Diversity, composition, and networking of saliva microbiota distinguish the severity of COVID-19 episodes as revealed by an analysis of 16S rRNA variable V1-V3 region sequences.

Studies on the role of the oral microbiome in SARS-CoV-2 infection and severity of the disease are limited. We aimed to characterize the bacterial communities present in the saliva of patients with varied COVID-19 severity to learn if there are differences in the characteristics of the microbiome among the clinical groups. We included 31 asymptomatic subjects with no previous COVID-19 infection or vaccination; 176 patients with mild respiratory symptoms, positive or negative for SARS-CoV-2 infection; 57 patients that required hospitalization because of severe COVID-19 with oxygen saturation below 92%, and 18 fatal cases of COVID-19. Saliva samples collected before any treatment were tested for SARS-CoV-2 by PCR. Oral microbiota in saliva was studied by amplification and sequencing of the V1-V3 variable regions of 16S gene using an Illumina MiSeq platform. We found significant changes in diversity, composition, and networking in saliva microbiota of patients with COVID-19, as well as patterns associated with severity of disease. The presence or abundance of several commensal species and opportunistic pathogens were associated with each clinical stage. Patterns of networking were also found associated with severity of disease: a highly regulated bacterial community (normonetting) was found in healthy people whereas poorly regulated populations (disnetting) were characteristic of severe cases. Characterization of microbiota in saliva may offer important clues in the pathogenesis of COVID-19 and may also identify potential markers for prognosis in the severity of the disease. IMPORTANCE SARS-CoV-2 infection is the most severe pandemic of humankind in the last hundred years. The outcome of the infection ranges from asymptomatic or mild to severe and even fatal cases, but reasons for this remain unknown. Microbes normally colonizing the respiratory tract form communities that may mitigate the transmission, symptoms, and severity of viral infections, but very little is known on the role of these microbial communities in the severity of COVID-19. We aimed to characterize the bacterial communities in saliva of patients with different severity of COVID-19 disease, from mild to fatal cases. Our results revealed clear differences in the composition and in the nature of interactions (networking) of the bacterial species present in the different clinical groups and show community-patterns associated with disease severity. Characterization of the microbial communities in saliva may offer important clues to learn ways COVID-19 patients may suffer from different disease severities.

Temporopolar amygdalohippocampectomy: seizure control and postoperative outcomes.

OBJECTIVE: The objective of this study was to evaluate the efficacy and safety of a modified surgical approach for the treatment of temporal lobe epilepsy secondary to hippocampal sclerosis (HS). This modified approach, called temporopolar amygdalohippocampectomy (TP-AH), includes a transsylvian resection of the temporal pole and subsequent amygdalohippocampectomy utilizing the limen insula as an anatomical landmark. METHODS: A total of 61 patients who were diagnosed with HS and underwent TP-AH between 2013 and 2017 were enrolled. Patients performed pre- and postoperative diffusion tensor imaging and were classified according to Engel's scale for seizure control. To evaluate the functional preservation of the temporal stem white-matter fiber tracts, the authors analyzed postoperative Humphrey perimetries and pre- and postoperative neurocognitive performance (Rey Auditory Verbal Learning Test [RAVLT], Weschler Memory Scale-Revised [WMS-R], intelligence quotient [IQ], Boston Naming Test [BNT], and semantic and phonemic fluency). Demographic data and surgical complications were also recorded and described. RESULTS: After a median follow-up of 36 ± 16 months, 46 patients (75.4%) achieved Engel class I, of whom 37 (60.6%) were Engel class IA. No significant changes in either the inferior frontooccipital fasciculus and optic radiation tractography were observed postoperatively for both left- and right-side surgeries. Reliable perimetry was obtained in 40 patients (65.6%), of whom 27 (67.5%) did not present any visual field defects (VFDs) attributable to surgery, while 12 patients (30%) presented with quadrant VFD, and 1 patient (2.5%) presented with hemifield VFD. Despite a significant decline in verbal memory (p = 0.007 for WMS-R, p = 0.02 for RAVLT recognition), there were significant improvements in both IQ (p < 0.001) and visual memory (p = 0.007). Semantic and phonemic fluency, and scores on the BNT, did not change postoperatively. CONCLUSIONS: TP-AH provided seizure control similar to historical temporal lobe approaches, with a tendency to preserve the temporal stem and a satisfactory incidence of VFD. Despite a significant decline in verbal memory, there were significant improvements in both IQ and visual memory, along with preservation of executive function. This approach can be considered a natural evolution of the selective transsylvian approach.

