Purifying viruses with a sheet of paper: Single-use steric exclusion chromatography as a capture platform for vaccine candidates

Steric exclusion chromatography (SXC) is a method in which a crude sample is mixed with polyethylene glycol (PEG) and fed to a hydrophilic stationary phase. Selectivity in SXC is strongly influenced by the target species’ size, so it is particularly well suited for purification of large biomolecules such as viruses and virus-like-particles. The product is captured without a direct chemical interaction thanks to the mutual steric exclusion of PEG between the product and the stationary phase (cellulose membranes with micron-sized pores). Product elution is achieved by removing the PEG from solution, and can theoretically be made in any buffer system. The low cost of the cellulose membranes allows this operation to be single-use. Using SXC, we have achieved virtually full recovery of several viruses produced in serum-free mammalian cell culture: influenza virus, yellow fever virus, and Modified Vaccinia Ankara (MVA) virus. For influenza virus, four different strains were produced separately in MDCK cell suspension cultures using either chemically defined medium or serum-free medium. Full recovery of all strains was observed using identical SXC conditions (loading with 8% PEG-6000) for both infectious and chemically inactivated virus particles. Coupling a nuclease treatment for DNA digestion prior to SXC, dsDNA was depleted \u3e99.98%. The column capacity in terms of the viral hemagglutinin antigen was at least 50 mg m−2. In the case of yellow fever virus, two attenuated strains used for commercial manufacture were produced separately in adherent Vero cells grown in serum-free medium. Full recovery of infective virus titer for both strains was attained using 10% PEG-6000 for sample load. The elution fraction was concentrated \u3e100-fold compared to the feed with the very high titer of 6×109 plaque forming units, equivalent to ≈100 000 doses. Total recovery was also observed for MVA virus loaded at 4% PEG-6000; produced in an avian cell line in chemically defined medium, the SXC elution pools contained ≈3.7×109 virions as estimated by TCID50 assay. In conclusion, SXC can drastically reduce process development in terms of time and equipment requirements. The convenience of purifying different virus strains using similar chromatography conditions is almost impossible to match by other methods, as are the high product recoveries typically achieved with SXC. The latter gives space to include additional polishing operations without risking low overall process yields. We deem membrane-based SXC as a promising platform technology for capturing viruses and virus-like particles in vaccine manufacturing

A single-use chromatographic purification platform for viral gene transfer vectors & viral vaccines

In steric exclusion chromatography (SXC), a crude sample is mixed with polyethylene glycol (PEG) and fed onto a single-use cellulose column. In this operation, selectivity is influenced strongly by the target species’ size, so SXC is well suited for purification of virus particles. The purified product is recovered at physiological pH and conductivity. We have observed recoveries above 95% for several cell culture-based virus particles used as viral gene transfer vectors or as viral vaccines, including: adeno-associated virus (AAV), Modified Vaccinia Ankara (MVA) virus, influenza virus, and yellow fever virus. Preliminary data for purification of lentiviruses suggests recoveries exceeding 60%. Host cell DNA and protein depletion are typically above 90% and infectivity is not compromised thanks to the inert character of PEG towards biomolecules and the mild elution conditions. Several AAV serotypes and display mutants were produced using HEK cells and purified with up to 95% recovery. Elution fractions had ≤2×1014 viral genomes·L−1 and, depending on the specific AVV particle, the purified viruses successfully transduced or induced gene knockdown in vitro. Elution pools from MVA virus produced in continuous bioreactors with an avian cell line contained about 3.7×109 infectious virions measured by TCID50. For influenza virus, four strains were produced in MDCK cells. Full recovery of all strains was observed using identical SXC conditions for both infectious and chemically inactivated viruses. The column capacity in terms of the viral hemagglutinin antigen was \u3e 50 mg·m−2. In the case of yellow fever virus, two attenuated strains were produced in Vero cells. Here, full recovery of infective titers was also achieved: the elution fraction was concentrated more than 100-fold to a titer of \u3e6×109 plaque forming units (≈100 000 doses). In summary, SXC capture with PEG and unmodified cellulose membranes seems to perform very well for a broad range of viruses from different production processes. Thanks to the high degree of success in a relatively narrow operational range, SXC can drastically reduce process development. The high recoveries obtained so far, enable subsequent polishing operations with minimum risk to low overall process yields

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

Generation of “OP7 chimera” defective interfering influenza A particle preparations free of infectious virus that show antiviral efficacy in mice

Influenza A virus (IAV) defective interfering particles (DIPs) are considered as new promising antiviral agents. Conventional DIPs (cDIPs) contain a deletion in the genome and can only replicate upon co-infection with infectious standard virus (STV), during which they suppress STV replication. We previously discovered a new type of IAV DIP “OP7” that entails genomic point mutations and displays higher antiviral efficacy than cDIPs. To avoid safety concerns for the medical use of OP7 preparations, we developed a production system that does not depend on infectious IAV. We reconstituted a mixture of DIPs consisting of cDIPs and OP7 chimera DIPs, in which both harbor a deletion in their genome. To complement the defect, the deleted viral protein is expressed by the suspension cell line used for production in shake flasks. Here, DIP preparations harvested are not contaminated with infectious virions, and the fraction of OP7 chimera DIPs depended on the multiplicity of infection. Intranasal administration of OP7 chimera DIP material was well tolerated in mice. A rescue from an otherwise lethal IAV infection and no signs of disease upon OP7 chimera DIP co-infection demonstrated the remarkable antiviral efficacy. The clinical development of this new class of broad-spectrum antiviral may contribute to pandemic preparedness

