Targeting the non-structural protein 1 from dengue virus to a dendritic cell population confers protective immunity to lethal virus challenge

Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1). In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs) are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb) directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (αDEC205 and αDCIR2) to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)), as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with αDEC-NS1 and αDCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-γ producing cells was higher in αDEC-NS1 immunized animals. In addition, mice immunized with the αDEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with αDCIR2-NS1 mAb. Protection was partially mediated by CD4(+) and CD8(+) T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205(+) DC population with poly (I:C) opens perspectives for dengue vaccine development.Brazilian National Research Council (CNPq)National Institute of Science and Technology Research Funding Agency (INCTV) - 15203*12São Paulo State Research Funding Agency (FAPESP) - 2007/08648-9, 2011/51761-6BNP-Paribas BankRio de Janeiro State Research Funding Agency (FAPERJ

Identification of blood ingested by the females of Aedes scapularis from the Tietê Ecological Park, São Paulo City, State of São Paulo, using the ELISA method

A Febre do Nilo Ocidental é doença emergente em diversas regiões do mundo. O agente etiológico é vírus da família Flaviviridae que infecta aves, eqüinos e, acidentalmente, o homem. No Brasil o vírus não foi encontrado até o momento, entretanto, vem sendo realizada a vigilância epidemiológica com vistas à detecção precoce da presença do agente viral no país. O conhecimento do hábito alimentar dos mosquitos é importante para estudos em Saúde Pública, pois indica a potencialidade desses organismos para veicular agentes infecciosos que podem causar doenças. Assim, o uso do teste imunoenzimático ELISA (Enzyme Linked ImmunoSorbent Assay) indireto para a identificação do sangue ingerido por fêmeas de culicídeos é ferramenta importante para a definição e discussão do hábito alimentar das espécies. O objetivo do estudo foi investigar o sangue ingerido por fêmeas de Aedes scapularis capturadas no Parque Ecológico do Tietê (PET), situado na região metropolitana da Grande São Paulo. O PET apresenta diversos ambientes que são adequados a múltiplas espécies de mosquitos. As coletas foram realizadas, mensalmente, no período de junho de 2005 a junho de 2006. Nas coletas, foram utilizados aspiradores de Nasci, movidos à bateria. Foram escolhidas 04 áreas do PET. Esses locais foram determinados com base em estudos anteriores. Dessa maneira, foi possível escolher os ambientes que apresentavam maior probabilidade de encontro de Aedes scapularis. Foram coletadas 843 fêmeas de Aedes scapularis. Dentre os insetos testados, os resultados obtidos foram: 67/336 (19,94%) positivos para homem; 20/336 (5,95%) positivos para aves; 129/336 (38,39%) positivos para cão, 17/336 (5,06%) positivos para rato, além de 103/336 (30,66%) positivos para repastes múltiplos. A baixa positividade para ingestão de sangue de rato e de aves aponta o possível envolvimento de outros hospedeiros como fonte alimentar. Os resultados para o repasto múltiplo demonstram que os mosquitos alimentam-se do sangue de vários hospedeiros antes de realizar a oviposição.The West Nile Fever is an emergent disease in several regions of the world. The etiologic agent is the virus from the Flaviviridae family which infected birds, equines and, accidentally, the man. The virus has not been found in Brazil so far; however the epidemiologic vigilance has been made to detect the premature presence from the virus agent in the country. The knowledge about the mosquitoes alimentary habits is important for the studies in Public Health, since it indicates the potentiality of these organisms to run infectious agents that can cause illness. Then, the use of the indirect ELISA method (Enzyme Linked ImmunoSorbent Assay) for the identification of the ingested blood by the females culicidae is an important tool to the definition and discussion of the mosquito alimentary habit. The purpose of the study was to investigate the ingested blood by females from Aedes scapularis captured in the Ecological Park of the Tietê (EPT), localized in the metropolitan area from São Paulo city. The EPT exhibits many regions which are suitable for multiple species of mosquitoes. Collections were held monthly during June 2005 to June 2006. The collections were done through Nasci\'s aspirators battery-operated. It was choose 04 areas in the EPT. These locations were determined in previous studies. In this manner, it was possible to choose the ambiences which featured by the highest probability of presence from Aedes scapularis. From the analyzed insects, the obtained results had been: 67/336 (19, 94) positive ones for man; 20/336 (5, 95) positive ones for birds; 129/336 (38, 39) positive ones for dog, 17/336 (5,06) positive ones for rat, beyond 103/336 (30, 66) positive for multiple feed. The low positive rate for ingestion of blood of rat and birds points the possible involvement of other hosts as alimentary source. The results for the multiple feed indicate that mosquitoes feed from blood of many hosts before to achieve the oviposition

Pre-clinical immunizations against malaria based on the Apical Membrane Antigen 1 (PvAMA-1): testing prime-boost homologous and heterologous protocols using DNA and/or recombinant protein

