Heidi Trusheim

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.

Human RNA Polymerase I-Driven Reverse Genetics for Influenza A Virus in Canine Cells

ABSTRACT 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.

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.

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

Abstract 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.

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.