scholarly journals Avian Cell Line DuckCelt®-T17 Is an Efficient Production System for Live-Attenuated Human Metapneumovirus Vaccine Candidate Metavac®

Vaccines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1190
Author(s):  
Caroline Chupin ◽  
Andrés Pizzorno ◽  
Aurélien Traversier ◽  
Pauline Brun ◽  
Daniela Ogonczyk-Makowska ◽  
...  
Keyword(s):  
Cell Line ◽  
Production System ◽  
Neutralizing Antibodies ◽  
Vaccine Candidate ◽  
Human Metapneumovirus ◽  
Efficient Production ◽  
Operational Parameters ◽  
Hmpv Infection ◽  
Viral Vaccine ◽  
Avian Cell

The development of a live-attenuated vaccine (LAV) for the prevention of human metapneumovirus (HMPV) infection is often hampered by the lack of highly efficient and scalable cell-based production systems that support eventual global vaccine production. Avian cell lines cultivated in suspension compete with traditional cell platforms used for viral vaccine manufacture. We investigated whether the DuckCelt®-T17 avian cell line (Vaxxel), previously described as an efficient production system for several influenza strains, could also be used to produce a new HMPV LAV candidate (Metavac®, SH gene-deleted A1/C-85473 HMPV). To that end, we characterized the operational parameters of MOI, cell density, and trypsin addition to achieve the optimal production of Metavac®, and demonstrated that the DuckCelt®-T17 cell line is permissive and well-adapted to the production of the wild-type A1/C-85473 HMPV and the Metavac® vaccine candidate. Moreover, our results confirmed that the LAV candidate produced in DuckCelt®-T17 cells conserves its advantageous replication properties in LLC-MK2 and 3D-reconstituted human airway epithelium models, and its capacity to induce efficient neutralizing antibodies in a BALB/c mouse model. Our results suggest that the DuckCelt®-T17 avian cell line is a very promising platform for the scalable in-suspension serum-free production of the HMPV-based LAV candidate Metavac®.

scholarly journals A novel effective live-attenuated human metapneumovirus vaccine candidate produced in the serum-free suspension DuckCelt®-T17 cell platform

2021 ◽  
Author(s):  
Caroline Chupin ◽  
Andrés Pizzorno ◽  
Aurélien Traversier ◽  
Pauline Brun ◽  
Daniela Ogonczyk-Makowska ◽  
...  
Keyword(s):  
Cell Line ◽  
Neutralizing Antibodies ◽  
Vaccine Candidate ◽  
Specific Treatment ◽  
Human Metapneumovirus ◽  
Respiratory Pathogen ◽  
Efficient Production ◽  
Serum Free ◽  
Viral Vaccine ◽  
Avian Cell

AbstractHuman metapneumovirus (HMPV) is a major pediatric respiratory pathogen for which there is currently no specific treatment or licensed vaccine. Different strategies have been evaluated to prevent this infection, including the use of live-attenuated vaccines (LAVs). However, further development of LAV approaches is often hampered by the lack of highly efficient and scalable cell-based production systems that support worldwide vaccine production. In this context, avian cell lines cultivated in suspension are currently competing with traditional cell platforms used for viral vaccine manufacturing. We investigated whether the DuckCelt®-T17 avian cell line (Vaxxel) we previously described as an efficient production system for several influenza strains could also be used to produce a new HMPV LAV candidate (Metavac®), an engineered SH gene-deleted mutant of the A1/C-85473 strain of HMPV. To that end, we characterized the operational parameters of multiplicity of infection (MOI), cell density, and trypsin addition to achieve optimal production of the LAV Metavac® in the DuckCelt®-T17 cell line platform. We demonstrated that the DuckCelt®-T17 cell line is permissive and is well adapted to the production of the wild-type A1/C-85473 HMPV and the Metavac® vaccine candidate. Moreover, our results confirmed that the LAV candidate produced in DuckCelt®-T17 cells conserves its advantageous replication properties in LLC-MK2 and 3D-reconstituted human airway epithelium models, as well as its capacity to induce efficient neutralizing antibodies in a mouse model. Our results suggest that the DuckCelt®-T17 avian cell line is a very promising platform for scalable in-suspension serum-free production of the HMPV-based LAV candidate Metavac®.

scholarly journals Immunogenicity profile in African green monkeys of a vaccine candidate based on a mutated form of human Interleukin-15

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yunier Rodríguez-Álvarez ◽  
Lino Gerardo Batista-Roche ◽  
Alexey Llopiz-Arzuaga ◽  
Pedro Puente-Pérez ◽  
Rafael Martínez-Castillo ◽  
...  
Keyword(s):  
Biological Activity ◽  
T Cell ◽  
Cell Line ◽  
Animal Behavior ◽  
Neutralizing Antibodies ◽  
Vaccine Candidate ◽  
Cytotoxic T Cell ◽  
The Third ◽  
Antibody Titers ◽  
Green Monkeys

