Perfusion process for the production of a new, VLP-based yellow fever vaccine candidate

Yellow fever (YF) is an acute viral hemorrhagic disease endemic in tropical areas of Africa, Central and South America, which is transmitted by the bite of infected mosquitoes. It is a “historically devastating disease” (Paules and Fauci, 2017) that killed during outbreaks in past centuries, before the introduction of the current vaccine, approximately 10% of the population of cities like Philadelphia (USA) and Barcelona (Spain). According to Garske et al. (2014), YF caused in 2013 78,000 deaths worldwide, which is a disease burden comparable to influenza. In the past few years, outbreaks in Angola (2016) and in Brazil (2017-2018) led to the depletion of the WHO vaccine stockpile and to the introduction of the emergency use of a fractional dose (1/5). Furthermore, the Angola outbreak in 2016 caused the first cases of YF ever to occur in Asia (11 imported cases to China), rising the concern about approximately 2 billion immunologically naïve people who would be at high risk in Asia in case local transmission of the virus starts to occur (Wilder-Smith et al., 2019). The urgent need for a new YF vaccine becomes evident from two major issues concerning the current vaccine, which consists of a live-attenuated virus propagated in chicken embryos: (i) vaccine shortage due to limitations in the manufacturing technology; (ii) rare, but fatal adverse effects. Therefore, this work focuses on the development of a safe, non-replicating YF vaccine, produced by a high-productivity perfusion process. Stable recombinant HEK293 cell lines constitutively expressing the structural proteins prM (pre-membrane) and E (envelope) of YFV were generated, enabling long-term production and secretion of recombinant virus-like particles (VLPs). FACS (fluorescence activated cell sorting) was used to sort the transfected population for high producer cells and allowed obtaining an enriched cell pool producing significantly higher amounts of VLPs. Small scale kinetic studies under intermittent perfusion (pseudoperfusion) were performed in order to investigate possible feeding strategies and to evaluate the use of short-chain fatty acids as productivity enhancers. Subsequently, perfusion runs were carried out in stirred-tank bioreactors in order to investigate optimal conditions for VLP production, as well as to evaluate different cell retention devices (e.g. inclined lamella settler and ATF-2). Partial retention of the VLPs in the perfusion bioreactor system occurred when the ATF-2 was used. VLPs produced by perfusion were purified by a two-step chromatographic process, and transmission electron microscopy (TEM) images confirmed the expected size and morphology of the VLPs, enabling their use in mouse immunogenicity studies. References: Garske T, Van Kerkhove MD, Yactayo S, Ronveaux O, Lewis RF, Staples JE, Perea W, Ferguson NM, Yellow Fever Expert Committee (2014). Yellow fever in Africa: estimating the burden of disease and impact of mass vaccination from outbreak and serological data. PLoS Medicine 11:e1001638. Paules CI, Fauci AS (2017), Yellow fever - once again on the radar screen in the Americas, N Engl J Med 376: 1397-1399. Wilder-Smith A, Lee V, Gubler DJ (2019), Yellow fever: is Asia prepared for an epidemic? The Lancet 19:241-242

Towards updatable, multivalent Covid vaccines: A platform process to produce trimeric spike protein of SARS-COV-2 variants expressed in HEK293 stable cell clones

Within 2.5 years since SARS-COV-2 emergence, COVID-19 pandemic has caused more than 6.3 million registered deaths and 530 million registered cases. The quick development of safe and effective vaccines was very important to mitigate the sanitary crisis. However, the continuous emergence of virus variants with increasing transmissibility continues to cause periodic outbreaks worldwide. The original vaccines keep protecting from severe disease and death caused by variants, but not from transmission and mild disease. Thus, new and improved vaccines are necessary, and updatable, multivalent pan-variant vaccines might be one way to control SARS-COV-2. The spike (S) protein of SARS-COV-2, a highly glycosylated and very large protein (1380 amino acids), is a key target for diagnostics, therapeutics (e.g. monoclonal antibodies) and vaccines. Very early in the pandemic, Wrapp et al. (https://doi.org/10.1126/science.abb2507) produced the recombinant spike protein as a trimer stabilized in the prefusion conformation and determined its structure by cryoelectron microscopy, providing evidence that it binds to the ACE-2 receptor. The stabilized spike trimer forms the basis of most approved COVID vaccines, across vaccine platforms. In the present work, we studied the expression and purification of the trimeric prefusion-stabilized spike protein in HEK293 cell lines and developed a platform process applicable to SARS-COV-2 variants (current - and potentially future - ones). Parental HEK293 cells (NRC, Canada or Thermofisher, USA) were transfected by lipofection using Lipofectamine 3000 (Thermofisher Scientific, USA) with a plasmid containing the gene encoding the spike protein. At first, for the ancestral strain (Wuhan), we used a plasmid intended for transient expression kindly provided by VRC/NIAID/NIH (USA). However, for the variants, we ordered synthetic genes (Genscript, USA) that were subcloned in a plasmid intended for stable expression. After genetic modification, stably transfected cells were maintained in the presence of G418 sulfate selection agent. After 3-4 weeks, stable cell pools were obtained and submitted to single cell deposit (FACS Aria, BD Biosciences) in order to obtain clonally-derived cell lines. Documented research cell banks of selected clones were cryopreserved. Batch and fed-batch cultivations were investigated in shake flasks and bioreactors, using the chemically defined HEK TF culture medium and HEK FS feed solution (both Sartorius Xell, Germany). For protein purification, different chromatographic techniques were investigated using Akta Purifier and Akta Pilot systems. Detection of the spike protein secreted in cell culture supernatant was performed by immunoblot, whereas UV280 (Nanodrop, Thermofisher, USA) was used for protein quantitation in purified samples. After first expressing the spike protein in February 2020 by transient transfection, we developed a stable cell pool by co-transfecting the same transient expression plasmid and an empty stable expression plasmid. This stable cell pool allowed the generation of Wuhan protein that was used to develop serological tests and a hyperimmune equine serum (Cunha et al., doi: 10.1016/j.isci.2021.103315; Alvim et al., in press) and was donated so far to over 90 laboratories in Brazil for basic or applied research. Please click Download on the upper right corner to see the full abstract

