Influenza A virus production in a single-use orbital shaken bioreactor with ATF or TFF perfusion systems

Driven by the concept of plug-and-play cell culture-based viral vaccine production using disposable bioreactors, we evaluated an orbital shaken bioreactor (OSB) for human influenza A virus production at high cell concentration. Therefore, the OSB model SB10-X was coupled to two hollow fiber-based perfusion systems, namely, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF). The AGE1.CR.pIX avian suspension cells grew to 50 × 10⁶ cells/mL in chemically defined medium, maintaining high cell viabilities with an average specific growth rate of 0.020 h⁻¹ (doubling time = 32 h). Maximum virus titers in the range of 3.28–3.73 log₁₀(HA units/100 µL) were achieved, corresponding to cell-specific virus yields of 1000–3500 virions/cell and productivities of 0.5–2.2 × 10¹² virions/L/d. This clearly demonstrates the potential of OSB operation in perfusion mode, as results achieved in a reference OSB batch cultivation were 2.64 log₁₀(HA units/100 µL), 1286 virions/cell and 1.4 × 10¹² virions/L/d, respectively. In summary, the SB10-X bioreactor can be operated with ATF and TFF systems, which is to our knowledge the first report regarding OSB operation in perfusion mode. Moreover, the results showed that the system is a promising cultivation system for influenza A virus vaccine production. The OSB disposable bioreactor has the potential for simplifying the scale-up from shake flasks to the large-scale bioreactor, facilitating rapid responses in the event of epidemics or pandemics.     

Comparison of influenza virus yields and apoptosis-induction in an adherent and a suspension MDCK cell line

Cell culture-based manufacturing of influenza vaccines is ideally based on easily scalable platforms using suspension cells that grow in chemically defined media. Consequently, different adherent cell lines selected for high virus yields were adapted to grow in suspension culture. This includes the MDCK suspension cell line MDCK.SUS2, which was shown to be a suitable substrate for influenza virus propagation in previous studies. In this study, we investigated options for further improvement of influenza A/PR/8 (H1N1) virus titres and cell-specific virus yields. Best results were achieved by performing a 1:2 dilution with fresh medium at time of infection. In shake flask cultivations, even for multiplicities of infection as low as 10⁻⁵, all cells were infected at 36 h post infection as determined by flow cytometry. In addition, these cells showed a better viability than cells infected without previous washing steps, which was reflected by a reduced level of apoptotic cells, and virus yields exceeding 3 log₁₀ HAU/100 μL. Comparison of bioreactor infections of MDCK.SUS2 cells to the parental adherent MDCK cells showed similar HA titres of 2.94 and 3.15 log₁₀ HAU/100 μL and TCID₅₀ of 1 × 10⁹ and 2.37 × 10⁹ infectious virions/mL. Surprisingly, virus-induced apoptosis differed between the two cell lines, with the MDCK.SUS2 cells showing a much stronger apoptosis induction than the adherent MDCK cells. Obviously, despite their resistance to anoikis, the suspension cells were more susceptible to virus-induced apoptosis. Whether this is related to the adaptation process itself and/or to changes in cell survival pathways influenced by adhesion molecules or influenza virus proteins needs to be clarified in additional studies.     

Efficient influenza A virus production in high cell density using the novel porcine suspension cell line PBG.PK2.1

Seasonal and pandemic influenza respiratory infections are still a major public health issue. Vaccination is the most efficient way to prevent influenza infection. One option to produce influenza vaccines is cell-culture based virus propagation. Different host cell lines, such as MDCK, Vero, AGE1.CR or PER.C6 cells have been shown to be a good substrate for influenza virus production. With respect to the ease of scale-up, suspension cells should be preferred over adherent cells. Ideally, they should replicate different influenza virus strains with high cell-specific yields. Evaluation of new cell lines and further development of processes is of considerable interest, as this increases the number of options regarding the design of manufacturing processes, flexibility of vaccine production and efficiency.Here, PBG.PK2.1, a new mammalian cell line that was developed by ProBioGen AG (Germany) for virus production is presented. The cells derived from immortal porcine kidney cells were previously adapted to growth in suspension in a chemically-defined medium. Influenza virus production was improved after virus adaptation to PBG.PK2.1 cells and optimization of infection conditions, namely multiplicity of infection and trypsin concentration. Hemagglutinin titers up to 3.24 log₁₀(HA units/100 µL) were obtained in fed-batch mode in bioreactors (700 mL working volume). Evaluation of virus propagation in high cell density culture using a hollow-fiber based system (ATF2) demonstrated promising performance: Cell concentrations of up to 50 × 10⁶ cells/mL with viabilities exceeding 95%, and a maximum HA titer of 3.93 log₁₀(HA units/100 µL). Analysis of glycosylation of the viral HA antigen expressed showed clear differences compared to HA produced in MDCK or Vero cell lines. With an average cell-specific productivity of 5000 virions/cell, we believe that PBG.PK2.1 cells are a very promising candidate to be considered for next-generation influenza virus vaccine production.     

