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Insect and yeast cells are considered the expression systems of choice for producing virus-like particles (VLPs), and numerous types of VLPs have been produced in these systems. However, previous studies were restricted to identifying the characteristics of individual VLP preparations. No direct comparison of the structures and immunogenic properties of insect and yeast-derived VLPs has so far been made. In the present study, the size distribution and immunogenic properties of human papillomavirus type 16 (HPV16) L1 VLPs produced in Spodoptera frugipedra-9 insect cells and Saccharomyces cerevisiae were compared. The insect cell-derived VLPs were larger than the yeast ones (P

There has been no attempt to apply protein-based markers of exfoliated cervical cells (ECCs) for primary screening of cervical cancer. In the present study, the levels of six tumor-associated protein [TAPs: Sialyl Lewis A (SLeA), Cancer Antigen 15-3 (CA 15-3), p53, heat shock protein (Hsp)70, Hsp27 and squamous cervical carcinoma antigen (SCCA)]and of two human papillomavirus (HPV) viral proteins (HPV16 E7 and HPV16 L1) of ECCs lysates were evaluated by enzyme-linked immunosorbent assays (ELISAs).The wells of 96-well plates were coated with the ECCs lysates from normal, cervical intraepithelial neoplasia (CIN) I, CIN II, CIN III and cancer groups, and candidate proteins were detected by relevant antibodies. SLeA level decreased with increasing severity of lesions, whereas the levels of other candidate proteins increased. SLeA, HPV16 L1 and p53 levels appeared more useful for detecting cervical lesions than the other candidates. The combination of ELISA-SLeA and ELISA-HPV16 L1 could efficiently detect cervical lesions from normal. The combination of ELISA-SLeA and ELISA-p53 could powerfully discriminate cancer from normal with 91.3% sensitivity and 96.7% specificity. The protein levels of ECCs have great potential as biomarkers for primary screening of cervical cancer.

Cervical cancer is the fourth most frequent cancer among women worldwide. Human papillomaviruses (HPVs) cause almost all cervical cancers in low-income countries. Three prophylactic HPV virus-like particle-based vaccines have been licensed to date, and they have all shown high efficacy and reliable safety profiles. However, isolated safety issues have resulted in a reluctance to use these vaccinations. In addition, the high prices of the vaccinations have caused the inequitable distribution of the vaccine: the prices are unaffordable for low-income countries. Meanwhile, great effort has been put into the development of therapeutic HPV vaccines, including protein/peptide-, live vector-, DNA- and cell-based vaccines. These new vaccines have considerable therapeutic potential but limited practical use. The development of immune checkpoint inhibitors and personalized immunotherapy remain challenges for future study. In this article, the current status of the licensed vaccines, therapeutic HPV vaccines and biosimilars, and new platforms for HPV vaccines, are reviewed, and safety issues related to the licensed vaccines are discussed. In addition, the prospects for HPV vaccines are considered.

Charge variants (acidic and basic) of recombinant monoclonal antibodies (Mabs) have received much attention due to their potential biological effects. C-terminal lysine variants are common in Mabs and their proportion is affected by the manufacturing process. In the present study, changes of trastuzumab charge variants brought about by carboxypeptidase B treatment and subsequent storage at 8 or 37 °C for up to 24 h were monitored by cation-exchange chromatography analysis to investigate the effects of C-terminal lysine cleavage and its subsequent reaction at 8 or 37 °C. C-terminal lysine cleavage at 8 °C reduced the fraction of basic species and had little effect on the fraction of acidic species. Analysis of individual peaks demonstrated that C-terminal lysine cleavage induced both increases and decreases in individual acidic variants, with the result that there was little overall change in the overall proportion of acidic species. It appeared that most of the basic variant Mab molecules but only a fraction of the acidic variant molecules had C-terminal lysines. Increasing the temperature to 37 °C appeared to increase the fraction of acidic species and decrease main species significantly, without a similar change in basic species. These results indicate that length of exposure to elevated temperature is a critical consideration in charge variant analysis.

Human papillomavirus (HPV) types 16 and 18 are the main targets in the field of prophylactic vaccines for preventing cervical cancer. L1 protein, the major capsid protein of HPV, selfassembles into virus-like particles (VLP), which are the major component of prophylactic vaccines. To obtain highly purified L1 protein, contaminants must be removed by several chromatography steps. However, this requires a great deal of time and labor, and results in loss of large amounts of the target protein. Therefore, we have sought to develop an efficient method for removing contaminants prior to chromatography during the purification of HPV18 L1 protein from Saccharomyces cerevisiae. For this purpose the contaminating proteins were removed by an ammonium sulfate precipitation step and further removed by a removal of precipitated contaminants step. Purification of the L1 protein by chromatography was significantly improved by the removal of precipitated contaminants step. In the present work we developed two one-step chromatography methods (heparin and cation-exchange chromatography), and HPV18 L1 proteins purified by both methods self-assembled into VLP. The two chromatographic purification methods are simpler and more convenient than previous methods and are widely applicable to work with VLPs.

