Reactivity of DENV-positive sera against recombinant envelope proteins produced in bacteria and eukaryotic cells.

Dengue is a mosquito-borne disease endemic in many tropical and subtropical countries. It is caused by the dengue virus (DENV) that can be classified into 4 different serotypes (DENV-1-4). Early diagnosis and management can reduce morbidity and mortality rates of severe forms of the disease, as well as decrease the risk of larger outbreaks. Hiperendemicity in some regions of the world and the possibility that some people develop a more severe form of disease after a secondary infection caused by antibody-dependent enhancement justify the need to understand more thoroughly the antibody response induced against the virus. Here, we successfully produced a recombinant DENV-2 envelope (E) protein and its domains (EDI/II and EDIII) in two distinct expression systems: the Drosophila S2 insect cell system and the BL21 (DE3) pLySs bacterial system. We then evaluated the reactivity of sera from patients previously infected with DENV to each recombinant protein and to each domain separately. Our results show that the E protein produced in Drosophila S2 cells is recognized more frequently than the protein produced in bacteria. However, the recognition of E protein produced in bacteria correlates better with the DENV-2 sera neutralization capacity. The results described here emphasize the differences observed when antigens produced in bacteria or eukaryotic cells are used and may be useful to gain more insight into the humoral immune responses induced by dengue infection.

Prime-boost with Chikungunya virus E2 envelope protein combined with Poly (I:C) induces specific humoral and cellular immune responses.

Chikungunya virus (CHIKV) is an arbovirus transmitted to humans mainly by the bite of infected Aedes aegypti and Aedes albopictus mosquitoes. CHIKV illness is characterized by fever and long-lasting arthritic symptoms, and in some cases it is a deadly disease. The CHIKV envelope E2 (E2CHIKV) glycoprotein is crucial for virus attachment to the cell. Furthermore, E2CHIKV is the immunodominant protein and the main target of neutralizing antibodies. To date, there is no available prophylactic vaccine or specific treatment against CHIKV infection. Here, we designed and produced a DNA vaccine and a recombinant protein containing a consensus sequence of E2CHIKV. C57BL/6 mice immunized twice with the E2CHIKV recombinant protein in the presence of the adjuvant Poly (I:C) induced the highest E2CHIKV-specific humoral and cellular immune responses, while the immunization with the homologous DNA vaccine pVAX-E2CHIKV was able to induce specific IFN-γ producing cells. The heterologous prime-boost strategy was also able to induce specific cellular and humoral immune responses that were, in general, lower than the responses induced by the homologous E2CHIKV recombinant protein immunization. Furthermore, recombinant E2CHIKV induced the highest titers of neutralizing antibodies. Collectively, we believe this is the first report to analyze E2CHIKV-specific humoral and cellular immune responses after immunization with E2CHIKV recombinant protein and DNA pVAX-E2CHIKV vaccine platforms.

The effect of memantine, an antagonist of the NMDA glutamate receptor, in in vitro and in vivo infections by Trypanosoma cruzi.

Chagas disease, caused by Trypanosoma cruzi, is a neglected tropical disease that affects 5-6 million people in endemic areas of the Americas. Presently, chemotherapy relies on two compounds that were proposed as trypanocidal drugs four decades ago: nifurtimox and benznidazole. Both drugs are able to eliminate parasitemia and to avoid seroconversion in infected people when used in the acute phase; however, their use in the chronic phase (the time when the majority of cases are diagnosed) is limited due to their serious side effects. Memantine is a glutamate receptor antagonist in the central nervous system of mammals that has been used for the treatment of Alzheimer's disease. Our group previously reported memantine as a trypanocidal drug that is able to induce apoptosis-like death in T. cruzi. In the present work, we further investigated the effects of memantine on the infection of RAW 264.7 macrophages and in vivo (in BALB/c mice). Here, we showed that memantine is able to diminish NO and Ca2+ entry in both LPS-activated and non-activated cells. These results, together with the fact that memantine was also able to reduce the infection of macrophages, led us to propose that this drug is able to activate a pro-oxidant non-NO-dependent cell defense mechanism. Finally, infected mice that were treated with memantine had diminished parasitemia, cardiac parasitic load, and inflammatory infiltrates. In addition, the treated mice had an increased survival rate. Taken together, these results indicate memantine to be a candidate drug for the treatment of Chagas disease.

Development and in vitro/in vivo evaluation of a novel benznidazole liquid dosage form using a quality-by-design approach.

OBJECTIVES: To develop an alcohol-free solution suitable for children of benznidazole, the drug of choice for treatment of Chagas disease. METHODS: In a quality-by-design approach, a systematic optimisation procedure was carried out to estimate the values of the factors leading to the maximum drug concentration. The formulations were analysed in terms of chemical and physical stability and drug content. The final preparation was subjected to an in vivo palatability assay. Mice were infected and treated orally in a murine model. RESULTS: The results showed that benznidazole solubility increased up to 18.38 mg/ml in the optimised co-solvent system. The final formulation remained stable at all three temperatures tested, with suitable drug content and no significant variability. Palatability of the preparation was improved by taste masking of BZL. In vivo studies showed that both parasitaemia and mortality diminished, particularly at a dose of 40 mg/kg/day. CONCLUSION: Quality by design was a suitable approach to formulate a co-solvent system of benznidazole. The in vivo studies confirmed the suitability of the optimised such solutions to diminish both parasitaemia and mortality. Thus, this novel alternative should be taken into account for further clinical evaluation in all age ranges.

Early dengue virus interactions: the role of dendritic cells during infection.

Dengue is an acute infectious disease caused by dengue virus (DENV) that affects approximately 400 million people annually, being the most prevalent human arthropod-borne disease. DENV infection causes a wide variety of clinical manifestations that range from asymptomatic to dengue fever, and in some cases may evolve to the more severe dengue hemorrhagic fever and dengue shock syndrome. The exact reasons why some patients do not have symptoms while others develop the severe forms of disease are still elusive, but gathered evidence showed correlation between a secondary infection with a heterologous DENV serotype and the occurrence of severe symptoms. Despite several advances, the mechanisms of DENV infection are still not completely elucidated, and efforts have been made to understand the development of immunity and/or pathology to DENV. When a mosquito transmits DENV, the virus is initially deposited in the skin, where mononuclear phagocytic cells, such as dendritic cells (DCs), become infected. DCs play a critical role in the induction of immune responses, as they are able to rapidly detect pathogen-associated molecular patterns, endocytose and process antigens, and efficiently activate naïve-T and B cells. Recent findings have shown that DCs serve as DENV targets, but they are also important mediators of immunity against the virus. In this review, we will briefly discuss DENV infection pathogenesis, and introduce DCs as central players in the induction of anti-DENV immune responses. Then, we will review in more detail how DENV interacts with and is sensed by DCs, with particular emphasis in two classes of receptors implicated in viral entry.