Engineering T7 bacteriophage as a potential DNA vaccine targeting delivery vector

Abstract 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

Displaying of GnRH Peptides on Bacteriophage T7 and Its Immunogenicity in Mice Model

T7 phage could be used as a perfect vector for peptides expression and haptens presentation. T7-3GnRH recombinant phage was constructed by inserting three copies of Gonadotrophin Releasing Hormone (GnRH) gene into the multiple cloning site of T7 Select 415-1b phage genome. The positive T7-3GnRH phage was selected by using polymerase chain reaction amplification, and the p10B-3GnRH fusion protein was verified by SDS-PAGE and Western-blotting assay. T7-3GnRH vaccine was made and immunized with 1010 pfu in 0.2 ml per dose in mice. Blood samples were collected at an interval in weeks, and anti-GnRH antibody and testosterone concentrations were detected by ELISA and radioimmunoassay, respectively. The results show that T7-3GnRH phage particles confer a high immunogenicity to the GnRH-derived epitope. Moreover, the T7-3GnRH vaccine induced higher level of anti-GnRH antibody than ImproVac®. However, the testosterone concentrations in both immunized groups were at a similar level, and the testis developments were significantly inhibited compared to controls. These findings demonstrated that the anti-GnRH antibody could neutralize the endogenous GnRH to down regulate testosterone level and limit testis development, highlighting the potential value of T7-3GnRH in the immunocastration vaccine research

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

Abstract 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

Long-term follow-up and exploration of the mechanism of stromal vascular fraction gel in chronic wounds

Abstract Background Chronic refractory wounds easily relapse and seriously affect the patients’ quality of life. Previous studies have shown that stromal vascular fraction gel (SVF-gel) significantly promotes the early healing of chronic wounds; however, the mechanisms of SVF-gel function per se remain unclear, and a long-term follow-up is lacking. This study aims to explore the mechanisms of SVF-gel promoting the healing of chronic wounds and follow up the long-term efficacy of SVF-gel. Methods Autologous SVF-gel transplantation was performed in 20 patients with chronic wounds (from March 2016 to September 2019), and the size of the wound before and after SVF-gel transplantation was observed. The conditioned medium (CM) was harvested from SVF-gel under serum-free, serum-deprivation and 10% fetal bovine serum (FBS) microenvironment in vitro, respectively. The concentration of the growth factors in the two kinds of gel-CM was tested, and their effects on the proliferation and migration of human dermal fibroblasts (HDFs) were detected. Results All patients had 100% wound closure eventually, and the average time to complete closure was 28.3 ± 9.7 days. The time of follow-up ranged from 2 to 6 years, and there was no wound recurrence. Interestingly, the concentrations of epidermal growth factor and transforming growth factor β1 of the CM were higher in serum-free and serum-deprivation condition than in 10% FBS microenvironment (p < 0.05). Correspondingly, the proliferation and migration ability of HDFs treated with gel-CM from serum-free condition were stronger than those treated with gel-CM from serum-deprivation (2% FBS) or 10% FBS microenvironment (p < 0.05). Conclusion These results indicate that it is safe, effective, and lasting in effect to treat chronic wounds with SVF-gel and mechanisms of action that include secreting various cytokines and promoting cell proliferation and migration ability. Trial registration: Chinese Clinical Trail Registry, ChiCTR2000034624. Registered 12 July 2020—Retrospectively registered, http://www.chictr.org.cn/showproj.aspx?proj=5605

Optimization of Heat-Resistance Technology for a Duck Hepatitis Lyophilized Live Vaccine

In this study, to improve the quality of a live attenuated vaccine for duck viral hepatitis (DHV), the lyophilization of a heat-resistant duck hepatitis virus vaccine was optimized. The optimized heat protectors were made of 10% sucrose, 1.2% pullulan, 0.5% PVP, and 1% arginine, etc., with a titer freeze-drying loss of &le;0.50 Lg. The vaccine product&rsquo;s valence measurements demonstrated the following: the vaccine could be stored at 2&ndash;8 &deg;C for 18 months with a virus titer loss &le;0.91 Lg; at 37 &deg;C for 10 days with a virus valence loss &le;0.89 Lg; and at 45 &deg;C for 3 days with a virus titer loss &le;0.90 Lg. Regarding safety, no deaths occurred in two-day-old ducklings immunized with a 10 times dose vaccine; their energy, diet, and weight gain were all normal, demonstrating that the DHV heat-resistant vaccines were safe for ducklings and did not cause any immune side effects. Duck viral hepatitis freeze-dried vaccine began to produce antibodies at 7 d after immunization, reached above 5.0 on 14 d, and reached above 7.0 on 21 d, showing a continuous upward trend. This indicates that duck viral hepatitis vaccine has a good immunogen level. The optimization of the freeze-drying process saves costs and also improves the quality of the freeze-drying products, which provides important theoretical and technical support for the further study of vaccine products