Use of MDCK cells for production of live attenuated influenza vaccine

To develop a cell-based live attenuated influenza vaccine (LAIV) manufacturing process, several different cell lines were evaluated by comparing the titer of viruses after infection with LAIV strains. While several cell lines have been reported to support influenza virus replication, the degree of replication and the ability to support replication of LAIV strains have not been systematically examined. MDCK cells, which have been considered as potential substrates for influenza vaccine production were evaluated in addition to Vero, MRC-5, WI-38 and FRhL cells. MRC-5, WI-38 and FRhL cells produced low to moderate titers of virus with titers equal or below 5.0 log₁₀ TCID₅₀/mL. Both Vero and MDCK cells could support a higher level of virus replication for certain strains, however, Vero cells only produced high titers when grown in the presence of serum. MDCK cells supported high levels of vaccine virus production for multiple different LAIV subtypes in both serum containing and serum-free media. These results suggest that MDCK cell-based production can be used as an alternative production platform to the currently used egg-based LAIV production system.     

Production of high-titer human influenza A virus with adherent and suspension MDCK cells cultured in a single-use hollow fiber bioreactor

Hollow fiber bioreactors (HFBRs) have been widely described as capable of supporting the production of highly concentrated monoclonal antibodies and recombinant proteins. Only recently HFBRs have been proposed as new single-use platforms for production of high-titer influenza A virus. These bioreactors contain multiple hollow fiber capillary tubes that separate the bioreactor in an intra- and an extra-capillary space. Cells are usually cultured in the extra-capillary space and can grow to a very high cell concentration. This work describes the evaluation of the single-use hollow fiber bioreactor PRIMER HF^® (Biovest International Inc., USA) for production of influenza A virus. The process was setup, characterized and optimized by running a total of 15 cultivations. The HFBRs were seeded with either adherent or suspension MDCK cells, and infected with influenza virus A/PR/8/34 (H1N1), and the pandemic strain A/Mexico/4108/2009 (H1N1). High HA titers and TCID₅₀ of up to 3.87 log₁₀ (HA units/100 μL) and 1.8 × 10¹⁰ virions/mL, respectively, were obtained for A/PR/8/34 influenza strain. Influenza virus was collected by performing multiple harvests of the extra-capillary space during a virus production time of up to 12 days. Cell-specific virus yields between 2,000 and 8,000 virions/cell were estimated for adherent MDCK cells, and between 11,000 and 19,000 virions/cell for suspension MDCK.SUS2 cells. These results do not only coincide with the cell-specific virus yields obtained with cultivations in stirred tank bioreactors and other high cell density systems, but also demonstrate that HFBRs are promising and competitive single-use platforms that can be considered for commercial production of influenza virus.     

Replication of live attenuated influenza vaccine viruses in human nasal epithelial cells is associated with H1N1 vaccine effectiveness