O Antígeno 1 de Membrana Apical (AMA-1) é um dos mais promissores candidatos a vacina contra a fase sanguínea da malária. No presente estudo, geramos uma nova proteína recombinante baseada no ectodomínio de AMA-1 de P. vivax (PvAMA-1) a partir do gene sintético com códon otimizado para expressão secretada na levedura Pichia pastoris. Por ELISA, a proteína PvAMA-1 foi reconhecida por 62,5% dos soros de pacientes da Amazônia brasileira infectados por P. vivax. A imunogenicidade de PvAMA-1 foi avaliada em camundongos BALB/c, utilizando protocolos homólogos e heterólogos de indução e reforço com DNA plasmidial e/ou proteína recombinante, emulsificada em Adjuvante de Freund. Os resultados mostraram que a eficiência da imunização é dependente da presença da proteína emulsificada em adjuvante forte. As imunizações subseqüentes foram realizadas com a proteína recombinante emulsificada em diferentes adjuvantes: sais de alumínio (Alum), Monophosphoryl Lipid A (MPLA) de Bordetella pertussis, flagelina FliC de Salmonella Typhimurium, saponina Quil A e Adjuvante Incompleto de Freund (AIF). Os resultados demonstraram que as imunizações na presença de Quil A e AIF foram capazes de induzir altos títulos de anticorpos IgG e uma resposta de isotipos de IgG mais balanceada, caracterizando um padrão do tipo Th1/Th2. Títulos de anticorpos mais baixos, mas também expressivos, foram obtidos na presença de Alum, MPLA, Alum + MPLA e Alum + FliC. A análise da resposta proliferativa, por citometria de fluxo utilizando marcação com CFSE, mostrou que células esplênicas CD3+CD4+, assim como CD3+CD8+, de animais imunizados com PvAMA-1 na presença dos adjuvantes Alum, Alum + MPLA, Quil A e AIF foram capazes de proliferar especificamente in vitro. Por imunofluorescência, soros policlonais de camundongos imunizados com o antígeno na presença de MPLA e Quil A foram capazes de reconhecer a proteína nativa expressa em isolados de P. Vivax da Ásia. Visando futuros testes pré clínicos em primatas não humanos, a proteína PvAMA-1 foi produzida em boas práticas de laboratório (BPL) por uma companhia especializada e o potencial imunogênico da proteína foi confirmado em imunizações posteriores. Em conjunto, nossos resultados demonstram que PvAMA-1 é um antígeno promissor para ser explorado em protocolos de imunização contra malária vivax, individualmente, ou em combinação com outros antígenos.The Apical Membrane Antigen 1 (AMA-1) is one of the most promissing vaccine candidates against the erythrocytic stage of malaria. In the present study we generated a new recombinant protein based on AMA-1 ectodomain of P. vivax (PvAMA-1) using a synthetic codon optimized gene for yeast secreted expression. By ELISA, the protein PvAMA-1 was recognized by 62,5% of the sera from Brazilian Amazonia patients infected by P. vivax. The immunogenicity of PvAMA-1 was evaluated in BALB/c mice using homologous and heterologous protocols of prime and boost with plasmidial DNA and/or recombinant protein emulsified in Freund adjuvant. The results showed that the efficiency of immunization is dependent on the presence of a strong adjuvant. The following immunizations were conducted using the protein emulsified in different adjuvants: aluminium salts (Alum), Monophosphoryl Lipid A (MPLA) of Bordetella pertussis, flagelin FliC of Salmonella Typhimurium, saponin Quil A and Incomplete Freund\'s Adjuvant (IFA). The results showed that immunizations in the presence of Quil A e IFA were able to induce high IgG titers and a more balanced IgG isotypes response, featuring a standard type Th1/Th2. Lower antibody titers, but also significant, were obtained in the presence of Alum, MPLA, Alum + MPLA and Alum + FliC. The proliferative cellular analysis, by flow cytometry using CFSE staining, showed that splenic cells CD3+CD4+, as well as CD3+CD8+ from immunized mice that received PvAMA-1 with Alum, Alum + MPLA, Quil A and IFA were specifically able to proliferate in vitro. By immunofluorescence, the polyclonal sera from mice immunized with the antigen in the presence of MPLA and Quil A were able to recognize native protein expressed in Asian P. vivax isolates. Aiming future pre-clinical assays in non-human primates, the protein PvAMA-1 was produced in good laboratory practices (GLP) conditions by a specialized company and its immunogenic efficiency was confirmed in later immunizations. Altogether, our results showed that PvAMA-1 is a promissor antigen to be explored in immunization protocols against vivax malaria, individually, or combined with other antigens

Intradermal Delivery of Dendritic Cell-Targeting Chimeric mAbs Genetically Fused to Type 2 Dengue Virus Nonstructural Protein 1

Targeting dendritic cells (DCs) by means of monoclonal antibodies (mAbs) capable of binding their surface receptors (DEC205 and DCIR2) has previously been shown to enhance the immunogenicity of genetically fused antigens. This approach has been repeatedly demonstrated to enhance the induced immune responses to passenger antigens and thus represents a promising therapeutic and/or prophylactic strategy against different infectious diseases. Additionally, under experimental conditions, chimeric αDEC205 or αDCIR2 mAbs are usually administered via an intraperitoneal (i.p.) route, which is not reproducible in clinical settings. In this study, we characterized the delivery of chimeric αDEC205 or αDCIR2 mAbs via an intradermal (i.d.) route, compared the elicited humoral immune responses, and evaluated the safety of this potential immunization strategy under preclinical conditions. As a model antigen, we used type 2 dengue virus (DENV2) nonstructural protein 1 (NS1). The results show that the administration of chimeric DC-targeting mAbs via the i.d. route induced humoral immune responses to the passenger antigen equivalent or superior to those elicited by i.p. immunization with no toxic effects to the animals. Collectively, these results clearly indicate that i.d. administration of DC-targeting chimeric mAbs presents promising approaches for the development of subunit vaccines, particularly against DENV and other flaviviruses