Abstract Background Interleukin (IL)-15 is a proinflammatory T-cell growth factor overexpressed in several autoimmune diseases such as rheumatoid arthritis. Our initial strategy to neutralize the increased levels of IL-15 consisted in a vaccine candidate based on the recombinant modified human IL-15 (mhIL-15) mixed with the alum adjuvant. A previous study in non-human primates Macaca fascicularis has shown that vaccination induces neutralizing antibodies against native IL-15, without affecting animal behavior, clinical status, or the percentage of IL-15-dependent cell populations. However, the mhIL-15 used as an antigen was active in the IL-2-dependent cytotoxic T-cell line CTLL-2, which could hinder its therapeutic application. The current article evaluated the immunogenicity in African green monkeys of a vaccine candidate based on IL-15 mutant D8SQ108S, an inactive form of human IL-15. Results IL-15 D8SQ108S was inactive in the CTLL-2 bioassay but was able to competitively inhibit the biological activity of human IL-15. Immunization with 200 µg of IL-15 mutant combined with alum elicited anti-IL-15 IgG antibodies after the second and third immunizations. The median values of anti-IL-15 antibody titers were slightly higher than those generated in animals immunized with 200 µg of mhIL-15. The highest antibody titers were induced after the third immunization in monkeys vaccinated with 350 µg of IL-15 D8SQ108S. In addition, sera from immunized animals inhibited the biological activity of human IL-15 in CTLL-2 cells. The maximum neutralizing effect was observed after the third immunization in sera of monkeys vaccinated with the highest dose of the IL-15 mutant. These sera also inhibited the proliferative activity of simian IL-15 in the CTLL-2 bioassay and did not affect the IL-2-induced proliferation of the aforementioned T-cell line. Finally, it was observed that vaccination neither affects the animal behavior nor the general clinical parameters of immunized monkeys. Conclusion Immunization with inactive IL-15 D8SQ108S mixed with alum generated neutralizing antibodies specific for human IL-15 in African green monkeys. Based on this fact, the current vaccine candidate could be more effective than the one based on biologically active mhIL-15 for treating autoimmune disorders involving an uncontrolled overproduction of IL-15.

scholarly journals The TZM-bl Reporter Cell Line Expresses Kynureninase That Can Neutralize 2F5-like Antibodies in the HIV-1 Neutralization Assay

2022 ◽  
Vol 23 (2) ◽  
pp. 641
Author(s):  
Vladimir Morozov ◽  
Sylvie Lagaye ◽  
Alexey Morozov
Keyword(s):  
Cell Line ◽  
Neutralizing Antibodies ◽  
False Negative ◽  
Neutralization Assay ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Negative Results ◽  
Viral Vaccine ◽  
Neutralizing Monoclonal Antibody ◽  
Hiv 1

Induction of broadly neutralizing antibodies targeting ectodomain of the transmembrane (TM) glycoprotein gp41 HIV-1 provides a basis for the development of a universal anti-viral vaccine. The HeLa cell-derived TZM-bl reporter cell line is widely used for the estimation of lentiviruses neutralization by immune sera. The cell line is highly permissive to infection by most strains of HIV, SIV, and SHIV. Here we demonstrated that TZM-bl cells express a 48 kDa non-glycosylated protein (p48) recognized by broadly neutralizing monoclonal antibody (mAb) 2F5 targeting the ELDKWA (aa 669–674) epitope of gp41TM of HIV-1. A significant amount of p48 was found in the cell supernatant. The protein was identified as human kynureninase (KYNU), which has the ELDKWA epitope. The protein is further called “p48 KYNU”. The HIV-1 neutralization by mAb 2F5 and 4E10 in the presence of p48KYNU was tested on Jurkat and TZM-bl cells. It was demonstrated that p48KYNU reduces neutralization by 2F5-like antibodies, but it has almost no effect on mAb 4E10. Therefore, p48KYNU can attenuate HIV-1 neutralization by 2F5-like antibodies and hence create false-negative results. Thus, previously tested immune sera that recognized the ELDKWA-epitope and demonstrated a “weak neutralization” of HIV-1 in TZM-bl assay should be reevaluated.

