On-Line influenza virus quantification for viral production processes thanks to affinity-based surface plasmon resonance biosensor

Influenza virus seasonal epidemics, associated with the constant threat of new pandemic outbreak, challenge vaccine manufacturers to develop responsive processes that can outreach the limitations of traditional egg-based technology. Recent progress made regarding cell culture bioprocesses allowed for numerous alternative strategies to developed future vaccine candidates, as for example the recombinant HA or Virus—like Particles (VLP) vaccines. However, while cell culture allows for more versatility than ovoculture, regarding process development and monitoring, these alternatives still require optimization to seriously concurrence the traditional process. To drive these developments, WHO and regulatory agencies underlined the need for developing better influenza vaccine potency assays1,2. Actual influenza vaccine formulation and lot release rely on single-radial immunodiffusion (SRID) assay, which requires strain-specific reference sera and antigen reagents. However, the annual preparation of these reagents takes between 2 to 6 months and constitutes a critical bottleneck for the release of vaccine lots3. Additionally, SRID is not implementable for process development as such technique cannot handle in-process low concentrated and non-purified material. We developed an assay for rapid and label-free quantification of influenza hemagglutinin (HA) antigen and influenza virus based on surface plasmon resonance (SPR). The method is based on affinity capture of hemagglutinin antigen by sialic-acid terminated glycans present at the surface of the fetuin-functionalized sensor. Conditions were optimized for the regeneration of the surface, in order to run multiple sequential analyses on a unique sensor. Two types of purified standard were used during the development of the assay. Commercial trivalent inactivated vaccine (“TIV”) has been used for the determination of optimal analytical conditions, while a stock of split inactivated H1N1 virus has been produced and calibrated in our laboratory to study the specific response obtained toward this HA subtype. This assay offers a quantification of influenza hemagglutinin within minutes with a wide dynamic range (30 ng/mL-20 µg/mL). Also, the technique provides a limit of detection (LOD) 100 times lower than SRID, and a better reproducibility than SRID and its potential alternatives recently proposed (1,4,5. Additionally, the applicability of this assay for an on-line vaccine production monitoring has been validated by off-line measurement of influenza H1N1 virus particles derived from cell culture supernatant. Such a test allowed to achieve a LOD of 106 Infectious Viral Particles/mL Thus, our assay provides an innovative tool to evaluate influenza new vaccine bioprocesses, from viral production kinetics in mammalian cell culture to vaccine potency evaluation

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

Drug Repurposing Approaches for the Treatment of Influenza Viral Infection: Reviving Old Drugs to Fight Against a Long-Lived Enemy

Influenza viruses still constitute a real public health problem today. To cope with the emergence of new circulating strains, but also the emergence of resistant strains to classic antivirals, it is necessary to develop new antiviral approaches. This review summarizes the state-of-the-art of current antiviral options against influenza infection, with a particular focus on the recent advances of anti-influenza drug repurposing strategies and their potential therapeutic, regulatory and economic benefits. The review will illustrate the multiple ways to reposition molecules for the treatment of influenza, from adventitious discovery to in silico-based screening. These novel antiviral molecules, many of which targeting the host cell, in combination with conventional antiviral agents targeting the virus, will ideally enter the clinics and reinforce the therapeutic arsenal to combat influenza virus infections

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

Gene Expression Signature-Based Screening Identifies New Broadly Effective Influenza A Antivirals

