Combination of E- and NS1-Derived DNA Vaccines: The Immune Response and Protection Elicited in Mice against DENV2.

The occurrence of dengue disease has increased radically in recent decades. Previously, we constructed the pE1D2 and pcTPANS1 DNA vaccines encoding the DENV2 envelope (E) and non-structural 1 (NS1) proteins, respectively. To decrease the number of plasmids in a tetravalent candidate vaccine, we constructed a bicistronic plasmid, pNS1/E/D2, encoding these two proteins simultaneously. We evaluated the protective immunity induced in mice vaccinated with the pNS1/E/D2 candidate and compared to the responses elicited by immunization with the former vaccines isolated or in combination. We transfected BHK-21 cells with the different plasmids and detected recombinant proteins by immunofluorescence and mass spectrometry assays to confirm antigen expression. BALB/c mice were inoculated with the DNA vaccines followed by a lethal DENV2 challenge. ELISA, PRNT50, and IFN-gamma ELISPOT assays were performed for the investigation of the humoral and cellular responses. We observed the concomitant expression of NS1 and E proteins in pNS1/E/D2-transfected cells. All E-based vaccines induced anti-E and neutralizing antibodies. However, anti-NS1 antibodies were only observed after immunization with the pcTPANS1 administered alone or combined with pE1D2. In contrast, splenocytes from pNS1/E/D2- or pcTPANS1 + pE1D2-vaccinated animals responded to NS1- and E-derived synthetic peptides. All the DNA vaccines conferred protection against DENV2.

T Cell Responses Induced by DNA Vaccines Based on the DENV2 E and NS1 Proteins in Mice: Importance in Protection and Immunodominant Epitope Identification.

The importance of the cellular immune response against DENV has been increasingly highlighted in the past few years, in particular for vaccine development. We have previously constructed two plasmids, pE1D2, and pcTPANS1, encoding the envelope (E) ectodomain (domains I, II, and III) and the non-structural 1 (NS1) protein of dengue virus serotype 2 (DENV2), respectively. In the present work, we analyzed the induction of the cellular response in mice immunized with these DNA vaccines and identified the immunogenic peptides. Vaccinated BALB/c mice became protected against a lethal challenge of DENV2. Depletion of CD4+ cells in vaccinated animals almost completely abolished protection elicited by both vaccines. In contrast, a significant number of pE1D2- and pcTPANS1-immunized mice survived virus challenge after depletion of CD8+ cells, although some animals presented morbidity. To identify immunogenic peptides recognized by T cells, we stimulated splenocytes with overlapping peptide libraries covering the E and NS1 proteins and evaluated the production of IFN-γ by ELISPOT. We detected two and three immunodominant epitopes in the E and NS1 proteins, respectively, and four additional NS1-derived peptides after virus challenge. Characterization by intracellular cytokine staining (ICS) revealed that both CD4+ and CD8+ T cells were involved in IFN-γ and TNF-α production. The IFN-γ ICS confirmed reaction of almost all E-derived peptides before challenge and identified other epitopes after infection. All NS1-derived peptides were able to elicit IFN-γ production in CD4+ cells, while only a few peptides induced expression of this cytokine in CD8+ T lymphocytes. Interestingly, we observed an increase in the frequency of either CD4+ or CD8+ T cells producing TNF-α after immunization with the pE1D2 and challenge with DENV2, while lymphocytes from pcTPANS1-vaccinated animals maintained ordinary TNF-α production after virus infection. We also assessed the recognition of E and NS1 immunogenic peptides in C57BL/6 mice due to the difference in MHC haplotype expression. Two NS1-derived epitopes featured prominently in the IFN-γ response with cells from both animal strains. Overall, our results emphasize the importance of the T cell response involved in protection against dengue induced by E and NS1 based DNA vaccines.

The effect of the dengue non-structural 1 protein expression over the HepG2 cell proteins in a proteomic approach.

