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.

cTo design and discover new antimicrobial peptides (AMPs) with high levels of antimicrobial activity, a number of machinelearning 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.A ntimicrobial peptides (AMPs) are produced by multicellular organisms to defend against microbial infections. Along with potent antimicrobial activity, many AMPs also have the ability to enhance immunity by functioning as immunomodulators (1-3). AMPs therefore have excellent therapeutic potential, especially in light of increased drug resistance to many conventional antibiotic therapies. A number of naturally occurring peptides and synthetic derivatives have been developed or are currently in development (2, 4-6). Although AMPs vary in length, amino acid composition, and structure, they share some similarities, such as electrical charge and amphipathicity (3, 7). To determine the characteristics that are important in antimicrobial activity, bioinformatic tools and prediction methods have been developed (8), all based to some extent on the sequence similarities between peptides (9-11).To optimize the antimicrobial activity of identified AMPs and to predict novel peptide sequences, we present a machine-learning method based on the concept of an antimicrobial activity contribution score for each amino acid. Here, we consider that each amino acid in a peptide sequence possesses a different level of importance for the biological activity of that peptide, and this is represented by an assigned score. By calculation of each amino acid's contribution score, we can predict the antimicrobial activity of an AMP.To verify our results, we tested some of our designed AMPs ...

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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.

Vaccines are critical tools for the prevention and treatment of several diseases. Adjuvants have been traditionally used to enhance immunity to vaccines and experimental antigens. In the present study, the adjuvant combination of CpG oligodeoxynucleotides (CpG ODN) and the innate defense regulator (IDR) peptide, IDR‑HH2, was evaluated for its ability to enhance and modulate the immune response when formulated with alum and the recombinant hepatitis B surface antigen (HBsAg). The CpG‑HH2 complex enhanced the secretions of tumor necrosis factor‑α, monocyte chemotactic protein 1 and interferon‑γ by human peripheral blood mononuclear cells and promoted murine bone marrow dentritic cell maturation. In addition, the present study demonstrated that IDR‑HH2 was chemotactic for human neutrophils, THP‑1 cells and RAW264.7 cells at concentrations between 2.5 and 40 µg/ml. The present study also observed that significantly higher anti‑HBs antibody titers, which were sustained at high levels for as long as 35 weeks following the boost immunization, were induced by the combination adjuvant, even when co‑administered with a commercial hepatitis B vaccine at a low antigen dose (0.1 µg HBsAg). Notably, the level of IgG2a was almost equal to the level of IgG1, indicating that a balanced T helper (Th)1/Th2 immune response was elicited by the novel vaccine, which was consistent with the ELISpot results. These data suggest that the CpG‑HH2 complex may be a potential effective adjuvant, which facilitates a reduction in the dose of antigen and induces long‑lasting, balanced immune responses.