Quantitative Disassembly and Reassembly of Human Papillomavirus Type 11 Viruslike Particles In Vitro

Abstract

The human papillomavirus (HPV) capsid is primarily composed of a structural protein denoted L1, which forms both pentameric capsomeres and capsids composed of 72 capsomeres. The L1 protein alone is capable of self-assembly in vivo into capsidlike structures referred to as viruslike particles (VLPs). We have determined conditions for the quantitative disassembly of purified HPV-11 L1 VLPs to the level of capsomeres, demonstrating that disulfide bonds alone are essential to maintaining long-term HPV-11 L1 VLP structure at physiological ionic strength. The ionic strength of the disassembly reaction was also important, as increased NaCl concentrations inhibited disassembly. Conversely, chelation of cations had no effect on disassembly. Quantitative reassembly to a homogeneous population of 55-nm, 150S VLPs was reliably achieved by the re-formation of disulfide linkages following removal of reducing agent at near-neutral pH and moderate NaCl concentration. HPV-11 L1 VLPs could also be dissociated by treatment with carbonate buffer at pH 9.6, but VLPs could not be regenerated following carbonate treatment. When probed with conformationally sensitive and/or neutralizing monoclonal antibodies, both capsomeres generated by disulfide reduction of purified VLPs and reassembled VLPs formed from capsomeres upon removal of reducing agents exhibited epitopes found on the surface of authentic HPV-11 virions. Antisera raised against either purified VLP starting material or reassembled VLPs similarly neutralized infectious HPV-11 virions. The ability to disassemble and reassemble VLPs in vitro and in bulk allows basic features of capsid assembly to be studied and also opens the possibility of packaging selected exogenous compounds within the reassembled VLPs.

Assembly of Human Papillomavirus Type-16 Virus-Like Particles: Multifactorial Study of Assembly and Competing Aggregation

“…The concentration of NaCl did not significantly affect the ELISA response, and neither did the presence of calcium chloride. Previous studies have shown that high NaCl concentrations can be beneficial in reassembly of VLPs expressed in insect cells (12) and improve the stability of VLPs assembled from L1 capsomeres expressed in yeast (17). Our analysis indicates NaCl does not significantly enhance the assembly process of L1 capsomeres expressed in E. coli, and it has little effect on aggregation, contradicting the trend in previous studies.…”

“…Our analysis indicates NaCl does not significantly enhance the assembly process of L1 capsomeres expressed in E. coli, and it has little effect on aggregation, contradicting the trend in previous studies. Calcium was also shown to play no significant role in assembly of HPV VLPs, which supports the findings of McCarthy et al (12).…”

“…Other solution conditions may affect assembly, including ionic strength, pH, and the presence of divalent cations. Low pH and moderately high concentrations of salt are considered beneficial for HPV-16 assembly (12), and the divalent cation calcium plays a role in bovine papillomavirus assembly (13). Studies on HPV-11 VLPs have shown that assembly can be promoted by diluting capsomeres into a buffer at pH 5.4 with 0.5 M NaCl (14).…”

“…For the large-scale manufacture of a VLP-based vaccine, it is important to optimize the assembly process using validated quantitative methods wherever possible. Previous studies of VLP assembly have relied on electron microscopy and sucrose gradient centrifugation for qualitative analysis of the assembly process (12,14). Our study focused on the environmental conditions that influence assembly of VLPs and applied an enzyme-linked immunosorbent assay (ELISA) using the conformational antibody H16V5 to quantify the degree of assembly.…”

Bioengineering virus‐like particles as vaccines

“…Assembly-incompetent capsomeres of papillomavirus and polyomavirus, produced in bacterial expression system, are suitable for heterologous epitope presentation as well as immunization against their cognate virus (Middelberg et al, 2011;Rose et al, 1998). As the structural building blocks of VLPs, capsomeres are often more stable than VLPs (McCarthy et al, 1998;Schmidt et al, 2000), and have high absolute stability as evidenced by recent MD simulations (Zhang et al, 2013). Since the demonstration that immunization with HPV capsomeres induced similar virus neutralization titers as immunization with HPV virions or recombinant VLPs (Rose et al, 1998), a number of capsomere-based vaccine candidates to HPV serotypes have been developed (Dell et al, 2006;Fligge et al, 2001;Wu et al, 2011;Yuan et al, 2001).…”