In the 2013–2014 and 2015–2016 influenza seasons, live attenuated influenza vaccine (LAIV) generated reduced vaccine effectiveness (VE) against circulating H1N1 strains. This reduced VE coincided with the introduction of pandemic 2009 H1N1 (A/H1N1pdm09) vaccine virus reassortants, in place of pre-2009 seasonal H1N1 strains. Here, we explored one specific hypothesis for reduced VE; decreased replicative fitness of A/H1N1pdm09 strains in humans. Two A/H1N1pdm09 strains with reduced VE, A/California/07/2009 (A/CA09) and A/Bolivia/559/2013 (A/BOL13), were compared to pre-2009 seasonal H1N1 strains, A/New Caledonia/20/1999 (A/NC99) and A/South Dakota/6/2007 (A/SD07). Initial results showed that A/H1N1pdm09 strains had reduced multi-cycle infectivity in Madin-Darby Canine Kidney (MDCK) cells, compared to their pre-2009 counterparts. The A/BOL13 viral titre was found to be 2.65 log₁₀/mL lower when measured by multi-cycle 50% tissue culture infectious dose (TCID₅₀) assay compared to single-cycle fluorescent focus assay (FFA). By contrast, clinically effective A/NC99 titres differed by only 0.54 log₁₀/mL. In human alveolar (A549) cells, A/H1N1pdm09 strains replicated less than pre-2009 strains, with A/CA09 and A/BOL13 generating lower peak viral titres over 5 days. This phenotype was corroborated in physiologically relevant, primary human nasal epithelial cells (hNECs). Here, peak titres for pre-2009 strains A/NC99 and A/SD07 were 8.43 log₁₀ TCID₅₀/mL and 8.52 log₁₀ TCID₅₀/mL, respectively, versus 6.89 log₁₀ TCID₅₀/mL and 6.06 log₁₀ TCID₅₀/mL for A/H1N1pdm09 strains A/CA09 and A/BOL13. This confirmed a reduced ability of A/H1N1pdm09 strains to sustain replication in human respiratory cells. Using this information, H1N1 candidate A/Slovenia/2903/2015 (A/SLOV15) was characterised for replacement of A/BOL13 in the 2017/18 LAIV. A/SLOV15 produced comparable single and multi-cycle infectivity titres (Δ 0.16 log₁₀/mL) and reached a peak titre 1.23 log₁₀ TCID₅₀/mL higher than that of A/BOL13 in hNEC cultures. Taken together, these data suggest a reduction in sustained multi-cycle replication in human cells as a plausible root cause for reduced A/H1N1pdm09 VE.     

Development of a live-attenuated influenza B ΔNS1 intranasal vaccine candidate

We discovered a unique, single amino acid mutation in the influenza B M1 protein promoting viral growth of NS1 truncation mutants in Vero cells. Due to this mutation, we were able to generate an influenza B virus lacking the complete NS1 open reading frame (ΔNS1-B virus) by reverse genetics, which was growing to titers of 8 log₁₀ TCID₅₀/ml in a Vero cell culture-based micro-carrier fermenter. The ΔNS1-B vaccine candidate was attenuated in IFN-competent hosts such as human alveolar epithelial cells (A549) similar to influenza A ΔNS1 viruses. In ferrets, the ΔNS1-B virus was replication-deficient and did not provoke any clinical symptoms. Importantly, a single intranasal immunization of ferrets at a dose as low as 6 log₁₀ TCID₅₀/animal induced a significant HAI response and provided protection against challenge with wild-type influenza B virus. So far, the lack of a ΔNS1-B virus component growing to high titers in cell culture has been limiting the possibility to formulate a trivalent vaccine based on deletion of the NS1 gene. Our study closes this gap and paves the way for the clinical evaluation of a seasonal, trivalent, live replication-deficient ΔNS1 intranasal influenza vaccine.     

Adaptation of Vero cells to suspension growth for rabies virus production in different serum free media

Vero cells are nowadays widely used in the production of human vaccines. They are considered as one of the most productive and flexible continuous cell lines available for vaccine manufacturing. However, these cells are anchorage dependent, which greatly complicates upstream processing and process scale-up. Moreover, there is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines that are affordable worldwide. The use of cell lines adapted to suspension growth contributes to reach this objective.The current work describes the adaptation of Vero cells to suspension culture in different serum free media according to multiple protocols based on subsequent passages. The best one that relies on cell adaption to IPT-AFM an in-house developed animal component free medium was then chosen for further studies. Besides, as aggregates have been observed, the improvement of IPT-AFM composition and mechanical dissociation were also investigated.In addition to IPT-AFM, three chemically defined media (CD293, Hycell CHO and CD-U5) and two serum free media (293SFMII and SFM4CHO) were tested to set up a serum free culture of the suspension-adapted Vero cells (VeroS) in shake flasks. Cell density levels higher than 2 × 10⁶ cells/mL were obtained in the assessed conditions. The results were comparable to those obtained in spinner culture of adherent Vero cells grown on Cytodex 1 microcarriers.Cell infection with LP-2061 rabies virus strain at an MOI (Multiplicity of Infection) of 0.1 and a cell density of 8 ± 0.5 × 10⁵ cells/mL resulted in a virus titer higher than 10⁷ FFU/mL in all media tested. Nevertheless, the highest titer equal to 5.2 ± 0.5 × 10⁷ FFU/mL, was achieved in IPT-AFM containing a reduced amount of Ca⁺⁺ and Mg⁺⁺. Our results demonstrate the suitability of the obtained VeroS cells to produce rabies virus at a high titer, and pave the way to develop VeroS cells bioreactor process for rabies vaccine production.     