Dengue is an important mosquito borne viral disease in the world. Dengue virus (DENV) encodes a polyprotein, which is cleaved in ten proteins, including the non-structural protein 1 (NS1). In this work, we analyzed the effect of NS1 expression in one hepatic cell line, HepG2, through a shotgun proteomic approach. Cells were transfected with pcENS1 plasmid, which encodes the DENV2 NS1 protein, or the controls pcDNA3 (negative control) and pMAXGFP (GFP, a protein unrelated to dengue). Expression of NS1 was detected by immunofluorescence, western blot and flow cytometry. We identified 14,138 peptides that mapped to 4,756 proteins in all analyzed conditions. We found 41 and 81 differentially abundant proteins when compared to cells transfected with plasmids pcDNA3 and pMAXGFP, respectively. Besides, 107 proteins were detected only in the presence of NS1. We identified clusters of proteins involved mainly in mRNA process and viral RNA replication. Down regulation expression of one protein (MARCKS), identified by the proteomic analysis, was also confirmed by real time PCR in HepG2 cells infected with DENV2. Identification of proteins modulated by the presence of NS1 may improve our understanding of its role in virus infection and pathogenesis, contributing to development of new therapies and vaccines. BIOLOGICAL SIGNIFICANCE: Dengue is an important viral disease, with epidemics in tropical and subtropical regions of the world. The disease is complex, with different manifestations, in which the liver is normally affected. The NS1 is found in infected cells associated with plasma membrane and secreted into the circulation as a soluble multimer. This protein is essential for virus viability, although its function is not elucidated. Some reports indicate that the NS1 can be used as a protective antigen for the development of a dengue vaccine, while others suggest its involvement in viral pathogenesis. In this work, we report an in-depth comprehensive proteomic profiling resulting from the presence of NS1 in HepG2 cells. These results can contribute to a better understanding of the NS1 role during infection.

Dynamic identification of H2 epitopes from Leishmania (Leishmania) amazonensis cysteine proteinase B with potential immune activity during murine infection.

Peptides from the COOH-terminal extension of cysteine proteinase B from Leishmania (Leishmania) amazonensis (cyspep) can modulate immune responses in vertebrate hosts. With this hypothesis as base, we used the online analysis tool SYFPEITHI to predict seven epitopes from this region with potential to bind H2 proteins. We performed proliferation tests and quantified reactive T lymphocytes applying a cytometry analysis, using samples from draining lymph node of lesions from L. (L.) amazonensis-infected mice. To define reactivity of T cells, we used complexes of DimerX (H2 D(b):Ig and H2 L(d):Ig) and the putative epitopes. Additionally, we applied surface plasmon resonance to verify real time interactions between the putative epitopes and DimerX proteins. Five peptides induced blastogenesis in BALB/c cells, while only two presented the same property in C57BL/6 mouse cells. In addition, our data indicate the existence of CD8+ T lymphocyte populations able to recognize each tested peptide in both murine strains. We observed an overlapping of results between the peptides that induced lymphocyte proliferation and those capable of binding to the DimerX in the surface plasmon resonance assays thus indicating that using these recombinant proteins in biosensing analyses is a promising tool to study real time molecular interactions in the context of major histocompatibility complex epitopes. The data gathered in this study reinforce the hypothesis that cyspep-derived peptides are important factors in the murine host infection by L. (L.) amazonensis.

SYBR Green-based real-time PCR targeting kinetoplast DNA can be used to discriminate between the main etiologic agents of Brazilian cutaneous and visceral leishmaniases.