Real-life Outcomes on Acute Promyelocytic Leukemia in Brazil - Early Deaths Are Still a Problem.

INTRODUCTION: Although a considerable improvement in survival of patients with acute promyelocytic leukemia (APL) has been seen over the past decades, real-life outcomes seem to be worse than those reported by prospective studies. We aim to describe clinical characteristics and outcomes of adult patients diagnosed with APL in an academic hospital from the University of Sao Paulo. PATIENTS AND METHODS: We retrospectively reviewed the medical charts of 61 patients with APL diagnosed between January 2007 and May 2017. Baseline clinical features and follow-up data were collected, focusing on early toxicity variables such as infection, bleeding, and thrombosis in the first 30 days from diagnosis. RESULTS: Among the 61 patients with APL, 54 received any chemotherapy. All patients also received all-trans retinoic acid (ATRA). Bleeding events were the main cause of death before receiving chemotherapy. Most patients belonged to the intermediate (43%) and high-risk (41%) groups, according to Sanz score. The '7 + 3 + ATRA' regimen was the most used regimen (n = 38). An early death rate of 20% was found, predominantly owing to sepsis. After a median follow-up of 5 years, only 1 relapse was diagnosed. The overall survival at 5 years was 59%. DISCUSSION: In comparison with prospective trials with ATRA-based regimens, we found an inferior overall survival, mostly on account of a high early-death rate. Our results are in line with other real-life retrospective reports published in the past decades. CONCLUSION: Results of real-life studies differ from those found by prospective trials. Accordingly, early actions and supportive care are still needed, aiming to decrease toxicity, especially in developing countries.

CUL5 is required for thalidomide-dependent inhibition of cellular proliferation.

Angiogenesis is essential for cancer metastasis, thus the discovery and characterization of molecules that inhibit this process is important. Thalidomide is a teratogenic drug which is known to inhibit angiogenesis and effectively inhibit cancer metastasis, yet the specific cellular targets for its effect are not well known. We discovered that CUL5 (previously identified as VACM-1), a scaffold protein in E3 ligase complexes, is involved in thalidomide-dependent inhibition of endothelial cell growth. Our results show that in human endothelial cells (HUVEC), thalidomide-dependent decrease in cell growth was associated with decreased nuclear localization of CUL5. In HUVEC transfected with anti-VACM-1 siRNA, thalidomide failed to decrease cell growth. Previously it was established that the antiproliferative effect of CUL5 is inhibited in rat endothelial cells (RAMEC) transfected with mutated CUL5 which is constitutively modified by NEDD8, a ubiquitin-like protein. In this study, the antiproliferative response to thalidomide was compromised in RAMEC expressing mutated CUL5. These results suggest that CUL5 protein is involved in the thalidomide-dependent regulation of cellular proliferation in vitro. Consequently, CUL5 may be an important part of the mechanism for thalidomide-dependent inhibition of cellular proliferation, as well as a novel biomarker for predicting a response to thalidomide for the treatment of disorders such as multiple myeloma and HIV infection.

Zebrafish Caudal Haematopoietic Embryonic Stromal Tissue (CHEST) Cells Support Haematopoiesis.

Haematopoiesis is an essential process in early vertebrate development that occurs in different distinct spatial locations in the embryo that shift over time. These different sites have distinct functions: in some anatomical locations specific hematopoietic stem and progenitor cells (HSPCs) are generated de novo. In others, HSPCs expand. HSPCs differentiate and renew in other locations, ensuring homeostatic maintenance. These niches primarily control haematopoiesis through a combination of cell-to-cell signalling and cytokine secretion that elicit unique biological effects in progenitors. To understand the molecular signals generated by these niches, we report the generation of caudal hematopoietic embryonic stromal tissue (CHEST) cells from 72-hours post fertilization (hpf) caudal hematopoietic tissue (CHT), the site of embryonic HSPC expansion in fish. CHEST cells are a primary cell line with perivascular endothelial properties that expand hematopoietic cells in vitro. Morphological and transcript analysis of these cultures indicates lymphoid, myeloid, and erythroid differentiation, indicating that CHEST cells are a useful tool for identifying molecular signals critical for HSPC proliferation and differentiation in the zebrafish. These findings permit comparison with other temporally and spatially distinct haematopoietic-supportive zebrafish niches, as well as with mammalian haematopoietic-supportive cells to further the understanding of the evolution of the vertebrate hematopoietic system.