Process intensification for Peste des Petites Ruminants Virus vaccine production

Process intensification for Peste des Petites Ruminants Virus (PPRV) vaccine production in anchorage dependent Vero cells is challenging, involving substantial amount of bioprocess development.In this study, we describe the implementation of a new, scalable bioprocess for PPRV vaccine production in Vero cells using serum-free medium (SFM), microcarrier technology in stirred-tank bioreactors (STB), in-situ cell detachment from microcarriers and perfusion. Vero cells were successfully adapted to ProVero™-1 SFM, reaching growth rates similar to serum-containing cultures (0.030 1/h vs 0.026 1/h, respectively). An in-situ cell detachment method was successfully implemented, with efficiencies above 85%. Up to 2.5-fold increase in maximum cell concentration was obtained using perfusion when compared to batch culture. Combining perfusion with the in-situ cell detachment method enabled the scale-up to 20 L STB directly from a 2 L STB, surpassing the need for a mid-scale platform (i.e. 5 L STB) and thus reducing seed train duration. Head-to-head comparison of cell growth and PPRV production in the 2 L and 20 L STB was performed, and no significant differences could be observed. Estimated infectious PPRV titers in Tissue Culture Infection Dose (TCID₅₀) (TCID₅₀/mL = 5 × 10⁶ and TCID₅₀/cell = 5) are within the log-range reported in literature for PPRV production in STB and SFM by Silva et al. (2008), thus confirming the feasibility and scalability of the seed train designed [1].The novel and scalable vaccine production process herein proposed has the potential to assist the upcoming Peste des Petites Ruminants (PPR) Global Eradication Program (targeted by FAAO for 2030) by providing African local and/or regional manufacturers with a platform capable of generating over 25,000 doses of Nigeria 75/1 strain in just 19 days using a 20 L STB.     

Propagation of Brazilian Zika virus strains in static and suspension cultures using Vero and BHK cells

The recent spread of Zika virus (ZIKV) in the Americas and the Pacific has reached alarming levels in more than 60 countries. However, relatively little is known about the disease on a virological and epidemiological level and its consequences for humans. Accordingly, a large demand for in vitro derived Brazilian ZIKV material to support in vitro and in vivo studies has arisen. However, a prompt supply of ZIKV and ZIKV antigens cannot be guaranteed as the production of this virus typically using Vero or C6/36 cell lines remains challenging.Here we present a production platform based on BHK-21 suspension (BHK-21_SUS) cells to propagate Brazilian ZIKV at larger quantities in perfusion bioreactors. Scouting experiments performed in tissue culture flasks using adherent BHK-21 and Vero cells have demonstrated similar permissivity and virus yields for four different Brazilian ZIKV isolates. The cell-specific yield of infectious virus particles varied between respective virus strains (1–48 PFU/cell), and the ZIKV isolate from the Brazilian state Pernambuco (ZIKV^PE) showed to be a best performing isolate for both cell lines. However, infection studies of BHK-21_SUS cells with ZIKV^PE in shake flasks resulted in poor virus replication, with a maximum titer of 8.9 × 10³ PFU/mL. Additional RT-qPCR measurements of intracellular and extracellular viral RNA levels revealed high viral copy numbers within the cell, but poor virus release. Subsequent cultivation in a perfusion bioreactor using an alternating tangential flow filtration system (ATF) under controlled process conditions enabled cell concentrations of about 1.2 × 10⁷ cells/mL, and virus titers of 3.9 × 10⁷ PFU/mL. However, while the total number of infectious virus particles was increased, the cell-specific yield (3.3 PFU/cell) remained lower than determined in adherent cell lines. Nevertheless, the established perfusion process allows to provide large amounts of ZIKV material for research and is a first step towards process development for manufacturing inactivated or live-attenuated ZIKV vaccines.     

