Enhanced immunogenicity and protective efficacy in mice following a Zika DNA vaccine designed by modulation of membrane-anchoring regions and its association to adjuvants

Zika virus (ZIKV) is a re-emerging pathogen with high morbidity associated to congenital infection. Despite the scientific advances since the last outbreak in the Americas, there are no approved specific treatment or vaccines. As the development of an effective prophylactic approach remains unaddressed, DNA vaccines surge as a powerful and attractive candidate due to the efficacy of sequence optimization in achieving strong immune response. In this study, we developed four DNA vaccine constructs encoding the ZIKV prM/M (pre-membrane/membrane) and E (envelope) proteins in conjunction with molecular adjuvants. The DNA vaccine candidate (called ZK_ΔSTP), where the entire membrane-anchoring regions were completely removed, was far more immunogenic compared to their counterparts. Furthermore, inclusion of the tPA-SP leader sequence led to high expression and secretion of the target vaccine antigens, therefore contributing to adequate B cell stimulation. The ZK_ΔSTP vaccine induced high cellular and humoral response in C57BL/6 adult mice, which included high neutralizing antibody titers and the generation of germinal center B cells. Administration of ZK-ΔSTP incorporating aluminum hydroxide (Alum) adjuvant led to sustained neutralizing response. In consistency with the high and long-term protective response, ZK_ΔSTP+Alum protected adult mice upon viral challenge. Collectively, the ZK_ΔSTP+Alum vaccine formulation advances the understanding of the requirements for a successful and protective vaccine against flaviviruses and is worthy of further translational studies

Avaliação do Efeito da Hiperglicemia na Via da Autofagia em Macrófagos de Camundongos Diabéticos

O Diabetes mellitus (DM) é um grupo de distúrbios metabólicos caracterizado pela hiperglicemia. O DM tipo 1 (DM1) se caracteriza pela destruição das células β pancreáticas, que consiste na maioria das vezes por um processo autoimune, promovendo uma deficiência de insulina. A hiperglicemia decorrente da falta de insulina ocasiona prejuízos para o sistema imunológico tornando indivíduos diabéticos mais susceptíveis a infecções quando comparado com indivíduos saudáveis. A alta susceptibilidade às infecções é, pelo menos em parte, em função de uma resposta imune inadequada. Além disto, a hiperglicemia per se desempenha um importante papel neste processo. Desta forma, o objetivo desta pesquisa foi de avaliar a hiperglicemia e seu envolvimento no processo da autofagia e na resposta induzida por lipopolissacarídeo (LPS) em populações de macrófagos de animais diabéticos e não diabéticos. Para tanto, utilizamos macrófagos derivados de medula óssea (BMDM) de camundongos C57BL/6 diabéticos (Aloxana 60mg/kg, i.v.) e não diabéticos. Os BMDMs foram mantidos em meio de cultura com concentração normal (5,5 mM) e uma alta concentração de glicose (25 mM) e foram estimuladas ou não com LPS na concentração de 100 ng/mL e Nigericina na concentração de 20µM, nos tempos de 30 minutos; 2; 4; 6; 24 horas, sendo dosadas as citocinas IL-6, IL-10, IL-1β e TNF-α. Em nossos estudos, identificamos alterações na secreção das citocinas pró-inflamatórias IL-6, IL-1β e TNF-α, em que os BMDMs de camundongos diabéticos e não diabéticos apresentaram aumento na secreção dessas citocinas quando estimulados por LPS+Nigericina. Além disso, foram observadas alterações na via de autofagia, onde o aumento da proteína autofágica LC3b e Beclin-1 ocorreu por macrófagos de animais não diabéticos em meio hiperglicêmico, sem estímulo. Os macrófagos de animais diabéticos apresentaram redução na expressão de LC3b e Beclin-1 em meio hiperglicêmcio, sugerindo um processo autofágico prejudicado nessas células. Os resultados obtidos permitem sugerir que a hiperglicemia altera as vias inflamatórias em macrófagos estimulados por LPS+Nigericina, desempenhando um papel importante na resposta inflamatória de indivíduos diabéticos.Diabetes mellitus (DM) is a group of metabolic disorders characterized by hyperglycemia. Type 1 DM (T1DM) is characterized by the pancreatic beta cells destruction, which most often consists of an autoimmune process, promoting an insulin deficiency. Hyperglycemia resulting from the lack of insulin leads to an impaired immune system, leading diabetic individuals more susceptible to infections when compared to healthy individuals. The high susceptibility is, at least in part, due to an inadequate immune response. In addition, hyperglycemia per se plays an important role in this process. Thus, the objective of this study was to evaluate the autophagy process and the lipopolysaccharide-induced response (LPS) in macrophage populations of diabetic and non-diabetic mice. For this, we used bone marrow-derived macrophages (BMDM) from diabetic (Aloxan 60mg/kg, i.v.) and non-diabetic C57BL/6 mice. BMDM were cultured in normal concentration (5.5 mM) and in a high concentration of glucose (25 mM) medium, and were stimulated or not with LPS at a concentration of 100 ng/mL and Nigericin at a concentration of 20 µM, at times 30 minutes; 2; 4; 6; 24 hours, followed by the measurement of the cytokines IL-6, IL-10, IL-1β and TNF-α. In our study, we identified changes in the levels of pro-inflammatory cytokines IL-6, IL-1β and TNF-α, where BMDM showed an increase in the secretion of these cytokines when stimulated by LPS + Nigericin. In addition, changes were observed in the autophagy pathway, where the increase in the autophagic protein LC3b and Beclin-1 occurred by macrophages of non-diabetic mice in hyperglycemic medium, without LPS stimulation. Macrophages from diabetic mice showed a reduction on the expression of LC3b and Beclin-1, suggesting an impaired autophagic process in these cells. The results obtained allow us to suggest that hyperglycemia alters the inflammatory pathways in macrophages stimulated by LPS, playing an important role in the inflammatory response of diabetic individuals

