A new T-antigen negative HEK293 cell line with improved AAV productivity.

Viral vectors for gene therapy, such as recombinant adeno-associated viruses, are produced in human embryonic kidney (HEK) 293 cells. However, the presence of the SV40 T-antigen-encoding CDS SV40GP6 and SV40GP7 in the HEK293T genome raises safety issues when these cells are used in manufacturing for clinical purposes. We developed a new T-antigen-negative HEK cell line from ExcellGene's proprietary HEKExpress,® using the CRISPR-Cas9 strategy. We obtained a high number of clonally-derived cell populations and all of them were demonstrated T-antigen negative. Stability study and AAV production evaluation showed that the deletion of the T-antigen-encoding locus did not impact neither cell growth nor viability nor productivity. The resulting CMC-compliant cell line, named HEKzeroT,® is able to produce high AAV titers, from small to large scale.

High-Titer Neutralizing Antibodies against the SARS-CoV-2 Delta Variant Induced by Alhydroxyquim-II-Adjuvanted Trimeric Spike Antigens.

Global control of COVID-19 will require the deployment of vaccines capable of inducing long-term protective immunity against SARS-CoV-2 variants. In this report, we describe an adjuvanted subunit candidate vaccine that affords elevated, sustained, and cross-variant SARS-CoV-2 neutralizing antibodies (NAbs) in multiple animal models. Alhydroxiquim-II is a Toll-Like Receptor (TLR) 7/8 small-molecule agonist chemisorbed on aluminum hydroxide (Alhydrogel). Vaccination with Alhydroxiquim-II combined with a stabilized, trimeric form of the SARS-CoV-2 spike protein (termed CoVac-II) resulted in high-titer NAbs in mice, with no decay in responses over an 8-month period. NAbs from sera of CoVac-II-immunized mice, horses and rabbits were broadly neutralizing against SARS-CoV-2 variants. Boosting long-term CoVac-II-immunized mice with adjuvanted spike protein from the Beta variant markedly increased levels of NAb titers against multiple SARS-CoV-2 variants; notably, high titers against the Delta variant were observed. These data strongly support the clinical assessment of Alhydroxiquim-II-adjuvanted spike proteins to protect against SARS-CoV-2 variants of concern. IMPORTANCE There is an urgent need for next-generation COVID-19 vaccines that are safe, demonstrate high protective efficacy against SARS-CoV-2 variants and can be manufactured at scale. We describe a vaccine candidate (CoVac-II) that is based on stabilized, trimeric spike antigen produced in an optimized, scalable and chemically defined production process. CoVac-II demonstrates strong and persistent immunity after vaccination of mice, and is highly immunogenic in multiple animal models, including rabbits and horses. We further show that prior immunity can be boosted using a recombinant spike antigen from the Beta variant; importantly, plasma from boosted mice effectively neutralize multiple SARS-CoV-2 variants in vitro, including Delta. The strong humoral and Th1-biased immunogenicity of CoVac-II is driven by use of Alhydroxiquim-II (AHQ-II), the first adjuvant in an authorized vaccine that acts through the dual Toll-like receptor (TLR)7 and TLR8 pathways, as part of the Covaxin vaccine. Our data suggest AHQ-II/spike protein combinations could constitute safe, affordable, and mass-manufacturable COVID-19 vaccines for global distribution.

Impact of 6-Month Exposure to Aerosols From Potential Modified Risk Tobacco Products Relative to Cigarette Smoke on the Rodent Gastrointestinal Tract.

Cigarette smoking causes adverse health effects that might occur shortly after smoking initiation and lead to the development of inflammation and cardiorespiratory disease. Emerging studies have demonstrated the role of the intestinal microbiome in disease pathogenesis. The intestinal microbiome is susceptible to the influence of environmental factors such as smoking, and recent studies have indicated microbiome changes in smokers. Candidate modified risk tobacco products (CMRTP) are being developed to provide substitute products to lower smoking-related health risks in smokers who are unable or unwilling to quit. In this study, the ApoE-/- mouse model was used to investigate the impact of cigarette smoke (CS) from the reference cigarette 3R4F and aerosols from two CMRTPs based on the heat-not-burn principle [carbon-heated tobacco product 1.2 (CHTP 1.2) and tobacco heating system 2.2 (THS 2.2)] on the intestinal microbiome over a 6-month period. The effect of cessation or switching to CHTP 1.2 after 3 months of CS exposure was also assessed. Next-generation sequencing was used to evaluate the impact of CMRTP aerosols in comparison to CS on microbiome composition and gene expression in the digestive tract of mice. Our analyses highlighted significant gene dysregulation in response to 3R4F exposure at 4 and 6 months. The findings showed an increase in the abundance of Akkermansiaceae upon CS exposure, which was reversed upon cessation. Cessation resulted in a significant decrease in Akkemansiaceae abundance, whereas switching to CHTP 1.2 resulted in an increase in Lactobacillaceae abundance. These microbial changes could be important for understanding the effect of CS on gut function and its relevance to disease pathogenesis via the microbiome.

Long-Distance Transport of Thiamine (Vitamin B1) Is Concomitant with That of Polyamines.