High-cell-density cultivations to increase MVA virus production

Increasing the yield and the productivity in cell culture-based vaccine manufacturing using high-cell-density (HCD) cultivations faces a number of challenges. For example, medium consumption should be low to obtain a very high concentration of viable host cells in an economical way but must be balanced against the requirement that accumulation of toxic metabolites and limitation of nutrients have to be avoided. HCD cultivations should also be optimized to avoid unwanted induction of apoptosis or autophagy during the early phase of virus infection. To realize the full potential of HCD cultivations, a rational analysis of the cultivation conditions of the appropriate host cell line together with the optimal infection conditions for the chosen viral vaccine strain needs to be performed for each particular manufacturing process.We here illustrate our strategy for production of the modified vaccinia Ankara (MVA) virus isolate MVA-CR19 in the avian suspension cell line AGE1.CR.pIX at HCD. As a first step we demonstrate that the adjustment of the perfusion rate strictly based on the measured cell concentration and the glucose consumption rate of cells enables optimal growth in a 0.8 L bioreactor equipped with an ATF2 system. Concentrations up to 57 × 10⁶ cells/mL (before infection) were obtained with a viability exceeding 95%, and a maximum specific cell growth rate of 0.019 h⁻¹ (doubling time = 36.5 h). However, not only the cell-specific MVA-CR19 virus yield but also the volumetric productivity was reduced compared to infections at conventional-cell-density (CCD).To facilitate optimization of the virus propagation phase at HCD, a larger set of feeding strategies was analyzed in small-scale cultivations using shake flasks. Densities up to 63 × 10⁶ cells/mL were obtained at the end of the cell growth phase applying a discontinuous perfusion mode (semi-perfusion) with the same cell-specific perfusion rate as in the bioreactor (0.060 nL/(cell d)). At this cell concentration, a medium exchange at time of infection was required to obtain expected virus yields during the first 24 h after infection. Applying an additional fed-batch feeding strategy during the whole virus replication phase resulted in a faster virus titer increase during the first 36 h after infection. In contrast, a semi-continuous virus harvest scheme improved virus accumulation and recovery at a rather later stage of infection. Overall, a combination of both fed-batch and medium exchange strategies resulted in similar cell-specific virus yields as those obtained for CCD processes but 10-fold higher MVA-CR19 titers, and four times higher volumetric productivity.     

Newcastle disease virus vectored infectious laryngotracheitis vaccines protect commercial broiler chickens in the presence of maternally derived antibodies

Newcastle disease virus (NDV) recombinants expressing the infectious laryngotracheitis virus (ILTV) glycoproteins B and D have previously been demonstrated to confer complete clinical protection against virulent ILTV and NDV challenges in naive chickens. We extended this study to assess whether maternally derived antibody (MDA) against NDV and ILTV would interfere with protection in vaccinated broiler chickens. Chickens with a mean NDV MDA hemagglutination inhibition (HI) titer of 6.4 (log₂) and detectable ILTV neutralization (VN) antibodies at hatch were vaccinated with rLS/ILTV-gB or rLS/ILTV-gD at 1 or 10 day of age (DOA) or on both days. Groups of birds vaccinated with the commercial ILT vaccines (FP-LT and CEO) or sham inoculated were also included in this study. All vaccinated birds were challenged with virulent ILTV strain at 21 DOA. By that time, NDV HI titers declined to 2.6 (log₂) in unvaccinated birds, whereas the HI titers in NDV vectored vaccine groups increased to 3.5–6.3 (log₂). At standard dosages, both vaccine candidates conferred significant clinical protection; however, the protection elicited by the rLS/ILTV-gD was superior to that of rLS/ILTV-gB. Recombinant rLS/ILTV-gD reduced ILTV shedding from tracheal and ocular tissues by approximately 3 log₁₀ TCID₅₀. Notably, there was no improvement in protection after booster vaccination at 10 DOA. Overall results indicate that the presence of maternal antibodies to NDV and ILTV did not significantly interfere with the ability of the NDV LaSota strain-vectored ILTV gB and gD vaccine candidates to elicit protective immunity against infectious laryngotracheitis.     