Classical antiviral therapies target viral proteins and are consequently subject to resistance. To counteract this limitation, alternative strategies have been developed that target cellular factors. We hypothesized that such an approach could also be useful to identify broad-spectrum antivirals. The influenza A virus was used as a model for its viral diversity and because of the need to develop therapies against unpredictable viruses as recently underlined by the H1N1 pandemic. We proposed to identify a gene-expression signature associated with infection by different influenza A virus subtypes which would allow the identification of potential antiviral drugs with a broad anti-influenza spectrum of activity. We analyzed the cellular gene expression response to infection with five different human and avian influenza A virus strains and identified 300 genes as differentially expressed between infected and non-infected samples. The most 20 dysregulated genes were used to screen the connectivity map, a database of drug-associated gene expression profiles. Candidate antivirals were then identified by their inverse correlation to the query signature. We hypothesized that such molecules would induce an unfavorable cellular environment for influenza virus replication. Eight potential antivirals including ribavirin were identified and their effects were tested in vitro on five influenza A strains. Six of the molecules inhibited influenza viral growth. The new pandemic H1N1 virus, which was not used to define the gene expression signature of infection, was inhibited by five out of the eight identified molecules, demonstrating that this strategy could contribute to identifying new broad anti-influenza agents acting on cellular gene expression. The identified infection signature genes, the expression of which are modified upon infection, could encode cellular proteins involved in the viral life cycle. This is the first study showing that gene expression-based screening can be used to identify antivirals. Such an approach could accelerate drug discovery and be extended to other pathogens

Accelerated mass production of influenza virus seed stocks in HEK-293 suspension cell cultures by reverse genetics

Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Viral Vaccine (CVV) as reported by the 2015 WHO Informal Consultation report titled “Influenza Vaccine Response during the Start of a Pandemic”. In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza A/Puerto Rico/8/34 H1N1 strain, and advances in the large-scale transfection of suspension cultured HEK-293 cells. Transfection in shake flasks was performed using 1ug of total plasmid and 1x106 cells/mL. The supernatant was harvested after 48 hpt and used to infect a new shake flasks at 1x106 cells/mL for virus amplification. 3-L bioreactor was inoculated and transfected at 1x106 cells/mL with 1ug of total plasmid and harvested after 48hpt and the virus generated was amplified in shake flask. Quantification by TCID50, SRID, Dot-blot and TRPS were performed as well as characterization by TEM and HA and NA sequencing. Small-scale transfection in shake flasks generated 1.5x105 IVP/mL after 48 hpt and 1x107 IVP/mL after 96 hpi. For large-scale experiment a 3-L controlled stirred tank bioreactor resulted in supernatant (P0) virus titer of 5x104 IVP/mL and 2.8x107 IVP/mL after only one amplification (P1) in HEK-293 suspension cells. We demonstrate the efficent generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CVV for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Thus, this innovative approach is better suited to rationally design and mass produce the CVV within timelines dictated by pandemic situations and produce effective responsiveness than previous methodolog

Preclinical evaluation of PHH-1V vaccine candidate against SARS-CoV-2 in non-human primates

SARS-CoV-2 emerged in December 2019 and quickly spread worldwide, continuously striking with an unpredictable evolution. Despite the success in vaccine production and mass vaccination programs, the situation is not still completely controlled, and therefore accessible second-generation vaccines are required to mitigate the pandemic. We previously developed an adjuvanted vaccine candidate coded PHH-1V, based on a heterodimer fusion protein comprising the RBD domain of two SARS-CoV-2 variants. Here, we report data on the efficacy, safety, and immunogenicity of PHH-1V in cynomolgus macaques. PHH-1V prime-boost vaccination induces high levels of RBD-specific IgG binding and neutralizing antibodies against several SARS-CoV-2 variants, as well as a balanced Th1/Th2 cellular immune response. Remarkably, PHH-1V vaccination prevents SARS-CoV-2 replication in the lower respiratory tract and significantly reduces viral load in the upper respiratory tract after an experimental infection. These results highlight the potential use of the PHH-1V vaccine in humans, currently undergoing Phase III clinical trials.Anna Moya and Mireia Muntada for the ELISA analysis; Clara Panosa and Ester Puigvert for her assistance in the production of the vaccine antigen; Glòria Pujol and Eduard Fossas for their assistance in review of the manuscript; and Adrián Lázaro-Frías from Evidenze Health España S.L. for providing medical writing support during the preparation of this paper funded by Hipra Scientific, S.L.U. This project was partially funded by the Centre for the Development of Industrial Technology (CDTI, IDI20210115), a public organization answering to the Spanish Ministry of Science and Innovation.info:eu-repo/semantics/publishedVersio