The aim of the study was to investigate the seroprevalence to HPV type 16 in Korean women with precancerous and cancerous lesions of the uterine cervix. The cases were 173 Korean women of whom 130 had high-grade cervical intraepithelial neoplasias (CIN), 43 cervical carcinomas and the control group was 106 women showing normal cervical cytology. Serologic assays were performed using HPV-16 VLPs as antigen in an ELISA. Specific antibodies against HPV-16 VLP were detected in 59.2% (77/130) of the patients with high-grade CIN, in 67.4% (29/43) of the patients with cervical cancer and in 20.8% (22/106) of control subjects. No difference of serologic response was found between high-grade CIN and cancer. HPV-16 seropositivity showed the significant association with patients' age and preoperative HPV DNA infection. Recurrence of high-grade CIN was not affected by the VLP-16 seropositivity. Recurrence of carcinoma showed the borderline association with HPV-16 seropositivity (P=0.06). The association between the cancer recurrence and seropositivity was not found in the logistic regression analysis. Two patients dying of cancer during the follow-up period were both seronegative (P=0.01). In conclusion, serologic testing for HPV-16 VLP antibody provides a disease indicator of cervical lesions and potential prognostic parameter of cervical carcinoma.

Cervical cancer caused by human papillomavirus (HPV) might be successfully prevented by HPV vaccination and screening. HPV vaccination and HPV serology assays have been investigated using HPV virus-like particles (VLPs). In this study we produced HPV18 L1 VLPs in Saccharomyces cerevisiae and purified them. The HPV18 L1 gene was cloned into the yeast expression vector YEGalpha-HIR525, and transformed into Saccharomyces cerevisiae. Expression of HPV18 L1 protein was demonstrated by Western blotting. The HPV18 L1 protein was purified by ultracentrifugation, size-exclusion chromatography and cation-exchange chromatography, and was up to 95% pure. We showed by transmission electron microscopy that the purified protein self-assembled into VLPs. These findings should be useful for establishing vaccine efficacy as well as characterizing vaccine candidates, and may provide an international reference standard for HPV serology assays.

Clerodendron trichotomum Tunberg Leaves (CTL) have been used for centuries in Chinese folk medicine for their anti-inflammatory properties. To investigate the molecular mechanism of anti-inflammation by CTL, we analyzed the regulation of TNF-alpha expression in RAW 264.7 cells, a key step in inflammation. The effect of CTL on the production and expression of tumor necrosis factor-alpha (TNF-alpha) was determined by enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). CTL inhibited the production and expression of TNF-alpha in LPS-stimulated RAW 264.7 cells in a dose-dependent manner. In addition, activation of NF-kappaB, which controls TNF-alpha expression, was inhibited in LPS-stimulated RAW 264.7 cells by CTL in a dose-dependent manner, as demonstrated by an electro phoretic mobility shift assay (EMSA). Furthermore, CTL inhibited activation of NF-kappaB through inhibition IkappaB degradation, as demonstrated by an western blot analysis of IkappaB-alpha. These results suggest that CTL inhibits the expression of the pro-inflammation gene through the inhibition of NF-kappaB dependent pathway in RAW 264.7 cells.

Amino acid sequences 200-225 and 255-278 of the F protein of human respiratory syncytial virus (HRSV) are T cell epitopes (Bourgeois et al., 1991; Corvaisier et al, 1993). Peptides corresponding to these two regions were synthesized and coupled with keyhole limpet haemocyanin (KLH). The two conjugated proteins were administered intranasally to BALB/c mice alone or together with cholera toxin B (CTB). ELISAs revealed that the mixture of the conjugates with CTB increased not only the systemic response but also the mucosal immune response of the saliva. The systemic response was lower and the mucosal immune response was undetectable in mice immunized with the conjugates on their own. These results suggest that these two peptide sequences are effective epitopes for inducing systemic and mucosal immune responses in conjunction with CTB, and may provide the basis for a nasal peptide vaccine against RSV for human use.

