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Li C.-M.,National Vaccine and Serum Institute
Chinese Journal of Biologicals | Year: 2012

Objective: To compare the purification efficacy of HBsAg expressed in Hansenula polymorpha by hydrophobic interaction chromatography using columns 3146VL, 3147L, 3148H and 3149VH with various butyl densities in WorkBeads 40/10000 Bus low series of Bio-Work and CIM Monoliths C4 of BIA. Methods: HBsAg was dissolved with various loading buffers, then loaded onto the column pre-filled with WorkBeads type 4 and C4 butyl chromatographic column, and eluated with various elution buffers. The flow-through and elution peaks were collected and determined for HBsAg content, based on which the flow-through and elution rates were calculated, and the optimal loading and elution buffers were screened. Results: The optimal column for purification of HBsAg was 3147L, while the optimal loading buffer was 20 mmol/L PB containing 4% ammonium sulfate. Neither 20 mmol/L PB nor 20 mmol/L PB containing 30% isopropanol was the ideal elution buffer. The optimal loading buffer on CIM Monoliths C4 was 50 mmol/L MOPS containing 3% ammonium sulfate and 20 mmol/L PB containing 4% ammonium sulfate, while the optimal elution buffer was 50 mmol/L MOPS containing 0.1% Triton X-100. Conclusion: It is feasible to introduce WorkBeads series which may be used repeatedly and CIM Monoliths C4 which is resistant to high flow rate into the purification of HBsAg expressed in Hansenula polymorpha.

Yang X.-Q.,National Vaccine and Serum Institute
Chinese Journal of Biologicals | Year: 2010

Objective: To analyze the regularity and characteristics of (suspected) adverse events following immunization (AEFI) of hepatitis B (HB) vaccine reported in documents. Methods: Based on CNKI Chinese database retrieval system, all cases of AEFI associated with the HB vaccine in the past 15 years were collected and analyzed by categorical statistics. Results: The clinical manifestations of (suspected) AEFI of HB vaccine were mainly allergic reactions (41.13%). The maximum percentage (48.39%) of (suspected) AEFI cases were induced by recombinant HB vaccine (Saccharomyces cerevisiae). The percentages of (suspected) AEFI cases appeared 0.5-24 h after immunization, after the second dose and at ages of less than one year were 35.48%, 37.90% and 49.19% respectively. Conclusion: The total case number of (suspected) AEFI of HB vaccine increased as compared with that reported previously in documents. However, it could not be concluded that the percentage of AEFI case increased, which should be further analyzed accurately.

Tang Y.-L.,National Vaccine and Serum Institute | Li Q.-M.,National Vaccine and Serum Institute
Chinese Journal of Biologicals | Year: 2011

Pneumococcal surface adhesin A (PsaA) is a lipoprotein and a ATP binding cassette transporter. As an important virulence factor and adhesion factor, it exists in all known serotypes of Streptococcus pneumoniae, of which the encoding genes are highly conserved, has multiple functions such as Mn 2+ transport and adherence. However, as an important surface antigen associated with virulence, Pneumococcal surface protein A (PspA) existed in almost all the clinical isolates of S. pneumoniae, of which the encoding genes are highly variable. Both PsaA and PspA are the protective antigens of S. pneumoniae and the hot spots of developing novel S. pneumoniae vaccines. This paper reviews the progress in research on structure, function, immunological characteristics and possible application of PsaA and PspA.

Wei J.-B.,National Vaccine and Serum Institute
Chinese Journal of Biologicals | Year: 2012

Objective: To develop a method for evaluation of vaccine-induced specific cellular killing activity based on flow cytometry(FCM) and perfect the method for assessment of vaccine and gene therapeutic drugs. Methods: Lymphocytes were labeled with CFSE (carboxyfluorescein diacetate, succinimidyl ester) and treated with TNFα to mimic the cytotoxic effect of cytotoxic killer cells, then determined by FCM after propidium iodide (PI) staining, based on which the concentrations of CFSE and PI and time for treatment were optimized. Mice were immunized with therapeutic hepatitis B vaccine with known high cellular immunity, of which the specific lymphocytes were isolated from spleen and stimulated with specific peptide. The lymphocytes of unimmunized mice were isolated as target cells and sensitized with specific antigenic peptide. A procedure was developed based on the optimization of conditions including the staining of target cells, the stimulation time of effector cells, as well as the action time between target and effector cells. Results: The cells were divided into CFSE+PI-, CFSE+PI+, CFSE -PI+ and CFSE-PI- groups by CFSE/PI staining, meanwhile, and survival and apoptotic cells were well distinguished. The optimal time for labeling of target cells with CFSE was 6 h, while that for stimulation of effector cells was 72 h, and the optimal action time between target and effector cells was 6 h. However, both the effector to target cell ratios of 100:1 and 50:1 might be adopted. Conclusion: A method for evaluation of vaccine-induced specific cellular killing activity based on FCM was successfully developed, which may be used for effective and precise assessment of CTL activity and perfection of method for assessment of vaccines and therapeutic drugs.

