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The main COVID-19 vaccine formulations used today are mainly based on the wild-type SARS-CoV-2 spike glycoprotein as an antigen. However, new virus variants capable of escaping neutralization activity of serum antibodies elicited in vaccinated individuals have emerged. The Omicron (B.1.1.529) variant caused epidemics in regions of the world in which most of the population has been vaccinated. In this study, we aimed to understand what determines individual's susceptibility to Omicron in a scenario of extensive vaccination. For that purpose, we collected nasopharynx swab (n = 286) and blood samples (n = 239) from flu-like symptomatic patients, as well as their vaccination history against COVID-19. We computed the data regarding vaccine history, COVID-19 diagnosis, COVID-19 serology and viral genome sequencing to evaluate their impact on the number of infections. As main results, we showed that vaccination in general did not reduce the number of individuals infected by Omicron, even with an increased immune response found among vaccinated non-infected individuals. Nonetheless, we found that individuals who received the third vaccine dose showed significantly reduced susceptibility to Omicron infections. A relevant evidence that support this finding was the higher virus neutralization capacity of serum samples of most patients who received the third vaccine dose. In summary, this study shows that boosting immune responses after a third vaccine dose reduces susceptibility to COVID-19 caused by the Omicron variant. Results presented in this study are useful for future formulations of COVID-19 vaccination policies. This article is protected by copyright. All rights reserved.

Psychological stress predisposes our body to several disorders. Understanding the cellular and molecular mechanisms involved in the physiological responses to psychological stress is essential for the success of therapeutic applications. New studies show, by using in vivo inducible Cre/loxP-mediated approaches in combination with pharmacological blockage, that sympathetic nerves, activated by psychological stress, induce brown adipocytes to produce IL-6. Strikingly, this cytokine promotes gluconeogenesis in hepatocytes, that results in the decline of tolerance to inflammatory organ damage. The comprehension arising from this research will be crucial for the handling of many inflammatory diseases. Here, we review recent advances in our comprehension of the sympathetic nerve-adipocyte axis in the tissue microenvironment.

Sensory neurons have recently emerged as components of the tumor microenvironment. Nevertheless, whether sensory neuronal activity is important for tumor progression remains unknown. Here we used Designer Receptors Exclusively Activated by a Designer Drug (DREADD) technology to inhibit or activate sensory neurons’ firing within the melanoma tumor. Melanoma growth and angiogenesis were accelerated following inhibition of sensory neurons’ activity and were reduced following overstimulation of these neurons. Sensory neuron-specific overactivation also induced a boost in the immune surveillance by increasing tumor-infiltrating anti-tumor lymphocytes, while reducing immune-suppressor cells. In humans, a retrospective in silico analysis of melanoma biopsies revealed that increased expression of sensory neurons-related genes within melanoma was associated with improved survival. These findings suggest that sensory innervations regulate melanoma progression, indicating that manipulation of sensory neurons’ activity may provide a valuable tool to improve melanoma patients’ outcomes. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-021-01273-9.

Diagnosis and prognosis of breast cancer is based on disease staging identified through histopathological and molecular biology techniques. Animal models are used to gain mechanistic insights into the development of breast cancer. C(3)1-TAg is a genetically engineered mouse model that develops mammary cancer. However, carcinogenesis caused by this transgene was characterized in the Friend Virus B (FVB) background. As most genetic studies are done in mice with C57BL/6 J background, we aimed to define the histological alterations in C3(1)-TAg C57BL/6 J animals. Our results showed that C3(1)-TAg animals with C57BL/6 J background develop solid-basaloid adenoid cystic carcinomas with increased fibrosis, decreased area of adipocytes, and a high proliferative index, which are triple-negative for progesterone, estrogen, and human epidermal growth factor receptor 2 (HER2) receptors. Our results also revealed that tumor development is slower in the C57BL/6 J background when compared with the FVB strain, providing a better model to study the different stages in breast cancer progression.