Oka strain VR-795 (Varicella Zoster Virus, VZV) of American Type Culture Collection (ATCC) has been used for chickenpox vaccine production. In order to use this strain for vaccine production, the strain must be identified and its stability must be confirmed. The identification of the Oka strain has been confirmed using Restriction Fragment Length Polymorphism (RFLP) and DNA sequence analysis of glycoprotein-II (gp-II). The amino acid sequences of Oka deduced from the DNA sequence of gp-II have changed at three amino acids against Ellen and at one amino acid against Webster. To prove the stability of the Oka strain during the passage, RFLP and DNA sequence analyses were also used with 11, 15 and 23 times of virus passage. We found that the Oka strain was stable at passages of up to 23 times, based on the RFLP and DNA sequence analyses. The confirmed Oka strain was renamed as BR-Oka for the purposes of chickenpox vaccine production.

To elucidate of role(s) of tTA as a repressor in the tTA-mediated gene repression system, we introduced mutations into the acidic domain of VP16 and examined the effects of such various mutations. In the transient repression experiment, a region containing 34 amino acids of the activation domain of VP16 (412-456) which interacts with TFIIB was found to be necessary and sufficient for the tTA-mediated repression of gene expression. However, in the experiment to investigate the fact that tTA-regulated repression is related to the activation function of VP 16, we found that the repression abilities of tTA derivatives did not correlate exactly with their activation abilities. Therefore, we conclude that increased mass of VP16 in tTA might be also important for efficient repression in addition to functional domain of VP16.

Previous studies have revealed that ingestion of bovine colostrum is effective in preventing pathogens from invading through the gastrointestinal tract (GI) and modulating the mucosal immunity of the GI tract, indicating that its effect is principally local. Thus it is unclear if ingestion of bovine colostrum can affect the systemic immune system. In this study, we investigated the effect of taking bovine colostrum (vs phosphate-buffered saline) for 14 days on the behavior of the immune cells of mice. Isolated splenocytes, which are pivotal cells of systemic immunity, were then stimulated with Escherichia coli lipopolysaccharide. Bovine colostrum significantly reduced NK cell and monocyte activities and lymphoproliferaltive responses to LPS stimulation. Thus dietary bovine colostrum renders immune cells less responsive to LPS stimulation. Dietary bovine colostrum thus affects the systemic immune system and may have anti-inflammatory actions.

We aimed to determine the prevalence of HPV-16/18 antibodies in Korean women with high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer. We conducted the hospital-based case-control study at the university hospital between 2003 and 2006. Cases were 130 high-grade CIN and 43 cervical cancer patients and the control group was 106 women showing normal cervical cytology. Enzyme-linked immunosorbent assays were performed for HPV-16/18 L1 virus-like particles (VLPs) as an antigen. Seropositivity for HPV-16 VLP and HPV-18 VLP was found in 67.4% and 30.2% of cancer patients and 59.2% and 20.0% of high-grade CIN patients, respectively. Seropositivity for HPV-16 with high-grade CIN (OR 6.91; 95% CI 3.74–12.76) and cervical cancer (OR 8.99; 95% CI 3.88–20.84) presented significant associations, as did seropositivity for HPV-18 (high-grade CIN: OR 3.64; 95% CI 1.67–7.95, cervical cancer: OR 6.82; 95% CI 2.52–18.45). Patients with both HPV-16 and 18 seropositivity were 9.38 times (95% CI 2.98–29.51) more likely to have high-grade CIN and 17.05 times (95% CI 4.55–63.87) more likely to have cancer. Both HPV 16 and 18 L1 VLP serology is the clear disease predictors of presence of high-grade CIN and cervical cancer.

Leishmania virus (LRV) 1–4 has been reported to produce a fusion of ORF2 and ORF3via a programmed +1 frameshift in the region where ORF2 and ORF3 overlap (Leeet al., 1996). However, the exact frameshift site has not been identified. In this study, we compared the frameshift efficiency of a 259bp (nt. 2565–2823), frameshift region of LRV1–4, and the 71bp (nt. 2605–2678) sub-region where ORF2 and ORF3 overlap. We then predicted the frameshift site using a new computer program (Pseudoviewer), and finally identified the specific region associated with the mechanism of the LRV1–4′s +1 frameshift by means of a mutational analysis based on the predicted structure of LRV1–4 RNA. The predicted structure was confirmed by biochemical analysis. In order to measure the frameshift efficiency, constructs, that generated, luciferase without a frameshift or with a +1 frameshift, were generated andin vitro transcription/translation analysis was performed. Measurements of the luciferase activity generated, showed that the frameshift efficiency was about 1% for both the 259bp (LRV1–4 259FS) and 71bp region (LRV1–4 71FS). Luciferase activity was strongly reduced in a mutant (LRV1–4 NH: nt. 2635–2670) with the entire hairpin deleted and in a mutant (LRV1–4, NUS: nt. 2644–2659) with the upper stem of the hairpin deleted. These results indicate that the frameshift site in LRV1–4′s is in the 71bp region where ORF2, and ORF3 overlap, and that nt. 2644–2659 (the upward hairpin stem) play a key role in generating the +1 frameshift.