Safety and immunogenicity of a live attenuated Rift Valley Fever recombinant arMP-12ΔNSm21/384 vaccine candidate for sheep,goats and calves

Rift Valley fever (RVF) causes serious health and economic losses to the livestock industry as well as a significant cause of human disease. The prevention of RVF in Africa is a global priority, however, available vaccines have only been partially effective. Therefore, the objective of this study was to evaluate the safety and immunogenicity of a live, attenuated recombinant RVFV arMP-12ΔNSm21/384 nucleotide deletion vaccine candidate in domestic ruminants. Evaluation involved testing to determine the infectivity titer of the vaccine virus in Vero cells for industrial scale up vaccine production. Safety experiments were conducted to determine the potential of the vaccine virus to revert to virulence by serial passages in sheep, the possibility of virus spread from vaccinated sheep and calves to unvaccinated animals, and the potential health effects of administering overdoses of the vaccine to sheep, goats and calves. The immunogenicity of 3 doses of 10⁴, 10⁵ and 10⁶ Tissue Culture Infectious Doses_50% (TCID₅₀) of the vaccine was assessed in 3 groups of 10 sheep and 3 groups of 10 goats, and doses of 10⁵, 10⁶ and 10⁷ TCID₅₀ was evaluated in 3 groups of 10 calves subcutaenous vaccintation. The results showed that the infectivity titer of the vaccine virus was 10^8.4 TCID₅₀/ml, that the vaccine did not spread from vaccinated to un-vaccinated animals, there was no evidence of reversion to virulence in sheep and the vaccine overdoses did not cause any adverse effects. The immunogenicity among sheep, goats and calves indicated that doses of 10⁴–10⁶ TCID₅₀ elicited detectable antibody by day 7 post-vaccination (PV) with antibody titers ranging from 0.6 log to 2.1 log on day 14 PV with sustained titers through day 28 PV. Overall, these findings indicated that the RVFV arMP-12ΔNSm21/384 vaccine is a promising candidate for the prevention of RVF among domestic ruminants.     

Safety and immunogenicity in man of a cell culture derived trivalent live attenuated seasonal influenza vaccine: A Phase I dose escalating study in healthy volunteers

Live attenuated influenza vaccine (LAIV) offers the promise of inducing a variety of immune responses thereby conferring protection to circulating field strains. LAIVs are based on cold adapted and temperature sensitive phenotypes of master donor viruses (MDVs) containing the surface glycoprotein genes of seasonal influenza strains. Two types of MDV lineages have been described, the Ann Arbor lineages and the A/Leningrad/17 and B/USSR/60 lineages. Here the safety and immunogenicity of a Madin Darby Canine Kidney – cell culture based, intranasal LAIV derived from A/Leningrad/17 and B/USSR, was evaluated in healthy influenza non-naive volunteers 18–50 years of age. In a double-blind, randomized, placebo-controlled design, single escalating doses of 1 × 10⁵, 1 × 10⁶, or 1 × 10⁷ tissue culture infectious dose 50% (TCID₅₀) of vaccine containing each of the three influenza virus re-assortants recommended by the World Health Organization for the 2008–2009 season were administered intranasally. A statistically significant geometric mean increase in hemagglutination inhibition titer was reached for influenza strain A/H3N2 after immunization with all doses of LAIV. For the A/H1N1 and B strains, the GMI in HI titer did not increase for any of the doses. Virus neutralization antibody titers showed a similar response pattern. A dose–response effect could not be demonstrated for any of the strains, neither for the HI antibody nor for the VN antibody responses. No influenza like symptoms, no nasal congestions, no rhinorrhea, or other influenza related upper respiratory tract symptoms were observed. In addition, no difference in the incidence or nature of adverse events was found between vaccine and placebo treated subjects. Overall, the results indicated that the LAIV for nasal administration is immunogenic (i.e. able to provoke an immune response) and safe both from the perspective of the attenuated virus and the MDCK cell line from which it was derived, and it warrants further development.     