Dataset on protein composition of a human plasma sub-proteome able to modulate the Dengue 2 virus infection in Huh 7.5 cells.

The four serotypes of dengue virus (DENV1-4) are the causal agents of the emerging disease Dengue Fever and its severe forms. DENV is inoculated into human blood through a mosquito bite. Thus, plasma is an important media for DENV dissemination in infected persons and several important interactions should take place for the virus with human plasma proteins that strongly influence or may determine the course of the infection. This dataset contains 239 proteins identified in the elution fractions of human plasma subjected to DE-52 anion exchange chromatography. Data on DENV2 infection of Huh 7.5 cells in presence of the human plasma fraction is also presented.

Novel interactions of domain III from the envelope glycoprotein of dengue 2 virus with human plasma proteins.

Blood cells and plasma are important media for the four serotypes of dengue virus (DENV1-4) spreading into an infected person. Thus, interactions with human plasma proteins are expected to be decisive in the course of the viral infection. Affinity purification followed by MS analysis (AP/MS) was used to isolate and identify plasma-derived proteins capable to interact with a recombinant protein comprising the domain III of the envelope protein of DENV2 (DIIIE2). The elution of the AP potently inhibits DENV2 infection. Twenty-nine proteins were identified using a label-free approach as specifically captured by DIIIE2. Of these, a direct interaction with C reactive protein, thrombin and Inter-alpha-inhibitor complexes was confirmed by ELISA. Results provide further evidence of a significant representation of proteins from complement and coagulation cascades on DENV2 interactome in human plasma and stand out the domain III of the viral envelope protein as participant on these interactions. A functional clustering analysis highlights the presence of three structural motifs among putative DIIIE2-binding proteins: hydroxylation and EGF-like calcium-binding- and Gla domains. BIOLOGICAL SIGNIFICANCE: Early cycles of dengue virus replication take place in human blood cells. Thus, the characterization of the interactome of dengue virus proteins in human plasma can lead to the identification of pivotal interactions for the infection that can eventually constitute the target for the development of methods to control dengue virus-caused disease. In this work we identified 29 proteins from human plasma that potentially interact with the envelope protein of dengue 2 virus either directly or through co-complex formation. C reactive protein, thrombin and Inter-alpha-inhibitor complexes were validated as interactors of the domain III of the envelope protein of dengue 2. Results highlight the presence of three structural motifs among putative DIIIE2-binding proteins: hydroxylation and EGF-like calcium-binding- and Gla domains. This finding together with the participation of domain III of the envelope protein on the interactions with human plasma proteins should contribute to a better understanding of dengue virus interactome in human plasma. Such knowledge can contribute to the development of more effective treatments to infected persons.

Receptor-activated human α2-macroglobulin interacts with the envelope protein of dengue virus and protects virions from temperature-induced inactivation through multivalent binding.

Based on the hypothesis that interactions between virions and serum components may influence the outcome of dengue virus (DENV) infections, we decided to use affinity chromatography with domain III from the envelope (E) protein of DENV2 (DIIIE2) as a ligand to isolate virus-binding proteins from human plasma. This approach yielded serum amyloid P (SAP) and α2-macroglobulin (α2M) as novel viral interactors. After confirming the specific binding of both SAP and α2M to DIIIE2 by ELISA, the latter interaction was examined in greater detail. We obtain evidence suggesting that the binding species was actually the receptor-activated form of α2M (α2M*), that α2M* could bind monovalently to recombinant domain III from all four DENV serotypes with affinities in the micromolar range ranking as DENV4>DENV1~DENV2>DENV3 and that this interaction exhibited a strong avidity effect when multivalent binding was favoured (KD 8 × 10(-8) M for DIIIE2). We also showed that α2M* bound to DENV virions of the four serotypes, protecting the virus from temperature-induced inactivation in the absence of serum and enhancing infectivity. The latter effect exhibited an ED50 of 2.9 × 10(-8) M, also suggesting an avidity effect due to multivalent binding. These results will further contribute to the characterization of the virus-host factor interaction network during human DENV infection.

Leukocyte attraction by CCL20 and its receptor CCR6 in humans and mice with pneumococcal meningitis.

