Shen Q.,Shanghai Zerun Biotech Co. |
Lei J.-Q.,Shanghai Zerun Biotech Co. |
Zhou C.-M.,Shanghai Zerun Biotech Co. |
Zhang G.-X.,Shanghai Zerun Biotech Co.
Chinese Journal of Biologicals | Year: 2012
Objective: To express human papillomavirus (HPV) 18 L1 protein in Pichia pastoris, purify the virus-like particles (VLPs) and determine its immunogenicity. Methods: Recombinant plasmid pHPV18 L1 was transformed to P. pastoris X-33 by electrotransformation and induced with methanol. The expressed recombinant HPV18 L1 protein was identified by SDS-PAGE and Western blot, then purified by ion exchange chromatography and molecular sieve chromatography, and analyzed for purity by SEC-HPLC, for particle size distribution and structure by dynamic light scattering and transmission electron microscopy, and for immunogenicity by determination of ED 50 in mice and antibody titer in rats. Results: HPV18 L1 protein, with a relative molecular mass of about 55 000, was effectively expressed in P. pastoris, which showed specific binding to mouse anti-HPV18 L1 monoclonal antibody. The purified HPV18 L1 protein reached a purity of 99%, of which the VLPs were even in size, at a diameter of about 50 nm. The HPV18 L1 protein after adsorption with aluminum hydroxide induced high antibody titer in rats, of which the ED 50 in mice was 0.006 68 μg. Conclusion: Self-assembled HPV18 L1 was highly expressed in P. pastoris, of which the VLPs showed high immunogenicity after purification and adsorption with aluminum hydroxide. It laid a foundation of industrial production of HPV18 L1 vaccine.
Mallajosyula V.V.,Indian Institute of Science |
Citron M.,Merck And Co. |
Ferrara F.,University of Kent |
Temperton N.J.,University of Kent |
And 4 more authors.
Frontiers in Immunology | Year: 2015
Seasonal epidemics caused by influenza A (H1 and H3 subtypes) and B viruses are a major global health threat. The traditional, trivalent influenza vaccines have limited efficacy because of rapid antigenic evolution of the circulating viruses. This antigenic variability mediates viral escape from the host immune responses, necessitating annual vaccine updates. Influenza vaccines elicit a protective antibody response, primarily targeting the viral surface glycoprotein hemagglutinin (HA). However, the predominant humoral response is against the hypervariable head domain of HA, thereby restricting the breadth of protection. In contrast, the conserved, subdominant stem domain of HA is a potential "universal" vaccine candidate. We designed an HA stem-fragment immunogen from the 1968 pandemic H3N2 strain (A/Hong Kong/1/68) guided by a comprehensive H3 HA sequence conservation analysis. The biophysical properties of the designed immunogen were further improved by C-terminal fusion of a trimerization motif, "isoleucine-zipper", or "foldon". These immunogens elicited cross-reactive, antiviral antibodies and conferred partial protection against a lethal, homologous HK68 virus challenge in vivo. Furthermore, bacterial expression of these immunogens is economical and facilitates rapid scale-up. © 2015 Mallajosyula, Citron, Ferrara, Temperton, Liang, Flynn and Varadarajan.