Immunization against influenza by the ocular route

The immunogenicity of influenza A strain A/Northern Territory/60/68 for CSL mice when delivered by the ocular, nasal and subcutaneous routes was determined according to the median protective dose, PD₅₀, i.e. the dose of infectious virus required to induce inhibition of multiplication of a standard intranasal challenge dose of 10^4.5 median tissue-culture-infectious doses (TCID₅₀) of homologous virus three weeks after vaccination (PD₅₀). For mice inoculated by the ocular route, an immunizing dose of 10^2.89TCID₅₀ per animal was required. For anaesthetized mice vaccinated intranasally and unanaesthetized mice vaccinated subcutaneously these figures are <10^2.00 and 10^6.00 TCID₅₀ per animal, respectively. The lower immunogenicity of virus delivered by the ocular route compared with the intranasal route can be correlated with a lowered capacity of ocularly administered virus to replicate in the murine respiratory tract. The immunogenicity of A/Ann Arbor/6/60-ca, administered in two identical doses, was also determined for (a) the intraocular route, (b) the intranasal route with anaesthetized animals and (c) the intranasal route with unanaesthetized animals, using the parental A/Ann Arbor/6/60 as the challenge virus. Two doses were required because ca viruses have been shown to be poor immunogens in the same animal model. The PD₅₀ for the ocular route was 10^2.83TCID₅₀ per animal compared with 10^2.71 for the intranasal route using unanaesthetized animals and 10^1.36 for the intranasal route using anaesthetized animals. Administration of living attenuated vaccine viruses by the ocular route is thus an efficient means of inducing immunity to influenza viruses in the respiratory tract of mice.     

Reduction of spiked porcine circovirus during the manufacture of a Vero cell-derived vaccine

Porcine circovirus-1 (PCV1) was recently identified as a contaminant in live Rotavirus vaccines, which was likely caused by contaminated porcine trypsin. The event triggered the development of new regulatory guidance on the use of porcine trypsin which shall ensure that cell lines and porcine trypsin in use are free from PCV1. In addition, manufacturing processes of biologicals other than live vaccines include virus clearance steps that may prevent and mitigate any potential virus contamination of product. In this work, artificial spiking of down-scaled models for the manufacturing process of an inactivated pandemic influenza virus vaccine were used to investigate inactivation of PCV1 and the physico-chemically related porcine parvovirus (PPV) by formalin and ultraviolet-C (UV-C) treatment as well as removal by the purification step sucrose gradient ultracentrifugation. A PCV1 infectivity assay, using a real-time PCR infectivity readout was established. The formalin treatment (0.05% for 48 h) showed substantial inactivation for both PCV1 and PPV with reduction factors of 3.0 log₁₀ and 6.8 log₁₀, respectively, whereas UV-C treatment resulted in complete PPV (≥5.9 log₁₀) inactivation already at a dose of 13 mJ/cm but merely 1.7 log₁₀ at 24 mJ/cm² for PCV1. The UV-C inactivation results with PPV were confirmed using minute virus of mice (MVM), indicating that parvoviruses are far more sensitive to UV-C than PCV1. The sucrose density gradient ultracentrifugation also contributed to PCV1 clearance with a reduction factor of 2 log₁₀. The low pH treatment during the production of procine trypsin was investigated and showed effective inactivation for both PCV1 (4.5 log₁₀) and PPV (6.4 log₁₀). In conclusion, PCV1 in general appears to be more resistant to virus inactivation than PPV. Still, the inactivated pandemic influenza vaccine manufacturing process provides for robust virus reduction, in addition to the already implemented testing for PCV1 to avoid any contaminations.     

Dose response effects of avian influenza (H7N7) vaccination of chickens: Serology,clinical protection and reduction of virus excretion

Knowledge of the relation between the antigen content of inactivated avian influenza (AI) vaccines, the serological response after vaccination and protection of vaccinated animals is important for the choice of optimal vaccines and vaccination regimes as well as for the assessment of criteria for the licensing of new AI-vaccines. We studied this relation in a dose response study using inactivated H7N7 avian influenza vaccines with varying antigen content. The serological response depended on the antigen content of the vaccines. Anti-AI antibodies were detected most frequently with ELISA, followed by the virus neutralisation test and the haemagglutination inhibition (HI) assay. Chickens with measurable HI-antibody titers, using homologous H7N7 antigen, were all protected against clinical disease after challenge with highly pathogenic A/chicken/Netherlands/621557/03 H7N7 virus. However, in these chickens high levels of virus could still be present on days 2–4 after challenge. The reduction of virus titers after challenge, depended on the antigen content of the vaccines as well as on the serum antibody titers. While 10 haemagglutinating units (HAU), equivalent to 0.8 μg haemagglutinin (HA) protein, per vaccine dose was sufficient for prevention of clinical disease, 128 HAU (9 μg HA) per dose was required for reduction of virus titers in all chickens to 10³ egg-infectious dose 50% (EID₅₀) or less. In order to reduce virus titers below 10³ EID₅₀ per swab a HI-antibody titer of 64 was required. After use of the vaccine with the highest antigen content, challenge still induced a booster of antibody titers which is indicative of replication of challenge virus.     

