Characterization and evaluation of the immune responses elicited by a novel human papillomavirus (HPV) therapeutic vaccine: HPV 16E7-HBcAg-Hsp65 fusion protein.

Human papillomaviruses (HPV), particularly HPV16, are associated with most cervical cancers. Currently, although prophylactic vaccines have been developed, there is still an urgent need to develop therapeutic HPV vaccines. In this study, a novel fusion protein, HPV 16 E7-HBcAg-Hsp65 (VR111), with the goal of increasing anti-HPV16 cellular immunity was developed. VR111 was analyzed using SDS-PAGE, western-blotting, capillary isoelectric focusing (cIEF), analytical ultracentrifugation (AUC) and dynamic light scattering (DLS). Gamma interferon (IFN-γ) secretion assay was performed by enzyme-linked immunospot (ELISPOT) and ELISA to test their ability to induce cellular immune response. Significant correlation between ELISPOT and ELISA was observed (r=0.8680, p<0.0001). It was shown that VR111 could induce a significant increase in E7-specific CD8(+) T cell responses. Humoral immune response was also observed. The antibody titer levels were measured by ELISA. These results indicated that VR111 was a promising therapeutic vaccine for treatment of cervical cancer with possible therapeutic potential in clinical settings.

[Expression and activity analysis of Enterovirus 71 3C protease in Escherichia coli].

The recombinant plasmid carrying the gene encoding 3C protease of Enterovirus 71 (EV71) was constructed, the recombinant protein was then expressed and purified, the functional activity was also measured. Firstly, the 3C protease gene was inserted into pET28a vector, the constructed recombinant plasmid was transformed into E. coli BL21 (DE3) for expression under the induction of IPTG. The expressed protein was purified by affinity chromatography (Ni-NTA) and the N-terminus His-tag was cleaved by enterokinase from 3C protease. The activity of 3C protease was evaluated with fluorescent peptide substrates. It was verified by restriction analysis and sequencing that recombinant plasmid pET28a-3C was constructed correctly and functionally expressed in E. coli BL21 (DE3) resulting in the production of recombinant 3C protease with a size of 22kD. Both His-tag and non-His-tag (cleaved by enterokinase) 3C protease exhibited similar enzyme activity to 3B-3C fluorescent peptide with Km, Vmax and Kcat values of 22 microM, 434nM. Min(-1) and 0.0669 Min(-1), respectively. The optimial pH and temperature were 7.0 and 30-37 degrees C, respectively. The acquirement of recombinant purified 3C protease with high activity has paved the way of further studies on anti-viral inhibitors, structural protein assembly, vaccine development and detection methods of EV71.