Multiple infectious diseases lead to impaired lung function. Revealing the cellular mechanisms involved in this impairment is crucial for the understanding of how the lungs shift from a physiologic to a pathologic state in each specific condition. In this context, we explored the pathogenesis of Paracoccidioidomycosis, which affects pulmonary functioning. The presence of cells expressing Nestin-GFP has been reported in different tissues, and their roles as tissue-specific progenitors have been stablished in particular organs. Here, we explored how Nestin-GFP+ cells are affected after lung infection by Paracoccidioides brasiliensis, a model of lung granulomatous inflammation with fibrotic outcome. We used Nestin-GFP transgenic mice, parabiosis surgery, confocal microscopy and flow cytometry to investigate the participation of Nestin-GFP+ cells in Paracoccidioides brasiliensis pathogenesis. We revealed that these cells increase in the lungs post-Paracoccidioides brasiliensis infection, accumulating around granulomas. This increase was due mainly to Nestin-GPF+ cells derived from the blood circulation, not associated to blood vessels, that co-express markers suggestive of hematopoietic cells (Sca-1, CD45 and CXCR4). Therefore, our findings suggest that circulating Nestin-GFP+ cells participate in the Paracoccidioides brasiliensis pathogenesis in the lungs.

COVID-19 continues to spread around the world despite significant progress in vaccine distribution and population immunity. The dynamics of the antiviral antibody response postvaccination is critical to evaluate vaccine effectiveness across different vaccine platforms and over time.

Background: This research addresses two questions: (1) how El Niño Southern Oscillation (ENSO) affects climate variability and how it influences dengue transmission in the Metropolitan Region of Recife (MRR), and (2) whether the epidemic in MRR municipalities has any connection and synchronicity. Methods: Wavelet analysis and cross-correlation were applied to characterize seasonality, multiyear cycles, and relative delays between the series. This study was developed into two distinct periods. Initially, we performed periodic dengue incidence and intercity epidemic synchronism analyses from 2001 to 2017. We then defined the period from 2001 to 2016 to analyze the periodicity of climatic variables and their coherence with dengue incidence. Results: Our results showed systematic cycles of 3-4 years with a recent shortening trend of 2-3 years. Climatic variability, such as positive anomalous temperatures and reduced rainfall due to changes in sea surface temperature (SST), is partially linked to the changing epidemiology of the disease, as this condition provides suitable environments for the Aedes aegypti lifecycle. Conclusion: ENSO may have influenced the dengue temporal patterns in the MRR, transiently reducing its main way of multiyear variability (3-4 years) to 2-3 years. Furthermore, when the epidemic coincided with El Niño years, it spread regionally and was highly synchronized.

Background Phylogenetic studies indicate bats as original hosts of SARS-CoV-2. However, it remains unclear whether other animals, including pets, are crucial in the spread and maintenance of COVID-19 worldwide. Methods In this study, we analyzed the first fatal case of a SARS-CoV-2 and FeLV co-infection in an eight-year-old male cat. We carried out a clinical evaluation and several laboratory analyses. Results As main results, we observed an animal presenting severe acute respiratory syndrome and lesions in several organs, which led to the animal’s death. RT-qPCR analysis showed a SARS-CoV-2 as the causative agent. The virus was detected in several organs, indicating a multisystemic infection. The virus was found in a high load in the trachea, suggesting that the animal may have contribute to the transmission of the virus. The whole-genome sequencing revealed an infection by SARS-CoV-2 Gamma VOC (P.1), and any mutations indicating host adaptation were observed. Conclusion Our data show that FeLV-positive cats are susceptible to SARS-CoV-2 infection and raise questions about the potential of immunocompromised FeLV-positive cats to act as a reservoir for SARS-CoV-2 new variants.

Background: Coronaviruses are recognized for their ability to cross the species barrier and infect new hosts. The coronavirus disease 2019 (COVID-19) is caused by the new coronavirus SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2). It remains unclear whether other animals, including pets, are crucial in the spread and maintenance of COVID-19 worldwide. Methods: In this study, we analysed the first fatal case of a SARS-CoV-2 and FeLV (Feline leukemia virus) co-infection of an eight-year-old male cat. We carried out a clinical evaluation, pathological analysis, and viral genomic analysis. Results: As main results, we observed an animal presenting severe acute respiratory syndrome and lesions in several organs, which led to animal’s death. The causative agent was confirmed to be SARS-CoV-2, variant of concern P.1 (Gamma). The virus presented a pattern of mutations potentially associated with feline infection. In addition, the virus was detected by RT-qPCR in the spleen, liver, heart, lungs, trachea, intestines and kidneys, indicating a multisystemic viral infection. The virus was found in a high load in the trachea, suggesting a capacity of transmitting the virus. Conclusion: Our data show that felines, such as FeLV-positive cats, are susceptible to SARS-CoV-2 infection, and may be intermediate hosts in this pandemic.

