Hepatitis C virus (HCV) infects 3% of world population being responsible for nearly half a million deaths annually urging the need for a prophylactic vaccine. Retrovirus like particles are commonly used scaffolds for antigens presentation being the core of diverse vaccine candidates. The immunogenicity of host proteins naturally incorporated in retrovirus was hypothesized to impact the performance of retrovirus based vaccines. In this work, the capacity of engineered retrovirus like particles devoided of host protein CD81 to display HCV envelope antigens was compared to non-engineered particles. A persistent inability of CD81 negative VLPs to incorporate HCV E2 protein as a result from the inefficient transport of HCV E2 to the plasma membrane, was observed. This work enabled the identification of a CD81-mediated transport of HCV E2 while stressing the importance of host proteins for the development of recombinant vaccines.

Develop an engineered cell line containing two flexible gene expression systems enabling the continuous production of tailor-made recombinant gammaretrovirus with predictable productivities through targeted integration.

Transthyretin (TTR) is a transport protein of retinol and thyroxine in serum and CSF, which is mainly secreted by liver and choroid plexus, and in smaller amounts in other cells throughout the body. The exact role of TTR and its specific expression in Central Nervous System (CNS) remains understudied. We investigated TTR expression and metabolism in CNS, through the intranasal and intracerebroventricular delivery of a specific anti-TTR Nanobody to the brain, unveiling Nanobody pharmacokinetics to the CNS. In TTR deficient mice, we observed that anti-TTR Nanobody was successfully distributed throughout all brain areas, and also reaching the spinal cord. In wild-type mice, a similar distribution pattern was observed. However, in areas known to be rich in TTR, reduced levels of Nanobody were found, suggesting potential target-mediated effects. Indeed, in wild-type mice, the anti-TTR Nanobody was specifically internalized in a receptor-mediated process, by neuronal-like cells, which were identified as motor neurons. Whereas in KO TTR mice Nanobody was internalized by all cells, for late lysosomal degradation. Moreover, we demonstrate that in vivo motor neurons also actively synthesize TTR. Finally, in vitro cultured primary motor neurons were also found to synthesize and secrete TTR into culture media. Thus, through a novel intranasal CNS distribution study with an anti-TTR Nanobody, we disclose a new cell type capable of synthesizing TTR, which might be important for the understanding of the physiological role of TTR, as well as in pathological conditions where TTR levels are altered in CSF, such as amyotrophic lateral sclerosis.

Metabolism plays an essential role in cell fate decisions. However, the methods used for metabolic characterization and for finding potential metabolic regulators are still based on characterizing cellular metabolic steady-state which is dependent on the extracellular environment. In this work, we hypothesized that the response dynamics of intracellular metabolic pools to extracellular stimuli is controlled in a cell type-specific manner. We applied principles of process dynamics and control to human induced pluripotent stem cells (hiPSC) and human neural stem cells (hNSC) subjected to a sudden extracellular glutamine step. The fold-changes of steady-states and the transient profiles of metabolic pools revealed that dynamic responses were reproducible and cell type-specific. Importantly, many amino acids had conserved dynamics and readjusted their steady state concentration in response to the increased glutamine influx. Overall, we propose a novel methodology for systematic metabolic characterization and identification of potential metabolic regulators.

A novel optical sensor probe combining monitoring of blood oxygen saturation (SpO₂) with contact pressure is presented. This is beneficial as contact pressure is known to affect SpO₂ measurement. The sensor consists of three plastic optical fibres (POF) used to deliver and collect light for pulse oximetry, and a fibre Bragg grating (FBG) sensor to measure contact pressure. All optical fibres are housed in a biocompatible epoxy patch which serves two purposes: (i) to reduce motion artefacts in the photoplethysmogram (PPG), and (ii) to transduce transverse loading into an axial strain in the FBG. Test results show that using a combination of pressure measuring FBG with a reference FBG, reliable results are possible with low hysteresis which are relatively immune to the effects of temperature. The sensor is used to measure the SpO₂ of ten volunteers under different contact pressures with perfusion and skewness indices applied to assess the quality of the PPG. The study revealed that the contact force ranging from 5 to 15 kPa provides errors of <2%. The combined probe has the potential to improve the reliability of reflectance oximeters. In particular, in wearable technology, the probe should find use in optimising the fitting of garments incorporating this technology.