Ren H.-M.,National Vaccine and Serum Institute
Chinese Journal of Biologicals | Year: 2012

Chitosan binds to antigen molecules and forms nanoparticles by various methods. As an immune adjuvant, chitosan is worthy to be studied because of the stable physiochemical properties and diverse immune routes of the nanoparticles. This paper reviews the physiochemical characters of chitosan, the method for preparation of nanoparticles, application of chitosan as an immune adjuvant, the immune route of chitosan nanoparticles as well as the type of induced immune response.

Wang J.,National Vaccine and Serum Institute
Chinese Journal of Biologicals | Year: 2011

Objective: To purify and identify the randomly recombinant multiepitope protein M. RCAg-I of Plasmodium falciparum vaccine. Methods: The fermentation product of recombinant E. coli BL21 (DE3)-M. RCAg-1/pDS-ex was purified, then investigated for physic-chemical property and determined for reactogenicity by Western blot. BALB/c mice and New Zealand rabbits were immunized with M. RCAg-1 protein at various dosages, and determined for antibody titer in sera by indirect ELISA. The recognition of natural parasite antigen with the immune sera was determined by IFA. The activation of mouse specific T lymphocytes and secretion of cytokines were determined by enzyme-linked immunosorbent spot test. The effect on growth of Plasmodium falciparum was observed by growth inhibition test in vitro. Results: The M. RCAg-I protein, with an isoelectric point of 5.0, contained about 30% of total somatic protein, reached a purity of more than 95% and showed specific reaction with mouse specific monoclonal antibody, of which the concentration was 1.5 mg/ml, the residual endotoxin content was less than 2.5 EU/mg, the residual host protein content of 0.08%, and the amino acid sequence at N-terminus was correct. The serum antibody titer increased with the increasing doses and dosages of M. RCAg-1 protein, while those after last immunization with 20 and 30 μg of M. RCAg-1 protein reached 1:1 031 707. All the immune sera of mice recognized the natural antigen of Plasmodium falciparum in a dose-dependent mode, while those induced by 10, 20 and 30 μg of M. RCAg-1 protein stimulated the proliferation of specific splenic lymphocytes (P < 0.01) as well as secretions of IFN-γ and IL-4 (both P < 0.01), and inhibited the growth of Plasmodium falciparum in vitro significantly. Conclusion: The purified M. RCAg-1 protein showed stable physic-chemical property and high immunogenicity, stimulated persistent high titer specific antibody and induced significant T cell response, which might be used as a potential candidate vaccine of good prospect of development.

Liu Y.,Northwest University, China | Liu Y.,National Vaccine and Serum Institute | Luo X.,Northwest University, China | Luo X.,National Vaccine and Serum Institute | And 3 more authors.
Vaccine | Year: 2011

Synthetic oligodeoxynucleotides containing unmethylated CpG-dinucleotides (CpG-ODNs) are immunostimulatory in a broad spectrum of species. Extensive studies provide evidence that CpG-ODNs are effective as immunotherapeutics and vaccine adjuvants in various clinical settings. Three major classes of immunostimulatory CpG-ODNs are well characterized according to their in vitro activities and chemical compositions. However, it remains largely unclear whether and how these differences translate in vivo and in particular when used as vaccine adjuvants. In the present study, a panel of CpG-ODNs, including four representative sequences respectively from each class, was used to characterize their adjuvant activities in mice. The results demonstrated that three CpG-ODN classes can differentially affect antigen-specific humoral and cellular immune responses. Specifically, the B- and C-class CpG-ODNs induce a potent Th1-biased immunity with comparable antibody levels as well as CD4 + and CD8 + T cell responses. In contrast, although the A-class CpG-ODNs can weakly enhance antibody titers and CD8 + T cell response regarding cytotoxic activity, they are not able to change the IgG1/IgG2a ratio or elicit antigen-specific, IFN-γ-secreting CD4 + and CD8 + T cells. Consistent with this, three CpG-ODN classes provide differential antigen-specific protection against Listeria monocytogenes, an intracellular bacterial infection. In conclusion, our study provides not only better knowledge about the adjuvant activities of three CpG-ODN classes but also implications for the rational design of CpG-ODN adjuvants. © 2011 Elsevier Ltd.