The four serotypes of Dengue virus (DENV1-4) are arboviruses (arthropod-borne viruses) that belong to the Flavivirus genus, Flaviviridae family. They are the causative agents of an infectious disease called dengue, an important global public health problem with significant social-economic impact. Thus, the development of safe and effective dengue vaccines is a priority according to the World Health Organization. Only one anti-dengue vaccine has already been licensed in endemic countries and two formulations are under phase III clinical trials. In this study, we aimed to compare the main anti-dengue virus vaccines, DENGVAXIA®, LAV-TDV, and TAK-003, regarding their antigens and potential to protect. We studied the conservation of both, B and T cell epitopes involved in immunological control of DENV infection along with vaccine viruses and viral isolates. In addition, we assessed the population coverage of epitope sets contained in each vaccine formulation with regard to different human populations. As main results, we found that all three vaccines contain the main B cell epitopes involved in viral neutralization. Similarly, LAV-TDV and TAK-003 contain most of T cell epitopes involved in immunological protection, a finding not observed in DENGVAXIA®, which explains main limitations of the only licensed dengue vaccine. In summary, the levels of presence and absence of epitopes that are target for protective immune response in the three main anti-dengue virus vaccines are shown in this study. Our results suggest that investing in vaccines that contain the majority of epitopes involved in protective immunity (cellular and humoral arms) is an important issue to be considered.

It is not clear if COVID-19 can be indirectly transmitted. It is not possible to conclude the role of environment in transmission of SARS-CoV-2 without studying areas in which people transit in great amounts, such as market areas. In this work we aimed to better understand the role of environment in the spread of COVID-19. We investigated the presence of SARS-CoV-2 in inanimate objects as well as in the air and in the sewage using RT-qPCR. We studied both, a reference market area and a COVID-19 reference hospital at Barreiras city, Brazil. We collected and analyzed a total of 268 samples from mask fronts, cell phones, paper moneys, card machines, sewage, air and bedding during the ascendant phase of the epidemiological curve of COVID-19 in Barreiras. As a result, we detected the human RNAse P gene in most of samples, which indicates the presence of human cells in specimens. However, we did not detect any trace of SARS-CoV-2 in all samples analyzed. To rule out the possibility of problems in sampling method we tested detection of SARS-CoV-2 by RT-qPCR in laboratory conditions to reproduce environmental temperature and humidity. As a result, we showed detection of the virus in different conditions. We conclude that our sampling method reliable and that, strikingly, the environment and inanimate materials do not have an important role in COVID-19 transmission.

Dengue virus represents the main arbovirus affecting humans, but there are no effective drugs or available worldwide licensed vaccine formulations capable of conferring full protection against the infection. Experimental studies and results generated after the release of the anti-DENV vaccine demonstrated that induction of high-titer neutralizing antibodies does not represent a unique protection correlate and that, indeed, T cell-based immune responses plays a relevant role in the establishment of an immune protective state. In this context, this study aimed to further demonstrate protective features of immune responses elicited in C57BL/6 mice immunized with three plasmids encoding DENV2 nonstructural proteins (NS1, NS3, and NS5), which were subsequently challenged with a DENV2 strain naturally capable of inducing lethal encephalitis in immunocompetent mouse strains. The animals were immunized intramuscularly with two doses of the DNA vaccine mix at a 2-week interval. Cytokine profiles generated by spleen or brain-infiltrating mononuclear cells revealed increased IFN-γ levels in vaccinated animals. Survival curves after lethal challenge with DENV2 showed complete protection only among immunized mice. The results confirm the pivotal role of cellular immune responses targeting nonstructural DENV proteins and validate the experimental model based on a DENV2 strain capable of infecting and killing immunocompetent mice as a tool for the evaluation of protective immunity induced by anti-DENV vaccines.

Glioma is the prevalent aggressive primary brain tumor, with a very poor prognosis. The absence of advanced understanding of the roles played by the cells within the glioma microenvironment limits the development of effective drugs. A recent study indicates that periostin expressed by pericytes is crucial for glioma angiogenesis. Here, we describe succinctly the results and implications of this discovery in what we know about pericytes within the glioma microenvironment. The emerging knowledge from this work will benefit the development of therapies for gliomas.