scholarly journals Gamma-irradiated SARS-CoV-2 vaccine candidate, OZG-38.61.3, confers protection from SARS-CoV-2 challenge in human ACEII-transgenic mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Raife Dilek Turan ◽  
Cihan Tastan ◽  
Derya Dilek Kancagi ◽  
Bulut Yurtsever ◽  
Gozde Sir Karakus ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Vaccine Candidate ◽  
Protective Efficacy ◽  
Phase 1 ◽  
Challenge Test ◽  
Viral Rna ◽  
Viral Vaccine ◽  
Gamma Irradiated

AbstractThe SARS-CoV-2 virus caused the most severe pandemic around the world, and vaccine development for urgent use became a crucial issue. Inactivated virus formulated vaccines such as Hepatitis A and smallpox proved to be reliable approaches for immunization for prolonged periods. In this study, a gamma-irradiated inactivated virus vaccine does not require an extra purification process, unlike the chemically inactivated vaccines. Hence, the novelty of our vaccine candidate (OZG-38.61.3) is that it is a non-adjuvant added, gamma-irradiated, and intradermally applied inactive viral vaccine. Efficiency and safety dose (either 1013 or 1014 viral RNA copy per dose) of OZG-38.61.3 was initially determined in BALB/c mice. This was followed by testing the immunogenicity and protective efficacy of the vaccine. Human ACE2-encoding transgenic mice were immunized and then infected with the SARS-CoV-2 virus for the challenge test. This study shows that vaccinated mice have lowered SARS-CoV-2 viral RNA copy numbers both in oropharyngeal specimens and in the histological analysis of the lung tissues along with humoral and cellular immune responses, including the neutralizing antibodies similar to those shown in BALB/c mice without substantial toxicity. Subsequently, plans are being made for the commencement of Phase 1 clinical trial of the OZG-38.61.3 vaccine for the COVID-19 pandemic.

scholarly journals Gamma-irradiated SARS-CoV-2 vaccine candidate, OZG-38.61.3, confers protection from SARS-CoV-2 challenge in human ACEII-transgenic mice

2020 ◽  
Author(s):  
Raife Dilek Turan ◽  
Cihan Tastan ◽  
Derya Dilek Kancagi ◽  
Bulut Yurtsever ◽  
Gozde Sir Karakus ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Vaccine Candidate ◽  
Protective Efficacy ◽  
Challenge Test ◽  
Viral Copy Number ◽  
Viral Vaccine ◽  
Viral Copy ◽  
Gamma Irradiated

AbstractThe SARS-CoV-2 virus caused the most severe pandemic around the world, and vaccine development for urgent use became a crucial issue. Inactivated virus formulated vaccines such as Hepatitis A, oral polio vaccine, and smallpox proved to be reliable approaches for immunization for prolonged periods. During the pandemic, we produced an inactivated SARS-CoV-2 vaccine candidate, having the advantages of being manufactured rapidly and tested easily in comparison with recombinant vaccines. In this study, an inactivated virus vaccine that includes a gamma irradiation process for the inactivation as an alternative to classical chemical inactivation methods so that there is no extra purification required has been optimized. The vaccine candidate (OZG-38.61.3) was then applied in mice by employing the intradermal route, which decreased the requirement of a higher concentration of inactivated virus for proper immunization, unlike most of the classical inactivated vaccine treatments. Hence, the novelty of our vaccine candidate (OZG-38.61.3) is that it is a non-adjuvant added, gamma-irradiated, and intradermally applied inactive viral vaccine. Efficiency and safety dose (either 1013 or 1014 viral copy per dose) of OZG-38.61.3 was initially determined in Balb/c mice. This was followed by testing the immunogenicity and protective efficacy of OZG-38.61.3. Human ACE2-encoding transgenic mice were immunized and then infected with a dose of infective SARS-CoV-2 virus for the challenge test. Findings of this study show that vaccinated mice have lower SARS-CoV-2 viral copy number in oropharyngeal specimens along with humoral and cellular immune responses against the SARS-CoV-2, including the neutralizing antibodies similar to those shown in Balb/c mice without substantial toxicity. Subsequently, plans are being made for the commencement of Phase 1 clinical trial of the OZG-38.61.3 vaccine for the COVID-19 pandemic.

scholarly journals Human Metapneumovirus Persists in BALB/c Mice despite the Presence of Neutralizing Antibodies

2004 ◽  
Vol 78 (24) ◽  
pp. 14003-14011 ◽  
Author(s):  
Rene Alvarez ◽  
Kevin S. Harrod ◽  
Wun-Ju Shieh ◽  
Sherif Zaki ◽  
Ralph A. Tripp
Keyword(s):  
Neutralizing Antibodies ◽  
Infectious Virus ◽  
Airway Remodeling ◽  
Neutralizing Antibody ◽  
Human Metapneumovirus ◽  
Respiratory Pathogen ◽  
Mucus Production ◽  
Hmpv Infection ◽  
Syncytial Virus ◽  
Tract Infections