Broad-Spectrum Antiviral Activity of Influenza A Defective Interfering Particles against Respiratory Syncytial, Yellow Fever, and Zika Virus Replication In Vitro

New broadly acting and readily available antiviral agents are needed to combat existing and emerging viruses. Defective interfering particles (DIPs) of influenza A virus (IAV) are regarded as promising options for the prevention and treatment of IAV infections. Interestingly, IAV DIPs also inhibit unrelated viral infections by stimulating antiviral innate immunity. Here, we tested the ability of IAV DIPs to suppress respiratory syncytial, yellow fever and Zika virus infections in vitro. In human lung (A549) cells, IAV DIP co-infection inhibited the replication and spread of all three viruses. In contrast, we observed no antiviral activity in Vero cells, which are deficient in the production of interferon (IFN), demonstrating its importance for the antiviral effect. Further, in A549 cells, we observed an enhanced type-I and type-III IFN response upon co-infection that appears to explain the antiviral potential of IAV DIPs. Finally, a lack of antiviral activity in the presence of the Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib was detected. This revealed a dependency of the antiviral activity on the JAK/signal transducers and activators of transcription (STAT) signaling pathway. Overall, this study supports the notion that IAV DIPs may be used as broad-spectrum antivirals to treat infections with a variety of IFN-sensitive viruses, particularly respiratory viruses

Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles.

In the present study, we generated a genetically engineered MDCK suspension cell line for production of a purely clonal DIP preparation that has a large deletion in its segment 1 (DI244) and is not contaminated with infectious STV as egg-derived material. First, the impact of the multiplicity of DIP (MODIP) per cell on DI244 yield was investigated in batch cultivations in shake flasks. Here, the highest interfering efficacy was observed for material produced at a MODIP of 1E-2 using an in vitro interference assay. Results of RT-PCR suggested that DI244 material produced was hardly contaminated with other defective particles. Next, the process was successfully transferred to a stirred tank bioreactor (500 mL working volume) with a yield of 6.0E+8 PFU/mL determined in genetically modified adherent MDCK cells. The produced material was purified and concentrated about 40-fold by membrane-based steric exclusion chromatography (SXC). The DI244 yield was 92.3% with a host cell DNA clearance of 97.1% (99.95% with nuclease digestion prior to SXC) and a total protein reduction of 97.2%. Finally, the DIP material was tested in animal experiments in D2(B6).A2G-Mx1r/r mice. Mice infected with a lethal dose of IAV and treated with DIP material showed a reduced body weight loss and all animals survived

Generation of “OP7 chimera” defective interfering influenza A particle preparations free of infectious virus that show antiviral efficacy in mice

Abstract Influenza A virus (IAV) defective interfering particles (DIPs) are considered as new promising antiviral agents. Conventional DIPs (cDIPs) contain a deletion in the genome and can only replicate upon co-infection with infectious standard virus (STV), during which they suppress STV replication. We previously discovered a new type of IAV DIP “OP7” that entails genomic point mutations and displays higher antiviral efficacy than cDIPs. To avoid safety concerns for the medical use of OP7 preparations, we developed a production system that does not depend on infectious IAV. We reconstituted a mixture of DIPs consisting of cDIPs and OP7 chimera DIPs, in which both harbor a deletion in their genome. To complement the defect, the deleted viral protein is expressed by the suspension cell line used for production in shake flasks. Here, DIP preparations harvested are not contaminated with infectious virions, and the fraction of OP7 chimera DIPs depended on the multiplicity of infection. Intranasal administration of OP7 chimera DIP material was well tolerated in mice. A rescue from an otherwise lethal IAV infection and no signs of disease upon OP7 chimera DIP co-infection demonstrated the remarkable antiviral efficacy. The clinical development of this new class of broad-spectrum antiviral may contribute to pandemic preparedness

An inactivated SARS-CoV-2 vaccine based on a Vero cell culture-adapted high-titer virus confers cross-protection in small animals

Abstract Rapidly waning immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires continued global access to affordable vaccines. Globally, inactivated SARS-CoV-2 vaccines have been widely used during the SARS-CoV-2 pandemic. In this proof-of-concept study we adapted an original-D614G SARS-CoV-2 virus to Vero cell culture as a strategy to enhance inactivated vaccine manufacturing productivity. A passage 60 (P60) virus showed enhanced fitness and 50-fold increased virus yield in a bioreactor compared to the original-D614G virus. It further remained susceptible to neutralization by plasma from SARS-CoV-2 vaccinated and convalescent individuals, suggesting exposure of relevant epitopes. Monovalent inactivated P60 and bivalent inactivated P60/omicron BA.1 vaccines induced neutralizing responses against original-D614G and BA.1 viruses in mice and hamsters, demonstrating that the P60 virus is a suitable vaccine antigen. Antibodies further cross-neutralized delta and BA.5 viruses. Importantly, the inactivated P60 vaccine protected hamsters against disease upon challenge with original-D614G or BA.1 virus, with minimal lung pathology and lower virus loads in the upper and lower airways. Antigenicity of the P60 virus was thus retained compared to the original virus despite the acquisition of cell culture adaptive mutations. Consequently, cell culture adaptation may be a useful approach to increase yields in inactivated vaccine antigen production