Development of a minicircular DNA vaccine against COVID-19

Nucleic acid vaccines have proven to be a promising technology in the fight against global threats such as coronavirus disease (COVID-19). Minicircle DNA (mcDNA) is an innovative vector more stable than messenger RNA and more efficient in cell transfection and transgene expression than conventional plasmid DNA. This work describes the construction of a parental plasmid (PP) vector encoding the receptorbinding domain (RBD) of the S protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the use of the Design of Experiments (DoE) to optimize PP recombination into mcDNA vector in an orbital shaker. First, the results revealed that host cells should be grown at 42 °C and the Terrific Broth (TB) medium should be replaced by Luria Broth (LB) medium containing 0.01% L-arabinose for the induction step. The antibiotic concentration, the induction time, and the induction temperature were used as DoE inputs to maximize the % of recombined mcDNA. The quadratic model was statistically significant (p-value 0.05) with a suitable coefficient of determination. The production of mcDNA was then maximized in a mini-bioreactor platform. The most favorable condition obtained in the bioreactor was obtained by applying 60% pO2 in the fermentation step during 5 h and 30% pO2 in the induction step, with 0.01% L-arabinose throughout 5 h. The application of delivery systems improves the DNA vaccines efficacy and allows their targeting when functionalized with specific ligands. In this work were explored two chitosan (Ch) polymers to formulate different Ch-TPP/R8 and R8-mannose based nanosystems for the delivery of a new mcDNA vaccine against COVID-19, encoding the receptor-binding domain (RBD) gene of severe acute respiratory syndrome coronavirus (SARS-CoV-2). For this purpose, different ratios of TPP, R8 and R8-mannose were evaluated. All systems were formulated using the ionotropic gelation technique and their size, surface charge, encapsulation efficiency and stability were subsequently evaluated. Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM) were also performed to ascertain the functional groups on the surface of the nanoparticles and their shape and morphology, respectively. Two cell lines, human fibroblasts (h-Fibro) and immature dendritic cells (JAWS II) were used in in vitro studies to evaluate the compatibility, transfection efficiency and gene expression of formulated systems. The 3-[4,5- dimethylthiazol2-yl]-2,5-diphenyltetrazolium (MTT) assays showed the biosafety of all Ch-based nanosystems. Subsequently, confocal microscopy studies were performed on dendritic cells (JAWSii), to verify the difference in internalization of non-mannosylated and mannosylated systems in APCs. Systems functionalized with R8-mannose showed better internalization into the cells.As vacinas de ácidos nucleicos provaram ser uma tecnologia promissora na luta contra ameaças globais como a doença coronavírus (COVID-19). O DNA minicircular (mcDNA) é um vetor inovador, mais estável do que o RNA mensageiro e mais eficiente na transfecção celular e expressão transgénica do que o DNA plasmídico convencional. Este trabalho descreve a construção de um vetor de plasmídeo parental (PP) que codifica o domínio de ligação ao recetor (RBD) da proteína S da Síndrome Respiratória Aguda Severa do coronavírus (SARS-CoV-2), e a utilização do Desenho Experimental (DoE) para otimizar a recombinação do PP em mcDNA no agitador orbital. Primeiro, os resultados revelaram que as células hospedeiras devem ser cultivadas a 42 °C com meio Terrific Broth (TB) e, posteriormente, deve ser substituído por meio Luria Broth (LB) contendo 0,01% de L-arabinose para a etapa de indução. A concentração de antibióticos, o tempo de indução, e a temperatura de indução foram utilizados como inputs para o DoE com o intuito de maximizar a % de mcDNA recombinado. O modelo quadrático foi estatisticamente significativo (p < 0,05) e apresentou um lack of fit não significativo (p > 0,05) com um coeficiente de determinação adequado. A produção de mcDNA foi então maximizada numa plataforma de mini-bioreactor. A condição mais favorável no biorreator foi obtida aplicando 60% pO2 na etapa de fermentação durante 5 h e 30% pO2 na etapa de indução, com 0,01% de L-arabinose durante 5 h. A aplicação de sistemas de entrega melhora a eficácia das vacinas de DNA e permite o seu direcionamento quando funcionalizadas com ligandos específicos. O quitosano (Ch) é um polímero natural catiónico, conhecido pelas suas propriedades biodegradáveis, biocompatíveis, mucoadesivas e de baixa citotoxicidade, e tem sido explorado na formulação de sistemas de entrega de biofármacos. A complementação com tripolifosfato (TPP) permite a criação de um sistema de estabilização reticulado através de interações eletrostáticas entre as cargas positivas de Ch e as cargas negativas do TPP e do DNA. A funcionalização com um péptido de penetração celular, como a octa-arginina (R8) melhora a capacidade de penetração e de entrega de biomoléculas. A decoração dos sistemas de entrega com ligandos de manose favorece o reconhecimento específico pelos recetores de manose sobrexpressos na superfície das células apresentadoras de antigénios (APCs). Neste trabalho foram explorados dois polímeros de Ch (HMW e 5 kDa) para formular diferentes nanosistemas baseados em Ch-TPP/R8 e R8-manose para a obtenção de uma nova vacina contra COVID-19, codificando o gene RBD do SARS-CoV-2. Para este efeito, foram avaliados diferentes rácios de TPP, R8 e R8-manose. Todos os sistemas foram formulados utilizando a técnica de ionotropic gelation e o seu tamanho, carga superficial, eficiência de encapsulação e estabilidade foram subsequentemente avaliados. A espectroscopia de infravermelho por transformada de Fourier (FTIR) e a microscopia eletrónica de varrimento (SEM) foram também realizadas para determinar os grupos funcionais presentes na superfície das nanopartículas e a sua forma e morfologia, respetivamente. Duas linhas celulares, fibroblastos humanos (h-Fibro) e células dendríticas imaturas (JAWS II), foram utilizadas em estudos in vitro para avaliar a biocompatibilidade, eficiência da transfecção e expressão génica dos sistemas formulados. Os ensaios de 3-[4,5- dimetiltiazol-2-il]-2,5-difeniltetrazólio (MTT) mostraram a biossegurança de todos os nanosistemas baseados em Ch. Posteriormente, foram efetuados estudos de microscopia confocal em células dendríticas (JAWSii), para verificar a diferença na internalização de sistemas não-manosilados e manosilados em APCs. Este trabalho revelou que a aplicação do biorreator aumentou fortemente o rendimento da biomassa do hospedeiro e simultaneamente melhorou os níveis de recombinação do PP em mcDNA. Adicionalmente, o uso de sistemas de entrega baseados em quitosano mostra um enorme potential para entrega da vacina de DNA, sendo que os sistemas manosilados permitem uma entrega direcionada às APCs melhoram a internalização da vacina de DNA