In the present study, we evaluated the immunological responses induced by dengue vaccines under experimental conditions after delivery via a transcutaneous (TC) route. Vaccines against type 2 Dengue virus particles (DENV2 New Guinea C (NGC) strain) combined with enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) were administered to BALB/c mice in a three-dose immunization regimen via the TC route. As a control for the parenteral administration route, other mouse groups were immunized with the same vaccine formulation via the intradermic (ID) route. Our results showed that mice vaccinated either via the TC or ID routes developed similar protective immunity, as measured after lethal challenges with the DENV2 NGC strain. Notably, the vaccine delivered through the TC route induced lower serum antibody (IgG) responses with regard to ID-immunized mice, particularly after the third dose. The protective immunity elicited in TC-immunized mice was attributed to different antigen-specific antibody properties, such as epitope specificity and IgG subclass responses, and cellular immune responses, as determined by cytokine secretion profiles. Altogether, the results of the present study demonstrate the immunogenicity and protective properties of a dengue vaccine delivered through the TC route and offer perspectives for future clinical applications.

Dengue Virus (DENV) is an arbovirus (arthropod-borne virus). Four serotypes of DENV are responsible for the infectious disease called dengue that annually affects nearly 400 million people worldwide. Although there is only one vaccine formulation licensed for use in humans, there are other vaccine formulations under development that apply different strategies. In this review, we present information about anti-dengue vaccine formulations regarding development, pre-clinical tests, and clinical trials. The improvement in vaccine development against dengue is much needed, but it should be considered that the correlate of protection is still uncertain. Neutralizing antibodies have been proposed as a correlate of protection, but this ignores the key role of T-cell mediated immunity in controlling DENV infection. It is important to confirm the accurate correlate of protection against DENV infection, and also to have other anti-dengue vaccine formulations licensed for use.

Aedes (Stegomyia) aegypti (A. aegypti) transmits arboviral diseases of high public health importance, including those caused by Zika virus (ZIKV), Dengue virus (DENV), Chikungunya virus (CHIKV) and Yellow fever virus (YFV). Barreiras is a city with 157,638 inhabitants in the West of the State of Bahia, Northeast of Brazil. The climate is dry, with well-determined and concentrated seasons of rains. The city is crossed by a Federal Highway and by the Rio Grande river. In this study, we aimed to understand the dynamics of mosquito vectors and arboviral diseases in Barreiras. We used correlation statistics to investigate a possible relationship among rains, mosquito abundance and transmission of diseases. In addition, as a preliminary population genetics estimate, we used geometric morphometrics to compare mosquitoes from areas limited by a highway and a river. We found that i) infestation occurs in rain-dependent cycles and that ii) both, the river and the highway segregate populations of A. aegypti in different areas of the studied city. Our results indicate that it is necessary to treat anthropic containers with mosquito breading capacity during both, the dry and rain seasons in urban areas similar to Barreiras.

Hematopoietic stem cells are the most illustrious inhabitants of the bone marrow. Direct visualization of endogenous hematopoietic stem cells in this niche is essential to study their functions. Until recently this was not possible in live animals. Recent studies, using state-of-the-art technologies, including sophisticated in vivo inducible genetic approaches in combination with two-photon laser scanning microscopy, allow the follow-up of endogenous hematopoietic stem cells’ behavior in their habitat. Strikingly, the new findings reveal that quiescent hematopoietic stem cells are more mobile than previously thought, and link their retained steady state within the niche to a mobile behavior. The arising knowledge from this research will be critical for the therapy of several hematological diseases. Here, we review recent progress in our understanding of hematopoietic stem cell biology in their niches.

Background The arboviruses Zika virus (ZIKV) and Dengue virus (DENV) have important epidemiological impact in Brazil and other tropical regions of the world. Recently, it was shown that previous humoral immunity to DENV enhances ZIKV replication in vitro, which may lead to more severe forms of the disease. Thus, traditional approaches of vaccine development aiming to control viral infection through neutralizing antibodies may induce cross-reactive enhancing antibodies. In contrast, cellular immune response was shown to be capable of controlling DENV infection independently of antibodies. The aim of the present study was to design a flavivirus NS5 protein capable of inducing a cellular immune response against DENV and ZIKV. Methods A consensus sequence of ZIKV NS5 protein was designed among isolates from various continents. Epitopes were predicted for the most prevalent alleles of class I and II HLA in the Brazilian population. Then, this epitopes were analyzed with regard to their conservation, population coverage and distribution along the whole antigen. Results Nineteen epitopes predicted to be more reactive (percentile rank

