Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome, potentially yielding safer and cheaper vaccine candidates. A handful of prophylactic VLP-based vaccines is currently commercialized worldwide: GlaxoSmithKline's Engerix (hepatitis B virus) and Cervarix (human papillomavirus), and Merck and Co., Inc.'s Recombivax HB (hepatitis B virus) and Gardasil (human papillomavirus) are some examples. Other VLP-based vaccine candidates are in clinical trials or undergoing preclinical evaluation, such as, influenza virus, parvovirus, Norwalk and various chimeric VLPs. Many others are still restricted to small-scale fundamental research, despite their success in preclinical tests. This article focuses on the essential role of VLP technology in new-generation vaccines against prevalent and emergent diseases. The implications of large-scale VLP production are discussed in the context of process control, monitorization and optimization. The main up- and down-stream technical challenges are identified and discussed accordingly. Successful VLP-based vaccine blockbusters are briefly presented concomitantly with the latest results from clinical trials and the recent developments in chimeric VLP-based technology for either therapeutic or prophylactic vaccination.

Virus-like particles (VLPs) hold tremendous potential as vaccine candidates. These innovative biopharmaceuticals present the remarkable advantages of closely mimicking the three-dimensional nature of an actual virus while lacking the virus genome packaged inside its capsid. As a result, an equally efficient but safer prophylaxis is anticipated as compared to inactivated or live attenuated viral vaccines. With the advent of successful cases of approved VLP-based vaccines, pharmaceutical companies are indeed redirecting their resources to the development of such products. This paper reviews the current choices and trends of large-scale production and purification of VLP-based vaccines generated through the baculovirus expression vector system using insect cells.

No abstract

Primary cultures of human hepatocyte spheroids are a promising in vitro model for longterm studies of hepatic metabolism and cytotoxicity. The lack of robust methodologies to culture cell spheroids, as well as a poor characterization of human hepatocyte spheroid architecture and liver-specific functionality, have hampered a widespread adoption of this three-dimensional culture format. In this work, an automated perfusion bioreactor was used to obtain and maintain human hepatocyte spheroids. These spheroids were cultured for 3-4 weeks in serum-free conditions, sustaining their phase I enzyme expression and permitting repeated induction during long culture times; rate of albumin and urea synthesis, as well as phase I and II drug-metabolizing enzyme gene expression and activity of spheroid hepatocyte cultures, presented reproducible profiles, despite basal interdonor variability (n 5 3 donors). Immunofluorescence microscopy of human hepatocyte spheroids after 3-4 weeks of long-term culture confirmed the presence of the liver-specific markers, hepatocyte nuclear factor 4a, albumin, cytokeratin 18, and cytochrome P450 3A. Moreover, immunostaining of the atypical protein kinase C apical marker, as well as the excretion of a fluorescent dye, evidenced that these spheroids spontaneously assemble a functional bile canaliculi network, extending from the surface to the interior of the spheroids, after 3-4 weeks of culture. Conclusion: Perfusion bioreactor cultures of primary human hepatocyte spheroids maintain a liver-specific activity and architecture and are thus suitable for drug testing in a long-term, repeated-dose format. (HEPATOLOGY 2012;55:1227-1236 T he liver-specific functions of hepatocytes, such as albumin secretion or drug-metabolizing activity, are rapidly down-regulated during in vitro primary cultures, limiting their use for drug development and toxicity tests. 1 For such assays, the current gold standard for long-term human hepatocyte culture is the collagen sandwich in vitro model. 2 The overlaying collagen layer increases cell-cell and cell-matrix contacts, providing a more three-dimensional (3D)-like architecture than a monolayer culture. For rat hepatocyte spheroids, where cell-cell interactions are maximized, liverspecific functions 3,4 and multicellular architecture 5,6 are increased, when compared to monolayer cultures.The use of microfluidic devices for primary cultures of hepatocytes is a promising approach to enable highthroughput screening in drug development. 7,8 However, the downscaling enabled by these technologies makes the culture environment harder to be controlled and limits the application of microfluidics for longterm primary cultures of hepatocytes. In fact, the most useful applications of microtechnologies for such cultures couple either microfluidic perfusion or coculture micropatterning to 12-9 or 24-well culture plates, 10Abbreviations: 2D, two-dimensional; 3D, three-dimensional; 7-EC, 7-ethoxycoumarin; 7-HC, 7-hydroxycoumarin; ANOVA, analysis of variance; aPKC, atypical protein ki...