The Effectiveness of Therapeutic Vaccines for the Treatment of Cervical Intraepithelial Neoplasia 3: A Systematic Review and Meta-Analysis

Cervical cancer (CC) is a disease that affects many women worldwide, especially in low-income countries. The human papilloma virus (HPV) is the main causative agent of this disease, with the E6 and E7 oncoproteins being responsible for the development and maintenance of transformed status. In addition, HPV is also responsible for the appearance of cervical intraepithelial neoplasia (CIN), a pre-neoplastic condition burdened by very high costs for its screening and therapy. So far, only prophylactic vaccines have been approved by regulatory agencies as a means of CC prevention. However, these vaccines cannot treat HPV-positive women. A search was conducted in several databases (PubMed, Scopus, Web of Science, and ClinicalTrials.gov) to systematically identify clinical trials involving therapeutic vaccines against CIN 3. Histopathological regression data, immunological parameters, safety, DNA clearance, and vaccine efficacy were considered from each selected study, and from the 102 articles found, 8 were selected based on the defined inclusion criteria. Histopathological regression from CIN 3 to CIN < 1 was 22.1% (95% CI: 0.627–0.967; p-value = 0.024), showing a vaccine efficacy of 23.6% (95% CI; 0.666–0.876; p-value < 0.001). DNA clearance was assessed, and the risk of persistent HPV DNA was 23.2% (95% CI: 0.667–0.885; p-value < 0.001). Regarding immunological parameters, immune responses by specific T-HPV cells were more likely in vaccinated women (95% CI: 1.245–9.162; p-value = 0.017). In short, these studies favored the vaccine group over the placebo group. This work indicated that therapeutic vaccines are efficient in the treatment of CIN 3, even after accounting for publication bias.info:eu-repo/semantics/publishedVersio

