Development of a water-in-oil-in-water adjuvant for foot-and-mouth disease vaccine based on ginseng stem-leaf saponins as an immune booster.

There has been an increasing interest in finding new formulations that qualify as vaccine adjuvants, which must be safe, stable, and have the capacity to stimulate a strong immune response. In this study, a basic formulation of a water-in-oil-in-water (W/O/W) adjuvant CV13 was developed, and ginseng stem-leaf saponins (GSLS) were added as an immune booster into oil phase. The physicochemical properties of the adjuvant were tested. Furthermore, the immune activity and the adjuvant effects, as indicated by the foot-and-mouth disease virus (FMDV) antigen were evaluated. The results showed that CV13 was similar in appearance to ISA 206 and could package FMDV antigen into a stable W/O/W emulsion. The FMD vaccine prepared with CV13 alone or CV13 containing GSLS achieved pharmaceutical characteristics comparable to a vaccine prepared with ISA 206, moreover the structural stability of the CV 13 vaccine was found to be better. Mice that were immunized with the FMD vaccine prepared with CV13 containing GSLS presented a significantly higher LPBE antibody titer and splenocyte proliferation rate than those immunized with a vaccine prepared with CV13 alone (p < 0.05). In addition, there was no significant difference between the groups that were immunized with FMD vaccine prepared with CV13 containing GSLS and ISA206 in terms of cellular and humoral immune response. In this paper, CV13 containing GSLS shows excellent immunologic adjuvant effect in mice model, and this new adjuvant may provide a potential choice for FMD vaccine production in the future.

Development of a novel oil-in-water emulsion and evaluation of its potential adjuvant function in a swine influenza vaccine in mice.

BACKGROUND: Vaccination is the principal strategy for prevention and control of diseases, and adjuvant use is an effective strategy to enhance vaccine efficacy. Traditional mineral oil-based adjuvants have been reported with post-immunization reactions. Developing new adjuvant formulations with improved potency and safety will be of great value. RESULTS: In the study reported herein, a novel oil-in-water (O/W) Emulsion Adjuvant containing Squalane (termed EAS) was developed, characterized and investigated for swine influenza virus immunization. The data show that EAS is a homogeneous nanoemulsion with small particle size (~ 105 nm), low viscosity (2.04 ± 0.24 cP at 20 °C), excellent stability (at least 24 months at 4 °C) and low toxicity. EAS-adjuvanted H3N2 swine influenza vaccine was administrated in mice subcutaneously to assess the adjuvant potency of EAS. The results demonstrated that in mice EAS-adjuvanted vaccine induced significantly higher titers of hemagglutination inhibition (HI) and IgG antibodies than water-in-oil (W/O) vaccines or antigen alone, respectively, at day 42 post vaccination (dpv) (P < 0.05). EAS-adjuvanted vaccine elicited significantly stronger IgG1 and IgG2a antibodies and higher concentrations of Th1 (IFN-γ and IL-2) cytokines compared to the W/O vaccine or antigen alone. Mice immunized with EAS-adjuvanted influenza vaccine conferred potent protection after homologous challenge. CONCLUSION: The O/W emulsion EAS developed in the present work induced potent humoral and cellular immune responses against inactivated swine influenza virus, conferred effective protection after homologous virus challenge and showed low toxicity in mice, indicating that EAS is as good as the commercial adjuvant MF59. The superiority of EAS to the conventional W/O formulation in adjuvant activity, safety and stability will make it a potential veterinary adjuvant.

Engineering T7 bacteriophage as a potential DNA vaccine targeting delivery vector.

BACKGROUND: DNA delivery with bacteriophage by surface-displayed mammalian cell penetrating peptides has been reported. Although, various phages have been used to facilitate DNA transfer by surface displaying the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide), no similar study has been conducted using T7 phage. METHODS: In this study, we engineeredT7 phage as a DNA targeting delivery vector to facilitate cellular internalization. We constructed recombinant T7 phages that displayed Tat peptide on their surface and carried eukaryotic expression box (EEB) as a part of their genomes (T7-EEB-Tat). RESULTS: We demonstrated that T7 phage harboring foreign gene insertion had packaged into infective progeny phage particles. Moreover, when mammalian cells that were briefly exposed to T7-EEB-Tat, expressed a significant higher level of the marker gene with the control cells infected with the wide type phage without displaying Tat peptides. CONCLUSION: These data suggested that the potential of T7 phage as an effective delivery vector for DNA vaccine transfer.

Expression and functional role of Cdx2 in intestinal metaplasia of cystitis glandularis.

