(620m) Engineering Yeast Saccharomyces Cerevisiae for Assembly of Enveloped Influenza Virus-like Particles

Virus-like particles (VLPs) are nanoscale biological structures consisting of viral proteins assembled in a morphology that mimics the native virion but do not contain the viral genetic material. The possibility to manipulate the proteins contained within VLPs makes them an attractive tunable system for synthesizing predictable nanoscale structures that have numerous applications such as bioactive materials, biocatalysts, and vaccines. VLPs are especially amenable to vaccine development because they present viral proteins to the immune system in their near-native conformation without risk of infection. The FDA has approved VLP-based vaccines for human papilloma, hepatitis B, and hepatitis E viruses, and clinical trials for several other vaccines containing VLPs are currently underway.

The choice of the host expression system for VLP assembly requires careful consideration. For industrial and research applications, it is often advantageous to express protein in the most primitive host system possible to efficiently produce high yields of protein while minimizing the cost. However, primitive host systems such as E. coli may not be able to correctly fold more complex viral proteins. For this reason, VLPs containing glycoproteins from more complex enveloped viruses, such as influenza and Ebola, have been almost exclusively produced in insect, mammalian, and plant cells. To our knowledge, no attempt has been made to produce enveloped VLPs displaying authentic viral glycoproteins in a yeast-based expression system despite yeasts’ ability to produce correctly folded glycoproteins of numerous enveloped viruses as well as their low cost and robustness in large-scale therapeutics production.

For the first time, we report that the yeast Saccharomyces cerevisiae is capable of assembling and producing enveloped influenza VLPs upon expression of hemagglutinin with or without the neuraminidase, M1, and M2 viral proteins from influenza subtypes H1N1 or H3N2. Budding of influenza VLPs off the yeast membrane was observed by examination of ultrathin sections of yeast cells using transmission electron microscopy (TEM). The influenza VLPs were shown to have key morphological and functional characteristics of influenza virus, including the presence of glycoprotein spikes on the VLP surface and the ability to agglutinate red blood cells. We are currently testing the effectiveness of vaccination with influenza VLPs in mouse models. These findings provide a proof-of-concept of using S. cerevesiae to produce complex enveloped VLPs for vaccine development. This system provides significant advantages over other higher eukaryotic systems because yeast cells can produce higher protein yields, are faster growing, are less expensive to maintain, and do not require the use of contaminating viral vectors.