Maximization of the Minicircle DNA Vaccine Production Expressing SARS-CoV-2 RBD

LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy-i4HB. Diana Costa acknowledges research program contract I(SFRH/BD/10201/2020. Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.Nucleic acid vaccines have been proven to be a revolutionary technology to induce an efficient, safe and rapid response against pandemics, like the coronavirus disease (COVID-19). Minicircle DNA (mcDNA) is an innovative vector more stable than messenger RNA and more efficient in cell transfection and transgene expression than conventional plasmid DNA. This work describes the construction of a parental plasmid (PP) vector encoding the receptor-binding domain (RBD) of the S protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the use of the Design of Experiments (DoE) to optimize PP recombination into mcDNA vector in an orbital shaker. First, the results revealed that host cells should be grown at 42◦C and the Terrific Broth (TB) medium should be replaced by Luria Broth (LB) medium containing 0.01% L-arabinose for the induction step. The antibiotic concentration, the induction time, and the induction temperature were used as DoE inputs to maximize the % of recombined mcDNA. The quadratic model was statistically significant (p-value 0.05) with a suitable coefficient of determination. The optimal point was validated using 1 h of induction, at 30◦C, without the presence of antibiotics, obtaining 93.87% of recombined mcDNA. Based on these conditions, the production of mcDNA was then maximized in a mini-bioreactor platform. The most favorable condition obtained in the bioreactor was obtained by applying 60% pO2 in the fermentation step during 5 h and 30% pO2 in the induction step, with 0.01% L-arabinose throughout 5 h. The yield of mcDNA-RBD was increased to a concentration of 1.15 g/L, when compared to the orbital shaker studies (16.48 mg/L). These data revealed that the bioreactor application strongly incremented the host biomass yield and simultaneously improved the recombination levels of PP into mcDNA. Altogether, these results contributed to improving mcDNA-RBD biosynthesis to make the scale-up of mcDNA manufacture simpler, cost-effective, and attractive for the biotechnology industry.publishersversionpublishe

Identification of a structural element of the hepatitis C virus minus strand RNA involved in the initiation of RNA synthesis

The replication of the genomic RNA of the hepatitis C virus (HCV) of positive polarity involves the synthesis of a replication intermediate of negative polarity by the viral RNA-dependent RNA polymerase (NS5B). In vitro and likely in vivo, the NS5B initiates RNA synthesis without primers. This de novo mechanism needs specific interactions between the polymerase and viral RNA elements. Cis-acting elements involved in the initiation of (–) RNA synthesis have been identified in the 3′ non-coding region and in the NS5B coding region of the HCV RNA. However, the detailed contribution of sequences and/or structures of (–) RNA involved in the initiation of (+) RNA synthesis has been less studied. In this report, we identified an RNA element localized between nucleotides 177 and 222 from the 3′-end of the (–) RNA that is necessary for efficient initiation of RNA synthesis by the recombinant NS5B. By site-directed mutagenesis experiments, we demonstrate that the structure rather than the primary sequence of this domain is important for RNA synthesis. We also demonstrate that the intact structure of this RNA element is also needed for efficient RNA synthesis when the viral NS5B functions in association with other viral and cellular proteins in cultured hepatic cells

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

How can patient preferences be used and communicated in the regulatory evaluation of medicinal products? Findings and recommendations from IMI PREFER and call to action