A tip-based fibreoptic localised surface plasmon resonance (LSPR) sensor is reported for the sensing of volatile organic compounds (VOCs). The sensor is developed by coating the tip of a multi-mode optical fibre with gold nanoparticles (size: 40 nm) via a chemisorption process and further functionalisation with the HKUST-1 metal-organic framework (MOF) via a layer-by-layer process. Sensors coated with different cycles of MOFs (40, 80 and 120) corresponding to different crystallisation processes are reported. There is no measurable response to all tested volatile organic compounds (acetone, ethanol and methanol) in the sensor with 40 coating cycles. However, sensors with 80 and 120 coating cycles show a significant redshift of resonance wavelength (up to ~9 nm) to all tested volatile organic compounds as a result of an increase in the local refractive index induced by VOC capture into the HKUST-1 thin film. Sensors gradually saturate as VOC concentration increases (up to 3.41%, 4.30% and 6.18% in acetone, ethanol and methanol measurement, respectively) and show a fully reversible response when the concentration decreases. The sensor with the thickest film exhibits slightly higher sensitivity than the sensor with a thinner film. The sensitivity of the 120-cycle-coated MOF sensor is 13.7 nm/% (R2 = 0.951) with a limit of detection (LoD) of 0.005% in the measurement of acetone, 15.5 nm/% (R2 = 0.996) with an LoD of 0.003% in the measurement of ethanol and 6.7 nm/% (R2 = 0.998) with an LoD of 0.011% in the measurement of methanol. The response and recovery times were calculated as 9.35 and 3.85 min for acetone; 5.35 and 2.12 min for ethanol; and 2.39 and 1.44 min for methanol. The humidity and temperature crosstalk of 120-cycle-coated MOF was measured as 0.5 ± 0.2 nm and 0.5 ± 0.1 nm in the humidity range of 50-75% relative humidity (RH) and temperature range of 20-25 °C, respectively.

Capillary refill time (CRT) refers to the time taken for body tissue to regain its colour after an applied blanching pressure is released. Usually, pressure is manually applied and not measured. Upon release of pressure, simple mental counting is typically used to estimate how long it takes for the skin to regain its colour. However, this method is subjective and can provide inaccurate readings due to human error. CRT is often used to assess shock and hydration but also has the potential to assess peripheral arterial disease which can result in tissue breakdown, foot ulcers and ultimately amputation, especially in people with diabetes. The aim of this study was to design an optical fibre sensor to simultaneously detect blood volume changes and the contact pressure applied to the foot. The CRT probe combines two sensors: a plastic optical fibre (POF) based on photoplethysmography (PPG) to measure blood volume changes and a fibre Bragg grating to measure skin contact pressure. The results from 10 healthy volunteers demonstrate that the blanching pressure on the subject's first metatarsal head of the foot was 100.8 ± 4.8 kPa (mean and standard deviation), the average CRT was 1.37 ± 0.46 s and the time to achieve a stable blood volume was 4.77 ± 1.57 s. For individual volunteers, the fastest CRT measured was 0.82 ± 0.11 and the slowest 1.94 ± 0.49 s. The combined sensor and curve fitting process has the potential to provide increased reliability and accuracy for CRT measurement of the foot in diabetic foot ulcer clinics and in the community.

Vaccination is the most effective method of disease prevention and control. Many viruses and bacteria that once caused catastrophic pandemics (e.g., smallpox, poliomyelitis, measles, and diphtheria) are either eradicated or effectively controlled through routine vaccination programs. Nonetheless, vaccine manufacturing remains incredibly challenging. Viruses exhibiting high antigenic diversity and high mutation rates cannot be fairly contested using traditional vaccine production methods and complexities surrounding the manufacturing processes, which impose significant limitations. Virus-like particles (VLPs) are recombinantly produced viral structures that exhibit immunoprotective traits of native viruses but are noninfectious. Several VLPs that compositionally match a given natural virus have been developed and licensed as vaccines. Expansively, a plethora of studies now confirms that VLPs can be designed to safely present heterologous antigens from a variety of pathogens unrelated to the chosen carrier VLPs. Owing to this design versatility, VLPs offer technological opportunities to modernize vaccine supply and disease response through rational bioengineering. These opportunities are greatly enhanced with the application of synthetic biology, the redesign and construction of novel biological entities. This review outlines how synthetic biology is currently applied to engineer VLP functions and manufacturing process. Current and developing technologies for the identification of novel target-specific antigens and their usefulness for rational engineering of VLP functions (e.g., presentation of structurally diverse antigens, enhanced antigen immunogenicity, and improved vaccine stability) are described. When applied to manufacturing processes, synthetic biology approaches can also overcome specific challenges in VLP vaccine production. Finally, we address several challenges and benefits associated with the translation of VLP vaccine development into the industry.