Effects of low-dose rapamycin on lymphoid organs of mice prone and resistant to accelerated senescence

Aging is a complex, natural, and irreversible phenomenon that subjects the body to numerous changes in the physiological process, characterized by a gradual decline in the organism’s homeostatic mechanisms, closely related to immunosenescence. Here, we evaluated the regulation of immunosenescence in lymphoid organs of senescence-accelerated prone 8 (SAM-P8) and senescence-accelerated resistant 1 (SAM-R1) mice treated with a low dose of rapamycin (RAPA). Mice were treated with a dose of 7.1 µg/kg RAPA for 2 months and had body mass and hematological parameters analyzed prior and during treatment. Cellular and humoral parameters of serum, bone marrow, thymus, and spleen samples were evaluated by ELISA, histology, and flow cytometry. Changes in body mass, hematological parameters, cell number, and in the secretion of IL-1β, IL-6, TNF-α, IL-7, and IL-15 cytokines were different between the 2 models used. In histological analyses, we observed that SAM-P8 mice showed faster thymic involution than SAM-R1 mice. Regarding the T lymphocyte subpopulations in the spleen, CD4+ and CD8+ T cell numbers were higher and lower, respectively, in SAM-P8 mice treated with RAPA, with the opposite observed in SAM-R1. Additionally, we found that the low dose of RAPA used did not trigger changes that could compromise the immune response of these mice and the administered dose may have contributed to changes in important lymphocyte populations in the adaptive immune response and the secretion of cytokines that directly collaborate with the maturation and proliferation of these cells

DataSheet_1_Enhanced immunogenicity and protective efficacy in mice following a Zika DNA vaccine designed by modulation of membrane-anchoring regions and its association to adjuvants.docx

