Novel avian DuckCeltTM-T17 cell line for production of viral vaccines : application to influenza viruses production

For the last 15 years, the viral vaccine manufacturing sector is looking for new producer cell lines, easily scalable, highly permissive to various viruses, and more effective in term of viral productivity. One critical characteristic for such cell lines is their ability to grow in suspension in serum free conditions at high cell densities. Regarding the pathogens under focus, influenza virus causing severe epidemics both in human and veterinary field is an important threat for world healthcare. The manufacturing sector is still demanding effective production processes to replace/supplement embryonated egg-based process and to provide efficient response to such threats. Cell-based production, with a focus on avian cell lines, is one of the promising solutions. Indeed, three avian cell lines ; namely duck EB66®cells (Vivalis), duck AGE.CR® cells (Probiogen) and quail QOR/2E11 cells (Baxter), are now competing with traditional mammalian cell platforms used for influenza vaccine productions (Vero and MDCK cells) and are currently at advance stage of commercial development for the manufacture of vaccine and biologicals [1]. The DuckCeltTM-T17 derived line presented here is a novel avian cell line developed by Transgene SA[2]. To generate immortalized duck cell lines, Transgene has used its proprietary DuckCelT technology which consisted in constitutively expressing the duck telomerase reverse transcriptase (dTERT) in primary embryo duck cells from spf eggs. DuckCeltTM-T17 cells were able to grow in batch suspension cultures and serum-free conditions up to 7 x 106 cell/ml and such growth was easily scalable in bioreactors up to 3L. Permissivity for different viruses including influenza has been evaluated. In the present study, DuckCeltTM-T17 cell line was tested for its abilities to produce various influenza strains from different origins; human, avian and porcine. All strains were satisfyingly produced with titres higher than 5.8 log TCID50/ml. H1N1 human strains and H5N2 and H7N1 avian strains were the most efficiently produced with highest titres reached of 8 log TCID50/ml. Porcine strains were also greatly rescued with titres of 4 to 7 log TCID50/ml depending of the subtypes. Interestingly, maximal titres are reached at 24h post-infection, allowing to have early harvest time. Process optimization on H1N1 2009 Human Pandemic strain allowed to identify best operating conditions for production (MOI, trypsin concentration, medium and density at infection) allowing to improve the production level by 2 log. 1. Meyer H-P, Scmidhalter DR: Industrial Scale Suspension Culture of Living Cells. 2014. 2. Balloul Jean-Marc, Duck cell line dedicated to the production of virus-based vaccines and therapeutic products BioProduction Optimization Workshop, September 22 & 23 2010 Frankfurt German

The nonstructural NS1 protein of influenza viruses modulates TP53 splicing through host factor CPSF4

International audienceInfluenza A viruses (IAV) are known to modulate and "hijack" several cellular host mechanisms, including gene splicing and RNA maturation machineries. These modulations alter host cellular responses and enable an optimal expression of viral products throughout infection. The interplay between the host protein p53 and IAV, in particular through the viral nonstructural protein NS1, has been shown to be supportive for IAV replication. However, it remains unknown whether alternatively spliced isoforms of p53, known to modulate p53 transcriptional activity, are affected by IAV infection and contribute to IAV replication. Using a TP53 minigene, which mimics intron 9 alternative splicing, we have shown here that the NS1 protein of IAV changes the expression pattern of p53 isoforms. Our results demonstrate that CPSF4 (cellular protein cleavage and polyadenylation specificity factor 4) independently and the interaction between NS1 and CPSF4 modulate the alternative splicing of TP53 transcripts, which may result in the differential activation of p53-responsive genes. Finally, we report that CPSF4 and most likely beta and gamma spliced p53 isoforms affect both viral replication and IAV-associated type I interferon secretion. All together, our data show that cellular p53 and CPSF4 factors, both interacting with viral NS1, have a crucial role during IAV replication that allows IAV to interact with and alter the expression of alternatively spliced p53 isoforms in order to regulate the cellular innate response, especially via type I interferon secretion, and perform efficient viral replication

Rescue of a H3N2 Influenza Virus Containing a Deficient Neuraminidase Protein by a Hemagglutinin with a Low Receptor-Binding Affinity

