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O tema central deste estudo é a análise dos instrumentos processuais de controle jurisdicional dos regulamentos de uma perspectiva comparada entre os ordenamentos jurídicos brasileiro e português, tema reconhecidamente atrativo e ainda de grande utilização prática. A metodologia aqui empregada consiste em um cotejo analítico entre os dois sistemas jurídicos acerca das ações judiciais com vistas a exercer o devido controle sobre os atos regulatórios, como forma de consolidação do estado democrático de direito.

Infectious bursal disease virus (IBDV) is a major threat to the productivity of the poultry industry due to morbidity, mortality, and immunosuppression that exacerbates secondary infections and reduces the efficacy of vaccination programs. Field strains of IBDV have a preferred tropism for chicken B cells, the majority of which reside in the bursa of Fabricius (BF). IBDV adaptation to adherent cell culture is associated with mutations altering amino acids in the hypervariable region (HVR) of the capsid protein, which affects immunogenicity and virulence. Until recently, this has limited both the application of reverse genetics systems for engineering molecular clones, and the use of in vitro neutralization assays, to cell-culture-adapted strains of IBDV. Here, we describe the rescue of molecular clones of IBDV containing the HVR from diverse field strains, along with a neutralization assay to quantify antibody responses against the rescued viruses, both using chicken B cells. These methods are readily adaptable to any laboratory with molecular biology expertise and negate the need to obtain wild-type strains. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: A chicken B-cell rescue system for IBDV Basic Protocol 2: A chicken B-cell neutralization assay for IBDV.

Eight infectious bursal disease virus (IBDV) genogroups have been identified based on the sequence of the capsid hypervariable region (HVR) (A1 to A8). Given reported vaccine failures, there is a need to evaluate the ability of vaccines to neutralize the different genogroups. To address this, we used a reverse genetics system and the chicken B-cell line DT40 to rescue a panel of chimeric IBDVs and perform neutralization assays. Chimeric viruses had the backbone of a lab-adapted strain (PBG98) and the HVRs from diverse field strains as follows: classical F52-70 (A1), U.S. variant Del-E (A2), Chinese variant SHG19 (A2), very virulent UK661 (A3), M04/09 distinct (A4), Italian ITA-04 (A6), and Australian variant Vic-01/94 (A8). Rescued viruses showed no substitutions at amino acid positions 253, 284, or 330, previously found to be associated with cell-culture adaptation. Sera from chickens inoculated with wild-type (wt) (F52-70) or vaccine (228E) A1 strains had the highest mean virus neutralization (VN) titers against the A1 virus (log

Eight infectious bursal disease virus (IBDV) genogroups have been identified based on the sequence of the capsid hypervariable region (HVR) (A1-8), yet many vaccines are based on A1 strains. Given reported vaccine failures, there is a need to evaluate the ability of vaccines to neutralize the different genogroups. To address this, we used a reverse genetics system and the chicken B-cell line DT40 to rescue a panel of chimeric IBDVs and perform neutralization assays. Chimeric viruses had the backbone of a lab-adapted strain (PBG98) and the HVRs from diverse field strains: classical F52-70 (A1), US-variant Del-E (A2), Chinese-variant SHG19 (A2), very-virulent UK661 (A3), M04/09 distinct (A4), Italian ITA-04 (A6), and Australian-variant Vic-01/94 (A8). Rescued viruses showed no substitutions at amino-acid positions 253, 284, or 330, previously associated with cell-culture adaptation. Sera from chickens inoculated with wt (F52-70) or vaccine (228E) A1 strains had the highest mean virus neutralization (VN) titers against the A1 virus (log2 15.41 and 12.66), and the lowest against A2 viruses (log2 7.41-7.91, p=0.0001-0.0274), consistent with A1 viruses being most antigenically distant from A2 strains, which correlated with the extent of differences in the predicted HVR structure. VN titers against the other genogroups ranged from log2 9.32-13.32, and A1 strains were likely more closely antigenically related to genogroups A3 and A4 than A6 and A8. Our data are consistent with field observations, validating our method, which can used to screen future vaccine candidates for breadth of neutralizing antibodies, and evaluate the antigenic relatedness of different genogroups.ImportanceThere is a need to evaluate the ability of vaccines to neutralize diverse IBDV genogroups, and to better understand the relationship between HVR sequence, structure, and antigenicity. Here, we used a chicken B cell-line to rescue a panel of chimeric IBDVs with the HVR from seven diverse IBDV field strains, and conduct neutralization assays and protein modelling. We evaluated the ability of serum from vaccinated or infected birds to neutralize the different genogroups. Our novel chicken B-cell rescue system and neutralization assay can be used to screen IBDV vaccine candidates, platforms, and regimens for the breadth of neutralizing antibody responses elicited, evaluate the antigenic relatedness of diverse IBDV strains, and when coupled with structural modelling, elucidate immunodominant and conserved epitopes to strategically design novel IBDV vaccines in the future.

