Shuling Liu

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.

Chimeric hepatitis B virus core particles carrying an epitope of anthrax protective antigen induce protective immunity against Bacillus anthracis

The major aim of present study is to develop and evaluate chimeric virus-like particles (VLPs) displaying a neutralizing epitope of anthrax protective antigen (PA) as a potential vaccine against anthrax. The truncated hepatitis B virus core (HBc) protein (aa 1-144) was used as a carrier, and the 2beta2-2beta3 loop of the PA domain 2 (aa 302-325) which has been shown contains a dominant neutralizing epitope was inserted into the major immunodominant region (MIR) of the HBc. The recombinant protein HBc-N144-PA-loop2 was expressed in Escherichia coli, and was able to form HBc-like particles confirmed by electron microscopy. The immunogenicity of these chimeric particles was evaluated in mice and guinea pigs. In mice the HBc-N144-PA-loop2 was able to induce PA-epitope specific antibodies; in guinea pigs it was able to induce PA-epitope specific antibodies and anthrax toxin-neutralizing antibodies regardless of whether alum adjuvant was used or not, and was able to partially protect the immunized guinea pigs against virulent anthrax spores challenge. This study suggests chimeric HBc particles carrying a neutralizing epitope of PA can induce protective immunity against Bacillus anthracis.

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.

The inhibition of the interaction between the anthrax toxin and its cellular receptor by an anti-receptor monoclonal antibody

The high affinity binding of the anthrax protective antigen (PA) to one of its receptors, capillary morphogenesis protein 2 (CMG2), is essential for the intoxication process of anthrax toxin. To acquire novel research tools to study the PA-CMG2 interaction, we generated several anti-CMG2 monoclonal antibodies (MAbs). We demonstrated that one of the MAbs, 4B5, could inhibit PA-CMG2 binding and could also protect the sensitive cells against an anthrax lethal toxin challenge. We identified the epitope recognized by 4B5 and confirmed that the key residues of the epitope were the residues (119)YI-LK(125) of CMG2. Based on our results, we propose that 4B5 binds to the E122 pocket of CMG2 and interrupts the interaction between the pocket and the PA 2beta3-2beta4 loop. To our knowledge, this is the first report to illustrate that an anti-CMG2 antibody could inhibit the PA-CMG2 interaction and therefore interfere with the intoxication of anthrax toxin.

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.

Monitoring luciferase-labeled human prostate stem cell antigen-expressing tumor growth in a mouse model

The aim of this study was to establish a tumor model in mice with the expression of luciferase (Luc) and human prostate stem cell antigen (PSCA), in order to evaluate the activities of anticancer drugs or vaccines for prostate cancer. RM-1 cells were stably transfected with pcDNA-Luc and pcDNA-PSCA plasmids. The Luc-expressing cells were examined using a luminometer and the PSCA-expressing cells were examined using a reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analysis. Male C57BL/6 mice were inoculated subcutaneously with the RM-PSCA/Luc cells, prior to the tumor growth and survival time of the mice being measured, respectively. In vivo bioluminescence imaging was used to detect Luc expression and immunohistochemical analysis was used to detect PSCA expression. Inoculation of the tumor cells into the C57BL/6 mice closely mimicked the tumor growth of prostate cancer. All of the inoculated mice exhibited a detectable tumor within two weeks. Tumor progression was able to be quantitatively monitored following the inoculation of 1×106 RM-PSCA/Luc cells. There was an excellent correlation (R2=0.9849) between the photon counts and tumor volume. The expression of PSCA in tumor tissues was confirmed using immunohistochemical analysis. The Luc and PSCA co-expression tumor model was successfully established in mice, which is likely to accelerate the understanding of the pathogenesis of prostate cancer and facilitate the development of novel antitumor drugs or vaccines for the disease.

Immunity induced by Staphylococcus aureus surface protein A was protective against lethal challenge of Staphylococcus aureus in BALB/c mice

Staphylococcus aureus is the most common cause of hospital‐acquired bacteremia. Due to emergence of antibiotic‐resistant strains, these infections present a serious public health threat. In this study, to develop a broadly protective vaccine, we tested whether immune responses induced by several proteins associated with S. aureus toxicity could protect mice from lethal challenge with human clinical S. aureus isolate USA300. We found that the surface protein A (SasA) of S. aureus could protect mice from lethal challenge of the bacteria.

Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.