Enhanced isolation of influenza viruses in qualified cells improves the probability of well-matched vaccines.

Influenza vaccines are utilised to combat seasonal and pandemic influenza. The key to influenza vaccination currently is the availability of candidate vaccine viruses (CVVs). Ideally, CVVs reflect the antigenic characteristics of the circulating virus, which may vary depending upon the isolation method. For traditional inactivated egg-based vaccines, CVVs are isolated in embryonated chicken eggs, while for cell-culture production, CVV's are isolated in either embryonated eggs or qualified cell lines. We compared isolation rates, growth characteristics, genetic stability and antigenicity of cell and egg CVV's derived from the same influenza-positive human clinical respiratory samples collected from 2008-2020. Influenza virus isolation rates in MDCK33016PF cells were twice that of eggs and mutations in the HA protein were common in egg CVVs but rare in cell CVVs. These results indicate that fully cell-based influenza vaccines will improve the choice, match and potentially the effectiveness, of seasonal influenza vaccines compared to egg-based vaccines.

Comparison of suspension MDCK cells, adherent MDCK cells, and LLC-MK2 cells for selective isolation of influenza viruses to be used as vaccine seeds.

BACKGROUND: Cell-based influenza vaccines can solve the problem of the frequent occurrence of egg adaptation-associated antigenic changes observed in egg-based vaccines. Seed viruses for cell-based vaccines can be prepared from clinical specimens by cell culture; however, clinical samples risk harboring respiratory viruses other than influenza virus. Therefore, it is necessary to investigate the patterns of co-infection in clinical samples and explore whether cell culture technology can selectively propagate influenza viruses from samples containing other respiratory viruses. METHODS: A total of 341 clinical specimens were collected from patients with influenza or influenza-like illness and analyzed by ResPlex II assay to detect 18 respiratory viruses. The patterns of co-infection were statistically analyzed with Fisher's exact test. The samples with double or triple infections were passaged in suspension MDCK cells (MDCK-S), adherent MDCK cells (MDCK-A), and LLC-MK2D cells. Cell-passaged samples were analyzed by ResPlex II assay again to investigate whether each cell line could amplify influenza viruses and eliminate other respiratory viruses. RESULTS: Double infections were detected in 8.5% and triple infections in 0.9% of the collected clinical specimens. We identified four pairs of viruses with significant correlation. For all samples with double and triple infection, MDCK-S and MDCK-A could selectively propagate influenza viruses, while eliminating all contaminating viruses. In contrast, LLC-MK2D showed lower isolation efficiency for influenza virus and higher isolation efficiency for coxsackievirus/echovirus than MDCK-S and MDCK-A. CONCLUSIONS: Both MDCK-S and MDCK-A are considered suitable for the preparation of influenza vaccine seed viruses without adventitious agents or egg-adaptation mutations.

Systematic evaluation of suspension MDCK cells, adherent MDCK cells, and LLC-MK2 cells for preparing influenza vaccine seed virus.

Suspension Madin-Darby canine kidney (MDCK) cells (MDCK-N), adherent MDCK cells (MDCK-C), and adherent rhesus monkey kidney LLC-MK2 cells (LLC-MK2D) were systematically evaluated for the preparation of influenza vaccine seed viruses for humans on the basis of primary virus isolation efficiency, growth ability, genetic stability of the hemagglutinin (HA) and neuraminidase (NA) genes, and antigenic properties in hemagglutination inhibition (HI) test of each virus isolate upon further passages. All the subtypes/lineages of influenza viruses (A(H1N1), A(H1N1)pdm09, A(H3N2), B-Victoria, and B-Yamagata) were successfully isolated from clinical specimens by using MDCK-N and MDCK-C, whereas LLC-MK2D did not support virus replication well. Serial passages of A(H1N1) viruses in MDCK-N and MDCK-C induced genetic mutations of HA that resulted in moderate antigenic changes in the HI test. All A(H1N1)pdm09 isolates from MDCK-C acquired amino acid substitutions at the site from K153 to N156 of the HA protein, which resulted in striking antigenic alteration. In contrast, only 30% of MDCK-N isolates showed amino acid changes at this site. The frequency of MDCK-N isolates with less than two-fold reduction in the HI titer was as high as 70%. A(H3N2) and B-Yamagata isolates showed high antigenic stability and no specific amino acid substitution during passages in MDCK-N and MDCK-C. B-Victoria isolates from MDCK-N and MDCK-C acquired genetic changes at HA glycosylation sites that greatly affected their antigenicity. When these cell isolates were applied to passages in hen eggs, A(H1N1), B-Victoria, and B-Yamagata viruses grew well in eggs, while none of the cell isolates of A(H3N2) viruses did. Thus, we demonstrate that MDCK-N might be useful for the preparation of influenza vaccine seed viruses.