Scalable production of influenza virus in HEK-293 cells for efficient vaccine manufacturing

Cell culture processes offer an attractive alternative to conventional chicken egg-based influenza vaccine production methods. However, most protocols still rely on the use of adherent cells, which makes process scale-up a challenging issue. In this study, it is demonstrated that the HEK-293 human cell line is able to efficiently replicate influenza virus. Production in serum-free suspension of HEK-293 cultures resulted in high titers of infectious influenza viruses for different subtypes and variants including A/H1, A/H3 and B strains. After virus adaptation and optimization of infection conditions, production in 3-L bioreactor resulted in titers of up to 10⁹ IVP/mL demonstrating the scale-up potential of the process.     

Immunogenicity and protective efficacy of clade 2.3.2.1c and clade 2.3.4.4c H5Nx avian influenza antigen bank vaccines in mice,Korea

• The immunogenicity and protective efficacy of inactivated clade 2.3.2.1c (rgKA435) and clade 2.3.4.4c (rgES2) H5Nx vaccines, which are representatives of an avian influenza antigen bank in Korea, were examined in mice. Mice were vaccinated twice and then challenged with homologous virus. Hemagglutinin inhibition and serum neutralizing antibody titers in the rgES2-vaccinated group were higher (4.4 ± 1.7 and 10.8 ± 2.3 log₂, respectively) than those in the rgKA435-vaccinated group (2.8 ± 1.1 and 2.5 ± 0.9 log₂, respectively). rgES2 conferred 100% protection, with no morbidity, no severe body weight loss, and no virus replication in any of the tissues tested. By contrast, 80% of mice in the rgKA435 group survived. One mouse in this group died at 10 dpi. Virus titers in the lung and turbinate were 10^2.5–3.5 TCID₅₀/0.1 ml at 3–7 dpi and 10^1.5 TCID₅₀/0.1 ml at 3–5 dpi, respectively. In particular, the viral titer in the turbinate from the rgKA435 group at 3 dpi was significantly lower than that in the equivalent control group (p < 0.05). The data suggest that both of these antigen bank vaccines are promising candidates for further evaluation in humans.

     

Evaluation of the attenuation,immunogenicity, and efficacy of a live virus vaccine generated by codon-pair bias de-optimization of the 2009 pandemic H1N1 influenza virus,in ferrets

Codon-pair bias de-optimization (CPBD) of viruses involves re-writing viral genes using statistically underrepresented codon pairs, without any changes to the amino acid sequence or codon usage. Previously, this technology has been used to attenuate the influenza A/Puerto Rico/8/34 (H1N1) virus. The de-optimized virus was immunogenic and protected inbred mice from challenge. In order to assess whether CPBD could be used to produce a live vaccine against a clinically relevant influenza virus, we generated an influenza A/California/07/2009 pandemic H1N1 (2009 pH1N1) virus with de-optimized HA and NA gene segments (2009 pH1N1-(HA + NA)^Min), and evaluated viral replication and protein expression in MDCK cells, and attenuation, immunogenicity, and efficacy in outbred ferrets. The 2009 pH1N1-(HA + NA)^Min virus grew to a similar titer as the 2009 pH1N1 wild type (wt) virus in MDCK cells (∼10⁶ TCID₅₀/ml), despite reduced HA and NA protein expression on western blot. In ferrets, intranasal inoculation of 2009 pH1N1-(HA + NA)^Min virus at doses ranging from 10³ to 10⁵ TCID₅₀ led to seroconversion in all animals and protection from challenge with the 2009 pH1N1 wt virus 28 days later. The 2009 pH1N1-(HA + NA)^Min virus did not cause clinical illness in ferrets, but replicated to a similar titer as the wt virus in the upper and lower respiratory tract, suggesting that de-optimization of additional gene segments may be warranted for improved attenuation. Taken together, our data demonstrate the potential of using CPBD technology for the development of a live influenza virus vaccine if the level of attenuation is optimized.