Influenza viruses possess at their surface two glycoproteins, the hemagglutinin and the neuraminidase, of which the antagonistic functions have to be well balanced for the virus to grow efficiently. Ferraris et al. isolated in 2003–2004 viruses lacking both a NA gene and protein (H3NA- viruses) (Ferraris O., 2006, Vaccine, 24(44–46):6656-9). In this study we showed that the hemagglutinins of two of the H3NA- viruses have reduced affinity for SAα2.6Gal receptors, between 49 and 128 times lower than that of the A/Moscow/10/99 (H3N2) virus and no detectable affinity for SAα2.3Gal receptors. We also showed that the low hemagglutinin affinity of the H3NA- viruses compensates for the lack of NA activity and allows the restoration of the growth of an A/Moscow/10/99 virus deficient in neuraminidase. These observations increase our understanding of H3NA- viruses in relation to the balance between the functional activities of the neuraminidase and hemagglutinin

Repurposing of Drugs as Novel Influenza Inhibitors From Clinical Gene Expression Infection Signatures

Influenza virus infections remain a major and recurrent public health burden. The intrinsic ever-evolving nature of this virus, the suboptimal efficacy of current influenza inactivated vaccines, as well as the emergence of resistance against a limited antiviral arsenal, highlight the critical need for novel therapeutic approaches. In this context, the aim of this study was to develop and validate an innovative strategy for drug repurposing as host-targeted inhibitors of influenza viruses and the rapid evaluation of the most promising candidates in Phase II clinical trials. We exploited in vivo global transcriptomic signatures of infection directly obtained from a patient cohort to determine a shortlist of already marketed drugs with newly identified, host-targeted inhibitory properties against influenza virus. The antiviral potential of selected repurposing candidates was further evaluated in vitro, in vivo, and ex vivo. Our strategy allowed the selection of a shortlist of 35 high potential candidates out of a rationalized computational screening of 1,309 FDA-approved bioactive molecules, 31 of which were validated for their significant in vitro antiviral activity. Our in vivo and ex vivo results highlight diltiazem, a calcium channel blocker currently used in the treatment of hypertension, as a promising option for the treatment of influenza infections. Additionally, transcriptomic signature analysis further revealed the so far undescribed capacity of diltiazem to modulate the expression of specific genes related to the host antiviral response and cholesterol metabolism. Finally, combination treatment with diltiazem and virus-targeted oseltamivir neuraminidase inhibitor further increased antiviral efficacy, prompting rapid authorization for the initiation of a Phase II clinical trial. This original, host-targeted, drug repurposing strategy constitutes an effective and highly reactive process for the rapid identification of novel anti-infectious drugs, with potential major implications for the management of antimicrobial resistance and the rapid response to future epidemic or pandemic (re)emerging diseases for which we are still disarmed

Biotechnological production of sialylated solid lipid microparticles as inhibitors of influenza A virus infection

International audienceAbstract Influenza viruses bind to their target through a multivalent interaction of their hemagglutinins (HAs) with sialosides at the host cell surface. To fight the virus, one therapeutic approach consists in developing sialylated multivalent structures that can saturate the virus HAs and prevent the binding to host cells. We describe herein the biotechnological production of sialylated solid lipid microparticles (SSLMs) in 3 steps: (i) a microbiological step leading to the large-scale production of sialylated maltodextrins by metabolic engineering of an Escherichia coli strain, (ii) a new in vitro glycosylation process using the amylomaltase MalQ, based on the transglycosylation of the terminal sialoside ligand of the sialylated maltodextrin onto a long-chain alkyl glucoside, and (iii) the formulation of the final SSLMs presenting a multivalent sialic acid. We also describe the morphology and structure of the SSLMs and demonstrate their very promising properties as influenza virus inhibitors using hemagglutination inhibition and microneutralization assays on the human A/H1N1 pdm09 virus

Role of p53/NF-\kappaB functional balance in respiratory syncytial virus-induced inflammation response

International audienceThe interplay between respiratory syncytial virus (RSV) and the p53 pathway has only been reported in a limited number of studies, yet the underlying abrogation mechanisms of p53 activity during the time course of infection, possibly involving viral proteins, remained unclear. Here, we demonstrate that RSV infection impairs global p53 transcriptional activity, notably via its proteasome-dependent degradation at late stages of infection. We also demonstrate that NS1 and NS2 contribute to the abrogation of p53 activity, and used different experimental strategies (e.g. siRNA, small molecules) to underline the antiviral contribution of p53 in the context of RSV infection. Notably, our study highlights a strong RSV-induced disequilibrium of the p53/NF-\kappaB functional balance, which appears to contribute to the up-regulation of the expression of several proinflammatory cytokines and chemokines

In vitro evaluation of antiviral activity of single and combined repurposable drugs against SARS-CoV-2

