia Pereira; Moreira, Gabriel José Lucas; Cardoso, Jamille Mirelle de Oliveira; Soares, Rodrigo Dian de Oliveira Aguiar; Reis, Alexandre Barbosa; Brito, Rory Cristiane Fortes de; Roatt, Bruno Mendes
In recent years, several advances have been observed in vaccinology especially for neglected tropical diseases (NTDs). One of the tools employed is epitope prediction by immunoinformatic approaches that reduce the time and cost to develop a vaccine. In this scenario, immunoinformatics is being more often used to develop vaccines for NTDs, in particular visceral leishmaniasis (VL) which is proven not to have an effective vaccine yet. Based on that, in a previous study, two predicted T-cell multi-epitope chimera vaccines were experimentally validated in BALB/c mice to evaluate the immunogenicity, central and effector memory and protection against VL. Considering the results obtained in the mouse model, we assessed the immune response of these chimeras in Mesocricetus auratus hamster, which displays, experimentally, similar pathological status to human and dog VL disease. Our findings indicate that both chimeras lead to a dominant Th1 response profile, inducing a strong cellular response by increasing the production of IFN-c and TNF-a cytokines associated with a decrease in IL-10. Also, the chimeras reduced the spleen parasite load and the weight a correlation between protector immunological mechanisms and consistent reduction of the parasitic load was observed. Our results demonstrate that both chimeras were immunogenic and corroborate with findings in the mouse model. Therefore, we reinforce the use of the hamster as a pre-clinical model in vaccination trials for canine and human VL and the importance of immunoinformatic to identify epitopes to design vaccines for this important neglected disease.
Ivabradine treatment lowers blood pressure and promotes cardiac and renal protection in spontaneously hypertensive rats.
(2022) Gomes, Fabiana Aparecida Rodrigues; Noronha, Sylvana Izaura Salyba Rendeiro de Noronha; Silva, Sabrina Carla Alves e; Machado Júnior, Pedro Alves; Ostolin, Thais Lopes Valentim Di Paschoale; Chírico, Máira Tereza Talma; Ribeiro, Marcelo C.; Reis, Alexandre Barbosa; Cangussú, Silvia Dantas; Montano, Nicola; Silva, Valdo Jose Dias da; Menezes, Rodrigo Cunha Alvim de; Silva, Fernanda Cacilda dos Santos; Chianca Júnior, Deoclécio Alves
Hypertension is linked to hyperpolarization-activated cyclic nucleotide-gated (HCN) function, expressed in excitable and non-excitable cells. Considering that the reduction in heart rate (HR) improves coronary perfusion and cardiac performance, ivabradine (IVA) emerged as an important drug for the treatment of cardiovascular diseases. Aim: Evaluate if IVA chronic treatment effect can mitigate hypertension and reverse the cardiac and renal damage in SHR. Main methods: Rats were divided into 4 groups treated for 14 days with PBS (1 ml/kg; i.p) or IVA (1 mg/kg; i.p): 1) WKY PBS; 2) SHR PBS; 3) WKY IVA; and 4) SHR IVA. The systolic blood pressure (SBP) was measured, indirectly, before and during the treatment period with IVA (day 0, 1, 7 and 11). Rats were subjected to artery cannulation for direct blood pressure (BP) measurement. Morphofunctional and gene expression were evaluated in the heart and kidneys. Key findings: IVA reduced SBP only in SHR on the 7th day. Direct blood pressure measurement showed that IVA chronic treatment reduced HR in the SHR. Interestingly, mean arterial pressure (MAP) was reduced in SHR IVA when compared to SHR PBS. Serum and urinary biochemical data were not altered by IVA. Moreover, IVA reduced the renal inflammatory infiltrates and increased glomerular density, besides preventing the cardiac inflammatory and hypertrophic responses. Significance: IVA treatment lowered blood pressure, improved cardiac remodeling and inflammation, as well as decreasing renal damage in SHR. Further, IVA increased renal HCN2 mRNA and reduced cardiac HCN4 mRNA.
A specific Leishmania infantum polyepitope vaccine triggers Th1-type immune response and protects against experimental visceral leishmaniasis.
