Practically the entire global population is infected by herpesviruses that establish lifelong latency and can be reactivated. Alpha-herpesviruses, herpes simplex viruses 1 and 2 (HSV-1/HSV-2) and ...varicella zoster virus (VZV), establish latency in sensory neurons and then reactivate to infect epithelial cells in the mucosa or skin, resulting in a vesicular rash. Licensed antivirals inhibit virus replication, but do not affect latency. On reactivation, VZV causes herpes zoster, also known as shingles. The 76-year-old first author of this paper published an autobiography of his own severe herpes zoster ophthalmicus (HZO) infection with orbital edema, which is considered an emergency condition. Acyclovir (ACV) treatment was complemented with an immunostimulatory viral therapy, which resolved most symptoms within a few days. The orally administered live-attenuated infectious bursal disease vaccine virus (IBDV) delivers its double-stranded RNA (dsRNA) cargo to host cells and activates the natural antiviral interferon (IFN) gene defense system from within the host cells. IBDV has already been demonstrated to be safe and effective against five different families of viruses, hepatitis A virus (HAV), hepatitis B and C virus (HBV/HCV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and varicella zoster virus (VZV). Here we propose a short phase I/II trial in elderly shingles patients who will be assigned to receive either ACV monotherapy or ACV combined with R903/78, an attenuated immunostimulatory IBDV strain. The primary endpoints will be safety, but the efficacy of the combination therapy against the ACV monotherapy also will be assessed.
Continuous cell culture-based influenza vaccine production could significantly reduce footprint and manufacturing costs compared to current batch processing. However, yields of influenza virus in ...continuous mode can be affected by oscillations in virus titers caused by periodic accumulation of defective interfering particles. The generation of such particles has also been observed previously in cascades of continuous stirred tank reactors (CSTRs) and is known as the "von Magnus effect". To improve virus yields and to avoid these oscillations, we have developed a novel continuous tubular bioreactor system for influenza A virus production. It was built using a 500 mL CSTR for cell growth linked to a 105 m long tubular plug-flow bioreactor (PFBR). Virus propagation took place only in the PFBR with a nominal residence time of 20 h and a production capacity of 0.2 mL/min. The bioreactor was first tested with suspension MDCK cells at different multiplicities of infection (MOI), and then with suspension avian AGE1.CR.pIX cells at a fixed nominal MOI of 0.02. Maximum hemagglutinin (HA) titers of 2.4 and 1.6 log10(HA units/100 μL) for suspension MDCK cells and AGE1.CR.pIX cells, respectively, were obtained. Flow cytometric analysis demonstrated that 100% infected cells with batch-like HA titers can be obtained at a MOI of at least 0.1. Stable HA and TCID50 titers over 18 days of production were confirmed using the AGE1.CR.pIX cell line, and PCR analysis demonstrated stable production of full-length genome. The contamination level of segments with deletions (potentially defective interfering particles), already present in the virus seed, was low and did not increase. Control experiments using batch and semi-continuous cultures confirmed these findings. A comparison showed that influenza virus production can be achieved with the tubular bioreactor system in about half the time with a space-time-yield up to two times higher than for typical batch cultures. In summary, a novel continuous tubular bioreactor system for cell culture-based influenza virus production was developed. One main advantage, an essentially single-passage amplification of viruses, should enable efficient production of vaccines as well as vectors for gene and cancer therapy.
Vaccines are complex products that are manufactured in highly dynamic processes. Cellular substrates are one critical component that can have an enormous impact on reactogenicity of the final ...preparation, level of attenuation of a live virus, yield of infectious units or antigens, and cost per vaccine dose. Such parameters contribute to feasibility and affordability of vaccine programs both in industrialized countries and developing regions. This review summarizes the diversity of cellular substrates for propagation of viral vaccines from primary tissue explants and embryonated chicken eggs to designed continuous cell lines of human and avian origin.
Mass vaccination of dogs as important rabies reservoir is proposed to most effectively reduce and eliminate rabies also in humans. However, a minimum coverage of 70% needs to be achieved for control ...of the disease in zoonotic regions. In numerous developing countries, dog vaccination rate is still dangerously low because of economic constraints and due to a high turnover in dog populations. Improved vaccine production processes may help to alleviate cost and supply limitations. In this work, we studied and optimized the replication and vaccine potency of PV rabies virus strain in the muscovy-duck derived AGE1.CR and AGE1.CR.pIX suspension cell lines. The BHK-21-adapted PV rabies virus strain replicated efficiently in the avian cell lines without requirement for prior passaging. CR.pIX was previously shown to augment heat shock responses and supported slightly higher infectious titers compared to the parental CR cell line. Both cell lines allowed replication of rabies virus also in absence of recombinant IGF, the only complex component of the chemically defined medium that was developed for the two cell lines. After scale-up from optimization experiments in shake flask to production in 7-l bioreactors peak virus titers of 2.4 x 10.sup.8 FFU/ml were obtained. The potency of inactivated rabies virus harvest according to the NIH test was 3.5 IU/ml. Perfusion with the chemically defined medium during the virus replication phase improved the potency of the vaccine twofold, and increased the number of doses 9.6 fold. This study demonstrates that a rabies vaccine for animal vaccination can be produced efficiently in the AGE1.CR.pIX suspension cell line in a scalable process in chemically defined medium.