We previously identified CCL20 as an early chemokine in the cerebrospinal fluid (CSF) of patients with pneumococcal meningitis but its functional relevance was unknown. Here we studied the role of CCL20 and its receptor CCR6 in pneumococcal meningitis. In a prospective nationwide study, CCL20 levels were significantly elevated in the CSF of patients with pneumococcal meningitis and correlated with CSF leukocyte counts. CCR6-deficient mice with pneumococcal meningitis and WT mice with pneumococcal meningitis treated with anti-CCL20 antibodies both had reduced CSF white blood cell counts. The reduction in CSF pleocytosis was also accompanied by an increase in brain bacterial titers. Additional in vitro experiments showed direct chemoattractant activity of CCL20 for granulocytes. In summary, our results identify the CCL20-CCR6 axis as an essential component of the innate immune defense against pneumococcal meningitis, controlling granulocyte recruitment.

Th2-Th1 shift with the multiantigenic formulation TERAVAC-HIV-1 in Balb/c mice.

In chronic HIV infection a progressive Th1 to Th2/Th0 cytokine-profile shift is related to disease progression. One of the possible benefits of a therapeutic vaccination might be to counterbalance this phenomenon to allow viral replication control under a Th1-type immune response. TERAVAC-HIV-1 is a multiantigenic formulation vaccine candidate against HIV-1 which comprises the recombinant protein CR3 that contains T cell epitopes and the surface and nucleocapsid antigens of Hepatitis B Virus (HBV). Previous studies showed that such virus like particles of the HBV provide a Th1 adjuvant effect. The present studies examined the capacity of TERAVAC to elicit a Th1 response in the presence of an ongoing HIV-specific Th2-type response in Balb/c mice. To examine this issue, we injected subcutaneously the animals with CR3 or viral lysate in alum which resulted in a Th2-type response. The CR3-specific Th2-type response was verified by induction of IL-4 and IL-10 secretion in ex vivo stimulated splenocytes without secretion of IFN-γ and IgG2a antibodies in serum. Further subcutaneous and simultaneous subcutaneous-nasal immunizations of the same mice with TERAVAC promoted IFN-γ secretion and production of IgG2a antibodies in accordance with a Th1-type response. This result suggests a therapeutic benefit of this vaccine candidate in the restoration of the Th1-type HIV-specific cellular response in seropositive patients.

Self-medication in ophthalmology: a questionnaire-based study in an Argentinean population.

PURPOSE: The aim of this study was to identify practices of self-medication in the treatment of ocular conditions and to identify a profile of patients who self-medicate. METHODS: We conducted a cross-sectional descriptive survey of patients, over the age of 17 years seen in our ophthalmology practice in Cordoba, Argentina. Self-medication was defined as the use of ophthalmic medicines which had not been prescribed by a health care specialist in the previous year. RESULTS: The sample included 379 subjects, 162 males (43%) and 217 females (57%); mean age 46.8 years. Prior to looking for medical attention in our institution, 97 patients (25.6%) reported self-medicating. The most frequently employed products included non-steroidal anti-inflammatory drops in combination with a vasoconstrictive agent (32%) followed by a combination of antibiotics and steroids (9%), however, 14% of patients did not remember the name or type of medication applied. A total of 31% of patients used drugs recommended by a pharmacist; 25% used drugs of their own choosing and 24% followed suggestions from a friend or family member. Only 12% of patients knew the drug's components and only 3% were aware of any possible side effects. There was no difference in behavior patterns related to educational level or age, however, there was a significant difference related to gender, with males misusing ophthalmic drops more frequently than women (P = 0.004). CONCLUSIONS: Patients commonly attempt to treat conditions that require ophthalmologic care by self-medicating with over-the-counter eye drops. Educational efforts to inform patients of the consequences of self-medication are necessary.

Regional neural activation defines a gateway for autoreactive T cells to cross the blood-brain barrier.

Although it is believed that neural activation can affect immune responses, very little is known about the neuroimmune interactions involved, especially the regulators of immune traffic across the blood-brain barrier which occurs in neuroimmune diseases such as multiple sclerosis (MS). Using a mouse model of MS, experimental autoimmune encephalomyelitis, we show that autoreactive T cells access the central nervous system via the fifth lumbar spinal cord. This location is defined by IL-6 amplifier-dependent upregulation of the chemokine CCL20 in associated dorsal blood vessels, which in turn depends on gravity-induced activation of sensory neurons by the soleus muscle in the leg. Impairing soleus muscle contraction by tail suspension is sufficient to reduce localized chemokine expression and block entry of pathogenic T cells at the fifth lumbar cord, suggesting that regional neuroimmune interactions may offer therapeutic targets for a variety of neurological diseases.