Foot-and-mouth disease virus (FMDV) vaccines protect animals from infection by inducing antibodies. The level of neutralising antibody induced in response to vaccination (or infection), as measured by a virus neutralisation test, is an important parameter with regards to the level of protection afforded against subsequent challenge. However, in addition to overall titre, antibody avidity also represents a crucial metric when assessing the protective efficacy of antibodies. In this project we investigated the use of biolayer interferometry (BLI) to measure the avidity of FMDV antibodies to FMDV antigens. Antibodies targeting site I of the FMDV particle were detected using a commercially synthesised biotinylated peptide. In contrast, the entire antigenic landscape of the FMDV particle was represented by biotinylated FMDV capsids. The antigens were loaded onto Octet streptavidin biosensors at an optimal concentration prior to dipping into antibodies. The sera from different animals varied in avidity, reflecting the quantitative differences in avidity that exist between individual animals in response to FMDV vaccines. Interestingly, the Kdis values obtained for site I vs the entire capsid were different, supporting the importance of other sites beyond site I. Similarly, monoclonal antibodies targeting distinct, known antigenic sites on the capsid surface also resulted in different avidities. The BLI methodology reported here offers a useful approach by which to investigate the strength of antibody interactions at specific sites. In conjunction with recombinant technology, BLI will help aid in investigations into the relative importance of the different antigenic sites with regards to inducing a protective response.

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic., Vaccines for SARS-COV-2 are needed in the ongoing pandemic. Here the authors characterize a vaccine candidate that presents the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on a synthetic VLP platform using SpyTag/SpyCatcher technology and show immunogenicity of a prime-boost regimen in mice and pigs.

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The present thesis has objectives divided into two chapters. Chapter 1 aims, through a systematic review with meta-analysis, to investigate whether diabetes mellitus type 1 (DM1) promotes changes in salivary levels of immunoglobulins (Ig) A, G and M when compared to that in individuals without DM1. The search strategy was performed in eight databases and gray literature, with nine studies being selected. Diabetic patients had a significant reduction in salivary flow (p=0.0008; Cohen's d = -0.19, 95% CI= -0.33, -0.05), but not significant enough to modify the IgA concentration (p=0.120; Cohen's d) = 0.58, 95% CI = -0.15, 1.32). However, DM1 increased IgA concentration by reducing salivary flow (Cohen's d = 0.84; 95% CI = 0.36, 1.32), with a strong estimate of effect (p=0.0006). Regarding IgG, no significant change was observed with DM1 in the patients' saliva (p=0.420). There was no significant variation in salivary IgM levels (p=0.300). Chapter 2 aimed to evaluate the association between salivary parameters: glucose (GS), flow, pH and proteins concentration; fasting and postprandial blood glucose; glycated hemoglobin; caries (CI), plaque (PI) and gingival bleeding (BI) indexes; and diet in children with (CDM1) and without (SDM1) DM1. The CDM1 and SDM1 groups were composed of 36 and 49 patients, respectively, matched for sex and age. For the evaluation of CI, PI and BI, a clinical examination was performed. The dietary pattern was obtained through the diet diary for the 24 hours prior to the appointment. Unstimulated total saliva was collected. Salivary protein concentration was analyzed by the bicinchoninic acid method. The Kolmogorov-Smirnov, Mann-Whitney and Spearman tests were used, considering p

In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p &lt, 0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (pex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

There is dire need for an effective and affordable vaccine against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a modular virus-like particle vaccine candidate displaying the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) using SpyTag/SpyCatcher technology (RBD-SpyVLP). Low doses of RBD-SpyVLP in a prime-boost regimen induced a strong neutralising antibody response in mice and pigs that was superior to convalescent human sera. We evaluated antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we showed that RBD-SpyVLP induced a polyclonal antibody response that recognised all key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. The induction of potent and polyclonal antibody responses by RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Moreover, RBD-SpyVLP is highly resilient, thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence.