Cell culture-derived influenza vaccines in the severe 2017-2018 epidemic season: a step towards improved influenza vaccine effectiveness.

The 2017-2018 seasonal influenza epidemics were severe in the US and Australia where the A(H3N2) subtype viruses predominated. Although circulating A(H3N2) viruses did not differ antigenically from that recommended by the WHO for vaccine production, overall interim vaccine effectiveness estimates were below historic averages (33%) for A(H3N2) viruses. The majority (US) or all (Australian) vaccine doses contained multiple amino-acid changes in the hemagglutinin protein, resulting from the necessary adaptation of the virus to embryonated hen's eggs used for most vaccine manufacturing. Previous reports have suggested a potential negative impact of egg-driven substitutions on vaccine performance. With BARDA support, two vaccines licensed in the US are produced in cell culture: recombinant influenza vaccine (RIV, Flublok™) manufactured in insect cells and inactivated mammalian cell-grown vaccine (ccIIV, Flucelvax™). Quadrivalent ccIIV (ccIIV4) vaccine for the 2017-2018 influenza season was produced using an A(H3N2) seed virus propagated exclusively in cell culture and therefore lacking egg adaptative changes. Sufficient ccIIV doses were distributed (but not RIV doses) to enable preliminary estimates of its higher effectiveness relative to the traditional egg-based vaccines, with study details pending. The increased availability of comparative product-specific vaccine effectiveness estimates for cell-based and egg-based vaccines may provide critical clues to inform vaccine product improvements moving forward.

Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing.

Cell culture is now available as a method for the production of influenza vaccines in addition to eggs. In accordance with currently accepted practice, viruses recommended as candidates for vaccine manufacture are isolated and propagated exclusively in hens' eggs prior to distribution to manufacturers. Candidate vaccine viruses isolated in cell culture are not available to support vaccine manufacturing in mammalian cell bioreactors so egg-derived viruses have to be used. Recently influenza A (H3N2) viruses have been difficult to isolate directly in eggs. As mitigation against this difficulty, and the possibility of no suitable egg-isolated candidate viruses being available, it is proposed to consider using mammalian cell lines for primary isolation of influenza viruses as candidates for vaccine production in egg and cell platforms. To investigate this possibility, we tested the antigenic stability of viruses isolated and propagated in cell lines qualified for influenza vaccine manufacture and subsequently investigated antigen yields of such viruses in these cell lines at pilot-scale. Twenty influenza A and B-positive, original clinical specimens were inoculated in three MDCK cell lines. The antigenicity of recovered viruses was tested by hemagglutination inhibition using ferret sera against contemporary vaccine viruses and the amino acid sequences of the hemagglutinin and neuraminidase were determined. MDCK cell lines proved to be highly sensitive for virus isolation. Compared to the virus sequenced from the original specimen, viruses passaged three times in the MDCK lines showed up to 2 amino acid changes in the hemagglutinin. Antigenic stability was also established by hemagglutination inhibition titers comparable to those of the corresponding reference virus. Viruses isolated in any of the three MDCK lines grew reasonably well but variably in three MDCK cells and in VERO cells at pilot-scale. These results indicate that influenza viruses isolated in vaccine certified cell lines may well qualify for use in vaccine production.

Synthetic generation of influenza vaccine viruses for rapid response to pandemics.