Wang N.,National Vaccine and Serum Institute
Chinese Journal of Biologicals | Year: 2011

Objective: To test for hepatitis E virus-like particles (HE VLPs) by size-exclusion HPLC (SE-HPLC). Methods: HE VLPs were prepared by ultracentrifugation and Sephacryl S-200HR gel chromatography respectively, and analyzed by SDS-PAGE, Western blot, electron microscopy, dynamic light scattering and SE-HPLC. based on which a SE-HPLC method for qualitative and quantitative assay of HE VLPs were developed and used for quantitative assay of HE VLPs in culture supernatants at various time points. Results: The HE VLPs prepared by the two methods showed target protein bands each with a relative molecular mass of about 50 000 on SDS-PAGE and Western blot profiles, of which the concentrations, purities and reactogenicities were basically in agreement. Electron microscopy showed that the HE VLPs prepared by ultracentrifugation were even hollowed particles at a mean diameter of about 30 nm, while those by chromatography consisted of mass irregular blocks and a small quantity of hollowed particles. Dynamic light scattering proved that the HE VLPs prepared by ultracentrifugation showed a single peak, at a mean diameter of 27.2 nm and a purity of more than 98%, while those by chromatography at radiuses ranged from 2 to 120 nm. SE-HPLC showed that the HE VLP content at a range of 1-40 μg was linearly related to the peak area. The developed method showed high precision and was suitable for the real-time quantitative assay of HE VLPs in cell culture of virus. Conclusion: SE-HPLC may be used for qualitative and quantitative assay of HE VLPs.

Wang X.,Nankai University | Yang X.,Nankai University | Yang C.,National Vaccine and Serum Institute | Wu Z.,National Vaccine and Serum Institute | And 2 more authors.
PLoS ONE | Year: 2011

NMB0315 is an outer membrane protein of Neisseria meningitidis serogroup B (NMB) and a potential candidate for a broad-spectrum vaccine against meningococcal disease. The crystal structure of NMB0315 was solved by single-wavelength anomalous dispersion (SAD) at a resolution of 2.4 Å and revealed to be a lysostaphin-type peptidase of the M23 metallopeptidase family. The overall structure consists of three well-separated domains and has no similarity to any previously published structure. However, only the topology of the carboxyl-terminal domain is highly conserved among members of this family, and this domain is a zinc-dependent catalytic unit. The amino-terminal domain of the structure blocks the substrate binding pocket in the carboxyl-terminal domain, indicating that the wild-type full-length protein is in an inactive conformational state. Our studies improve the understanding of the catalytic mechanism of M23 metallopeptidases. © 2011 Wang et al.

Li X.,University of Saskatchewan | Li X.,National Vaccine and Serum Institute | Huang H.,University of Saskatchewan | Zhang X.,University of Saskatchewan | And 5 more authors.
American Journal of Respiratory Cell and Molecular Biology | Year: 2010

In mouse models of asthma, therapeutic use of allergen-presenting IL-10-differentiated dendritic cells (DCs) can abrogate airway hyper-responsiveness, and reduce other asthma-related responses to near background. Analogous human DCs can suppress human T cell responses in vitro, but the operative mechanisms are poorly defined. We investigated the ability of IL-10-treated human DCs to induce tolerance among autologous T cells of subjects with asthma and the mechanisms by which they do this. CD14+ monocyte-derived DCs were differentiated in the presence of IL-10 (DC10) ex vivo from 11 donors with asthma and 4 control donors, and characterized for relevant markers. They were pulsed with specific or irrelevant allergen, and cultured with autologous peripheral blood CD4+ T cells, either alone or together with autologous immunostimulatory DCs (DC-TNF), and the impact of this treatment on the T-cell responses was assessed for each donor. The DC10 expressed reduced levels of some relevant markers (CD40, CD80, human leukocyte antigen-DR) and stimulatory cytokines (IL-6 and IL-12), but augmented levels of Ig-like transcript-22/CD85j and IL-10 relative to DC-TNF. In cocultures, they dampened DC-TNF-driven T helper (Th) type 2 cell proliferation and cytokine (IL-4, -5, and -13) secretion. They also drove the development from atopic CD4+CD25loFoxp3lo cells of a population of IL-10-secreting CD25+Foxp3+LAG-3+CTLA-4 + regulatory T cells (Tregs). These Tregs suppressed stimulatory DC-induced autologous Th2 cell proliferation and cytokine secretion in a contact-dependent manner. Our data indicate that IL-10-treated human DCs induce Th2 cell allergen tolerance ex vivo by driving the differentiation of Tregs.

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