Objective: Patients have unique insights and are (in-)directly affected by each decision taken throughout the life cycle of medicinal products. Patient preference studies (PPS) assess what matters most to patients, how much, and what trade-offs patients are willing to make. IMI PREFER was a six-year European public-private partnership under the Innovative Medicines Initiative that developed recommendations on how to assess and use PPS in medical product decision-making, including in the regulatory evaluation of medicinal products. This paper aims to summarize findings and recommendations from IMI PREFER regarding i) PPS applications in regulatory evaluation, ii) when and how to consult with regulators on PPS, iii) how to reflect PPS in regulatory communication and iv) barriers and open questions for PPS in regulatory decision-making.Methods: PREFER performed six literature reviews, 143 interviews and eight focus group discussions with regulators, patient representatives, industry representatives, Health Technology Assessment bodies, payers, academics, and clincians between October 2016 and May 2022.Results: i) With respect to PPS applications, prior to the conduct of clinical trials of medicinal products, PPS could inform regulators’ understanding of patients’ unmet needs and relevant endpoints during horizon scanning activities and scientific advice. During the evaluation of a marketing authorization application, PPS could inform: a) the assessment of whether a product meets an unmet need, b) whether patient-relevant clinical trial endpoints and outcomes were studied, c) the understanding of patient-relevant effect sizes and acceptable trade-offs, and d) the identification of key (un-)favorable effects and uncertainties. ii) With respect to consulting with regulators on PPS, PPS researchers should ideally have early discussions with regulators (e.g., during scientific advice) on the PPS design and research questions. iii) Regarding external PPS communication, PPS could be reflected in the assessment report and product information (e.g., the European Public Assessment Report and the Summary of Product Characteristics). iv) Barriers relevant to the use of PPS in regulatory evaluation include a lack of PPS use cases and demonstrated impact on regulatory decision-making, and need for (financial) incentives, guidance and quality criteria for implementing PPS results in regulatory decision-making. Open questions concerning regulatory PPS use include: a) should a product independent broad approach to the design of PPS be taken and/or a product-specific one, b) who should optimally be financing, designing, conducting, and coordinating PPS, c) when (within and/or outside clinical trials) to perform PPS, and d) how can PPS use best be operationalized in regulatory decisions.Conclusion: PPS have high potential to inform regulators on key unmet needs, endpoints, benefits, and risks that matter most to patients and their acceptable trade-offs. Regulatory guidelines, templates and checklists, together with incentives are needed to foster structural and transparent PPS submission and evaluation in regulatory decision-making. More PPS case studies should be conducted and submitted for regulatory assessment to enable regulatory discussion and increase regulators’ experience with PPS implementation and communication in regulatory evaluations

The Lancaster Care Charter

In the fall of 1991 the Munich Design Charter was published in Design Issues. This charter was written as a design-led “call to arms” on the future nations and boundaries of Europe. The signatories of the Munich Design Charter saw the problem of Europe, at that time, as fundamentally a problem of form that should draw on the creativity and expertise of design. Likewise, the Does Design Care…? workshop held at Imagination, Lancaster University in the autumn of 2017 brought together a multidisciplinary group of people from 16 nations across 5 continents, who, at a critical moment in design discourse saw a problem with the future of Care. The Lancaster Care Charter has been written in response to the vital question “Does Design Care…?” and via a series of conversations, stimulated by a range of presentations that explored a range of provocations, insights, and more questions, provides answers for the contemporary context of Care. With nation and boundary now erased by the flow of Capital the Charter aims to address the complex and urgent challenges for Care as both the future possible and the responsibility of design. The Lancaster Care Charter presents a collective vision and sets out new pragmatic encounters for the design of Care and the care of Design

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio

Cohort profile : demographic and clinical characteristics of the MILESTONE longitudinal cohort of young people approaching the upper age limit of their child mental health care service in Europe

Purpose: The presence of distinct child and adolescent mental health services (CAMHS) and adult mental health services (AMHS) impacts continuity of mental health treatment for young people. However, we do not know the extent of discontinuity of care in Europe nor the effects of discontinuity on the mental health of young people. Current research is limited, as the majority of existing studies are retrospective, based on small samples or used non-standardised information from medical records. The MILESTONE prospective cohort study aims to examine associations between service use, mental health and other outcomes over 24 months, using information from self, parent and clinician reports. Participants: Seven hundred sixty-three young people from 39 CAMHS in 8 European countries, their parents and CAMHS clinicians who completed interviews and online questionnaires and were followed up for 2 years after reaching the upper age limit of the CAMHS they receive treatment at. Findings to date: This cohort profile describes the baseline characteristics of the MILESTONE cohort. The mental health of young people reaching the upper age limit of their CAMHS varied greatly in type and severity: 32.8% of young people reported clinical levels of self-reported problems and 18.6% were rated to be ‘markedly ill’, ‘severely ill’ or ‘among the most extremely ill’ by their clinician. Fifty-seven per cent of young people reported psychotropic medication use in the previous half year. Future plans: Analysis of longitudinal data from the MILESTONE cohort will be used to assess relationships between the demographic and clinical characteristics of young people reaching the upper age limit of their CAMHS and the type of care the young person uses over the next 2 years, such as whether the young person transitions to AMHS. At 2 years follow-up, the mental health outcomes of young people following different care pathways will be compared. Trial registration number: NCT03013595