During the 2009 H1N1 influenza pandemic, vaccines for the virus became available in large quantities only after human infections peaked. To accelerate vaccine availability for future pandemics, we developed a synthetic approach that very rapidly generated vaccine viruses from sequence data. Beginning with hemagglutinin (HA) and neuraminidase (NA) gene sequences, we combined an enzymatic, cell-free gene assembly technique with enzymatic error correction to allow rapid, accurate gene synthesis. We then used these synthetic HA and NA genes to transfect Madin-Darby canine kidney (MDCK) cells that were qualified for vaccine manufacture with viral RNA expression constructs encoding HA and NA and plasmid DNAs encoding viral backbone genes. Viruses for use in vaccines were rescued from these MDCK cells. We performed this rescue with improved vaccine virus backbones, increasing the yield of the essential vaccine antigen, HA. Generation of synthetic vaccine seeds, together with more efficient vaccine release assays, would accelerate responses to influenza pandemics through a system of instantaneous electronic data exchange followed by real-time, geographically dispersed vaccine production.

Exploring synergies between academia and vaccine manufacturers: a pilot study on how to rapidly produce vaccines to combat emerging pathogens.

BACKGROUND: In spring 2009, a new swine-origin influenza A (H1N1) virus emerged in Mexico. During the following weeks the virus spread worldwide, prompting the World Health Organization to declare the first influenza pandemic of the 21st century. Sustained human-to-human transmission and severe disease progression observed in some patients urged public health authorities to respond rapidly to the disease outbreak and vaccine manufacturers to develop pandemic influenza vaccines for mass distribution. With the onset of the pandemic we began to explore the potential of academic/industrial collaboration to accelerate the production of vaccines during an outbreak of an emerging virus by combining the use of an academic BSL-4 laboratory with the expertise of a commercial vaccine manufacturer. METHODS AND RESULTS: To obtain virus seed stocks used for the production of a vaccine to combat the pandemic H1N1 2009 influenza virus (H1N1pdm), we followed various strategies: (i) optimization of cell culture conditions for growth of wild-type H1N1pdm isolates; (ii) classical reassortment of H1N1pdm and standard influenza vaccine donor strain PR8; and (iii) generation of corresponding reassortant viruses using reverse genetics. To ensure a rapid transition to production, the entire potential seed stock development process was carried out in a certified canine kidney suspension cell line (MDCK 33016-PF) under Good Manufacturing Practice (GMP) conditions. CONCLUSIONS: The outcome of this study indicates that a combination of different experimental strategies is the best way to cope with the need to develop vaccines rapidly in the midst of an emerging pandemic.

Safety of MDCK cell culture-based influenza vaccines.

After more than 60 years, the conventional production of influenza vaccines employing fertilized chicken eggs has reached its limits - both in terms of temporal flexibility and vaccine production volume. This problem is compounded by the fact that the pandemic-driven situation in 2009 has roughly doubled the overall vaccine demand. Modern cell culture technology has significant advantages over the conventional method of manufacturing influenza vaccines employing embryonated chicken eggs, and enables manufacturers to respond rapidly to the increasing worldwide seasonal and pandemic-driven need for influenza vaccines. Recent articles in the popular press claiming that cell culture-based influenza vaccines can cause tumors have fomented uncertainty among the general population and physicians, and also discredit officially accepted test results and product licensing. This article provides an overview of the safety profile of the cell culture technology, of the cells and of the final vaccine product.

Mutations at positions 186 and 194 in the HA gene of the 2009 H1N1 pandemic influenza virus improve replication in cell culture and eggs.

Obtaining suitable seed viruses for influenza vaccines poses a challenge for public health authorities and manufacturers. We used reverse genetics to generate vaccine seed-compatible viruses from the 2009 pandemic swine-origin influenza virus. Comparison of viruses recovered with variations in residues 186 and 194 (based on the H3 numbering system) of the viral hemagglutinin showed that these viruses differed with respect to their ability to grow in eggs and cultured cells. Thus, we have demonstrated that molecular cloning of members of a quasispecies can help in selection of seed viruses for vaccine manufacture.

[Safety of cell culture-based influenza vaccines]. / Sicherheit von Influenza-Impfstoffen auf Zellkulturbasis.

