Shiqi Sun

Hollow mesoporous silica nanoparticles for intracellular delivery of fluorescent dye

In this study, hollow mesoporous silica nanoparticles (HMSNs) were synthesized using the sol-gel/emulsion approach and its potential application in drug delivery was assessed. The HMSNs were characterized, by transmission electron microscopy (TEM), Scanning Electron Microscopy (SEM), nitrogen adsorption/desorption and Brunauer-Emmett-Teller (BET), to have a mesoporous layer on its surface, with an average pore diameter of about 2 nm and a surface area of 880 m2/g. Fluorescein isothiocyanate (FITC) loaded into these HMSNs was used as a model platform to assess its efficacy as a drug delivery tool. Its release kinetic study revealed a sequential release of FITC from the HMSNs for over a period of one week when soaked in inorganic solution, while a burst release kinetic of the dye was observed just within a few hours of soaking in organic solution. These FITC-loaded HMSNs was also found capable to be internalized by live human cervical cancer cells (HeLa), wherein it was quickly released into the cytoplasm within a short period of time after intracellular uptake. We envision that these HMSNs, with large pores and high efficacy to adsorb chemicals such as the fluorescent dye FITC, could serve as a delivery vehicle for controlled release of chemicals administered into live cells, opening potential to a diverse range of applications including drug storage and release as well as metabolic manipulation of cells.

Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli.

BackgroundPorcine circovirus 2 (PCV2) is a serious problem to the swine industry and can lead to significant negative impacts on profitability of pork production. Syndrome associated with PCV2 is known as porcine circovirus closely associated with post-weaning multisystemic wasting syndrome (PMWS). The capsid (Cap) protein of PCV2 is a major candidate antigen for development of recombinant vaccine and serological diagnostic method. The recombinant Cap protein has the ability to self-assemble into virus-like particles (VLPs) in vitro, it is particularly opportunity to develop the PV2 VLPs vaccine in Escherichia coli,(E.coli ), because where the cost of the vaccine must be weighed against the value of the vaccinated pig, when it was to extend use the VLPs vaccine of PCV2.ResultsIn this report, a highly soluble Cap-tag protein expressed in E.coli was constructed with a p-SMK expression vector with a fusion tag of small ubiquitin-like modifiers (SUMO). The recombinant Cap was purified using Ni2+ affinity resins, whereas the tag was used to remove the SUMO protease. Simultaneously, the whole native Cap protein was able to self-assemble into VLPs in vitro when viewed under an electron microscope. The Cap-like particles had a size and shape that resembled the authentic Cap. The result could also be applied in the large-scale production of VLPs of PCV2 and could be used as a diagnostic antigen or a potential VLP vaccine against PCV2 infection in pigs.Conclusionwe have, for the first time, utilized the SUMO fusion motif to successfully express the entire authentic Cap protein of PCV2 in E. coli. After the cleavage of the fusion motif, the nCap protein has the ability to self-assemble into VLPs, which can be used as as a potential vaccine to protect pigs from PCV2-infection.

Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD50) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.

Immunization of mice by Hollow Mesoporous Silica Nanoparticles as carriers of Porcine Circovirus Type 2 ORF2 Protein

BackgroudPorcine circovirus type 2 (PCV2) is a primary etiological agent of post-weaning multi-systemic wasting syndrome (PMWS), which is a disease of increasing importance to the pig industry worldwide. Hollow mesoporous silica nanoparticles (HMSNs) have gained increasing interest for use in vaccines.MethodsTo study the potential of HMSNs for use as a protein delivery system or vaccine carriers. HMSNs were synthesized by a sol–gel/emulsion(oil-in-water/ethanol) method, purified PCV2 GST-ORF2-E protein was loaded into HMSNs, and the resulting HMSN/protein mixture was injected into mice. The uptake and release profiles of protein by HMSNs in vitro were investigated. PCV2 GST-ORF2-E specific antibodies and secretion of IFN-γ were detected by enzyme-linked immunosorbent assays, spleen lymphocyte proliferation was measured by the MTS method, and the percentage of CD4+ and CD8+ were determined by flow cytometry.ResultsHMSNs were found to yield better binding capacities and delivery profiles of proteins; the specific immune response induced by PCV2 GST-ORF2-E was maintained for a relatively long period of time after immunization with the HMSN/protein complex.ConclusionThe findings suggest that HMSNs are good protein carriers and have high potential for use in future applications in therapeutic drug delivery.

