Changming Yu

Silencing SARS-CoV Spike protein expression in cultured cells by RNA interference

The severe acute respiratory syndrome (SARS) has been one of the most epidemic diseases threatening human health all over the world. Based on clinical studies, SARS‐CoV (the SARS‐associated coronavirus), a novel coronavirus, is reported as the pathogen responsible for the disease. To date, no effective and specific therapeutic method can be used to treat patients suffering from SARS‐CoV infection. RNA interference (RNAi) is a process by which the introduced small interfering RNA (siRNA) could cause the degradation of mRNA with identical sequence specificity. The RNAi methodology has been used as a tool to silence genes in cultured cells and in animals. Recently, this technique was employed in anti‐virus infections in human immunodeficiency virus and hepatitis C/B virus. In this study, RNAi technology has been applied to explore the possibility for prevention of SARS‐CoV infection. We constructed specific siRNAs targeting the S gene in SARS‐CoV. We demonstrated that the siRNAs could effectively and specifically inhibit gene expression of Spike protein in SARS‐CoV‐infected cells. Our study provided evidence that RNAi could be a tool for inhibition of SARS‐CoV.

Vaccination with a DNA vaccine based on human PSCA and HSP70 adjuvant enhances the antigen-specific CD8+ T-cell response and inhibits the PSCA+ tumors growth in mice

DNA vaccines have been shown to be an effective approach to induce antigen‐specific cellular and humoral immunity. However, the lower immune intensity in clinical trials limits the application of DNA vaccine. Here we intend to develop a new DNA vaccine based on prostate stem‐cell antigen (PSCA), which has been suggested as a potential target for prostate cancer therapy, and enhance the DNA vaccine potency with heat shock proteins (HSPs) as adjuvant.

siRNA targeting the Leader sequence of SARS-CoV inhibits virus replication

SARS-CoV (the SARS-Associated Coronavirus) was reported as a novel virus member in the coronavirus family, which was the cause of severe acute respiratory syndrome. Coronavirus replication occurs through a unique mechanism employing Leader sequence in the transcripts when initiating transcription from the genome. Therefore, we cloned the Leader sequence from SARS-CoV(BJ01), which is identical to that identified from SARS-CoV(HKU-39849), and constructed specific siRNA targeting the Leader sequence. Using EGFP and RFP reporter genes fused with the cloned SARS-CoV Leader sequence, we demonstrated that the siRNA targeting the Leader sequence decreased the mRNA abundance and protein expression levels of the reporter genes in 293T cells. By stably expressing the siRNA in Vero E6 cells, we provided data that the siRNA could effectively and specifically decrease the mRNA abundance of SARS-CoV genes as analyzed by RT-PCR and Northern blot. Our data indicated that the siRNA targeting the Leader sequence inhibited the replication of SARS-CoV in Vero E6 cells by silencing gene expression. We further demonstrated, via transient transfection experiments, that the siRNA targeting the Leader sequence had a much stronger inhibitory effect on SARS-CoV replication than the siRNAs targeting the Spike gene or the antisense oligodeoxynucleotides did. This report provides evidence that targeting Leader sequence using siRNA could be a powerful tool in inhibiting SARS-CoV replication.

Monoclonal Antibody Targeting Staphylococcus aureus Surface Protein A (SasA) Protect Against Staphylococcus aureus Sepsis and Peritonitis in Mice

Epidemic methicillin-resistant Staphylococcus aureus (MRSA) imposes an increasing impact on public health. Due to multi-antibiotics resistance in MRSA strains, there is an urgent need to develop novel therapeutics such as effective monoclonal antibodies (mAbs) against MRSA infections. Staphylococcus aureus surface protein A (SasA), a large surface-located protein (~240 kDa), is one of MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) and a potential target for immunotherapeutic approaches against S. aureus infections. In the present study, we analyzed the sequence of SasA with bioinformatics tools and generated a protective monoclonal antibody (2H7) targeting the conserved domain of SasA. 2H7 was shown to recognize wild-type S. aureus and promote opsonophagocytic killing of S. aureus. In both sepsis and peritoneal infection models, prophylactic administration of 2H7 improved the survival of BALB/c mice challenged by S. aureus strain USA300 and ST239 (prevalent MRSA clones in North America and Asian countries, respectively) and enhanced bacterial clearance in kidneys. Additionally, 2H7 prophylaxis prevented the formation of intraperitoneal abscess in a murine model of peritoneal infection and therapeutic administration of 2H7 showed protective efficacy in a murine sepsis model. Our results presented here provide supporting evidences that an anti-SasA mAb might be a potential component in an antibody-based immunotherapeutic treatment of MRSA infections.