Optimization on Preparation Conditions of Salidroside Liposome and Its Immunological Activity on PCV-2 in Mice

The aim of this study was to optimize the preparation conditions of salidroside liposome with high encapsulation efficiency (EE) and to study the immunological enhancement activity of salidroside liposome as porcine circovirus type 2 virus (PCV-2) vaccine adjuvant. Response surface methodology (RSM) was selected to optimize the conditions for the preparation of salidroside liposome using Design-Expert V8.0.6 software. Three kinds of salidroside liposome adjuvants were prepared to study their adjuvant activity. BALB/c mice were immunized with PCV-2 encapsulated in different kinds of salidroside liposome adjuvants. The PCV-2-specific IgG in immunized mice serum was determined with ELISA. The results showed that when the concentration of ammonium sulfate was 0.26 mol·L−1, ethanol volume 6.5 mL, temperature 43°C, ethanol injection rate 3 mL·min−1, and salidroside liposome could be prepared with high encapsulation efficiency of 94.527%. Salidroside liposome as adjuvant could rapidly induce the production of PCV-2-specific IgG and salidroside liposome I adjuvant proved to provide the best effect among the three kinds of salidroside liposome adjuvants

Lycium barbarum polysaccharide protects HSF cells against ultraviolet-induced damage through the activation of Nrf2

Abstract Background Lycium barbarum polysaccharide (LBP) is considered an antioxidant agent. NF-E2-related factor-2 (Nrf2) is an important regulator for protection against UV damage. In this study, we verified the performance of LBP and the correlation between LBP and Nrf2. Methods HSF cells were treated with LBP to determine dose and time dependencies. An antioxidant response element (ARE) reporter was designed to monitor the activity of the Nrf2 antioxidant pathway. Results For HSF cells, the optimal LBP treatment was 300 μg/ml for 3 h. The ARE-reporter assay showed that LBP could increase the robustness of p-Nrf2. Treatments with genistein and LY294002 reduced of nuclear p-Nrf2 after 24 h. LBP increased the level of nuclear Nrf2, which functions by both phosphorylation and nuclear translocation. Silencing Nrf2 led to increased reactive oxygen species (ROS) levels, lower cell viability, and decreased superoxide dismutase (SOD) and glutathione peroxidase (GSP-PX) levels. This induced a higher level of lipid peroxide (LPO). However, LBP could decrease the levels of ROS and LPO and enhance the levels of SOD and GSP-PX. Conclusion LBP protects HSF cells against UV damage via the regulation of Nrf2

DataSheet_1_Evaluation of dendritic cell-targeting T7 phages as a vehicle to deliver avian influenza virus H5 DNA vaccine in SPF chickens.docx

IntroductionThere is a growing demand for effective technologies for the delivery of antigen to antigen-presenting cells (APCs) and their immune-activation for the success of DNA vaccines. Therefore, dendritic cell (DC)-targeting T7 phages were used as a vehicle to deliver DNA vaccine.MethodsIn this study, a eukaryotic expression plasmid pEGFP-C1-HA2-AS containing the HA2 gene derived from the avian H5N1 virus and an anchor sequence (AS) gene required for the T7 phage packaging process was developed. To verify the feasibility of phage delivery, the plasmid encapsulated in DC-targeting phage capsid through the recognition of AS was evaluated both in vitro and in vivo. The pEGFP-C1-HA2-AS plasmid could evade digestion by DNase I by becoming encapsulated into the phage particles and efficiently expressed the HA2 antigen in DCs with the benefit of DC-targeting phages.ResultsFor chickens immunized with the DC-targeting phage 74 delivered DNA vaccine, the levels of IgY and IgA antibodies, the concentration of IFN-γ and IL-12 cytokines in serum, the proliferation of lymphocytes, and the percentage of CD4+/CD8+ T lymphocytes isolated from peripheral blood were significantly higher than chickens which were immunized with DNA vaccine that was delivered by non-DC-targeting phage or placebo (pConclusionThis study provides a strategy for development of a novel avian influenza DNA vaccine and demonstrates the potential of DC-targeting phage as a DNA vaccine delivery vehicle.</p

Datasets on Irregular Migration and Irregular Migrants in the EU

The evidence produced during the recent migration crisis in Europe is often based on datasets that have intrinsic limitations of coverage and availability, and that capture the complex phenomenon of migration from different perspectives. Simple questions such as “What is the number of migrants in the European Union (EU)?” cannot be answered by providing one single number, but a set of numbers where each number tells a different part of the story. Besides trying to expand the availability of data on migration, it is important to be aware of the characteristics of the existing datasets since knowledge of this determines the type of analysis and conclusions that can be drawn from the data. This paper describes the main datasets that can be used to quantify trends of irregular migration and indirectly also the stock of irregular migrants in the EU. The review covers only datasets that are openly available and have supra-national relevance.JRC.E.6-Demography, Migration and Governanc