Self-assembly of virus-like particles of canine parvovirus capsid protein expressed from Escherichia coli and application as virus-like particle vaccine.

Canine parvovirus disease is an acute infectious disease caused by canine parvovirus (CPV). Current commercial vaccines are mainly attenuated and inactivated; as such, problems concerning safety may occur. To resolve this problem, researchers developed virus-like particles (VLPs) as biological nanoparticles resembling natural virions and showing high bio-safety. This property allows the use of VLPs for vaccine development and mechanism studies of viral infections. Tissue-specific drug delivery also employs VLPs as biological nanomaterials. Therefore, VLPs derived from CPV have a great potential in medicine and diagnostics. In this study, small ubiquitin-like modifier (SUMO) fusion motif was utilized to express a whole, naturalVP2 protein of CPV in Escherichia coli. After the cleavage of the fusion motif, the CPV VP2 protein has self-assembled into VLPs. The VLPs had a size and shape that resembled the authentic virus capsid. However, the self-assembly efficiency of VLPs can be affected by different pH levels and ionic strengths. The mice vaccinated subcutaneously with CPV VLPs and CPV-specific immune responses were compared with those immunized with the natural virus. This result showed that VLPs can effectively induce anti-CPV specific antibody and lymphocyte proliferation as a whole virus. This result further suggested that the antigen epitope of CPV was correctly present on VLPs, thereby showing the potential application of a VLP-based CPV vaccine.

Development and validation of an ELISA using a protein encoded by ORF2 antigenic domain of porcine circovirus type 2.

BackgroundThe capsid protein (ORF2) is a major structural protein of porcine circovirus type 2 (PCV2). A simple and reliable diagnostic method based on ORF2 protein immunoreactivity would serve as a valuable diagnostic method for detecting serum antibodies to PCV2 and monitoring PCV infection. Here, we reported an indirect enzyme-linked immunosorbent assay (I-ELISA) by using an antigenic domain (113-147AA) of ORF2-encoded antigen, expressed in E. coli, for diagnosis of PCV infection.ResultsThe ELISA was performed on 288 serum samples collected from different porcine herds and compared with an indirect immunofluorescent assay (IFA). In total, 262 of 288 samples were positive as indicated by both I-ELISA and IFA. The specificity and sensitivity of I-ELISA were 87.7% and 93.57%.ConclusionsThis ELISA is suitable for detection and discrimination of PCV2 infection in both SPF and farm antisera.

Upconversion nanoparticles modified with aminosilanes as carriers of DNA vaccine for foot-and-mouth disease

The potential of the upconversion nanoparticles NaYF4:Yb/Er@silica(UCPs)/plasmid DNA (pcDNA3.1/VP1-GFP) complex in inducing immune responses was evaluated using the UCPs as carriers of the foot-and-mouth disease virus (FMDV AsiaI/Jiangsu2005) DNA vaccine. The UCPs protection against DNaseI degradation was measured using an in vitro inhibition assay. The expression of the plasmid in vivo was determined via confocal microscopy. Its biocompatibility was evaluated through cytotoxicity assay. Based on the results, the aminosilane-modified UCPs can electrostatically bind, condense, and protect plasmid DNA. Cell viability assays demonstrated that the cytotoxicity of the UCPs/plasmid DNA complex is lower than that of the cationic lipid/plasmid DNA complex, and that the transfection efficiency of UCPs is the same as that of the cationic lipid. Furthermore, the UCP/plasmid DNA complex was intramuscularly administered to guinea pigs. Humoral and cellular immune responses were detected using indirect enzyme-linked immunosorbent assay (ELISA), micro-neutralization assay, and T-lymphocyte proliferation assay. Anti-FMDV specific antibodies, neutralizing antibodies, and T-lymphocyte proliferation responses were induced after vaccination. In the challenge test, all the guinea pigs vaccinated with the UCPs/plasmid DNA complex were fully protected from the FMDV challenge. The current study encourages the use of UCPs as an effective nanosystem for gene delivery to cells for in vitro and in vivo vaccination, and other therapeutic applications.