International audienceIn response to the current pandemic caused by the novel SARS-CoV-2, identifying and validating effective therapeutic strategies is more than ever necessary. We evaluated the in vitro antiviral activities of a shortlist of compounds, known for their cellular broad-spectrum activities, together with drugs that are currently under evaluation in clinical trials for COVID-19 patients. We report the antiviral effect of remdesivir, lopinavir, chloroquine, umifenovir, berberine and cyclosporine A in Vero E6 cells model of SARS-CoV-2 infection, with estimated 50% inhibitory concentrations of 0.99, 5.2, 1.38, 3.5, 10.6 and 3 μM, respectively. Virus-directed plus host-directed drug combinations were also investigated. We report a strong antagonism between remdesivir and berberine, in contrast with remdesivir/diltiazem, for which we describe high levels of synergy, with mean Loewe synergy scores of 12 and peak values above 50. Combination of host-directed drugs with direct acting antivirals underscore further validation in more physiological models, yet they open up interesting avenues for the treatment of COVID-19

Novel calixarene-based surfactant enables low dose split inactivated vaccine protection against influenza infection

International audienceInfluenza A viruses cause major morbidity and represent a severe global health problem. Current influenza vaccines are mainly egg-based products requiring the split of whole viruses using classical detergents such as Triton X-100, which implies certain limitations. Here, we report the use of the novel calixarene-based surfactant CALX133ACE as an alternative to classical detergents for influenza inactivated split vaccine preparation. We confirmed that CALX133ACE-based split HA antigens are fully functional and quantifiable by the "gold standard" method SRID. Additionally, as in the case of the Triton X-100-based split, the CALX133ACE-based split antigens are stable for at least 6 months at 4 °C. Moreover, immunization of mice with CALX133ACE-based split NYMC X-179A (H1N1) antigens harboring 10 to 30-fold less antigen than the commercialized trivalent inactivated vaccines Vaxigrip® or Fluviral® induced comparable efficient protection and neutralizing antibody responses against A(H1N1)pdm09 infection. Taken together, our results demonstrate for the first time the use of a calixarene-based detergent as an efficient splitting agent for the production of optimized influenza split antigens, paving the way for significant improvement in the vaccine manufacturing process, notably with regard to the current regulation on the prohibition of endocrine disruptors, such as Triton X-100

Characterization of cellular transcriptomic signatures induced by different respiratory viruses in human reconstituted airway epithelia

International audienceAcute respiratory infections, a large part being of viral origin, constitute a major public health issue. To propose alternative and/or new therapeutic approaches, it is necessary to increase our knowledge about the interactions between respiratory viruses and their primary cellular targets using the most biologically relevant experimental models. In this study, we used RNAseq to characterize and compare the transcriptomic signature of infection induced by different major respiratory viruses (Influenza viruses, hRSV and hMPV) in a model of reconstituted human airway epithelia. Our results confirm the importance of several cellular pathways commonly or specifically induced by these respiratory viruses, such as the innate immune response or antiviral defense. A very interesting common feature revealed by the global virogenomic signature shared between hRSV, hMPV and influenza viruses is the global downregulation of cilium-related gene expression, in good agreement with experimental evaluation of mucociliary clearance. Beyond providing new information about respiratory virus/host interactions, our study also underlines the interest of using biologically relevant experimental models to study human respiratory viruses. Acute respiratory infections (ARI) constitute a leading cause of acute illness worldwide and a major cause of death among young children, with nearly 2 million deaths per year 1-3. Among a panoply of different viral and bacterial pathogens, respiratory viruses, such as influenza A and B viruses (IAV and IBV), respiratory syncytial virus (hRSV-A and hRSV-B), human metapneumovirus (hMPV-A and hMPV-B) or rhinoviruses (RV), represent the main etiologic agents of these infections 2,4,5. In that regard, the limited or non-existent prophylactic and therapeutic arsenal available, coupled with the emergence of antiviral resistance, highlight the public health burden imposed by these respiratory pathogens. It appears therefore urgent to develop alternative and/or new therapeutic approaches, for which it is a necessary condition to increase our knowledge of the interactions between respiratory viruses and their primary cellular targets, namely respiratory epithelial cells. The last decade has witnessed the development of high-throughput "omics" approaches that have contributed to deepen our understanding of the multiple levels of interplay between respiratory viruses and the host cell. Numerous studies have described the impact of infection on host gene expression in vitro or in vivo, mainly in the context of influenza viruses or hRSV and to a lesser extent for other respiratory viruses such hMPV 6-10. For example , several mRNA profiling studies, including ours, have highlighted the role of NF-kB, p53, or MAPK cellula