Effect of Early Enteral Feeding on Apolipoprotein AI Levels and High-Density Lipoprotein Heterogeneity in Preterm Infants

Background/Aim: We have previously shown that infants receiving total parenteral nutrition have low apolipoprotein Al levels which are associated with high-density lipoprotein (HDL) class distributions as in lecithin:cholesterol acyltransferase deficiency. This study investigates the influence of early enteral feedings on apolipoprotein Al and HDL subclasses. Methods: Apolipoprotein Al and HDL distributions were determined in 15 total parenterally fed preterm infants (TPN group) receiving early feedings, in 28 enterally fed preterm infants (ENT group), and in 26 term infants at birth and on day 5. The HDL subclasses were determined by gradient gel electrophoresis. Results: In the TPN group, the apolipoprotein Al levels increased significantly postnatally (from 73 +/- 16 to 104 +/- 23 mg/dl) to levels found in the term and ENT groups on day 5 (88 +/- 16 and 96 +/- 19 mg/dl). The HDL subclass distributions at birth and on day 5 were similar in both TPN and ENT groups with more large HDL2b and less small HDL3c than in term infants. Whereas the HDL subclass distribution of term infants remained unchanged, in TPN and ENT infants, a shift from HDL2b to HDL3c was observed, with no difference between term and preterm infants on day 5. Conclusion: In contrast to exclusively parenterally fed infants, infants receiving early enteral feedings exhibited a significant rise of apolipoprotein Al and HDL subclass distributions as fully enterally fed preterm infants. Copyright (C) 2002 S. Karger AG, Basel

Production of a fusogenic oncolytic rVSV-NDV virus: Cell-line screening and process development in small-scale suspension cultures

Fusogenic oncolytic viruses represent a novel class of immunotherapeutics, which offer hope for the treatment of otherwise incurable cancers. Their enhanced intratumoral spread through syncytia formation allows for a potent mechanism of tumor cell death and induction of antitumor immune responses [1]. While the ability of these viruses to induce cell-cell fusion reactions offers numerous beneficial properties, it also presents unique challenges for large-scale clinical-grade manufacturing. Infected cells rapidly fuse with surrounding cells, resulting in large multinucleated syncytia, which quickly die before high titers of the virus can be produced or released [2]. Here, we evaluated the production of a novel hyper-fusogenic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV) in four different suspension cell lines. Cell growth, metabolism, and virus productivity were characterized for each candidate respectively. Permissiveness was evaluated based on extracellular infectious virus titer and cell-specific virus yields (CSVY). For the purpose of process intensification, virus adaptation, and multiplicity of infection (MOI) screenings were conducted in small-scale and confirmed in a 1 L bioreactor. BHK-21 and HEK293SF were identified as promising candidates for rVSV-NDV production, yielding infectious titers at infection cell concentrations of 2.0 E06 cells/mL of up to 3.0 E08 TCID50/mL and 7.5 E07 TCID50/mL, and CSVYs of 153 and 9, respectively. Oncolytic potency was not affected by production in suspension cultures compared to the reference stock produced in adherent AGE1.CR.pIX cultures. Overall, promising suspension cell substrates were identified for a highly efficient and scalable production process of this fusogenic rVSV-NDV. This paves the way for an efficient large-scale manufacturing process, which can be further intensified towards high cell density production in order to provide sufficient virus material for conducting a phase I clinical trial of oncolytic VSV-NDV in cancer patients. 1. Krabbe, T. and J. Altomonte, Fusogenic Viruses in Oncolytic Immunotherapy. Cancers (Basel), 2018. 10(7). 2. Abdullahi, S., et al., A Novel Chimeric Oncolytic Virus Vector for Improved Safety and Efficacy as a Platform for the Treatment of Hepatocellular Carcinoma. J Virol, 2018. 92(23)