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

Eduarda Mansilla: la Biografía, Redes familiares y amicales. Los Epistolarios. Los escritos dispersos. Hacia un estudio crítico integral

El estudio de la vida y obra de Eduarda Mansilla se enmarca en la crítica sobre literatura argentina decimonónica escrita por mujeres, así como en la historia de la vida privada y su interconexión con el mapa político-social de la época, especialmente visible en las grandes familias de la clase dirigente. El corpus específicamente literario de Mansilla aún no había sido efectivamente cerrado y determinado. Se sabía, sobre todo por las memorias de su hijo Daniel García-Mansilla (1950), de la existencia de obras varias, y de escritos de crítica de arte que no han podido ubicarse. Nos constaba además, por compulsas ya realizadas, que diversos textos de la autora subsisten dispersos en publicaciones periódicas de la época, nacionales y en el extranjero. Tanto ese material, como cuanto se pudiera conseguir en lo que hace a epistolarios, crónicas, memorias, diarios y escritos testimoniales de todo tipo guardados en archivos públicos y privados, era precario y se hallaba expuesto a la desaparición y al deterioro. Hasta el presente los asedios a la vida/obra de nuestra autora se han mantenido dentro de marcos más o menos acotados, subordinados a un objetivo mayor: una edición textual, la presentación de un panorama femenino (“argentinas”, “mujer romántica”), la historia de la composición musical en la Argentina, en el que esta figura singular se engarza, pero no constituyeron investigaciones de envergadura autónoma. Lo antedicho confirmó la necesidad de localizar y rescatar la producción total de la autora a través de la compulsa de archivos públicos y privados, lo que nos permitió establecer una biografía confiable de Eduarda Mansilla que, aspiramos, se constituya en obra de consulta. Los resultados de esta investigación se publicaron en la Colección EALA de la editorial Corregidor.Our study of Eduarda Mansilla´s life and works is carried on within the field of criticism about Argentine Nineteenth-Century literature written by women, as well as in the history of private lifeand its interconnection with the political and social map of that period, which was especially visiblein certain traditional families. The specifically literary corpus of Mansilla has not yet been closednor clearly determined. Thanks to the memories written by one of her sons, Daniel García-Mansilla(1950), it is known that Mansilla has produced several literary pieces that disappeared and are notavailable to us nowadays. In addition, there are many other diverse literary works that have beenoriginally published nationally and abroad in a series of periodicals, newspapers and literary journalsthat are still inaccessible to the researchers. These materials, along with memoires, travel journals,personal notes, and any other disperse writing production by Eduarda are scattered, hidden inlibraries, unpublished and in danger of material destruction. Until today, research about Mansillahas been framed within certain fields such as the study of romanticism, Women writings, musicalcomposition, always with the goal of pursuing a critical edition. But none of them constitutes initself an independent research project. The above-mentioned confirmed the necessity of localizingand rescuing the total production of this writer nowadays scattered in public as well as in privatearchives, with the goal of achieving a serious and reliable biography of Eduarda Mansilla which,we expect, will become a referent in our discipline. The results of this research were published inthe EALA Collection Corregidor publisher

Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs

Life-threatening `breakthrough' cases of critical COVID-19 are attributed to poor or waning antibody response to the SARS- CoV-2 vaccine in individuals already at risk. Pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; however, their contribution to hypoxemic breakthrough cases in vaccinated people remains unknown. Here, we studied a cohort of 48 individuals ( age 20-86 years) who received 2 doses of an mRNA vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Antibody levels to the vaccine, neutralization of the virus, and auto- Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal antibody response to the vaccine. Among them, ten (24%) had auto-Abs neutralizing type I IFNs (aged 43-86 years). Eight of these ten patients had auto-Abs neutralizing both IFN-a2 and IFN-., while two neutralized IFN-omega only. No patient neutralized IFN-ss. Seven neutralized 10 ng/mL of type I IFNs, and three 100 pg/mL only. Seven patients neutralized SARS-CoV-2 D614G and the Delta variant (B.1.617.2) efficiently, while one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only 100 pg/mL of type I IFNs neutralized both D61G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating antibodies capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a significant proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population