Search

Currently virus-like particles (VLPs) are receiving much attention as platforms for next generation vaccines. However, chromatography-based methods for purifying VLPs remain challenging. Unlike traditional methods using density gradient for purifying VLPs, there have been few advances in explaining how assembled particles can be obtained by chromatography. Nervous necrosis virus (NNV) infects over 30 species of fish and leads to large economic losses in the farmed fish industry. Previously we developed a heparin chromatography-based method for purifying red-spotted grouper NNV (RGNNV) VLPs. However it is unclear how the assembled RGNNV VLPs are obtained by this method. It is known that assembly of NNV capsid proteins depends on calcium ions. In the present study, we found that the yield of purified RGNNV capsid protein in heparin chromatography was enhanced when calcium ions were present during binding. Also, it appears that the capsid protein of RGNNV undergoes partial disassembly and reassembly during sample preparation prior to heparin chromatography and the protein finally undergoes assembly during the chromatography. Therefore, our results indicated that heparin-binding affinity of RGNNV capsid protein is linked to its ability for VLP formation. The assembly of RGNNV capsid proteins recombinantly produced is a good model for explaining VLP formation during chromatography-based purification processes.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0296-3) contains supplementary material, which is available to authorized users.

PCRs for reporter genes in HPV16 PsVs from fractions I, II and III. A shows the pYSEAP contents of PsVs from fraction I, II and III. B confirms the specificity of the PCR for the relevant region of the pYSEAP construct. Buffer only and pcDNA3.1 are negative controls. To measure pYSEAP content per particle, in panel A, identical amounts of PsVs from fraction I, II, and III were used (20 ng based on L1 content). 25, 30 or 38 cycles of PCR were carried out. Thirty cycles of PCR were performed for panel B. The same primer set used in the quantitative real time PCR for amplifying the 80 bp region of pYSEAP was used (see the quantitative real time PCR section in Methods). Amplicons were analyzed on a 1.5 % agarose gel. (DOCX 406 kb)

L2 protein contents of HPV16 PsVs from fraction I, II and III. To detect the L1 and L2 proteins, 100 or 200Â ng of PsVs was loaded per well (based on the L1 amount). The L2 protein was detected by Western blotting using the anti-RG1-4MAP mouse serum (1:500 dilution) with HRP conjugated goat anti-mouse IgG antibody (Bethyl Laboratories). The proteins on SDS-PAGE gels were visualized by silver staining. The quantities of the L2 protein per PsV particle were similar for all three types of PsV. (DOCX 153 kb)

Schematic diagram of heparin chromatography to separate HPV16 PsVs into fraction I, II and III. (DOCX 75 kb)

TEM analysis of HPV16 PsVs from fraction I, II and III. Magnification x 234,000 (bars are 50 or 100Â nm). (DOCX 1656 kb)

Reacitivities of HPV16 PsVs from fraction I, II and III with H16.V5 or H16.E70 MAbs. The reactivities of fraction I PsVs were set at 100 %. The values are the mean ± SD of quadriplicate assays. (DOCX 43 kb)

Lymphoproliferative responses following four immunizations with HPV16 PsVs from fraction I, II, or III. The mice were immunized four times with 50 ng of PsVs per dose at 2-week intervals. Mouse splenocytes were obtained 5 days after the fourth immunization. Mouse splenocytes were labeled with carboxyfluorescein succinimidyl ester (CFSE), stimulated with purified HPV16 L1 VLPs, and cultured for 4 days. The splenocytes were stained with allophycocyanin (APC)-conjugated anti-CD4 antibody (eBioscience, USA) and examined with a FACSCalibur flow cytometer (BD Bioscience, USA). To count CD4+ cells, the cells were gated according to forward and side scatter, and the upper-left segment of each graph was counted on FITC and APC scatter plots. Panel A shows the flow cytometry results for three individual mice. The value in panel B represents the mean ± SEM (n = 3). (DOCX 171 kb)

SDS-PAGE analysis of heparin chromatography fractions containing HPV16 PsVs. Mature HPV16 PsVs purified by SEC were further separated by heparin chromatography. The binding buffer for heparin chromatography contains 0.65Â M NaCl. LS, FT and W refer to loading sample, flow-through, and wash, respectively. M indicates a protein marker. The PsVs in the flow-through and wash fractions were designated fraction I. PsVs in fraction II and III were eluted from the heparin resin by successive additions of 0.8 and 1Â M NaCl. (DOCX 197 kb)

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 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.