Association between C-reactive protein with all-cause mortality in ELSA-Brasil cohort

Background: High-sensitive C-reactive protein (hsCRP) has been proposed as a marker of incident cardiovascular disease and vascular mortality, and it may also be a marker of non-vascular mortality. However, most evidence comes from either North American or European cohorts. The present proposal aims to investigate the association of high-sensitive C-reactive protein with the risk of all-cause mortality in a multi-ethnic Brazilian population Methods: Cohort data from baseline (2008–2010) of 14 792 subjects participating in the Brazilian Longitudinal Study of Adult Health were used. HsCRP was assayed with Immunochemistry. The association of baseline covariates with all-cause mortality was calculated by Cox regression for univariate model and adjusted for different confounders after mean follow-up of 8.0 ± 1.1 years. The final model was adjusted for age, sex, self-rated race/ethnicity, schooling, health behaviours and prevalent chronic disease. Results: The risk of death increased steadily by quartiles of hsCRP from 1.45 (95% Confidence Interval: 1.05, 2.01) in Quartile 2 to 1.95 (1.42, 2.69) in Quartile 4 compared to Quartile 1. Furthermore, the persistence of a significant graded association after the exclusion of deaths in the first year of follow-up suggests that these results are unlikely to be due to reverse causality. Finally, the hazard ratios were unaffected by the exclusion of participants that had self-reported past medical history for diabetes, cancer and chronic obstructive pulmonary disease. Conclusions: Our study shows that hsCRP levels is associated with mortality in a highly admixed population, independently of a large set of lifestyle and clinical variables

Pastagens consorciadas: procedimentos, técnicas e cuidados no estabelecimento.

bitstream/item/65397/1/COT-32-Pastagens-consorciadas.pd

Simplification of State Transition Diagrams in Average Unavailability Analysis by Using Generalized Perturbation Theory

Safety analysis studies in nuclear engineering, more specifically system reliability, usually handle a great number of components, so that computational difficulties may arise. To face the problem of many component systems a method for simplifying the state transition diagram in Markovian reliability analyses has been proposed, using the edges which can be cut, since these latter have a smaller influence on system failure probability. In order to extend the application of GPT (Generalized Perturbation Theory), this work uses GPT formalism to reduce the number of states in a transition diagram, not considering the state probability as the integral quantity of interest, but the mean system unavailability instead. Therefore, after simplifying the original diagram, the mean unavailability for the new system was calculated and the results were very close to those of the original diagram integral quantity (giving a relative error of about 2%), showing that the proposed simplification is quite reasonable and simple to apply

Sustainability improvement of a composite materials' industry through recycling re-engineering process approaches

This case study was aimed at measuring and assessing the potential improvements that couldbe made on the eco-efficiency performance of a composite materials' industry, specifically aglass fibre reinforced plastic (GFRP) pultrusion manufacturing company. For this purpose, allthe issues involved in the pultrusion process of GFRP profiles were analysed, the current ecoefficiency performance of the company was determined, all the procedures applied in theproduction process were revised, and improvement strategies were planned and investigatedwith basis on the performed analysis. The new eco-efficiency ratios were estimated takinginto account the implementation of new proceedings and procedures through re-engineeringthe manufacturing process and recycling approaches. These features lead to significantimprovements on the sequent assessed eco-efficiency ratios, yielding to a more sustainableproduct and manufacturing process of pultruded GFRP profiles

Avaliação de genótipos de feijoeiro comum do grupo comercial carioca cultivados nas épocas das águas e do inverno em Uberlândia, Estado de Minas Gerais.

O feijoeiro é uma das principais culturas do país, e o Estado de Minas Gerais é um dos maiores produtores, representando cerca de 15% da produção nacional. O objetivo deste trabalho foi testar o desempenho de linhagens de feijoeiro comum cultivadas nas épocas das águas e do inverno, na região do Triângulo Mineiro, Minas Gerais. No cultivo das águas (semeadura em dezembro de 2005) e do inverno (semeadura em maio de 2006), as linhagens/cultivares testadas foram BRS Horizonte, CNFC 8059, CNFC 8065, CNFC 8075, VC-6, VC-7, VC-8, VC-9, VC-10, VC-11, VC-12, CNFC 10443, CNFC 10453, CNFC 10476, MA-I-2.5, MA-I-8.9, MA-I-18.13, RC-I-8, CV-46, CV-55 e Z-22, além das testemunhas BRSMG Majestoso, Pérola e BRSMG Talismã e VC-3. Em cada experimento, o delineamento experimental foi o de blocos casualizados com 25 tratamentos, em três repetições, a parcela útil foi de 4 m². No cultivo das águas, os rendimentos das linhagens mais produtivas variaram entre 2,69 e 2,78 t ha-1 (VC-9 e VC-10, respectivamente) e, no inverno, alcançaram 3 t ha-1 (3,01 e 3,00 t ha-1, CNFC 8065 e Z-22, respectivamente). De maneira geral, as linhagens foram mais produtivas no cultivo do inverno, embora a interação entre linhagens e época de cultivo tenha indicado desempenho distinto entre elas em relação à época