Exploiting the yeast L-A viral capsid for the in vivo assembly of chimeric VLPs as platform in vaccine development and foreign protein expression.
A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae) dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs) as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8(+) memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i) a heterologous model protein (GFP), (ii) a per se toxic protein (K28 alpha-subunit), and (iii) a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A). Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.
Powilleit
Frank
F
Angewandte Molekularbiologie, Universität des Saarlandes, Saarbrücken, Germany.
Breinig
Tanja
T
Schmitt
Manfred J
MJ
D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006801:Humans; D007156:Immunologic Memory; D046529:Microscopy, Electron, Transmission; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014612:Vaccines / Q000235:genetics* / Q000276:immunology; D014771:Virion / Q000235:genetics* / Q000276:immunology
D011993:Recombinant Fusion Proteins; D014612:Vaccines