Zika virus (ZIKV) is a re-emerging pathogen with high morbidity associated to congenital infection. Despite the scientific advances since the last outbreak in the Americas, there are no approved specific treatment or vaccines. As the development of an effective prophylactic approach remains unaddressed, DNA vaccines surge as a powerful and attractive candidate due to the efficacy of sequence optimization in achieving strong immune response. In this study, we developed four DNA vaccine constructs encoding the ZIKV prM/M (pre-membrane/membrane) and E (envelope) proteins in conjunction with molecular adjuvants. The DNA vaccine candidate (called ZK_ΔSTP), where the entire membrane-anchoring regions were completely removed, was far more immunogenic compared to their counterparts. Furthermore, inclusion of the tPA-SP leader sequence led to high expression and secretion of the target vaccine antigens, therefore contributing to adequate B cell stimulation. The ZK_ΔSTP vaccine induced high cellular and humoral response in C57BL/6 adult mice, which included high neutralizing antibody titers and the generation of germinal center B cells. Administration of ZK-ΔSTP incorporating aluminum hydroxide (Alum) adjuvant led to sustained neutralizing response. In consistency with the high and long-term protective response, ZK_ΔSTP+Alum protected adult mice upon viral challenge. Collectively, the ZK_ΔSTP+Alum vaccine formulation advances the understanding of the requirements for a successful and protective vaccine against flaviviruses and is worthy of further translational studies.</p

Molecular characterization of the gastrointestinal eukaryotic virome in elderly people in Belem, Para, Brazil

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001 / Fonds Wetenschappelijk Onderzoek (FWO) (1S78019N)Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Programa de Pós-Graduação em Virologia. Ananindeua, PA, BrasilKU Leuven-University of Leuven. Rega Institute Department of Microbiology, Immunology and Transplantation. Laboratory of Clinical and Epidemiological Virology. Leuven, BelgiumKU Leuven-University of Leuven. Rega Institute Department of Microbiology, Immunology and Transplantation. Laboratory of Clinical and Epidemiological Virology. Leuven, BelgiumKU Leuven-University of Leuven. Rega Institute Department of Microbiology, Immunology and Transplantation. Laboratory of Clinical and Epidemiological Virology. Leuven, BelgiumMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Programa de Pós-Graduação em Virologia. Ananindeua, PA, BrasilMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Programa de Iniciação Científica Cnpq - Fapespa. Ananindeua, PA, BrasilMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Programa de Iniciação Científica Cnpq - Fapespa. Ananindeua, PA, BrasilMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Programa de Iniciação Científica Cnpq - Fapespa. Ananindeua, PA, BrasilFederal University of Pará. Institute of Biological Sciences. Biology of Infectious and Parasitic Agents Graduate Program,. Belém, PA, BrazilState University of Pará. Belém, PA, BrazilMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, BrasilMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, BrasilMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, BrasilKU Leuven-University of Leuven. Rega Institute Department of Microbiology, Immunology and Transplantation. Laboratory of Clinical and Epidemiological Virology. Leuven, BelgiumMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, BrasilAcute gastroenteritis is one of the main causes of mortality and morbidity worldwide, affecting mainly children, the immunocompromised and elderly people. Enteric viruses, especially rotavirus A, are considered important etiological agents, while long-term care facilities are considered favorable environments for the occurrence of sporadic cases and outbreaks of acute gastroenteritis. Therefore, it is important to monitor the viral agents present in nursing homes, especially because studies involving the elderly population in Brazil are scarce, resulting in a lack of available virological data. As a result, the causative agent remains unidentified in a large number of reported acute gastroenteritis cases. However, the advent of next-generation sequencing provides new opportunities for viral detection and discovery. The aim of this study was to identify the viruses that circulate among elderly people with and without acute gastroenteritis, living in residential care homes in Belém, Pará, Brazil, between 2017 and 2019. Ninety-three samples were collected and screened by immunochromatography and qPCR. After, the samples were analyzed individually or in pools by next generation sequencing to identify the viruses circulating in this population. In 26 sequenced samples, members of 13 eukaryotic virus families were identified. The most abundantly present virus families were Parvoviridae, Genomoviridae and Smacoviridae. Contigs displaying similarity to pegiviruses were also detected. Furthermore, a near-complete rotavirus A genome was obtained and could be classified as G3P[8] genotype with the equine DS-1-like genetic background. Complete sequences of the VP4 and VP7 genes of a rotavirus C were also detected, belonging to G4P[2]. This study demonstrates the first characterization of the gastrointestinal virome in elderly in Northern Brazil. A diversity of viruses was found to be present in patients with and without diarrhea, reinforcing the need to monitor elderly people residing in long-term care facilities, especially in cases of acute gastroenteritis