The Flavivirus genus is composed by viral serocomplexes with relevant global epidemiological impact. Many areas of the world present both, vector fauna and geographical conditions compatible with co-circulation, importing, emergence, and epidemics of flaviviruses of different serocomplexes. In this study, we aimed to identify both, immunological determinants and patterns of immune response possibly involved in flavivirus serocomplex cross-protection. We searched B and T cells epitopes which were thoroughly shown to be involved in flavivirus immunological control. Such epitopes were analyzed regarding their conservation, population coverage, and location along flavivirus polyprotein. We found that epitopes capable of eliciting flavivirus cross-protective immunity to a wide range of human populations are concentrated in proteins E, NS3, and NS5. Such identification of both, immunological determinants and patterns of immune response involved in flavivirus cross-protective immunity should be considered in future vaccine development. Moreover, cross-reactive epitopes presented in this work may be involved in dynamics of diseases caused by flaviviruses worldwide.

Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses.

Generating neutralizing antibodies have been considered a prerequisite to control dengue virus (DENV) infection. However, T lymphocytes have also been shown to be important in a protective immune state. In order to investigate the contribution of both humoral and cellular immune responses in DENV immunity, we used an experimental model in which a non-lethal DENV2 strain (ACS46) is used to intracranially prime Balb/C mice which develop protective immunity against a lethal DENV2 strain (JHA1). Primed mice generated envelope-specific antibodies and CD8(+) T cell responses targeting mainly non-structural proteins. Immune sera from protected mice did not confer passive protection to naïve mice challenged with the JHA1 strain. In contrast, depletion of CD4(+) and CD8(+) T lymphocytes significantly reduced survival of ACS46-primed mice challenged with the JHA1 strain. Collectively, results presented in this study show that a cellular immune response targeting non-structural proteins are a promising way in vaccine development against dengue.

Heat-labile toxins (LTs) have ADP-ribosylation activity and induce the secretory diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains in different mammalian hosts. LTs also act as adjuvants following delivery via mucosal, parenteral, or transcutaneous routes. Previously we have shown that LT produced by human-derived ETEC strains encompass a group of 16 polymorphic variants, including the reference toxin (LT1 or hLT) produced by the H10407 strain and one variant that is found mainly among bacterial strains isolated from pigs (LT4 or pLT). Herein, we show that LT4 (with six polymorphic sites in the A (K4R, K213E, and N238D) and B (S4T, A46E, and E102K) subunits) displays differential in vitro toxicity and in vivo adjuvant activities compared with LT1. One in vitro generated LT mutant (LTK4R), in which the lysine at position 4 of the A subunit was replaced by arginine, showed most of the LT4 features with an ∼10-fold reduction of the cytotonic effects, ADP-ribosylation activity, and accumulation of intracellular cAMP in Y1 cells. Molecular dynamic studies of the A subunit showed that the K4R replacement reduces the N-terminal region flexibility and decreases the catalytic site crevice. Noticeably, LT4 showed a stronger Th1-biased adjuvant activity with regard to LT1, particularly concerning activation of cytotoxic CD8(+) T lymphocytes when delivered via the intranasal route. Our results further emphasize the relevance of LT polymorphism among human-derived ETEC strains that may impact both the pathogenicity of the bacterial strain and the use of these toxins as potential vaccine adjuvants.

Here, we report the complete polyprotein sequence of a dengue virus 2 strain isolated in Brazil. This virus belongs to the American genotype and has the ability to cause neurovirulence in immunocompetent adult mice. The data presented here may help understand the genetic determinants responsible for neurovirulence.

Dengue virus (DENV) causes dengue fever, a widely distributed endemic disease transmitted by mosquitoes. The complex interaction of DENV with the human immune system has complicated the development of an effective vaccine. This may be attributed, at least in part, to the lack of a suitable animal model capable to reproduce symptoms observed in humans. Mouse models are simple but usually rely on host-adapted virus strains or immunodeficient mouse lineages. Recent evidences indicated that some natural DENV strains are capable to infect immunocompetent mice. In addition, humanized mouse lineages can more faithfully reproduce some of the symptoms observed in humans. Such experimental models are valuable tools for the study of DENV biology.

Dengue nonstructural protein 1 (NS1) is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2), which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV) protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections.