Thiamine (vitamin B1) is ubiquitous and essential for cell energy supply in all organisms as a vital metabolic cofactor, known for over a century. In plants, it is established that biosynthesis de novo is taking place predominantly in green tissues and is furthermore limited to plastids. Therefore, transport mechanisms are required to mediate the movement of this polar metabolite from source to sink tissue to activate key enzymes in cellular energy generating pathways but are currently unknown. Similar to thiamine, polyamines are an essential set of charged molecules required for diverse aspects of growth and development, the homeostasis of which necessitates long-distance transport processes that have remained elusive. Here, a yeast-based screen allowed us to identify Arabidopsis (Arabidopsis thaliana) PUT3 as a thiamine transporter. A combination of biochemical, physiological, and genetic approaches permitted us to show that PUT3 mediates phloem transport of both thiamine and polyamines. Loss of function of PUT3 demonstrated that the tissue distribution of these metabolites is altered with growth and developmental consequences. The pivotal role of PUT3 mediated thiamine and polyamine homeostasis in plants, and its importance for plant fitness is revealed through these findings.

ABC1K1/PGR6 kinase: a regulatory link between photosynthetic activity and chloroplast metabolism.

Arabidopsis proton gradient regulation (pgr) mutants have high chlorophyll fluorescence and reduced non-photochemical quenching (NPQ) caused by defects in photosynthetic electron transport. Here, we identify PGR6 as the chloroplast lipid droplet (plastoglobule, PG) kinase ABC1K1 (activity of bc1 complex kinase 1). The members of the ABC1/ADCK/UbiB family of atypical kinases regulate ubiquinone synthesis in bacteria and mitochondria, and impact various metabolic pathways in plant chloroplasts. Here, we demonstrate that abc1k1 has a unique photosynthetic and metabolic phenotype that is distinct from that of the abc1k3 homolog. The abc1k1/pgr6 single mutant is specifically deficient in the electron carrier plastoquinone, as well as in ß-carotene and the xanthophyll lutein, and is defective in membrane antioxidant tocopherol metabolism. After 2 days of continuous high light stress, abc1k1/pgr6 plants suffer extensive photosynthetic and metabolic perturbations, strongly affecting carbohydrate metabolism. Remarkably, however, the mutant acclimates to high light after 7 days together with a recovery of carotenoid levels and a drastic alteration in the starch-to-sucrose ratio. Moreover, ABC1K1 behaves as an active kinase and phosphorylates VTE1, a key enzyme of tocopherol (vitamin E) metabolism in vitro. Our results indicate that the ABC1K1 kinase constitutes a new type of regulatory link between photosynthetic activity and chloroplast metabolism.

A chloroplast ABC1-like kinase regulates vitamin E metabolism in Arabidopsis.

In bacteria and mitochondria, ABC1 (for Activity of bc1 complex)-like kinases regulate ubiquinone synthesis, mutations causing severe respiration defects, including neurological disorders in humans. Little is known about plant ABC1-like kinases; in Arabidopsis (Arabidopsis thaliana), five are predicted in mitochondria but, surprisingly, six are located at lipid droplets in chloroplasts. These are a known site of prenylquinone (including tocopherol [vitamin E], phylloquinone [vitamin K] and plastoquinone) metabolism and contain a large proportion of the tocopherol cyclase (VTE1) required for vitamin E synthesis and recycling. Therefore, ABC1-like kinases may be involved in the regulation of chloroplast prenylquinone metabolism. Using a nontargeted lipidomics approach, we demonstrate that plants lacking the plastoglobule ABC1-like kinase ABC1K3 are defective both for the production of plastochromanol-8 (a plastoquinone-derived lipid antioxidant) and the redox recycling of α-tocopherol, whereas tocopherol production is not affected. All of these pathways require VTE1 activity. However, in the abc1k3 mutant, VTE1 levels are strongly reduced posttranscriptionally. We provide evidence that the ABC1-like kinase ABC1K3 phosphorylates VTE1, possibly stabilizing it at plastoglobules. However, ABC1K3 may also have other targets and be involved in a wider chloroplast regulatory network.

A novel method for prenylquinone profiling in plant tissues by ultra-high pressure liquid chromatography-mass spectrometry.

BACKGROUND: Prenylquinones are key compounds of the thylakoid membranes in chloroplasts. To understand the mechanisms involved in the response of plants to changing conditions such as high light intensity, the comprehensive analysis of these apolar lipids is an essential but challenging step. Conventional methods are based on liquid chromatography coupled to ultraviolet and fluorescence detection of a single or limited number of prenylquinones at a time. Here we present an original and rapid approach using ultra-high pressure liquid chromatography-atmospheric pressure chemical ionization-quadrupole time-of-flight mass spectrometry (UHPLC-APCI-QTOFMS) for the simultaneous profiling of eleven prenylquinones in plant tissues, including α-tocopherol, phylloquinone, plastochromanol-8 and plastoquinone-9. RESULTS AND DISCUSSION: Mass spectrometry and chromatography parameters were optimized using pure standards. Sample preparation time was kept to minimum and different extraction solvents were evaluated for yield, ability to maintain the redox state of prenylquinones, and compatibility with chromatography. In addition to precise absolute quantification of 5 prenyllipids for which standards were available, relative quantification of 6 other related compounds was possible thanks to the high identification power of QTOFMS. Prenylquinone levels were measured in leaves of Arabidopsis grown under normal and high light intensities. Quantitatively, the obtained results were consistent with those reported in various previous studies, demonstrating that this new method can profile the full range of prenylquinones in a very short time. CONCLUSION: The new profiling method proves faster, more sensitive and can detect more prenylquinones than current methods based on measurements of selected compounds. It enables the extraction and analysis of twelve samples in only 1.5 h and may be applied to other plant species or cultivars.