Characterization of human papillomavirus type 16 pseudovirus containing histones.

BACKGROUND: Pseudoviruses (PsVs) that encapsidate a reporter plasmid DNA have been used as surrogates for native human papillomavirus (HPV), whose continuous production is technically difficult. HPV PsVs have been designed to form capsids made up of the major capsid protein L1 and the minor capsid proteins L2. HPV PsVs have been produced in 293TT cells transfected with plasmid expressing L1 and L2 protein and plasmid containing the reporter gene. Several studies have suggested that naturally occurring HPV virions contain cellular histones, and histones have also been identified in mature HPV PsVs. However, the effect of the histones on the properties of the PsVs has not been investigated. Using heparin chromatography, we separated mature HPV type 16 PsVs into three fractions (I, II, and III) according to their heparin-binding affinities. RESULTS: The amounts of cellular histone and cellular nucleotides per PsV were found to increase in the order fraction I, II and III. It appeared that PsVs in fraction I contains just small amount of cellular histone in Western blot analysis. The proportions of the three fractions in PsV preparations were 83.4, 7.5, and 9.1 % for fraction I, II, and III PsVs, respectively. In the electron microscope PsVs in fraction I appeared to have a more condensed structure than those in fractions II and III. Under the electron microscope fraction II and III PsVs appeared to be covered by substantial amounts of cellular histone while there was no visible histone covering PsVs of fraction I. Also the levels of reporter gene expression in infections of fraction II and III PsVs to 293TT cells were significantly lower than those in infections of fraction I PsV, and fraction II and III particles had significantly reduced immunogenicity. CONCLUSIONS: Our findings suggest that the involvement of large amounts of cellular histones during PsV formation interferes with the structural integrity of the PsVs and affects their immunogenicity. The fraction I particle therefore has the most suitable characteristics for use as an HPV PsV.

Codon optimization of the human papillomavirus type 58 L1 gene enhances the expression of soluble L1 protein in Saccharomyces cerevisiae.

The effect of codon optimization of L1 gene on the production of the L1 protein of human papillomavirus (HPV) was investigated in a yeast expression system. Saccharomyces cerevisiae was transformed with a plasmid containing either the wild type (WS)-HPV type 58 L1 (HPV58 L1) gene or a codon-optimized (MO)-HPV58 L1 gene. The proportion of soluble L1 protein expressed from MO-HPV58 L1 was significantly higher than that expressed from WS-HPV58 L1. Moreover, the amount of purified MO-HPV58 L1 protein recovered was 2.5-fold higher than the amount of WS-HPV58 L1 protein. Codon optimization of HPV58 L1 gene thus increases the proportion of soluble L1 protein and the amount of purified product that can be used as antigen to generate vaccines.

The composition of the carbon source and the time of cell harvest are critical determinants of the final yield of human papillomavirus type 16 L1 protein produced in Saccharomyces cerevisiae.

The L1 protein is a major component of prophylactic human papillomavirus (HPV) vaccines. Little effort has been made to improve the productivity of L1 protein by optimizing cell culture conditions although the high price of the vaccines is considered one of the factors limiting its widespread use. In biopharmaceutical manufacturing, strategies for optimizing culture conditions tend to focus on improvements in upstream processing rather than final yield because of the complexities of purification procedures. In this study, we investigated L1 protein productivity as a function of the composition of the carbon source and the point of cell harvesting in the Saccharomyces cerevisiae expression system. We performed 44 series of purifications and achieved the highest productivity when the cells were cultured in a medium composed of 7% glucose and 1% galactose for 144 h. The final yield of L1 protein was markedly affected by the glucose: galactose ratio and the point at which cells were harvested: there was a 15-fold difference between the lowest and highest yields. We believe that optimization of the composition of the carbon source and the time of cell harvest have considerable potential for reducing the cost of production of HPV L1 protein.

The choice of resin-bound ligand affects the structure and immunogenicity of column-purified human papillomavirus type 16 virus-like particles.

Cell growth conditions and purification methods are important in determining biopharmaceutical activity. However, in studies aimed at manufacturing virus-like particles (VLPs) for the purpose of creating a prophylactic vaccine and antigen for human papillomavirus (HPV), the effects of the presence of a resin-bound ligand during purification have never been investigated. In this study, we compared the structural integrity and immunogenicity of two kinds of VLPs derived from HPV type 16 (HPV16 VLPs): one VLP was purified by heparin chromatography (hHPV16 VLP) and the other by cation-exchange chromatography (cHPV16 VLP). The reactivity of anti-HPV16 neutralizing monoclonal antibodies (H16.V5 and H16.E70) towards hHPV16 VLP were significantly higher than the observed cHPV16 VLP reactivities, implying that hHPV16 VLP possesses a greater number of neutralizing epitopes and has a greater potential to elicit anti-HPV16 neutralizing antibodies. After the application of heparin chromatography, HPV16 VLP has a higher affinity for H16.V5 and H16.E70. This result indicates that heparin chromatography is valuable in selecting functional HPV16 VLPs. In regard to VLP immunogenicity, the anti-HPV16 L1 IgG and neutralizing antibody levels elicited by immunizations of mice with hHPV16 VLPs were higher than those elicited by cHPV16 VLP with and without adjuvant. Therefore, the ability of hHPV16 VLP to elicit humoral immune responses was superior to that of cHPV16 VLP. We conclude that the specific chromatographic technique employed for the purification of HPV16 VLPs is an important factor in determining the structural characteristics and immunogenicity of column-purified VLPs.

The production and immunogenicity of human papillomavirus type 58 virus-like particles produced in Saccharomyces cerevisiae.

Human papillomavirus (HPV) is the cause of most cases of cervical cancer. HPV type 58 (HPV58) is the second most frequent cause of cervical cancer and high-grade squamous intraepithelial lesions (HSIL) in Asia and South / Central America, respectively. However, there is no vaccine against HPV58, although there are commercially available vaccines against HPV16 and 18. In this study, we produced HPV58 L1 protein from Saccharomyces cerevisiae, and investigated its immunogenicity. We first determined the optimum period of culture for obtaining HPV58 L1. We found that a considerable portion of the HPV58 L1 resulting from 48 h culture cannot be recovered by purification, while the HPV58 L1 resulting from 144 h culture is recovered efficiently: the yield of HPV58 L1 finally recovered from 144 h culture was 2.3 times higher than that from 48 h culture, although the production level of L1 protein from 144 h culture was lower than that from 48 h culture. These results indicate that the proportion of functional L1 protein from 144 h-cultured cells is significantly higher than that of 48 h-cultured cells. The HPV58 L1 purified from the 144 h culture was correctly assembled into structures similar to naturally occurring HPV virions. Immunization with the HPV58 L1 efficiently elicited anti-HPV58 neutralizing antibodies and antigen-specific CD4+ and CD8+ T cell proliferations, without the need for adjuvant. Our findings provide a convenient method for obtaining substantial amounts of highly immunogenic HPV58 L1 from S. cerevisiae.