Two mucosal-parenteral schedules to coadminister a multiantigenic formulation against HIV-1 in Balb/c mice.

Previous studies showed that simultaneous immunization through the nasal (IN) and subcutaneous (SC) route of a multiantigenic formulation induced a Th1 anti-HIV humoral and cellular immune responses. The formulation was comprised of a recombinant protein of HIV-1 (named CR3; Cellular Response number 3) and the surface and nucleocapsid antigens of hepatitis B virus. This study asks whether four times simultaneous administration through the IN and SC routes (SC+IN) of the multiantigenic formulation induces a similar systemic and mucosal immune responses than two sequential IN priming and two SC boosting (2IN&2SC) inoculations in mice. To answer this question, we tested the same total dose of each antigen per animal in both schedules of inoculation. We found that SC+IN and 2IN&2SC coadministration induced comparable levels of CR3(HIV)-specific IFN-γ-secreting cells and CD8+ cells proliferation in the systemic compartment of animals. Consistent with these findings, a similar Th1 profile considering anti-CR3 IgG1:IGg2a ratio was observed. Additionally, the level of IgG antibodies and the frequency of seroconverting animals in vagina were not different. However, in the case of IgA antibodies the same parameters were significantly higher in the SC+IN group. We also found important level of HBsAg-specific antibodies in serum and vaginal washes.

Digestive enzymatic activity on tropical gar (Atractosteus tropicus) larvae fed different diets.

Digestive enzymatic activity and growth performance on tropical gar (Atractosteus tropicus) larvae fed Artemia nauplii (LF), frozen adult Artemia (AB), an artificial diet (AF) with 46% protein and 16% lipids and a starvation group (SG) from first feeding (5 days after hatching-5 DAH) to 34 DAH were studied. All larvae under starvation (SG) died at 15 DAH. By the end of the experimental period, morphological variables (total length, wet weight and specific growth rate) were significant in larvae fed AF compared to LF and AB. All enzymes studied in the experiment were present since the start of exogenous feeding (including pepsin) and the enzymatic activity varied with the diets. Low levels of enzymatic activity were observed until the 29 DAH; however, after this moment, there was a significant increase (eightfold), particularly for the AF treatment. In vitro protein digestibility tests performed with enzymatic extracts showed that artificial diets with 52% protein and 14% lipids were better digested by larvae before 30 DAH, while diets with 45% protein and 11% lipids were better digested after this age. Taking into account the better growth performance, higher enzymatic activity and better protein digestibility obtained, artificial diets can be used since the start of exogenous feeding on tropical gar larvae, as in other lepisosteids.

Resveratrol enhances anti-proliferative effect of VACM-1/cul5 in T47D cancer cells.

Vasopressin-activated calcium-mobilizing (VACM-1) protein is a cul-5 gene product that forms complexes with a subclass of ubiquitin E3 ligases involved in proteasomal protein degradation. The expression of VACM-1 cDNA in the T47D breast cancer cell line inhibits growth and decreases phosphorylation of mitogen activated protein kinase. Factors that regulate expression or stability of VACM-1 protein have not been identified, however. In our search to identify drugs/substances that may control VACM-1 protein expression, we examined the effects of resveratrol (trans-3,5,4'-trihydroxystilbene), a natural component in the human diet which inhibits tumor initiation and promotion. CMV vector and VACM-1 cDNA stably transfected T47D breast cancer-derived cells were treated with resveratrol and cell growth and VACM-1 protein concentrations were measured. Since the cellular mechanism of resveratrol-dependent inhibition of cell growth also involves the regulation of estrogen receptors, the effect of 17-ß-estradiol and resveratrol on ERα levels and on cell growth was examined in control and in VACM-1 cDNA transfected cells. Our results demonstrate that antiproliferative effect of resveratrol observed in the control T47D cancer cells was significantly enhanced in VACM-1 cDNA transfected T47D cells. Western blot results indicated that resveratrol increased VACM-1 protein concentration. Finally, treatment with resveratrol for 24 and 48 h attenuated 17-ß-estradiol induced increase in cell growth both in control and in VACM-1 cDNA transfected cells. The effect was significantly higher in the VACM-1 cDNA transfected cells when compared to controls. These results indicate that the antiproliferative effect of resveratrol may involve induction of VACM-1/cul5.

Phosphorylation of VACM-1/Cul5 by protein kinase A regulates its neddylation and antiproliferative effect.

