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.

The importance of Cre recombinase to minimize helper vector (HV) contamination during helper-dependent adenovirus vectors (HDVs) production is well documented. However, Cre recombinase, by inducing DNA double-strand breaks (DSBs), can cause a reduced proliferation and genotoxic effects in cultured cells. In this work, Cre-expressing cell stability, co-infection and their relation to adenovirus amplification/HV contamination were evaluated to develop a production protocol for HD canine adenovirus type 2 (CAV-2) vectors. Long-term Cre expression reduced the capacity of MDCK-E1-Cre cells to produce CAV-2 by 7-fold, although cell growth was maintained. High HDV/HV MOI ratio (5:0.1) led to low HV contamination without compromising HDV yields. Indeed, such MOI ratio was sufficient to reduce HV levels, as these were similar either in MDCK-E1 or MDCK-E1-Cre cells. This raises the possibility of producing HDVs without Cre-expressing cells, which would circumvent the negative effects that this recombinase holds to the production system. Here, we show how Cre and MOI ratio impact adenovirus vectors yields and infectivity, providing key-information to design an improved manufacturing of HDV. Potential mechanisms to explain how Cre is specifically impacting cell productivity without critically compromising its growth are presented.

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

Adenovirus vectors have been extensively studied through the manipulation of viral genome. However, little attention is being paid to their producer cell-lines; cells are selected according to virus yields, neglecting the expression profile of transcomplementing gene products underlying cell performance. This work evaluates the impact of E1 (E1A and E1B) and Cre recombinase levels in the production of E1-deleted and helper-dependent canine adenovirus type 2 (CAV-2) vectors using MDCK cells. E1A and E1B gene expression and Cre activity were evaluated in different cell clones and compared with the corresponding cell productivity and susceptibility to oxidative stress injury. CAV-2 production was proportional to E1A expression (the highest levels of E1A corresponding to productivities of 3000-5000 I.P./cell), while E1B prolonged host cell viability after infection, conferring protection against apoptosis. Cre recombinase counteracted E1B anti-apoptotic properties, however viral production was maintained under high levels of Cre. Yet, Cre recombinase side effects can be reduced using cell lines with lower Cre-activities, without compromising the excision efficiency of helper vector packaging signal. These results highlight the influence of transcomplementing gene products on CAV-2 producer cell line performance, and the ability to express high levels of E1A and E1B as an important feature for cell line establishment and high adenovirus titers.

Cell-based assays are essential for virus functional characterization in fundamental and applied research. Overcoming the limitations of virus-labelling strategies while allowing functional assessment of critical viral enzymes, virus-induced cell-based biosensors constitute a powerful approach. Herein, we designed and characterized different cell-based switch-on split GFP sensors reporting viral proteolytic activity and virus infection. Crucial to these sensors is the effective-yet reversible-fluorescence off-state, through protein distortion. For that, single (protein embedment or intein-mediated cyclization) or dual (coiled-coils) distortion schemes prevent split GFP self-assembly, until virus-promoted proteolysis of a cleavable sequence. All strategies showed their applicability in detecting viral proteolysis, although with different efficiencies depending on the protease. While for tobacco etch virus protease the best performing sensor was based on coiled-coils (signal-to-noise ratio, SNR, 97), for adenovirus and lentivirus proteases it was based on GFP11 cyclization (SNR 3.5) or GFP11 embedment distortion (SNR 6.0), respectively. When stably expressed, the sensors allowed live cell biosensing of adenovirus infection, with sensor fluorescence activation 24 h post-infection. The structural distortions herein studied are highly valuable in the development of cellular biosensing platforms. Additionally highlighted, selection of the best performing strategy is highly dependent on the unique properties of each viral protease.