PURPOSE: Cdx2 is an essential transcription factor in intestinal epithelial cell differentiation and proliferation. However, to our knowledge the expression and role of Cdx2 in the development of intestinal cystitis glandularis, a metaplastic lesion induced by chronic inflammation, remained to be explored. MATERIALS AND METHODS: Real-time polymerase chain reaction was used to examine Cdx2, LI-cadherin and villin expression in typical and intestinal cystitis glandularis, and normal bladder tissue. Cdx2 cDNA was subcloned to the retroviral vector pLNCX2 for subsequent transfection into human bladder urothelium cells and rat bladder urothelium. Cdx2 mRNA and protein levels, and cell morphology and proliferation were assessed after transfection using real-time polymerase chain reaction, phase contrast microscopy, transmission electron microscopy and MTT assay, respectively. RESULTS: Higher mRNA levels of Cdx2, villin and LI-cadherin were detected in intestinal cystitis glandularis compared to normal bladder and typical cystitis glandularis. Only Cdx2 groups attained statistical significance (p <0.001). Retroviral over expression of Cdx2 resulted in increased mRNA and protein expression of Cdx2 as well as villin and LI-cadherin levels, and increased cell proliferation. A distinct change in cellular morphology, in which cells resembled intestinal-like cells, was also observed in vitro and in vivo. CONCLUSIONS: Cdx2 may have a critical role in regulating intestinal metaplasia in cystitis glandularis. Further studies are planned to assess the potential of using Cdx2 as a marker and therapeutic target for cystitis glandularis.

Interleukin-6 levels in female rats with protamine sulfate-induced chronic cystitis treated with hyaluronic acid.

OBJECTIVES: To measure interleukin-6 levels in a protamine sulfate-induced chronic cystitis rat model treated with hyaluronic acid, and to study the correlation among interleukin-6, bladder inflammatory degree and voiding frequency. METHODS: A chronic cystitis model was created in female rats by using long-term intermittent intravesical protamine sulfate (0.5 mL, 30 mg/mL). Then, hyaluronic acid (0.5 mL, 0.8 mg/mL) was also instilled intravesically in the rats. Interleukin-6 levels were analyzed with immunohistochemistry, real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Hematoxylin-eosin staining was carried out to examine bladder inflammatory degree based on a four-point scoring system (from 0 - none to 3 - severe). Voiding patterns were investigated by cystometrography. RESULTS: According to cystometrography, protamine sulfate-induced rats had significantly shorter intercontraction intervals and less bladder capacity (P < 0.001). The bladder tissue of the rats showed severe chronic inflammation. Immunohistochemistry, reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay showed significantly higher expression of interleukin-6 (P < 0.001). After intravesical administration of hyaluronic acid, both intercontraction intervals and bladder capacity increased significantly (P < 0.001), whereas both bladder inflammatory degree and interleukin-6 levels decreased significantly (P < 0.001). Furthermore, there was a strong correlation between interleukin-6 levels and inflammatory degree (r = 0.727, P < 0.001), and also between interleukin-6 levels and voiding frequency (r = -0.761, P < 0.001). CONCLUSIONS: Intravesical administration of hyaluronic acid decreases interleukin-6 levels, as well as the severity of bladder inflammation and voiding frequency in a rat model of chronic cystitis. Interleukin-6 levels closely correlate with the inflammatory degree and voiding frequency. Thus, they can be regarded as an assessment measure of therapeutic impact.

[Construction and immunogenicity of chimeric peptide antigen composed of M2e and NP genes of avian influenza virus].

OBJECTIVE: In order to overcome the defect of traditional avian influenza vaccine that lacks cross-protection among different serotypes, we developed a universal anti-influenza vaccine. METHODS: Based on the gene analysis for A IV matrix protein 2 and two cytotoxic T-lymphocyte epitopes, we constructed four prokaryotic expression vectors. The target gene was induced by IPTG and the fusion protein was mixed with Freund's adjuvant; then used to immunize 20-day-old chicken by intramuscular injection and boosted 3 weeks later. Blood samples were collected weekly following the primary vaccination. The anti-M2e antibody was detected with ELISA coated by synthesized peptide; the neutralizing ability of anti-serum was evaluated on MDCK cell line and chick embryo; the CD4+ and CD8+ T lymphocyte amounts in peripheral blood of immunized chicken were measured by flow cytometry. RESULTS: The fusion protein induced immunological reaction, and the antibody bound with the viral M2 protein expressed on the surface of MDCK cells. Serum could only inhibit the replication without neutralizing the virus. Flow cytometry results showed that CD4+ and CD8+ T lymphocyte in peripheral blood increased obviously following immunization (P<0.05), which possessed the character of cell immunization. CONCLUSION: Chimeric peptides kept good immuno-genicity and provided useful probes for the control of avian influenza.