Enhanced expression of soluble recombinant tetanus neurotoxin Hc in Escherichia coli as a tetanus vaccine candidate.

The expression of the carboxyl fragment of the heavy chain of tetanus neurotoxin (TeNT-Hc) in Escherichia coli has been hampered by the unusually high AT content and the presence of rarely used codons by Clostridium. The gene encoding TeNT-Hc was optimized for E. coli by replacing rare codons and decreasing the AT pairs from 72.57% to 52.47%. The reconstructed gene was expressed in E. coli BL21(DE3) and resulted in a soluble product which was about 46% of the total bacterial protein. TeNT-Hc produced in a 42 L fermentor was purified to >95% at 87 g/kg of cell paste (approximately 333 mg/L). BALB/c mice vaccinated with three bi-weekly doses of TeNT-Hc with Freunds adjuvant were fully protected against an intraperitoneally challenge of 2 × 10(3) 50% lethal doses (LD(50)s) of tetanus neurotoxin. NIH mice vaccinated with TeNT-Hc adsorbed to aluminum hydroxide gel adjuvant demonstrated a potency of 168 IU/mL, which was 2 times higher than the national standard for tetanus vaccines. These results suggest that TeNT-Hc may be a promising second-generation vaccine candidate for clinical use against tetanus neurotoxin.

Protection against anthrax and plague by a combined vaccine in mice and rabbits

The protective antigen (PA) of Bacillus anthracis and the Fraction 1 Capsular Antigen (F1 antigen), V antigen of Yersinia pestis have been demonstrated to be potential immunogens and candidate vaccine sub-units against anthrax and plague respectively. In this study, the authors have investigated the antibody responses and the protective efficacy when the antigens were administered separately or in combination intramuscularly formulation adsorbed to an aluminum hydroxide adjuvant. Results show that immunized rF1 + rV and rPA antigen together was as effective as separately for induction of serological antibody response, and these titers were maintained for over 1 year in mice. An isotype analysis of the serum indicates that the co-administration of these antigens did not influence the antigen-specific IgG1/IgG2a ratio which was consistent with a Th2 bias. Furthermore, the combined vaccine comprising the protein antigens rF1 + rV + rPA has been demonstrated to protect mice from subcutaneous challenge with 10(7) colony-forming units (CFU) virulent Y. pestis strain, and to fully protect rabbit against subcutaneous challenge with 1.2x10(5) colony-forming units (CFU) virulent B. anthracis spores. These data show that the protective efficacy was unaffected when the antigens were administered in combination.

Fusion protein of Δ27LFn and EFn has the potential as a novel anthrax toxin inhibitor

MINT‐7014735, MINT‐7014747, MINT‐7014761: PA63 (uniprotkb:P13423) and LF (uniprotkb:P15917) bind (MI:0407) by surface plasmon resonance (MI:0107)

Identification and Functional Characterization of Glycosylation of Recombinant Human Platelet-Derived Growth Factor-BB in Pichia pastoris

