Chaoheng Yu

In Vitro and In Vivo Activities of Antimicrobial Peptides Developed Using an Amino Acid-Based Activity Prediction Method

ABSTRACT To design and discover new antimicrobial peptides (AMPs) with high levels of antimicrobial activity, a number of machine-learning methods and prediction methods have been developed. Here, we present a new prediction method that can identify novel AMPs that are highly similar in sequence to known peptides but offer improved antimicrobial activity along with lower host cytotoxicity. Using previously generated AMP amino acid substitution data, we developed an amino acid activity contribution matrix that contained an activity contribution value for each amino acid in each position of the model peptide. A series of AMPs were designed with this method. After evaluating the antimicrobial activities of these novel AMPs against both Gram-positive and Gram-negative bacterial strains, DP7 was chosen for further analysis. Compared to the parent peptide HH2, this novel AMP showed broad-spectrum, improved antimicrobial activity, and in a cytotoxicity assay it showed lower toxicity against human cells. The in vivo antimicrobial activity of DP7 was tested in a Staphylococcus aureus infection murine model. When inoculated and treated via intraperitoneal injection, DP7 reduced the bacterial load in the peritoneal lavage solution. Electron microscope imaging and the results indicated disruption of the S. aureus outer membrane by DP7. Our new prediction method can therefore be employed to identify AMPs possessing minor amino acid differences with improved antimicrobial activities, potentially increasing the therapeutic agents available to combat multidrug-resistant infections.

Synergistic effects of antimicrobial peptide DP7 combined with antibiotics against multidrug-resistant bacteria

Antibiotic-resistant bacteria present a great threat to public health. In this study, the synergistic effects of antimicrobial peptides (AMPs) and antibiotics on several multidrug-resistant bacterial strains were studied, and their synergistic effects on azithromycin (AZT)-resistance genes were analyzed to determine the relationships between antimicrobial resistance and these synergistic effects. A checkerboard method was used to evaluate the synergistic effects of AMPs (DP7 and CLS001) and several antibiotics (gentamicin, vancomycin [VAN], AZT, and amoxicillin) on clinical bacterial strains (Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli). The AZT-resistance genes (ermA, ermB, ermC, mefA, and msrA) were identified in the resistant strains using quantitative polymerase chain reaction. For all the clinical isolates tested that were resistant to different antibiotics, DP7 had high antimicrobial activity (≤32 mg/L). When DP7 was combined with VAN or AZT, the effect was most frequently synergistic. When we studied the resistance genes of the AZT-resistant isolates, the synergistic effect of DP7–AZT occurred most frequently in highly resistant strains or strains carrying more than two AZT-resistance genes. A transmission electron microscopic analysis of the S. aureus strain synergistically affected by DP7–AZT showed no noteworthy morphological changes, suggesting that a molecular-level mechanism plays an important role in the synergistic action of DP7–AZT. AMP DP7 plus the antibiotic AZT or VAN is more effective, especially against highly antibiotic-resistant strains.

Effective inhibition of melanoma tumorigenesis and growth via a new complex vaccine based on NY-ESO-1-alum-polysaccharide-HH2.

BackgroundA safe and effective adjuvant plays an important role in the development of a vaccine. However, adjuvants licensed for administration in humans remain limited. Here, for the first time, we developed a novel combination adjuvant alum-polysaccharide-HH2 (APH) with potent immunomodulating activities, consisting of alum, polysaccharide of Escherichia coli and the synthetic cationic innate defense regulator peptide HH2.MethodsThe adjuvant effects of APH were examined using NY-ESO-1 protein-based vaccines in prophylactic and therapeutic models. We further determined the immunogenicity and anti-tumor effect of NY-ESO-1-APH (NAPH) vaccine using adoptive cellular/serum therapy in C57/B6 and nude mice. Cell-mediated and antibody-mediated immune responses were evaluated.ResultsThe APH complex significantly promoted antigen uptake, maturation and cross-presentation of dendritic cells and enhanced the secretion of TNF-α, MCP-1 and IFN-γ by human peripheral blood mononuclear cells compared with individual components. Vaccination of NAPH resulted in significant tumor regression or delayed tumor progression in prophylactic and therapeutic models. In addition, passive serum/cellular therapy potently inhibited tumor growth of NY-ESO-1-B16. Mice treated with NAPH vaccine produced higher antibody titers and greater antibody-dependent/independent cellular cytotoxicity. Therefore, NAPH vaccination effectively stimulated innate immunity, and boosted both arms of the adaptive humoral and cellular immune responses to suppress tumorigenesis and growth of melanoma.ConclusionsOur study revealed the potential application of APH complex as a novel immunomodulatory agent for vaccines against tumor refractory and growth.

