Vaccine 26 (2008) 3778–3782 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine High titer growth of human and avian influenza viruses in an immortalized chick embryo cell line without the need for exogenous proteases Kristen A. Smith a,∗ , Christopher J. Colvin a , Patty S.D. Weber a , Stephen J. Spatz b , Paul M. Coussens a a Molecular Pathogenesis Laboratory, Department of Animal Science, Michigan State, University, East Lansing, Michigan, USA b Endemic Poultry Viral Diseases Research Unit, USDA, Athens, Georgia, USA article info Article history: Received 20 November 2007 Received in revised form 5 April 2008 Accepted 9 April 2008 Available online 8 May 2008 Keywords: PBS-1 Influenza Vaccine abstract The current method of growing influenza virus for vaccine production is through the use of embryonated chicken eggs. This manufacturing system yields a low concentration of virus per egg, requires significant downstream production for purification, and demands a considerable amount of time for production. We have demonstrated an immortalized chick embryo cell line, termed PBS-1, is capable of growing unmodified recent isolates of human and avian influenza A and B viruses to extremely high titers. In many cases, PBS-1 cells out perform primary chick embryo kidney (CEK) cells, Madin-Darby Canine Kidney (MDCK) cells and African green monkey kidney cells (Vero) in growth of recent influenza isolates. PBS- 1 cells are free of any exogenous agents, are non-tumorigenic, and are readily adaptable to a variety of culture conditions, including growth on microcarrier beads. Influenza viruses grown in PBS-1 cells are released into the culture fluid without the need for exogenous proteases, thus simplifying downstream processing. In addition to offering a significant improvement in vaccine production, PBS-1 cells should prove valuable in diagnostics and as a cell line of choice for influenza virus research. © 2008 Elsevier Ltd. All rights reserved. 1. Introduction Current influenza vaccine production technology is largely restricted to growth in embryonated hens’ eggs and often requires one to two eggs per dose [1], which makes this process expen- sive and requires months of preparation. In addition, this scheme requires significant downstream processing to purify virus away from egg components and is subject to significant loss of product if the eggs are found to be contaminated with exogenous agents. There are also concerns that individuals who are allergic to eggs may experience adverse reactions to egg-derived vaccines [2,3]. The process of selecting vaccine viruses to be included in the year’s vaccine to actual production can take as long as six to nine months. Many scientists and vaccine experts have expressed concern that this is far too long in the face of a potential influenza pandemic [1,4]. Due to safety, time constraints, and consistency issues with egg-derived influenza vaccines, there has been a push from both ∗ Corresponding author at: Department of Animal Science, B215 Anthony Hall, Michigan State University, East Lansing, MI 48824, USA. Tel.: +1 517 432 1379; fax: +1 517 353 1699. E-mail address: smith277@msu.edu (K.A. Smith). regulatory agencies and major vaccine manufacturers to adopt a continuous cell culture-based influenza vaccine production sys- tem [5]. While influenza viruses generally grow efficiently on primary chick embryo kidney (CEK) cells [6,7], this system would be subject to many of the same concerns and issues surround- ing egg-derived vaccines, particularly the potential presence of harmful contaminating pathogens. The existing continuous cell lines that are known to grow influenza virus, such as Madin-Darby Canine Kidney (MDCK) cells, African green monkey kidney cells (Vero) and human retinal cells (PER.C6) [8,9], require the addition of exogenous proteases, such as tosyl phenylalanyl chloromethyl ketone (TPCK) treated trypsin, for propagation of the virus. Viruses grown in this manner are typically modified through reassortment and/or reverse genetics to allow high-titer growth. Thus, a well- characterized continuous cell line which could be used to establish a master cell bank that is non-tumorigenic and free of exogenous pathogens and adventitious agents is highly desirable [5]. Here, we report the use of an immortalized chick embryo cell line, termed PBS-1, which is capable of growing unmodified recent isolates of human and avian influenza A and B viruses to extremely high titers (>10 7 PFU/ml). PBS-1 cells are derived from an 11-day-old line C/CE chick embryos, which were chick helper factor negative (no env gene product) and specific pathogen-free [10]. The primary cells were passaged once, and then immortal- 0264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2008.04.048