After more than 60 years, the conventional production of influenza vaccines employing fertilized chicken eggs has reached its limits - both in terms of temporal flexibility and vaccine production volume. This situation is compounded by the fact that the present pandemic-driven situation has roughly doubled the overall vaccine demand virtually "overnight". Modem cell culture technology has significant advantages over the conventional method of manufacturing influenza vaccines employing embryonated chicken eggs, and enables manufacturers to respond rapidly to the exploding worldwide seasonal and pandemic-driven need for influenza vaccines. Recent articles in the popular press claiming that cell culture-based influenza vaccines can cause tumours raised uncertainty among physicians and the general population, and also discredit officially accepted assessments and product licensing by the relevant authorities. The present article provides an overview on the cell culture technology and on the safety profile of the cells and of the vaccine product.

Human RNA polymerase I-driven reverse genetics for influenza a virus in canine cells.

We have established a human RNA polymerase I (pol I)-driven influenza virus reverse genetics (RG) system in the Madin-Darby canine kidney 33016-PF cell line, which is approved for influenza vaccine manufacture. RNA pol I polymerases are generally active only in cells of species closely related to the species of origin of the polymerases. Nevertheless, we show that a nonendogenous RNA pol I promoter drives efficient rescue of influenza A viruses in a canine cell line. Application of this system allows efficient generation of virus strains and presents an alternative approach for influenza vaccine production.

[Prion safety of medicinal products using the example of an influenza vaccine produced in a cell line]. / Prionen-Sicherheit von Arzneimitteln am Beispiel eines Zellkultur-Impfstoffs.

Prions are pathogenic proteins and are the cause for spongiform encephalopathies. Pathogenic prions differ from physiologically common non-pathogenic prions only in their sterical structure. Upon infection by a pathogenic prion protein, a series of reactions is initiated in which common non-pathogenic prion proteins are transformed into pathogenic prions. Animals, mainly ruminants like cattle, sheep and goats are susceptible to prions, but also man. Prions are very robust and it is difficult to inactivate them. During the production processes of pharmaceuticals, the risk for contamination by infectious prions can be reduced by careful choice of animal material, the replacement of animal material and by appropriate production procedures. For instance biologicals like influenza vaccines can be produced by a permanent canine cell line, whose prion safety has been proven by useful methods (standard scrapy cell assay). Mandatory guidelines ensure that the risk for contamination by pathogenic prions has to be considered and excluded in the production of bio-pharmaceuticals.

Current challenges in implementing cell-derived influenza vaccines: implications for production and regulation, July 2007, NIBSC, Potters Bar, UK.

A meeting was held at NIBSC, UK in July 2007 to discuss the implications of progress in the use of cell culture systems for the manufacture of vaccines against influenza. Issues discussed included the effect of using eggs and different cell types in strain selection, development of seed viruses to be used in production and the nature of the reagents to be used in determining vaccine potency. Future studies to progress the field were reviewed.

Canine MDCK cell lines are refractory to infection with human and mouse prions.

Influenza vaccine production in embryonated eggs is associated with many disadvantages, and production in cell culture systems is a viable alternative. Madin Darby canine kidney (MDCK) cells are permissive for a variety of orthomyxoviruses and have proven particularly suitable for vaccine mass production. However, mammalian cells harboring the Prnp gene can theoretically acquire prion infections. Here, we have attempted to infect MDCK cells and substrains thereof with prions. We found that MDCK cells did not produce any protease-resistant PrP(Sc) upon exposure to brain homogenates derived from humans suffering from Creutzfeldt-Jakob disease (CJD) or from mice infected with Rocky Mountain Laboratory (RML) scrapie prions. Further, transmission of MDCK lysates to N2aPK1 cells did not induce formation of PrP(Sc) in the latter. PrP(C) biogenesis and processing in MDCK cells were similar to those of prion-sensitive N2aPK1 cells. However, steady-state levels of PrP(C) were very low, and PrP(C) did not partition with detergent-resistant membranes upon density gradient analysis. These factors may account for their resistance to infection. Alternatively, prion resistance may be related to the specific sequence of canine Prnp, as suggested by the lack of documented prion diseases in dogs.

Extracellular RNA constitutes a natural procoagulant cofactor in blood coagulation.