The purification of virus particles and viral vectors for vaccine and gene therapy applications is gaining increasing importance in order to deliver a fast, efficient, and reliable production process. Ultrafiltration (UF) is a widely employed unit operation in bioprocessing and its use is present in several steps of the downstream purification train of biopharmaceuticals. However, to date few studies have thoroughly investigated the performance of several membrane materials and cut-offs for virus concentration/diafiltration. The present study aimed at developing a novel class of UF cassettes for virus concentration/diafiltration. A detailed study was conducted to evaluate the effects of (i) membrane materials, namely polyethersulfone (PES), regenerated cellulose (RC), and highly cross-linked RC (xRC), (ii) nominal cut-off, and (iii) UF device geometry at different production scales. The results indicate that the xRC cassettes with a cut-off of approximately 500 kDa are able to achieve a 10-fold concentration factor with 100% recovery of particles with a process time twice as fast as that of a commercially available hollow fiber. DNA and host cell protein clearances, as well as hydraulic permeability and fouling behavior, were also assessed.

Canine adenovirus vector type 2 (CAV2) represents an alternative to human adenovirus vectors for certain gene therapy applications, particularly neurodegenerative diseases. However, more efficient production processes, assisted by a greater understanding of the effect of infection on producer cells, are required. Combining [1,2-(13)C]glucose and [U-(13)C]glutamine, we apply for the first time (13)C-Metabolic flux analysis ((13)C-MFA) to study E1-transformed Madin-Darby Canine Kidney (MDCK) cells metabolism during growth and CAV2 production. MDCK cells displayed a marked glycolytic and ammoniagenic metabolism, and (13)C data revealed a large fraction of glutamine-derived labelling in TCA cycle intermediates, emphasizing the role of glutamine anaplerosis. (13)C-MFA demonstrated the importance of pyruvate cycling in balancing glycolytic and TCA cycle activities, as well as occurrence of reductive alphaketoglutarate (AKG) carboxylation. By turn, CAV2 infection significantly upregulated fluxes through most central metabolism, including glycolysis, pentose-phosphate pathway, glutamine anaplerosis and, more prominently, reductive AKG carboxylation and cytosolic acetyl-coenzyme A formation, suggestive of increased lipogenesis. Based on these results, we suggest culture supplementation strategies to stimulate nucleic acid and lipid biosynthesis for improved canine adenoviral vector production.

No abstract available

The malaria asexual blood-stage antigen PfRipr and its most immunogenic fragment PfRipr5 have recently risen as promising vaccine candidates against this infectious disease. Continued development of high-yielding, scalable production platforms is essential to advance the malaria vaccine research. Insect cells have supplied the production of numerous vaccine antigens in a fast and cost-effective manner; improving this platform further could prove key to its wider use. In this study, insect (Sf9 and High Five) and human (HEK293) cell hosts as well as process-optimizing strategies (new baculovirus construct designs and a culture temperature shift to hypothermic conditions) were employed to improve the production of the malaria asexual blood-stage vaccine candidate PfRipr5. Protein expression was maximized using High Five cells at CCI of 2 × 106 cell/mL and MOI of 0.1 pfu/cell (production yield = 0.49 mg/ml), with high-purity PfRipr5 binding to a conformational anti-PfRipr monoclonal antibody known to hold GIA activity and parasite PfRipr staining capacity. Further improvements in the PfRipr5 expression were achieved by designing novel expression vector sequences and performing a culture temperature shift to hypothermic culture conditions. Addition of one alanine (A) amino acid residue adjacent to the signal peptide cleavage site and a glycine-serine linker (GGSGG) between the PfRipr5 sequence and the purification tag (His6) induced a 2.2-fold increase in the expression of secreted PfRipr5 over using the expression vector with none of these additions. Performing a culture temperature shift from the standard 27-22°C at the time of infection improved the PfRipr5 expression by up to 1.7 fold. Notably, a synergistic effect was attained when combining both strategies, enabling to increase production yield post-purification by 5.2 fold, with similar protein quality (i.e., purity and binding to anti-PfRipr monoclonal antibody). This work highlights the potential of insect cells to produce the PfRipr5 malaria vaccine candidate and the importance of optimizing the expression vector and culture conditions to boost the expression of secreted proteins.