(2022) Ostolin, Thais Lopes Valentim Di Paschoale; Gusmão, Miriã Rodrigues; Mathias, Fernando Augusto Siqueira; Cardoso, Jamille Mirelle de Oliveira; Roatt, Bruno Mendes; Soares, Rodrigo Dian de Oliveira Aguiar; Ruiz, Jeronimo Conceição; Resende, Daniela de Melo; Brito, Rory Cristiane Fortes de; Reis, Alexandre Barbosa
The development of an immunogenic, effective, and safe vaccine is essential as an alternative for disease control. The present study aimed to evaluate the immunogenicity and efficacy potential of a polyepitope T-cell antigen candidate against visceral leishmaniasis in a murine model. BALB/c mice were immunized with three doses subcutaneously with Poly-T Leish alone or adjuvanted with Saponin plus Monophosphoryl lipid A, with 15-day intervals between doses, and challenged with 107 stationary-phase Leishmania infantum promastigotes via tail vein. Immunogenicity and parasitism in spleen and liver of immunized mice were evaluated 45 days postchallenge. Our results revealed that the immunization with Poly-T Leish and Poly-T Leish/SM increases the percentage of specific T (CD4+ and CD8+) lymphocytes proliferation in vitro after antigen-specific stimulation. Also, Poly-T Leish and Poly-T Leish/SM groups showed a high percentage of IFN-γ and TNF-α-producing T cells, meanwhile, the Poly-T Leish/SM group also showed an increased percentage of multifunctional T cells producing double and triple-positive (IFN-γ+TNF-α+IL-2+) cytokines. The immunization with Poly-T Leish or Poly-T Leish/ SM stimulated a decreased IL-4 and IL-10 compared to the Saline and adjuvant group. Poly-T Leish/SM immunized mice exhibit a noteworthy reduction in the parasite burden (spleen and liver) through real-time PCR (96%). Moreover, we observed higher nitrite secretion in 120-hour stimulated-culture supernatant using Griess method. We demonstrated that the Poly-T Leish/SM candidate was potentially immunogenic, providing enhancement of protective immune mechanisms, and conferred protection reducing parasitism. Our candidate was considered potential against visceral leishmaniasis, and eventually, could be tested in phase I and II clinical trials in dogs.
The use of an adjuvant system improves innate and adaptive immune response when associated with a Leishmania (Viannia) braziliensis antigen in a vaccine candidate against L. (Leishmania) infantum infection.
(2023) Mathias, Fernando Augusto Siqueira; Ostolin, Thais Lopes Valentim Di Paschoale; Reis, Levi Eduardo Soares; Cardoso, Jamille Mirelle de Oliveira; Brito, Rory Cristiane Fortes de; Soares, Rodrigo Dian de Oliveira Aguiar; Roatt, Bruno Mendes; Vieira, Paula Melo de Abreu; Reis, Alexandre Barbosa
Background: The adjuvants’ optimal dose and the administration route can directly influence the epitope recognition patterns and profiles of innate response. We aimed to establish the effect and the optimal dose of adjuvant systems for proposing a vaccine candidate to be employed with Leishmania (Viannia) braziliensis. Methods: We evaluated the adjuvants saponin (SAP), monophosphoryl lipid A (MPL) and resiquimod (R-848) isolated and combined as adjuvant systems in a lower dose corresponding to 25%, 33%, and 50% of each adjuvant total dose. Male outbred BALB/c mice were divided into 13 groups, SAP, MPL, and R-848 isolated, and the adjuvant systems SAP plus MPL (SM), SAP plus R-848 (SR), and MPL plus R-848 (MR). Results: SM50 increased levels of all chemokines analyzed and TNF production, while it presented an increased inflammatory cell infiltrate in the skin with macrophage recruitment. Thus, we proposed a vaccine candidate employing L. (V.) braziliensis antigen associated with the SM adjuvant system against experimental L. (Leishmania) infantum challenge. We observed a significant increase in the frequency of cells expressing the central and effector memory CD4+ T cells phenotype in immunized mice with the LBSM50. In the liver, there was a decreased parasite load when mice received LBSM50. Conclusions: When combined with L. (V.) braziliensis antigen, SM50 increases TNF and IFN-γ, which generates central and effector memory CD4+ T cells. Therefore, using an adjuvant system can promote an effective innate immune response with the potential to compose future vaccines.