Ad-endostatin treatment combined with low-dose irradiation in a murine lung cancer model

Radiation therapy is a conventional strategy for treating advanced lung cancer yet is accompanied by serious side-effects. Its combination with other strategies, such as antiangiogenesis and gene therapy, has shown excellent prospects. As one of the potent endogenous vascular inhibitors, endostatin has been widely used in the antiangiogenic gene therapy of tumors. In the present study, LL/2 cells were infected with a recombinant adenovirus encoding endostatin (Ad-endostatin) to express endostatin. The results showed that LL/2 cells infected with the Ad-endostatin efficiently and longlastingly expressed endostatin. In order to further explore the role of Ad-endostatin combined with irradiation in the treatment of cancer, a murine lung cancer model was established and treated with Ad-endostatin combined with low-dose irradiation. The results showed that the combination treatment markedly inhibited tumor growth and metastasis, and prolonged the survival time of the tumor-bearing mice. Furthermore, this significant antitumor activity was associated with lower levels of microvessel density and anoxia factors in the Ad-Endo combined with irradiation group, and with an increased apoptotic index of tumor cells. In addition, no serious side-effects were noted in the combination group. Based on our findings, Ad-endostatin combined with low-dose irradiation may be a rational alternative treatment for lung cancer and other solid tumors.

The novel complex combination of alum, CpG ODN and HH2 as adjuvant in cancer vaccine effectively suppresses tumor growth in vivo

Single-component adjuvant is prone to eliciting a specific type of Th1 or Th2 response. So, the development of combinatorial adjuvants inducing a robust mixed Th1/Th2 response is a promising vaccination strategy against cancer. Here, we describe a novel combination of aluminum salts (alum), CpG oligodeoxynucleotide (CpG) and innate defense regulator peptide HH2 for improving anti-tumor immune responses. The CpG-HH2 complex significantly enhanced the production of IFN-γ, TNF-α and IL-1β, promoted the uptake of antigen and strengthened the activation of p38, Erk1/2 and NF-κB in vitro, compared to CpG or HH2 alone. Immunization with NY-ESO-1 antigen plus alum-CpG-HH2 combinatorial adjuvant effectively inhibited tumor growth and reduced tumor burden in prophylactic and therapeutic tumor models and even in passive serum or cellular therapy. In addition, co-administration of NY-ESO-1 with alum-CpG-HH2 combinatorial adjuvant markedly activated NK cell cytotoxicity, induced antibody-dependent cellular cytotoxicity (ADCC), dramatically elicited cytotoxic T lymphocytes (CTLs) response, and increased infiltrating lymphocytes in tumors. Moreover, in vivo depletion of CD8+ T cells completely and depletion of NK cells partially blocked the anti-tumor activity of NY-ESO-1-alum-CpG-HH2 immunization. Overall, our results demonstrate a novel adjuvant combination for cancer vaccine with efficient immunomodulation by stimulating innate immunity and mediating adaptive immunity.

Delivery of a Modified mRNA Encoding IL-22 Binding Protein (IL-22BP) for Colon Cancer Gene Therapy

As an alternative form of genetic material, mRNA lacks a CpG island and can function without crossing the nuclear membrane. These properties make mRNA less of a potential immune stimulant than plasmid DNA. Therefore, chemically modified mRNA is an effective alternative to plasmid DNA for gene therapy. In this study, cationic liposomes were used as a vector to transport mRNA complexes that had been compressed using protamine and to obtain high mRNA transport and expression efficiency. The liposome-protamine-IL-22BP mRNA complex can strongly inhibit the growth of C26 tumour cells by inducing apoptosis, inhibiting tumour angiogenesis and increasing the number of immune cells that infiltrate the tumour microenvironment. The obvious tumour regression and safety of this approach were observed in peritoneal metastasis and subcutaneous xenograft models. The antitumour effect of the liposome protamine mRNA complex was as good as that for a plasmid DNA complex, and demonstrated the potential for mRNA-based gene therapy.