Yeast Pichia pastoris is a widely used system for heterologous protein expression. However, post-translational modifications, especially glycosylation, usually impede pharmaceutical application of recombinant proteins because of unexpected alterations in protein structure and function. The aim of this study was to identify glycosylation sites on recombinant human platelet-derived growth factor-BB (rhPDGF-BB) secreted by P. pastoris, and investigate possible effects of O-linked glycans on PDGF-BB functional activity. PDGF-BB secreted by P. pastoris is very heterogeneous and contains multiple isoforms. We demonstrated that PDGF-BB was O-glycosylated during the secretion process and detected putative O-glycosylation sites using glycosylation staining and immunoblotting. By site-directed mutagenesis and high-resolution LC/MS analysis, we, for the first time, identified two threonine residues at the C-terminus as the major O-glycosylation sites on rhPDGF-BB produced in P. pastoris. Although O-glycosylation resulted in heterogeneous protein expression, the removal of glycosylation sites did not affect rhPDGF-BB mitogenic activity. In addition, the unglycosylated PDGF-BBΔGly mutant exhibited the immunogenicity comparable to that of the wild-type form. Furthermore, antiserum against PDGF-BBΔGly also recognized glycosylated PDGF-BB, indicating that protein immunogenicity was unaltered by glycosylation. These findings elucidate the effect of glycosylation on PDGF-BB structure and biological activity, and can potentially contribute to the design and production of homogeneously expressed unglycosylated or human-type glycosylated PDGF-BB in P. pastoris for pharmaceutical applications.

Creation of a Six-fingered Artificial Transcription Factor That Represses the Hepatitis B Virus HBx Gene Integrated into a Human Hepatocellular Carcinoma Cell Line

Chronic hepatitis B virus (HBV) infection is an independent risk factor for the development of hepatocellular carcinoma (HCC). The HBV HBx gene is frequently identified as an integrant in the chromosomal DNA of patients with HCC. HBx encodes the X protein (HBx), a putative viral oncoprotein that affects transcriptional regulation of several cellular genes. Therefore, HBx may be an ideal target to impede the progression of HBV infection–related HCC. In this study, integrated HBx was transcriptionally downregulated using an artificial transcription factor (ATF). Two three-fingered Cys2-His2 zinc finger (ZF) motifs that specifically recognized two 9-bp DNA sequences regulating HBx expression were identified from a phage-display library. The ZF domains were linked into a six-fingered protein that specified an 18-bp DNA target in the Enhancer I region upstream of HBx. This DNA-binding domain was fused with a Krüppel-associated box (KRAB) transcriptional repression domain to produce an ATF designed to downregulate HBx integrated into the Hep3B HCC cell line. The ATF significantly repressed HBx in a luciferase reporter assay. Stably expressing the ATF in Hep3B cells resulted in significant growth arrest, whereas stably expressing the ATF in an HCC cell line lacking integrated HBx (HepG2) had virtually no effect. The targeted downregulation of integrated HBx is a promising novel approach to inhibiting the progression of HBV infection–related HCC.

Human Prostate Stem Cell Antigen and HSP70 Fusion Protein Vaccine Inhibits Prostate Stem Cell Antigen-Expressing Tumor Growth in Mice

Prostate stem cell antigen (PSCA) has been considered a potentially worthwhile target for prostate cancer therapy with its overexpression in both androgen-dependent and androgen-independent prostate cancers. However, PSCA is an autoantigen that can evoke immunological tolerance and hardly incite effective immunologic response. In this study, we sought to construct the fusion protein vaccines based on PSCA and heat shock protein 70 (HSP70) and to evaluate their immune responses and therapeutic efficacy. A series of recombinant proteins were prepared, and then, the male C57BL/6 mice were immunized subcutaneously by inoculation with RM-PSCA/Luc cells. The PSCA-specific cellular immune responses were monitored with ELISPOT and intracellular cytokines staining assay, and ELISA assay was used to detect humoral immune responses. The tumor growth was observed by in vivo bioluminescence imaging. The results showed that the mice vaccinated with PSCA-HSP could induce the PSCA-specific cellular and humoral immune responses. Tumor progression could be quantitatively monitored by in vivo bioluminescence imaging. Animal experiments showed that PSCA-HSP could inhibit the growth of PSCA-expressing tumors and prolong the survival time of vaccinated mice. This study supported and confirmed the potential of HSP70 as a chaperone for protein vaccines, and PSCA-HSP could be of potential value for prostate cancer treatment.