Temperature and medium composition were changed with the aim of increasing growth and erythropoietin (EPO) production in EPO-producing Chinese hamster ovary (CHO) cells. We used the CHO cell line, IBE, and its derivative, CO5, which over-expresses the first two enzymes of the urea cycle, carbamoyl phosphate synthetase I (CPS I) and ornithine transcarbamoylase (OTC). When supplements were added to the medium at 33 degrees C, the growth of IBE and CO5 cells increased by 27% and 26%, respectively and the maximum yield of EPO was increased by 40% in both cell lines. The absolute EPO concentration in the CO5 cells was always 55-60% higher than in the IBE cells. In addition, when the two cell lines were continuously cultured with supplements at 33 degrees C until their growth rates approached those at 37 degrees C, the growth rates of both IBE and CO5 cells increased by 54% and their maximum EPO levels increased by up to 73% and 56%, respectively. Therefore, the growth and EPO expression levels of CO5 cells increased 2.2-fold and 2.6-fold, respectively, compared to those of the IBE cells. These results indicate that adaptation to lower temperature as well as medium supplementation could be important for improving cell growth and EPO production.

Clerodendron trichotomum Thunberg Leaves (CTL) have been used for centuries in Chinese folk medicine for their anti-inflammatory properties. We have studied the anti-inflammatory effects of CTL extracts in rats, mice and in Raw 264.7 cells. 1 mg/kg solutions of the 30% and 60% methanol extracts of CTL were used and a 1 mg/kg of indomethacin was used as a positive anti-inflammatory standard; these were then administrated to rats. Carrageenan was injected subcutaneously to induce hind paw edema in rats. The result of carrageenan-induced rat paw oedema showed that a 1 mg/kg of the 30%, and 60% methanol fraction of CTL and 1 mg/kg of indomethacin inhibited the hind paw edema by 19.5%, 23.0%, and 20.5% respectively. The effect of CTL on inflammation in mice by a capillary permeability assay was examined by detecting Evans blue leakage from capillaries after the intraperitoneal injection of acetic acid, a potent inflammatory stimulus. The 60% methanol fraction of CTL inhibited Evans blue dye leakage by 47.0%, which was 10% higher than that of the inhibition of 1 mg/kg of indomethacin. Also, the 60% methanol fraction of CTL suppressed the prostaglandin E2 (PGE2) generation in RAW 264.7 macrophage cells after treatment with lipopolysaccharide (LPS) by as much as the inhibition of 1 mg/kg of indomethacin and this led to the synthesis of PGE2 by COX-2 induction. The inhibition of the carrageenan-induced rat paw oedema, vascular permeability and the PGE2 generation demonstrates that the 60% methanol fraction of CTL contains a potent anti-inflammatory activity.

Traditional taxonomic methods used for the identification and differentiation of ginsengs rely primarily on morphological observations or physiochemical methods, which cannot be used efficiently when only powdered forms or shredded material is available. Randomly amplified polymorphic DNA (RAPD) was used to determine the unique DNA profiles that are characteristic not only of the genus Panax but also of various Panax subgroups collected from five different countries. RAPD results of OP-5A primer showed a specific single band that is characteristic of all ginseng samples. RAPD results of OP-13B primer demonstrated that OP-13B primer could be used as a unique RAPD marker to differentiate Panax species. These results support that this approach could be applied to distinguish Korean Ginseng (Panax ginseng) from others at the molecular level.

The tetracycline-controlled transactivator (tTA)-mediated gene activation system was examined in virus infected cells to determine its role in the control of gene expression. In the presence of tTA, the gene expression from the tetO-modified minimal promoter was efficiently activated in the uninfected cells, whereas essentially no activation was observed from the only minimal promoter without the seven direct repeats of 42 bp tetO sequences. However, essentially no activation was observed when only the minimal promoter was used, without the seven direct repetitions of the 42 bp tetO sequences. On the other hand, in the infected cells, a substantial background of beta-glucuronidase expression was detected in the absence of tTA, even though tTA stimulated the gene expression by approximately 7-fold. This background expression indicates that the sequences within or nearby tetO are involved in the background stimulation of the gene expression by HCMV and HSV-1. These results suggest that the application of the tTA-mediated gene activation system may not be extremely useful for studying the biological roles of HCMV and HSV genes in the viral replicative cycles, because of the basal activity of the gene expression.