ABSTRACT Human metapneumovirus (HMPV) has emerged as an important human respiratory pathogen causing upper and lower respiratory tract infections in young children and older adults. Recent epidemiological evidence indicates that HMPV may cocirculate with respiratory syncytial virus, and HMPV infection has been associated with other respiratory diseases. In this study, we show that BALB/c mice are susceptible to HMPV infection, the virus replicates in the lungs with biphasic growth kinetics in which peak titers occur at days 7 and 14 postinfection (p.i.), and infectious HMPV can be recovered from lungs up to day 60 p.i. In addition, we show that genomic HMPV RNA can be detected in the lungs for ≥180 days p.i. by reverse transcription-PCR; however, neither HMPV RNA nor infectious virus can be detected in serum, spleen, kidneys, heart, trachea, and brain tissue. Lung histopathology revealed prevalent mononuclear cell infiltration in the interstitium beginning at day 2 p.i. and peaking at day 4 p.i. which decreased by day 14 p.i. and was associated with airway remodeling. Increased mucus production evident at day 2 p.i. was concordant with increased bronchial and bronchiolar inflammation. HMPV-specific antibodies were detected by day 14 p.i., neutralizing antibody titers reached ≥6.46 log2 end-point titers by day 28 p.i., and depletion of T cells or NK cells resulted in increased HMPV titers in the lungs, suggesting some immune control of viral persistence. This study shows that BALB/c mice are amenable for HMPV studies and indicates that HMPV persists as infectious virus in the lungs of normal mice for several weeks postinfection.

Endothelial Cell Protein S Synthesis Is Upregulated by the Complex of IL-6 and Soluble IL-6 Receptor

1997 ◽  
Vol 77 (05) ◽  
pp. 1014-1019 ◽  
Author(s):  
W Craig Hooper ◽  
Donald J Phillips ◽  
Bruce L Evatt
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Line ◽  
Neutralizing Antibodies ◽  
Hepatoma Cell ◽  
Protein S ◽  
Oncostatin M ◽  
Cell Protein ◽  
Microvascular Endothelial Cell ◽  
Hepatoma Cell Line

SummaryWe have recently demonstrated that the proinflammatory cytokine, interleukin-6 (IL-6), could upregulate the production of protein S in the human hepatoma cell line, HepG-2, but not in endothelial cells. In this study, we have demonstrated that the combination of exogenous IL-6 and soluble IL-6 receptor (sIL-6R) could significantly upregulate protein S production in both primary human umbilical vein endothelial cells (HUVEC) and in the immortalized human microvascular endothelial cell line, HMEC-1. The IL-6/sIL-6R complex was also able to rapidly induce tyrosine phosphorylation of the IL-6 transducer, gpl30. Neutralizing antibodies directed against either IL-6 or gpl30 blocked protein S upregulation by the IL-6/sIL-6R complex. It was also observed that exogenous sIL-6R could also upregulate protein S by forming a complex with IL-6 constitutively produced by the endothelial cell. Two other cytokines which also utilize the gpl30 receptor, oncostatin M (OSM) and leukemia inhibitory factor (LIF), were also able to upregulate endothelial cell protein S. This study demonstrates a mechanism that allows endothelial cells to respond to IL-6 and also illustrates the potential importance of circulating soluble receptors in the regulation of the anticoagulation pathway.

scholarly journals Tracking changes in adaptation to suspension growth for MDCK cells: cell growth correlates with levels of metabolites, enzymes and proteins

2021 ◽  
Vol 105 (5) ◽  
pp. 1861-1874
Author(s):  
Sabine Pech ◽  
Markus Rehberg ◽  
Robert Janke ◽  
Dirk Benndorf ◽  
Yvonne Genzel ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Line ◽  
Mdck Cells ◽  
Suspension Cell ◽  
Regulatory Mechanisms ◽  
Adherent Cell ◽  
Holistic View ◽  
Cellular Processes ◽  
Viral Vaccine ◽  
Analytical Approaches

Abstract Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth. Key points • Less and more efficient glucose utilization for suspension cell growth • Concerted alteration of metabolic enzyme activity and protein expression • Protein candidates to interfere glycolytic activity in MDCK cells

An avian cell line designed for production of highly attenuated viruses

Vaccine ◽  
2009 ◽  
Vol 27 (5) ◽  
pp. 748-756 ◽  
Author(s):  
Ingo Jordan ◽  
Ad Vos ◽  
Stefanie Beilfuß ◽  
Andreas Neubert ◽  
Sabine Breul ◽  
...  
Keyword(s):  
Cell Line ◽  
Avian Cell ◽  
Attenuated Viruses
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