BackgroundCoronavirus disease 2019 (COVID-19) vaccination has substantially altered the course of the pandemic, saving tens of millions of lives globally. The problem is that despite such spectacular ...results, vaccination alone will not be able to control the COVID-19 pandemic because of the rapid evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) even in vaccinated human populations. Therefore, the development of a post-infection, broad-based, orally administered antiviral therapy that would complement vaccination efforts is urgently needed.MethodologyThe so-called viral superinfection therapy (SIT) administers a nonpathogenic attenuated double-stranded RNA (dsRNA) vaccine virus drug candidate, the infectious bursal disease virus serotype R903/78 (IBDV-R903/78) that activates the interferon (IFN) genes, which are the natural, antiviral defense system of host cells.ResultsHere we present two cases of properly vaccinated (with BNT162b2-Pfizer) and booster-dosed COVID-19 patients with vaccine breakthrough infections whose disease duration was shortened to a few days by oral SIT.ConclusionsSIT has already been demonstrated to be safe and effective against five different families of viruses, hepatitis A virus, hepatitis B virus, hepatitis C virus, SARS-CoV-2, and herpes zoster virus. The R903/78 drug candidate is simple to manufacture and easy to administer in an outpatient setting. The expected cost of SIT will be affordable even in resource-limited countries.
Modified vaccinia virus Ankara is a versatile vaccine vector, well suited for transgene delivery, with an excellent safety profile. However, certain transgenes render recombinant MVA (rMVA) ...genetically unstable, leading to the accumulation of mutated rMVA with impaired transgene expression. This represents a major challenge for upscaling and manufacturing of rMVA vaccines. To prevent transgene-mediated negative selection, the continuous avian cell line AGE1.CR pIX (CR pIX) was modified to suppress transgene expression during rMVA generation and amplification. This was achieved by constitutively expressing a tetracycline repressor (TetR) together with a rat-derived shRNA in engineered CR pIX PRO suppressor cells targeting an operator element (tetO) and 3' untranslated sequence motif on a chimeric poxviral promoter and the transgene mRNA, respectively. This cell line was instrumental in generating two rMVA (isolate CR19) expressing a
papillomavirus type 3 (MfPV3) E1E2E6E7 artificially-fused polyprotein following recombination-mediated integration of the coding sequences into the DelIII (CR19 M-DelIII) or TK locus (CR19 M-TK), respectively. Characterization of rMVA on parental CR pIX or engineered CR pIX PRO suppressor cells revealed enhanced replication kinetics, higher virus titers and a focus morphology equaling wild-type MVA, when transgene expression was suppressed. Serially passaging both rMVA ten times on parental CR pIX cells and tracking E1E2E6E7 expression by flow cytometry revealed a rapid loss of transgene product after only few passages. PCR analysis and next-generation sequencing demonstrated that rMVA accumulated mutations within the E1E2E6E7 open reading frame (CR19 M-TK) or deletions of the whole transgene cassette (CR19 M-DelIII). In contrast, CR pIX PRO suppressor cells preserved robust transgene expression for up to 10 passages, however, rMVAs were more stable when E1E2E6E7 was integrated into the TK as compared to the DelIII locus. In conclusion, sustained knock-down of transgene expression in CR pIX PRO suppressor cells facilitates the generation, propagation and large-scale manufacturing of rMVA with transgenes hampering viral replication.
Vectored vaccines based on highly attenuated modified vaccinia Ankara (MVA) are reported to be immunogenic, tolerant to pre-existing immunity, and able to accommodate and stably maintain very large ...transgenes. MVA is usually produced on primary chicken embryo fibroblasts, but production processes based on continuous cell lines emerge as increasingly robust and cost-effective alternatives. An isolate of a hitherto undescribed genotype was recovered by passage of a non-plaque-purified preparation of MVA in a continuous anatine suspension cell line (CR.pIX) in chemically defined medium. The novel isolate (MVA-CR19) replicated to higher infectious titers in the extracellular volume of suspension cultures and induced fewer syncytia in adherent cultures. We now extend previous studies with the investigation of the point mutations in structural genes of MVA-CR19 and describe an additional point mutation in a regulatory gene. We furthermore map and discuss an extensive rearrangement of the left telomer of MVA-CR19 that appears to have occurred by duplication of the right telomer. This event caused deletions and duplications of genes that may modulate immunologic properties of MVA-CR19 as a vaccine vector. Our characterizations also highlight the exceptional genetic stability of plaque-purified MVA: although the phenotype of MVA-CR19 appears to be advantageous for replication, we found that all genetic markers that differentiate wildtype and MVA-CR19 are stably maintained in passages of recombinant viruses based on either wildtype or MVA-CR.