Induction and Amelioration of Methotrexate-Induced Gastrointestinal Toxicity are Related to Immune Response and Gut Microbiota

As a widely used anticancer and immunosuppressive agent, methotrexate (MTX) can induce multiple adverse drug reactions (ADRs), such as gastrointestinal toxicity, the mechanisms are poorly understood. Gut microbiota has been widely reported to be associated with the onset of multiple diseases as well as treatment outcomes of different drugs. In this study, mucosal injury was observed in MTX-treated mice, leading to significant changes in macrophages (i.e., M1/M2 ratio, P < 0.05) but not in dendritic cells. Moreover, the population, diversity and principal components of the gut microbiota in mice were dramatically altered after MTX treatment in a time-dependent manner, and Bacteroidales exhibited the most distinct variation among all the taxa (P < 0.05). Bacteroides fragilis was significantly decreased with MTX treatment (P < 0.01) and tended to decrease proportionately with increasing macrophage density. Gavage of mice with B. fragilis ameliorated MTX-induced inflammatory reactions and modulate macrophage polarization. In conclusion, our results delineate a strong impact of the gut microbiota on MTX-induced intestinal mucositis and provide a potential method for the prevention of such ADRs.

Abstract LB-202: A novel neoepitope screening platform for generating effective and safe neoantigen-based vaccines and immunotherapeutics in a hepatocellular carcinoma model

Immunotherapy has emerged as a novel and promising strategy for cancer treatment, while available markers for immunotherapy have poor effectiveness and safety. The advance of next-generation sequencing (NGS) technology enables us to identify tumor-specific neoantigen which could be more effective and safer for personalized immunotherapy. In this study, we demonstrated a novel workflow for the development of liver cancer personalized vaccine. Xenograft models for liver cancer were built in mice using Hepa1-6 cell lines. Tumor-specific candidate neoantigen were determined by performed whole exome sequencing (WES) and used a comprehensive bioinformatics pipeline integrating multiple factors such as MHCI-binding affinity fold change (FC), variant allele frequencies (VAFs) of somatic mutation, mRNA expression and hydrophobicity of peptides. The results showed that peptide vaccine of Gpr55 neoantigen not only triggered striking neo-antigen specific CD8+ T cell response, but also significantly inhibited the growth of tumors in both prophylactic and therapeutic models. Moreover, our results demonstrated that VAFs and FC were the effective and safety property for immunogenicity. The identified Gpr55 neoantigen was an ideal vaccine for Hepa1-6 induced xenograft models considering the immunogenicity, anti-tumor effect and safety evaluation. It was also possible to identify and develop highly effective personalized cancer vaccine by our workflow within 13 weeks. The results illustrated huge potential of tumor-specific neoantigen as a potent vaccine for clinical tumor therapy and the workflow built in this study could be used to develop more specific, safer, more effective treatments to all types of cancer patients. Note: This abstract was not presented at the meeting. Citation Format: Cheng-chi Chao, Fengjiao Yuan, Geng Liu, Guanglei Li, Bo Li, Lian Lu, Yaomei Tian, Dongli Li, Bailing Zhou, Ting An, Yingzi Fan, Chongming Jiang, Yang Shu, Honggang Lin, Heng Xu, Hanshuo Yang, Rui zhang, Xueyan Zhang, Zhen Cheng, Rong Huang, Michael Dean, Ke Men, Xiuqing Zhang, Chaoheng Yu, Jian Wang, Shuang Cheng, Huanming Yang, Yantai Wang, Yong Hou, Yuquan Wei, Li Yang. A novel neoepitope screening platform for generating effective and safe neoantigen-based vaccines and immunotherapeutics in a hepatocellular carcinoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-202. doi:10.1158/1538-7445.AM2017-LB-202

Development of a Recombinant Xenogeneic Tumor Necrosis Factor Alpha Protein Vaccine To Protect Mice from Experimental Colitis

ABSTRACT Previous studies have highlighted the efficacy of tumor necrosis factor alpha (TNF-α) inhibitors, including monoclonal antibodies and soluble receptors, in the treatment and management of intestinal bowel disease (IBD). However, because of the immunogenicity of xenogeneic TNF-α inhibitors, antidrug antibodies (ADAs) can be triggered after repeated administration. An alternative way to target TNF-α is active immunization to elicit the production of high titers of neutralizing antibodies. In this study, we prepared a xenogeneic TNF-α protein vaccine and studied the protective effects in experimental colitis models. The xenogeneic TNF-α protein vaccine could overcome self-tolerance and induce TNF-α-specific neutralizing antibody. Moreover, the xenogeneic TNF-α protein vaccine could protect mice from acute and chronic colitis induced by dextran sodium sulfate (DSS). One possible explanation for this protective effect is the production of TNF-α-specific neutralizing antibody, which absorbed the biological activity of mouse TNF-α (mTNF-α) and failed to induce T lymphocyte apoptosis. In summary, use of the xenogeneic TNF-α protein vaccine may be a potent therapeutic strategy for IBD.