BACKGROUND: Leishmaniases control has been hampered by the unavailability of rapid detection methods and the lack of suitable therapeutic and prophylactic measures. Accurate diagnosis, which can distinguish between Leishmania isolates, is essential for conducting appropriate prognosis, therapy and epidemiology. Molecular methods are currently being employed to detect Leishmania infection and categorize the parasites up to genus, complex or species level. Real-time PCR offers several advantages over traditional PCR, including faster processing time, higher sensitivity and decreased contamination risk. RESULTS: A SYBR Green real-time PCR targeting the conserved region of kinetoplast DNA minicircles was able to differentiate between Leishmania subgenera. A panel of reference strains representing subgenera Leishmania and Viannia was evaluated by the derivative dissociation curve analyses of the amplified fragment. Distinct values for the average melting temperature were observed, being 78.95 °C ± 0.01 and 77.36 °C ± 0.02 for Leishmania and Viannia, respectively (p < 0.05). Using the Neighbor-Joining method and Kimura 2-parameters, the alignment of 12 sequences from the amplified conserved minicircles segment grouped together L. (V.) braziliensis and L. (V.) shawii with a bootstrap value of 100%; while for L. (L.) infantum and L. (L.) amazonensis, two groups were formed with bootstrap values of 100% and 62%, respectively. The lower dissociation temperature observed for the subgenus Viannia amplicons could be due to a lower proportion of guanine/cytosine sites (43.6%) when compared to species from subgenus Leishmania (average of 48.4%). The method was validated with 30 clinical specimens from visceral or cutaneous leishmaniases patients living in Brazil and also with DNA samples from naturally infected Lutzomyia spp. captured in two Brazilian localities. CONCLUSIONS: For all tested samples, a characteristic amplicon melting profile was evidenced for each Leishmania subgenus, corroborating the data from reference strains. Therefore, the analysis of thermal dissociation curves targeting the conserved kinetoplast DNA minicircles region is able to provide a rapid and reliable method to identify the main etiologic agents of cutaneous and visceral leishmaniases in endemic regions of Brazil.

In silico predicted epitopes from the COOH-terminal extension of cysteine proteinase B inducing distinct immune responses during Leishmania (Leishmania) amazonensis experimental murine infection.

BACKGROUND: Leishmania parasites have been reported to interfere and even subvert their host immune responses to enhance their chances of survival and proliferation. Experimental Leishmania infection in mice has been widely used in the identification of specific parasite virulence factors involved in the interaction with the host immune system. Cysteine-proteinase B (CPB) is an important virulence factor in parasites from the Leishmania (Leishmania) mexicana complex: it inhibits lymphocytes Th1 and/or promotes Th2 responses either through proteolytic activity or through epitopes derived from its COOH-terminal extension. In the present study we analyzed the effects of Leishmania (Leishmania) amazonensis CPB COOH-terminal extension-derived peptides on cell cultures from murine strains with distinct levels of susceptibility to infection: BALB/c, highly susceptible, and CBA, mildly resistant. RESULTS: Predicted epitopes, obtained by in silico mapping, displayed the ability to induce cell proliferation and expression of cytokines related to Th1 and Th2 responses. Furthermore, we applied in silico simulations to investigate how the MHC/epitopes interactions could be related to the immunomodulatory effects on cytokines, finding evidence that specific interaction patterns can be related to in vitro activities. CONCLUSIONS: Based on our results, we consider that some peptides from the CPB COOH-terminal extension may influence host immune responses in the murine infection, thus helping Leishmania survival.

Cysteine proteinases from promastigotes of Leishmania (Viannia) braziliensis.

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.

Purification and Characterization of a Population of EGFP-Expressing Cells from the Developing Pancreas of a Neurogenin3/EGFP Transgenic Mouse.

Neurogenin 3 (ngn3) is a basic helix loop helix transcription factor that is transiently expressed in the developing mouse pancreas with peak expression around E15. In mice lacking the ngn3 gene the endocrine cells of the pancreas fail to develop suggesting that the ngn3-positive cell may represent a progenitor cell for the endocrine pancreas. In order to purify and characterize this cell in detail we have generated a transgenic mouse, in which the ngn3 promoter drives expression of enhanced green fluorescent protein (EGFP). In the E15.5 embryo EGFP was expressed in the dorsal and ventral pancreas, the duodenum, and lower intestine as well as in the brain. This pattern of expression was in keeping with the known expression profile of the endogenous ngn3 gene. Within the pancreas EGFP was localized in close proximity to cells that stained positive for ngn3, insulin, and glucagon, but was absent from regions of the pancreas that stained positive for amylase. EGFP was also present in the pancreas at E18.5, although there was no detectable expression of ngn3. At this stage EGFP did not colocalize with any of the hormones or exocrine markers. EGFP(+) cells were FACS purified (96%) from the E15 pancreas yielding approximately 10,000 cells or 1.6% of the total pancreatic cells from one litter. RT/PCR analysis confirmed that the purified cells expressed EGFP, ngn3, insulin, glucagon, somatostatin and pancreatic polypeptide. The ability to purify ngn3(+) cells provides an invaluable source of material for charactering in detail their properties.