Upon vascular injury, locally controlled haemostasis prevents life-threatening blood loss and ensures wound healing. Intracellular material derived from damaged cells at these sites will become exposed to blood components and could contribute to blood coagulation and pathological thrombus formation. So far, the functional and mechanistic consequences of this concept are not understood. Here, we present in vivo and in vitro evidence that different forms of eukaryotic and prokaryotic RNA serve as promoters of blood coagulation. Extracellular RNA was found to augment (auto-)activation of proteases of the contact phase pathway of blood coagulation such as factors XII and XI, both exhibiting strong RNA binding. Moreover, administration of exogenous RNA provoked a significant procoagulant response in rabbits. In mice that underwent an arterial thrombosis model, extracellular RNA was found associated with fibrin-rich thrombi, and pretreatment with RNase (but not DNase) significantly delayed occlusive thrombus formation. Thus, extracellular RNA derived from damaged or necrotic cells particularly under pathological conditions or severe tissue damage represents the long sought natural "foreign surface" and provides a procoagulant cofactor template for the factors XII/XI-induced contact activation/amplification of blood coagulation. Extracellular RNA thereby reveals a yet unrecognized target for antithrombotic intervention, using RNase or related therapeutic strategies.

A positively charged cluster in the epidermal growth factor-like domain of Factor VII-activating protease (FSAP) is essential for polyanion binding.

FSAP (Factor VII-activating protease) is a novel plasma-derived serine protease that regulates haemostasis as well as vascular cell proliferation. FSAP undergoes autoactivation in the presence of polyanionic macromolecules such as heparin and RNA. Competition experiments suggest that RNA and heparin bind to the same or overlapping interaction sites. A proteolysis approach, where FSAP was hydrolysed into smaller fragments, was used to identify the polyanion-binding site. The EGF (epidermal growth factor)-like domains EGF2 and EGF3 of FSAP are the major interaction domains for RNA. The amino acids Arg170, Arg171, Ser172 and Lys173 within the EGF3 domain were essential for this binding. This is also the region with the highest positive net charge in the protein and is most probably located in an exposed loop. It is also highly conserved across five species. Disruption of disulphide bridges led to the loss of RNA and heparin binding, indicating that the three-dimensional structure of the EGF3 domain is essential for binding to negatively charged heparin or RNA. The identification of polyanion-binding sites will help to define the role of FSAP in the vasculature.

Evidence for an RNA chaperone function of polypyrimidine tract-binding protein in picornavirus translation.

The cellular polypyrimidine tract-binding protein (PTB) is recruited by the genomic RNAs of picornaviruses to stimulate translation initiation at their internal ribosome entry site (IRES) elements. We investigated the contribution of the individual RNA recognition motif (RRM) domains of PTB to its interaction with the IRES of foot-and-mouth disease virus (FMDV). Using a native gel system, we found that PTB is a monomer, confirming recent reports that challenged the previous view that PTB is a dimer. Mapping the spatial orientation of PTB relative to the bound IRES RNA, we found that the two C-terminal RRM domains III and IV of PTB bind in an oriented way to the IRES. Domain III contacts the IRES stem-loop 2, while domain IV contacts the separate IRES 3' region. PTB domain I appears not to be involved directly in RNA binding, but domain II stabilizes the RNA binding conferred by domains III and IV. A PTB protein containing only these two C-terminal PTB domains is sufficient to enhance the entry of initiation factor eIF4G to the IRES and stimulate IRES activity, and the long-lived PTB-IRES interaction stabilized by domain II is not a prerequisite for this function. Thus, PTB most likely acts as an RNA chaperone to stabilize IRES structure and, in that way, augment IRES activity.

Extracellular RNA is a natural cofactor for the (auto-)activation of Factor VII-activating protease (FSAP).