Influenza viruses have been successfully propagated using a variety of animal cell lines in batch, fed-batch, and perfusion culture. For suspension cells, most studies reported on membrane-based cell ...retention devices typically leading to an accumulation of viruses in the bioreactor in perfusion mode. Aiming at continuous virus harvesting for improved productivities, an inclined settler was evaluated for influenza A virus (IAV) production using the avian suspension cell line AGE1.CR.pIX. Inclined settlers present many advantages as they are scalable, robust, and comply with cGMP regulations, e.g., for recombinant protein manufacturing. Perfusion rates up to 3000 L/day have been reported. In our study, successful growth of AGE1.CR.pIX cells up to 50 × 10
6
cells/mL and a cell retention efficiency exceeding 96% were obtained with the settler cooled to room temperature. No virus retention was observed. A total of 5.4–6.5 × 10
13
virions were produced while a control experiment with an ATF system equaled to 1.9 × 10
13
virions. For infection at 25 × 10
6
cells/mL, cell-specific virus yields up to 3474 virions/cell were obtained, about 5-fold higher than for an ATF based cultivation performed as a control (723 virions/cell). Trypsin activity was shown to have a large impact on cell growth dynamics after infection following the cell retention device, especially at a cell concentration of 50 × 10
6
cells/mL. Further control experiments performed with an acoustic settler showed that virus production was improved with a heat exchanger of the inclined settler operated at 27°C. In summary, cell culture-based production of viruses in perfusion mode with an inclined settler and continuous harvesting can drastically increase IAV yields and possibly the yield of other viruses. To our knowledge, this is the first report to show the potential of this device for viral vaccine production.
For the improved production of vaccines and therapeutic proteins, a detailed understanding of the metabolic dynamics during batch or fed-batch production is requested. To study the new human cell ...line AGE1.HN, a flexible metabolic flux analysis method was developed that is considering dynamic changes in growth and metabolism during cultivation. This method comprises analysis of formation of cellular components as well as conversion of major substrates and products, spline fitting of dynamic data and flux estimation using metabolite balancing. During batch cultivation of AGE1.HN three distinct phases were observed, an initial one with consumption of pyruvate and high glycolytic activity, a second characterized by a highly efficient metabolism with very little energy spilling waste production and a third with glutamine limitation and decreasing viability. Main events triggering changes in cellular metabolism were depletion of pyruvate and glutamine. Potential targets for the improvement identified from the analysis are (i) reduction of overflow metabolism in the beginning of cultivation, e.g. accomplished by reduction of pyruvate content in the medium and (ii) prolongation of phase 2 with its highly efficient energy metabolism applying e.g. specific feeding strategies. The method presented allows fast and reliable metabolic flux analysis during the development of producer cells and production processes from microtiter plate to large scale reactors with moderate analytical and computational effort. It seems well suited to guide media optimization and genetic engineering of producing cell lines.
Analytical methods fr direct quantitative
-glycan analysis require a sequence of sample preparation and clean-up steps that result in reduced glycan recovery. Therefore, we aimed to combine glycan ...release and labeling steps. Based on the hypothesis that the reaction mechanism for oxidative chemical glycan release comprises a stable glycan isocyanate intermediate, we investigated whether this could be exploited for the in-situ preparation of fluorescent glycan conjugates. ANTS-labeled
-glycans were derived from chicken ovalbumin via an in-situ chemical release/coupling approach and by standard Peptide-
-Glycosidase F (PNGase F) digestion/reductive amination. Synoptic fluorescence-assisted carbohydrate electrophoresis with UV detection (FACE-UV) analysis yielded matching patterns of fluorescent
-glycan bands in the expected electrophoretic mobility range between hexose units GU-5 and GU-11 of the standard. Anthranilamide (2-AB)-glycan conjugates prepared from a test glycoprotein carrying a predominant Core-F glycan gave single predominant peaks in hydrophilic interaction chromatography with fluorescence detection (HILIC-FLD) and electrospray ionization mass spectrometry (ESI-MS) spectra in agreement with sodiated triply charged Core-F-AB conjugates for both the standard and the in-situ coupling methods. The Core-F-AB conjugate prepared by the in-situ coupling approach had a slightly elevated retention time on HILIC-FLD and an ESI-MS
/
peak in line with a urea-bonded glycan-AB conjugate, with closed pyran ring structures on the glycan moiety. Glycan isocyanates intermittently formed during chemical glycan release, which could be utilized to prepare labeled glycan samples directly from glycoproteins and fluorescent dyes bearing a primary amine functional group.
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