The use of non-standard culture conditions has proven efficient to increase cell performance and recombinant protein production in different cell hosts. However, the establishment of high-producing cell populations through adaptive laboratory evolution (ALE) has been poorly explored, in particular for insect cells. In this study, insect High Five cells were successfully adapted to grow at a neutral culture pH (7.0) through ALE for an improved production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs). A stepwise approach was used for the adaptation process, in which the culture pH gradually increased from standard 6.2 to 7.0 (ΔPh = 0.2-0.3), and cells were maintained at each pH value for 2-3 weeks until a constant growth rate and a cell viability over 95% were observed. These adapted cells enabled an increase in cell-specific HA productivity up to three-fold and volumetric HA titer of up to four-fold as compared to non-adapted cells. Of note, the adaptation process is the element driving increased specific HA productivity as a pH shift alone was inefficient at improving productivities. The production of HA-VLPs in adapted cells was successfully demonstrated at the bioreactor scale. The produced HA-VLPs show the typical size and morphology of influenza VLPs, thus confirming the null impact of the adaptation process and neutral culture pH on the quality of HA-VLPs produced. This work strengthens the potential of ALE as a bioprocess engineering strategy to improve the production of influenza HA-VLPs in insect High Five cells.

Oncolytic virus therapy is currently considered as a promising therapeutic approach for cancer treatment. Adenovirus is well-known and extensively characterized as an oncolytic agent. The increasing number of clinical trials using this virus generates the demand for the development of a well-established purification approach. Triton X-100 is commonly used in cell lysis buffer preparations. The addition of this surfactant in the list of substances with the very high concern of the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) regulation promoted the research for effective alternatives.

The widely applied capillary refill time (CRT) measurement is currently performed by manually applying pressure and using a stopwatch to record the time taken for the skin to recover its normal colour after a blanching pressure is applied. This method is highly subjective and observer-dependent. This paper presents a new, integrated optical sensor probe, combining monitoring of the capillary refilling process with the blanching pressure applied. The sensor consists of an optical fibre-based reflectance photoplethysmography (PPG) sensor to measure the reflected light signal, as well as a fibre Bragg grating (FBG) to measure the applied blanching pressure and to indicate the time when pressure is released. This sensor was applied to calculate the CRT (1.38 ± 0.66 s) of 10 healthy adult volunteers with (55.2 ± 21.8 kPa) blanching pressures. The form of the capillary refilling data was investigated by fitting using an exponential regression model (R2 > 0.96). The integrated probe has the potential to improve the reliability of CRT measurements by standardising the optimum duration and magnitude of the pressure.

Few studies have assessed the effectiveness of the Protected Area networks on the conservation status of target species. Here, we assess the effectiveness of the Portuguese Natura 2000 (the European Union network of protected areas) in maintaining a species included in the Annex I of the Bird Directive, namely the population of a priority farmland bird, the little bustard Tetrax tetrax.

Mucins are high-molecular weight glycoproteins (0.25-20 MDa) containing one or more domains that are heavily O-glycosylated. Their implications as targets for cancer treatment have increased the interest in these glycoproteins, mainly in the fields of vaccines and antibodies. However, mucins present high heterogeneity, posing challenges that affect purification processes and quality control analysis. In that sense, it is necessary to develop and improve downstream processes and analytical methods to characterize these products. Here a tool based on biolayer interferometry analysis to improve mucin's detection and quantification in a fast, simple and label free-way is presented. Taking advantage of lectin recognition of mucins' carbohydrate structures, several lectins were evaluated and immobilized on streptavidin biosensors. Different assay conditions were optimized and the most suitable lectin, Aleuria aurantia lectin (AAL), was selected. Bovine Submaxillary Gland and human MUC5B mucins were used as proof of concept and were successfully detected and quantified at different stages of purification. High sensitivity levels were achieved with LOD and LOQ of 3.8 μg mL-1 and 11.7 μg mL-1 for BSM, and 0.2 μg mL-1 and 0.6 μg mL-1 for MUC5B. AAL binding specificity was also confirmed with fucose competition assays. Our method represents an advance on mucins detection and quantification since the existing methods present several disadvantages for process development. Hereafter, it can be applied to the optimization of new or already established downstream processes for mucins' purification.