Production of a fusogenic oncolytic rVSV-NDV virus in perfusion processes

Oncolytic viruses (OVs), as a therapeutic vaccine, offer an elegant approach to cancer therapy. On the one side they have the ability to cause direct tumor cell lysis, on the other side they can stimulate immune responses directed against the tumor. By expressing endogenous or heterologous fusion glycoproteins, an enhanced intratumoral spread via syncytia formation can be achieved. Rapid and efficient fusion of infected cells may result in large multinucleated syncytia, in which cells quickly die before high titers are reached [1]. Prospective treatment with OVs will require manufacturing processes that enable the production of a very high number of doses with high titers. As a first step towards this goal, suspension cell substrates were identified to develop a highly efficient and scalable production process of a novel hyper-fusogenic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV). Please click Download on the upper right corner to see the full abstract

Integrated end-to-end MVA viral vector production: Perfusion culture shows economical advantage over batch culture

Modified Vaccinia Ankara (MVA) virus is a promising viral vector for gene therapy. Several pre-clinical and clinical trials are currently being conducted with MVA as a live vector vaccine against COVID-19, Ebola disease, influenza or various types of cancers. For most applications, a large amount of the vector will be required (\u3e108 infectious virus per dose). High cell concentrations are favorable for developing high-yield MVA vector production systems. Efficient production of MVA in an avian suspension cell line (AGE1.CR.pIX) cultivated in perfusion mode with a membrane-based cell retention system has previously been demonstrated. However, up to now a direct harvest through the membrane for a continuous integrated process was not feasible. Please click Additional Files below to see the full abstract

Scale-down of an orbital shaken bioreactor: High cell density cultivation in perfusion mode and virus production

Application of single-use bioreactors has been commonly shown for several cell culture-based production systems including commercial vaccine production. Compared to stainless steel bioreactors, competitive cell growth characteristics as well as virus yields can be reached [1]. In addition to conventional stirred tank reactors (STR), wave bioreactors or orbital shaken bioreactors (OSBs) are available that rely on alternative mixing regimes. For small-scale screening of clones and media, cell maintenance and process optimization, OSBs are the most widely used system. Besides their simple design and ease of handling, OSBs allow for robust processes due to reduced mechanical stress caused by stirring and aeration [2]. Furthermore, scale-up (£ 2500 L) is simplified as larger OSBs rely on the same basic principles for mixing and aeration (e.g. bubble-free surface gassing). Particularly for high cell density (HCD) processes, high oxygen transfer rates, short mixing times, and low shear stress are beneficial. Until now, the step from spin tubes or shake flasks into larger OSBs was rather large, as only the OSB SB10-X (Kühner AG, Switzerland) with a minimum working volume (wv) of 4-5 L was available. In this study, a novel scale-down 3 L vessel module (wv = 1-3 L) for the OSB SB10-X was evaluated for cultivation of suspension BHK-21 cells (CEVA, Germany) in perfusion mode to HCD. Cultivation was carried out in serum-free medium in a 3 L and 10 L single-use standard bag with 3 L and 5 L initial wv and 100 and 70 rpm shaking frequency with a shaking diameter of 50 mm, respectively. For perfusion, an alternating tangential flow system (ATF2, Repligen) with a cut-off of 0.4 µm (SB10-X) and 0.5 µm (SB3-X), respectively, was used. Following an initial batch phase of 2-3 days, perfusion was initiated. After a complete media exchange, cells in the 3 L vessel module were infected with a fusogenic oncolytic virus (rVSV-NDV, recombinant vesicular stomatitis virus-Newcastle disease virus) at a cell concentration of 44.5x106 cells/mL at a multiplicity of infection (MOI) of 10-4. The obtained data were compared to a cultivation of BHK-21 cells in the standard SB10-X module (infection at a cell concentration of 12.5x106 cells/mL with yellow fever virus WHO 17D-213/77 with an MOI of 10-3) and to a cultivation in a 1 L STR. The novel 3 L vessel module allowed for a successful and direct scale-down utilizing the SB10-X backbone without the need for further optimization. For both the SB10-X and the 3 L vessel module, the ATF system was successfully coupled and cell concentrations of 32.7x106 cells/mL and 45.9x106 cells/mL were reached with high viabilities above 98%, respectively. A faster doubling time (tD=22 h) was observed in the 3 L vessel module compared to the SB10-X system (tD=27 h). For rVSV-NDV production, similar infectious virus titers were reached compared to perfusion cultivations of BHK-21 cells in a 1 L STR. Volumetric media consumption was significantly reduced in the 3 L vessel module, facilitating the implementation of OSB systems in non-industrial research environments. All in all, we demonstrated the adaptability and scalability of the single-use OSB system for the production of various viruses in HCD perfusion mode. References [1] Gallo-Ramirez, L. E., A. Nikolay, Y. Genzel, and U. Reichl. 2015. Bioreactor concepts for cell culture-based viral vaccine production. Expert Rev Vaccines 14 (9):1181-95. doi: 10.1586/14760584.2015.1067144 [2] Klöckner W, Diederichs S, Büchs J. Orbitally shaken single-use bioreactors. Adv Biochem Eng Biotechnol. 2014;138:45-60. doi: 10.1007/10_2013_188. PMID: 23604207

