Analysis of Modified Human Papillomavirus Type 16 L1 Capsomeres: the Ability To Assemble into Larger Particles Correlates with Higher Immunogenicity

Abstract

L1 capsomeres purified from Escherichia coli represent an economic alternative to the recently launched virus-like particle (VLP)-based prophylactic vaccines against infection with human papillomavirus types 16 and 18 (HPV-16and HPV-18), which are causative agents of cervical cancer. It was recently reported that capsomeres are much less immunogenic than VLPs. Numerous modifications of the L1 protein leading to the formation of capsomeres but preventing capsid assembly have been described, such as the replacement of the cysteine residues that form capsid-stabilizing disulfide bonds or the deletion of helix 4. So far, the influence of these modifications on immunogenicity has not been thoroughly investigated. Here, we describe the purification of eight different HPV-16 L1 proteins as capsomeres from Escherichia coli. We compared them for yield, structure, and immunogenicity in mice. All L1 proteins formed almost identical pentameric structures yet differed strongly in their immunogenicity, especially regarding the humoral immune responses. Immunization of TLR4 ؊/؊ mice and DNA immunization by the same constructs confirmed that immunogenicity was independent of different degrees of contamination with copurifying immune-stimulatory molecules from E. coli. We hypothesize that immunogenicity correlates with the intrinsic ability of the capsomeres to assemble into larger particles, as only assembly-competent L1 proteins induced high antibody responses. One of the proteins (L1⌬N10) proved to be the most immunogenic, inducing antibody titers equivalent to those generated in response to VLPs. However, preassembly prior to injection did not increase immunogenicity. Our data suggest that certain L1 constructs can be used to produce highly immunogenic capsomeres in bacteria as economic alternatives to VLP-based formulations.Certain types of human papillomavirus (HPV) are the cause of cervical cancer, most frequently HPV types 16 and 18 (HPV-16 and HPV-18), which are responsible for about 50% and 20% of cases, respectively (8,15,16). Recently, two vaccines that prevent infection with HPV-16 and HPV-18 have been introduced to the market. These vaccines are based on the viral major structural protein L1, which can spontaneously self-assemble in vitro into empty virus-like particles (VLPs) that resemble the native virions in size and shape. VLPs have been shown to be highly immunogenic, as they can induce high titers of neutralizing antibodies (29,30). HPV virions and VLPs consist of 72 L1 pentamers, also called capsomeres, which are arranged in an icosahedral Tϭ7 particle lattice with a diameter of 55 nm. Cryo-electron microscopic analysis has revealed the presence of 60 hexavalent and 12 pentavalent capsomeres (4).Capsid assembly has been reported to be optimal at low pH (pH 5.4) and high ionic strength, whereas both high pH (pH 8.2) and the presence of reducing agents favor disassembly into capsomeres, the latter because the viral particles are stabilized by intercapsomeric disulfide bonds between two conserved cysteine resi...

A Cell-Free Assembly System for Generating Infectious Human Papillomavirus 16 Capsids Implicates a Size Discrimination Mechanism for Preferential Viral Genome Packaging

“…4B). Smaller or distorted particles should float more than mature particles (44)(45)(46). For example, when Cap/L2 particles were run on the OptiPrep gradient, the L1-containing fractions were dispersed throughout the gradient but were mostly in the upper half of the gradient.…”

Influence of Oxidation and Multimerization on the Immunogenicity of a Thioredoxin-L2 Prophylactic Papillomavirus Vaccine

“…Despite the encouraging results obtained with the prototype TrxL2 vaccine, a detailed knowledge of all the factors (especially the higher-order multimerization and aggregation states of the antigen) that potentially influence immunogenicity and virus neutralization capacity is an important aspect to consider in further vaccine development. In fact, in various subunit vaccine settings, including L1-based vaccines, where VLPs are superior to pentameric L1 capsomeres in terms of immunogenicity (19,20), antigen multiplicity and assembly states have been shown to be important determinants of vaccine immunogenicity and efficacy. Thus, the multim-erization state of the L1 antigen is likely to be a major factor influencing immunogenicity, as has been observed with other antigens (21,22).…”

Expression of HPV-16 L1 capsomeres with glutathione-S-transferase as a fusion protein in tobacco plastids: An approach for a capsomere-based HPV vaccine

“…Modified L1 gene (L1_2xCysM) alone leads to the assembly of L1 protein to pentameric capsomeres. 32,55 The GST gene is a widely-used affinity tag gene which encodes evolutionarily conserved detoxification enzymes. 44 These enzymes have the ability to conjugate a broad range of potentially harmful xenobiotics, 45 hence rendering plants to detoxify directly the products of oxidative stress.…”