Viroporin activity and membrane topology of classic swine fever virus p7 protein

Viroporins are a group of viral proteins that participate in viral replication cycles, including modification of membrane permeability and promotion of viral release. Although biological data have been accumulated on viroporion-like proteins of other viruses belonging to family Flaviviridae, the viroporin activity and membrane topology of p7 protein from classical swine fever virus (CSFV), a member of the genus Pestivirus of the family Flaviviridae, are largely unknown. In this study, sequence analysis of the primary structure of p7 polypeptide demonstrates that p7 contains two putative transmembrane regions connected by a short hydrophilic segment. Expression of p7 protein in Escherichia coli leads to the permeabilization of bacterial cells to small molecules. The p7 protein also enhances the permeability of mammalian cells, increasing the intracellular Ca(2+) concentration and the permeability of cells to the translation inhibitor Hygromycin B. This protein is an integral membrane protein and can form homo-oligomers. It mainly localizes to the ER at the early stage of the expression and can be transferred to the plasma membrane at the late stage of the expression. Detergent permeabilization assays confirmed that the p7 protein is a 2-pass transmembrane protein and its N and C termini are exposed to the ER lumen. Deletion analysis showed that amino acid residues 41-63 may be essential for the viroporin activity of the protein. Our studies demonstrate that CSFV p7 possesses properties commonly associated with viroporins, which could be a potential target for the development of a therapeutic intervention for classic swine fever virus infection.

Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein.

Viroporins are a family of low-molecular-weight hydrophobic transmembrane proteins that are encoded by various animal viruses. Viroporins form transmembrane pores in host cells via oligomerization, thereby destroying cellular homeostasis and inducing cytopathy for virus replication and virion release. Among the Picornaviridae family of viruses, the 2B protein encoded by enteroviruses is well understood, whereas the viroporin activity of the 2B protein encoded by the foot-and-mouth disease virus (FMDV) has not yet been described. An analysis of the FMDV 2B protein domains by computer-aided programs conducted in this study revealed that this protein may contain two transmembrane regions. Further biochemical, biophysical and functional studies revealed that the protein possesses a number of features typical of a viroporin when it is overexpressed in bacterial and mammalian cells as well as in FMDV-infected cells. The protein was found to be mainly localized in the endoplasmic reticulum (ER), with both the N- and C-terminal domains stretched into the cytosol. It exhibited cytotoxicity in Escherichia coli, which attenuated 2B protein expression. The release of virions from cells infected with FMDV was inhibited by amantadine, a viroporin inhibitor. The 2B protein monomers interacted with each other to form both intracellular and extracellular oligomers. The Ca2+ concentration in the cells increased, and the integrity of the cytoplasmic membrane was disrupted in cells that expressed the 2B protein. Moreover, the 2B protein induced intense autophagy in host cells. All of the results of this study demonstrate that the FMDV 2B protein has properties that are also found in other viroporins and may be involved in the infection mechanism of FMDV.

Biological function of Foot-and-mouth disease virus non-structural proteins and non-coding elements

Foot-and-mouth disease virus (FMDV) represses host translation machinery, blocks protein secretion, and cleaves cellular proteins associated with signal transduction and the innate immune response to infection. Non-structural proteins (NSPs) and non-coding elements (NCEs) of FMDV play a critical role in these biological processes. The FMDV virion consists of capsid and nucleic acid. The virus genome is a positive single stranded RNA and encodes a single long open reading frame (ORF) flanked by a long structured 5ʹ-untranslated region (5ʹ-UTR) and a short 3ʹ-UTR. The ORF is translated into a polypeptide chain and processed into four structural proteins (VP1, VP2, VP3, and VP4), 10 NSPs (Lpro, 2A, 2B, 2C, 3A, 3B1–3, 3Cpro, and 3Dpol), and some cleavage intermediates. In the past decade, an increasing number of studies have begun to focus on the molecular pathogenesis of FMDV NSPs and NCEs. This review collected recent research progress on the biological functions of these NSPs and NCEs on the replication and host cellular regulation of FMDV to understand the molecular mechanism of host–FMDV interactions and provide perspectives for antiviral strategy and development of novel vaccines.