Baseline characteristics of 14 weeks old male BL6 mice treated with GlcNAc or kept on ad libitum standard chow.

<p>A) and B) Baseline creatinine and urea values (control n = 5, GlcNAc i.p. n = 5, GlcNAc gavage n = 6) C) Plasma GlcNAc levels 2 hours after gavage of 250 μl of a 10% GlcNAc solution (n = 6) and in control animals with ad libitum access to water and standard chow (n = 5). D) Weight curves of control mice (n = 10) and mice kept on a 4 weeks ad libitum diet of chow enriched with 0.5% GlcNAc (n = 9).</p

TUNEL stainings of mouse kidneys.

<p>A) Control kidney without IR damage. B) and C) Mouse kidneys 24 hours after IR. B) Control mouse on standard ad libitum chow and drinking water. C) Mouse treated with twice oral gavage of 250 μl of 10% GlcNAc solution.</p

Histology before and after IR.

<p>A) Damage score after inspection of 5 HPF at the corticomedullary border (control baseline n = 5, control 24 hours n = 6, GlcNAc baseline n = 11, GlcNAc 24 hours n = 14). Sections were evaluated in a blinded manner by an experienced nephropathologist. B)–D) Representative PAS stainings from kidneys before and after IR. (X 200) B) Uninephrectomy section from undamaged kidney. C) and D) Kidney sections 24 hours after the end of ischemia. Asterix marks vanishing or missing nuclei. Black arrows mark tubular casts consisting of tubular cells. Yellow arrows show necrotic areas and denuded tubuli with regions just consisting of naked basement membrane. C) Kidney from animal treated with twice oral gavage of 10% GlcNAc solution before IR. (D) Kidney from control animal with twice oral gavage of PBS before IR.</p

Kidney failure 24 hours after ischemia-reperfusion injury in preconditioned and control animals.

<p>Creatinine and urea values after unilateral nephrectomy followed by 40 minutes of ischemia and 24 hours of reperfusion (IR) of the contralateral kidney. Different dosing regimens for GlcNAc were used. A) and D) Mice were kept on a four-week ad libitum diet enriched with 0.5% GlcNAc (n = 17) or received standard chow ad libitum (n = 15). B) and E) Mice received a 250 μl PBS twice, 24 and 2 hours before IR by gavage (n = 5). In the verum group PBS contained 10% GlcNAc (n = 10). C) and F) Verum mice were injected 20 mg GlcNAc in PBS either one hour before surgery i.p (n = 3) or immediately at the end of ischemia at the beginning of reperfusion (n = 4) directly into the abdominal cavity. Baselines represent control animals from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161315#pone.0161315.g001" target="_blank">Fig 1A and 1B</a> without ischemia-reperfusion injury A) and D) for untreated animals (n = 5), B) and E) for mice after gavage of 10% GlcNAc solution (n = 6) and C) and F) for mice after i.p. injection of 10 mg GlcNAc (n = 5). After four weeks of caloric restriction (n = 4) with standard chow mice are protected against ischemia-reperfusion damage compared to controls having ad libitum access to standard chow (n = 5) (G+H).</p