Laser irradiation of polymeric materials has drawn great attention as a fast, simple, and cost-effective method for the formation of porous graphene films that can be subsequently fabricated into low-cost and flexible electronic and energy-storage devices. In this work, we report a systematic study of the formation of laser-induced graphene (LIG) with sheet resistances as low as 9.4 Ω/sq on parylene-C ultrathin membranes under a CO2 infrared laser. Raman analysis proved the formation of the multilayered graphenic material, with ID/IG and I2D/IG peak ratios of 0.42 and 0.65, respectively. As a proof of concept, parylene-C LIG was used as the electrode material for the fabrication of ultrathin, solid-state microsupercapacitors (MSCs) via a one-step, scalable, and cost-effective approach, aiming at future flexible and wearable applications. The produced LIG-MSC on parylene-C exhibited good electrochemical behavior, with a specific capacitance of 1.66 mF/cm2 and an excellent cycling stability of 96% after 10 000 cycles (0.5 mA/cm2). This work allows one to further extend the knowledge in LIG processes, widening the group of precursor materials as well as promoting future applications. Furthermore, it reinforces the potential of parylene-C as a key material for next-generation biocompatible and flexible electronic devices.

Virus-like particles (VLPs) constitute a promising platform in vaccine development and targeted drug delivery. To date, most applications use simple nonenveloped VLPs as human papillomavirus or hepatitis B vaccines, even though the envelope is known to be critical to retain the native protein folding and biological function. Here, we present tagged enveloped VLPs (TagE-VLPs) as a valuable strategy for the downstream processing and monitoring of the in vivo production of specific-site-functionalized enveloped influenza VLPs. This two-step procedure allows bioorthogonal functionalization of azide-tagged nascent influenza type A hemagglutinin proteins in the envelope of VLPs through a strain-promoted [3 + 2] alkyne-azide cycloaddition reaction. Importantly, labeling does not influence VLP production and allows for construction of functionalized VLPs without deleterious effects on their biological function. Refined discrimination and separation between VLP and baculovirus, the major impurity of the process, is achieved when this technique is combined with flow cytometry analysis, as demonstrated by atomic force microscopy. TagE-VLPs is a versatile tool broadly applicable to the production, monitoring, and purification of functionalized enveloped VLPs for vaccine design trial runs, targeted drug delivery, and molecular imaging.

Lentiviral vectors (LVs) are excellent tools for gene transfer into mammalian cells. It is noteworthy that the first gene therapy treatment using LVs was approved for commercialization in 2017. The G glycoprotein from rhabdovirus vesicular stomatitis virus (VSV-G) is the glycoprotein most used to pseudotype LVs, due to its high efficiency in transducing several cell types and its resistance to viral vector purification and storage conditions. However, VSV-G expression induces cytotoxicity, which limits LV production to short periods. As alternative to VSV-G, γ-retrovirus glycoproteins (4070A derived, GaLV derived, and RD114 derived) have been used to pseudotype both γ-retroviral vectors (RVs) and LVs. These glycoproteins do not induce cytotoxicity, allowing the development of stable LV producer cells. Additionally, these LV pseudotypes present higher transduction efficiencies of hematopoietic stem cells when compared to VSV-G. Here, new 4070A-, RD114-TR-, and GaLV-TR-derived glycoproteins were developed with the aim of improving its cytoplasmic tail R-peptide cleavage and thus increase LV infectious titers. The new glycoproteins were tested in transient LV production using the wild-type or the less active T26S HIV-1 protease. The GaLV-TR-derived glycoproteins were able to overcome titer differences observed between LV production using wild-type and T26S protease. Additionally, these glycoproteins were even able to increase LV titers, evidencing its potential as an alternative glycoprotein to pseudotype LVs.