FSAP (Factor VII-activating protease) is a new plasma-derived serine protease with putative dual functions in haemostasis, including activation of coagulation Factor VII and generation of urinary-type plasminogen activator (urokinase). The (auto-)activation of FSAP is facilitated by polyanionic glycosaminoglycans, such as heparin or dextran sulphate, whereas calcium ions stabilize the active form of FSAP. In the present study, extracellular RNA was identified and characterized as a novel FSAP cofactor. The conditioned medium derived from various cell types such as smooth muscle cells, endothelial cells, osteosarcoma cells or CHO (Chinese-hamster ovary) cells contained an acidic factor that initiated (auto-)activation of FSAP. RNase A, but not other hydrolytic enzymes (proteases, glycanases and DNase), abolished the FSAP cofactor activity, which was subsequently isolated by anion-exchange chromatography and unequivocally identified as RNA. In purified systems, as well as in plasma, different forms of natural RNA (rRNA, tRNA, viral RNA and artificial RNA) were able to (auto-)activate FSAP into the two-chain enzyme form. The specific binding of FSAP to RNA (but not to DNA) was shown by mobility-shift assays and UV crosslinking, thereby identifying FSAP as a new extracellular RNA-binding protein, the K(D) estimated to be 170-350 nM. Activation of FSAP occurred through an RNA-dependent template mechanism involving a nucleic acid size of at least 100 nt. In a purified system, natural RNA augmented the FSAP-dependent Factor VII activation several-fold (as shown by subsequent Factor Xa generation), as well as the FSAP-mediated generation of urokinase. Our results provide evidence for the first time that extracellular RNA, present at sites of cell damage or vascular injury, can serve an important as yet unrecognized cofactor function in haemostasis by inducing (auto-)activation of FSAP through a novel surface-dependent mechanism.

Altered signaling and cell cycle regulation in embryonal stem cells with a disruption of the gene for phosphoinositide 3-kinase regulatory subunit p85alpha.

The p85alpha regulatory subunit of class I(A) phosphoinositide 3-kinases (PI3K) is derived from the Pik3r1 gene, which also yields alternatively spliced variants p50alpha and p55alpha. It has been proposed that excess monomeric p85 competes with functional PI3K p85-p110 heterodimers. We examined embryonic stem (ES) cells with heterozygous and homozygous disruptions in the Pik3r gene and found that wild type ES cells express virtually no monomeric p85alpha. Although, IGF-1-stimulated PI3K activity associated with insulin receptor substrates was unaltered in all cell lines, p85alpha-null ES cells showed diminished protein kinase B activation despite increased PI3K activity associated with the p85beta subunit. Furthermore, p85alpha-null cells demonstrated growth retardation, increased frequency of apoptosis, and altered cell cycle regulation with a G(0)/G(1) cell cycle arrest and up-regulation of p27(KIP), whereas signaling through CREB and MAPK was enhanced. These phenotypes were reversed by re-expression of p85alpha via adenoviral gene transfer. Surprisingly, all ES cell lines could be differentiated into adipocytes. In these differentiated ES cells, however, compensatory p85beta signaling was lost in p85alpha-null cells while increased signaling by CREB and MAPK was still observed. Thus, loss of p85alpha in ES cells induced alterations in IGF-1 signaling and regulation of apoptosis and cell cycle but no defects in differentiation. However, differentiated ES cells partially lost their ability for compensatory signaling at the level of PI3K, which may explain some of the defects observed in mice with homozygous deletion of the Pik3r1 gene.

DUG is a novel homologue of translation initiation factor 4G that binds eIF4A.

To elucidate the molecular mechanisms of cell death, we have cloned a new gene, designated death-upregulated gene (DUG), from rat insulinoma cells. DUG is constitutively expressed at very low levels in normal cells but is dramatically upregulated in apoptotic cells following serum/glucose starvation or death receptor ligation by Fas ligand. The DUG mRNA is present in two splicing forms: a long form that encodes a protein of 469 amino acids and a short form that gives rise to a polypeptide of 432 amino acids. The predicted DUG protein sequence contains two putative nuclear localization signals and multiple phosphorylation sites for protein kinases and two conserved MA3 domains. Importantly, DUG is homologous to eukaryotic translation initiation factor (eIF) 4G and binds to eIF4A presumably through MA3 domains. Upon transfection, DUG inhibits both intrinsic and extrinsic pathways of apoptosis. Thus, DUG is a novel homologue of eIF4G that regulates apoptosis.