Objective To assess the difference in the salivary levels of immunoglobulins between patients with type 1 diabetes mellitus (DM1) and healthy controls. Design This systematic review was registered on the PROSPERO (CRD42020159198) database. All references were cross-checked and the risk of bias assessment was conducted using the Newcastle-Ottawa Scale. The Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) approach was used to appraise the quality of evidence. The standardized mean difference and Cohen's d as the effect size were used in the meta-analysis. I-square statistics was used to estimate heterogeneity. Analysis was performed using the RevMan® software (p Results Of the total 92 articles, 9 were selected for this study. The meta-analysis included 333 DM1 patients and 325 healthy controls. DM1 patients showed a significant reduction in salivary flow (p = 0.0008; Cohen’s d= −0.19, CI 95 %= −0.33, −0.05), although not significant enough to modify the IgA concentration (p = 0.120; Cohen’s d = 0.58, CI 95 %= −0.15, 1.32). However, DM1 increased IgA concentration by reducing salivary flow (Cohen’s d = 0.84; CI 95 % = 0.36, 1.32), with a strong estimate of effect (p = 0.0006). Regarding IgG, no significant change was noted with DM1 in the patient’s saliva (p = 0.420). Furthermore, there was no significant variation in the salivary IgM levels (p = 0.300). Conclusions The data suggest that the salivary levels of the evaluated immunoglobulins do not seem to be altered in DM1 patients when compared to that in healthy controls. However, the increase in IgA salivary concentration was dependent on total protein estimation.

Congenital Zika syndrome (CZS) constitutes a recently identified malformation caused by Zika virus infection during pregnancy. Limited data is available to date on the facial dysmorphic features of these patients. This study evaluated the facial dysmorphisms of children with CZS, compared with clinically healthy children, using clinical examination and standardized photographic images. Sixty-three children with CZS (9.70 ± 3.2 months-age), and 31 Controls (8.67 ± 6.2 months-age) joined the study. Seven out of 15 indices differed between groups: midfacial height (MFH)/horizontal facial reference (HFR) (p = .0003), interalar distance/HFR (p = .0027), nasal root depth/MFH (p = .0030), posterior nasal length/MFH (p = .0002), vertical position of the ear/MFH (p

Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.

Diagnostic tests for foot-and-mouth disease (FMD) include the detection of antibodies against either the viral nonstructural proteins or the capsid. The detection of antibodies against the structural proteins (SP) of the capsid can be used to monitor seroconversion in both infected and vaccinated animals. However, SP tests need to be tailored to the individual FMD virus (FMDV) serotype and their sensitivity may be affected by antigenic variability within each serotype and mismatching between test reagents., Diagnostic tests for foot-and-mouth disease (FMD) include the detection of antibodies against either the viral nonstructural proteins or the capsid. The detection of antibodies against the structural proteins (SP) of the capsid can be used to monitor seroconversion in both infected and vaccinated animals. However, SP tests need to be tailored to the individual FMD virus (FMDV) serotype and their sensitivity may be affected by antigenic variability within each serotype and mismatching between test reagents. As a consequence, FMD reference laboratories are required to maintain multiple type-specific SP assays and reagents. A universal SP test would simplify frontline diagnostics and facilitate large-scale serological surveillance and postvaccination monitoring. In this study, a highly conserved region in the N terminus of FMDV capsid protein VP2 (VP2N) was characterized using a panel of intertype-reactive monoclonal antibodies. This revealed a universal epitope in VP2N which could be used as a peptide antigen to detect FMDV-specific antibodies against all types of the virus. A VP2-peptide enzyme-linked immunosorbent assay (VP2-ELISA) was optimized using experimental and reference antisera from immunized, convalescent, and naïve animals (n = 172). The VP2-ELISA is universal and simple and provided sensitive (99%) and specific (93%) detection of antibodies to all FMDV strains used in this study. We anticipate that this SP test could have utility for serosurveillance during virus incursions in FMD-free countries and as an additional screening tool to assess FMD virus circulation in countries where the disease is endemic.