Expression of the VACM-1/cul5 gene in endothelial and in cancer cell lines in vitro inhibits cellular proliferation and decreases phosphorylation of MAPK. Structure-function analysis of the VACM-1 protein sequence identified consensus sites specific for phosphorylation by protein kinases A and C (PKA and PKC) and a Nedd8 protein modification site. Mutations at the PKA-specific site in VACM-1/Cul5 ((S730A)VACM-1) sequence resulted in increased cellular growth and the appearance of a Nedd8-modified VACM-1/Cul5. The aim of this study was to examine if PKA-dependent phosphorylation of VACM-1/Cul5 controls its neddylation status, phosphorylation by PKC, and ultimately growth. Our results indicate that in vitro transfection of rat adrenal medullary endothelial cells with anti-VACM-1-specific small interfering RNA oligonucleotides decreases endogenous VACM-1 protein concentration and increases cell growth. Western blot analysis of cell lysates immunoprecipitated with an antibody directed against a PKA-specific phosphorylation site and probed with anti-VACM-1-specific antibody showed that PKA-dependent phosphorylation of VACM-1 protein was decreased in cells transfected with (S730A)VACM-1 cDNA when compared with the cytomegalovirus-transfected cells. This change was associated with increased modification of VACM-1 protein by Nedd8. Induction of PKA activity with forskolin reduced modification of VACM-1 protein by Nedd8. Finally, rat adrenal medullary endothelial cells transfected with (S730A)VACM-1/cul5 cDNA and treated with phorbol 12-myristate 13-acetate (10 and 100 nm) to induce PKC activity grew significantly faster than the control cells. These results suggest that the antiproliferative effect of VACM-1/Cul5 is dependent on its posttranslational modifications and will help in the design of new anticancer therapeutics that target the Nedd8 pathway.

Immunization with a DNA vaccine candidate in chronic hepatitis C patients is safe, well tolerated and does not impair immune response induction after anti-hepatitis B vaccination.

BACKGROUND: In the present study, we evaluated the safety of CIGB-230, a novel vaccine candidate based on the mixture of a plasmid for DNA immunization, expressing hepatitis C virus (HCV) structural antigens, with a recombinant HCV Core protein. METHODS: Fifteen HCV chronically-infected volunteers with detectable levels of HCV RNA genotype 1b, who were nonresponders to previous treatment with interferon plus ribavirin, were intramuscularly injected with CIGB-230 on weeks 0, 4, 8, 12, 16 and 20. Individuals were also immunized at weeks 28, 32 and 36 with a recombinant vaccine against hepatitis B. Adverse events were recorded and analyzed. Blood samples were taken every 4 weeks up to month 12 for hematological, biochemical, virological and immunological analysis. RESULTS: All patients completed the treatment with CIGB-230. Adverse events were only slight (83.6%) or moderate (16.4%). No significant differences in hematological and biochemical parameters, including serum aminotransferases, were detected between the baseline and post-treatment state. Induction of a CD4+ T lymphocyte response against a particular region in HCV E1, spanning amino acids 230-312 in HCV polyprotein, was detected in 42.8% of patients during treatment with CIGB-230. The ability of T cells to proliferate in response to mitogenic stimulation was not weakened. Most individuals (78.6%) were seroprotected after anti-hepatitis B vaccination and 42.8% were hyper-responders (antibody titers > 100 UI/ml). No anti-mitochondrial, anti-nuclear and anti-extractable nuclear antigen antibodies were generated during immunization with CIGB-230. CONCLUSIONS: Vaccination with CIGB-230 in HCV chronically-infected individuals was safe, well tolerated and did not impair the ability to respond to non-HCV antigens.

In vitro assembly of nucleocapsid-like particles from purified recombinant capsid protein of dengue-2 virus.

The capsid protein is one of the three structural proteins of flaviviruses and is the building block of the nucleocapsid. It has also a predominant role in the replication of dengue virus. To obtain nucleocapsid-like particles from recombinant dengue-2 capsid protein produced in E. coli, a purification process using cation exchange chromatography was established. The purified protein exhibited a molecular mass corresponding to a dimer; therefore, similar to that reported for alphaviruses, an in vitro assembly reaction using single-stranded DNA was performed. In all cases, particles were obtained independently of the specificity and the length of the oligonucleotides used. The present work is the first report of in vitro assembly of the recombinant dengue capsid protein, which could constitute a powerful tool in the development of vaccine candidates.