Speed Matters: Directed Assembly of Icosahedral HPV Virus-Like Particles

Virus-like particles (VLPs) are an important vaccine modality. They self-assemble from virus capsid proteins, and the optimum immunogenic response is expected from VLPs with the same structure as the native virion. However, without the guardrails that guide the assembly of native viruses, off-target structures often result. Here, we show that assembly of HPV (human papillomavirus) VLPs can be redirected to form almost exclusively the native structure, a 21 MDa pseudo-T = 7 icosahedron consisting of 72 pentamers of the L1 capsid protein. HPV VLP assembly products were interrogated using charge detection mass spectrometry and resistive pulse sensing. Under standard conditions, the measured size distributions show that the assembly reaction stalls before most of the particles reach the T = 7 icosahedron, leading to a broad distribution of VLP sizes with some much more abundant than others. The preferred sizes correlate with particularly stable structures found in simulations. These results point to an assembly mechanism where spherical morphology is retained, and error-correction allows particles to fall into kinetic traps provided by particularly stable arrangements of pentamers. By accelerating the assembly reaction, conditions can be found where the 21 MDa T = 7 particles form almost exclusively. We hypothesize that accelerating assembly thwarts error-correction, so that the assembling particles skip over the kinetic traps. The T = 7 particles formed by accelerated assembly are more resistant to dissociation than the heterogeneous particles generated by slower assembly. The generation of high-stability particles that are uniform in size and shape and resemble the morphology of the naturally occurring virion has important benefits.