The dengue virus (DENV) non-structural 1 (NS1) protein plays a critical role in viral RNA replication and has a central position in DENV pathogenesis. DENV NS1 is a glycoprotein expressed in infected mammalian cells as soluble monomers that dimerize in the lumen of the endoplasmic reticulum; NS1 is subsequently transported to the cell surface, where it remains membrane associated or is secreted into the extracellular milieu as a hexameric complex. During the last three decades, the DENV NS1 protein has also been intensively investigated as a potential target for vaccines and antiviral drugs. In addition, NS1 is the major diagnostic marker for dengue infection. This review highlights some important issues regarding the role of NS1 in DENV pathogenesis and its biotechnological applications, both as a target for the development of safe and effective vaccines and antiviral drugs and as a tool for the generation of accurate diagnostic methods.

O presente trabalho propõe o desenvolvimento e a caracterização de uma estratégia vacinal de caráter profilático contra o vírus da dengue (VD), baseada nas proteínas NS1 e domínio III da proteína E (EIII), empregando proteínas recombinantes em ensaios de imunização por via sub-cutânea em modelo murino. Estes antígenos foram obtidos pela clonagem e expressão de suas sequências de DNA codificadoras em sistema procarioto (E. coli). Além disso, formas atóxicas da toxina termo-lábil (LTG33D e LTK63) de E. coli enterotoxigência (ETEC) foram obtidas e incorporadas como adjuvantes às formulações vacinais. As respostas celulares e humorais anti-NS1 e anti-EIII foram monitoradas por ELISA para anticorpos e citocinas, ICS (do inglês intracellular citokine staining) e atividade citotóxica in vivo. Observamos que animais imunizados com a NS1 recombinante adicionada da LTG33D foram capazes de gerar respostas imunológicas com produção de anticorpos específicos e alta afinidade pelo antígeno. Em ensaios de desafio realizados para avaliar a proteção vacinal conferida à infecção por uma linhagem referência do o VD tipo 2 (NGC) observamos que essa formulação conferiu uma proteção de 50% aos animais imunizados. Paralelamente a esses resultados, demonstramos que a EIII não é um bom antígeno vacinal e que pode induzir anticorpos capazes de acentuar a infecção do VD. Descrevemos ainda a obtenção e a caracterização genética e patológica de um isolado clínico de VD tipo 2 naturalmente letal para camundongos Balb/C. A nova cepa viral (JHA1) demonstrou ser capaz de induzir perda de peso corporal, dano tecidual geral, e distúrbios hematológicos similares aos observados em humanos infectados pelo VD, podendo ser aplicada como modelo de infecção na avaliação de candidatos vacinais. Os resultados obtidos neste trabalho representam uma importante contribuição na área de desenvolvimento de estratégias vacinais contra a dengue e representam uma base importante para futuros estudos sobre a patologia da dengue. The present study proposes the development and characterization of a strategy for prophylactic vaccination against dengue virus (VD) based on the NS1 protein and the domain III of the envelope glycoprotein (EIII), using recombinant proteins in subcutaneous immunization in a murine model. These antigens were obtained by cloning and expression of their DNA coding sequences in prokaryotic system (E. coli). In addition, the s non-toxic forms of the heat-labile toxin from enterotoxigenic E. coli (ETEC) (LTK63 and LTG33D) were obtained and incorporated as adjuvants to vaccine formulations. Anti-NS1 and anti-EIII cellular and humoral immune responses were monitored by antibody and cytokine ELISA, , intracellular citokine staining (ICS) and in vivo cytotoxic activity. We observed that animals immunized with the recombinant NS1 and LTG33D were capable to induce immune responses including specific antibodies with high affinity for the antigen. In challenge assays performed to evaluate the immunization protective efficacy such vaccine conferred protection of 50% against infection with a reference type 2 VD (VD2) strain(NGC). Alongside to these results, we demonstrated that EIII is not a good vaccine antigen and can induce the generation of antibodies that enhance DENV infection. We also described the isolation and the genetic and pathological characterization of a VD2 clinical isolate naturally lethal to immunocompetent Balb/c mice. The new strain was shown to cause weight loss, general tissue damage, and hematological disturbances similar to those observed in VDinfected humans, and therefore, may be applied as infection model to evaluate vaccine candidates. The results obtained in this study represent an important contribution to DENV vaccine development and established an important background for future studies of the dengue pathology.