Employing ATP as a New Adjuvant Promotes the Induction of Robust Antitumor Cellular Immunity by a PLGA Nanoparticle Vaccine.

Tumor vaccines based on synthetic human papillomavirus (HPV) oncoprotein E7 and/or E6 peptides have shown encouraging results in preclinical model studies and human clinical trials. However, the clinical efficacy may be limited by the disadvantages of vulnerability to enzymatic degradation and low immunogenicity of peptides. To further improve the potency of vaccine, we developed a poly(lactide-co-glycolide)-acid (PLGA) nanoparticle, which encapsulated the antigenic peptide HPV16 E744-62, and used adenosine triphosphate (ATP), one of the most important intracellular metabolites and an endogenous extracellular danger signal for the immune system, as a new adjuvant component. The results showed that PLGA nanoparticles increased the in vivo stability, lymph node accumulation, and dendritic cell (DC) uptake of the E7 peptide; in addition, ATP further increased the migration, nanoparticle uptake, and maturation of DCs. Preventive immunization with ATP-adjuvanted nanoparticles completely abolished the growth of TC-1 tumors in mice and produced long-lasting immunity against tumor rechallenge. When tumors were fully established, therapeutic immunization with ATP-adjuvanted nanoparticles still significantly inhibited tumor progression. Mechanistically, ATP-adjuvanted nanoparticles significantly improved the systemic generation of antitumor effector cells, boosted the local functional status of these cells in tumors, and suppressed the generation and tumor infiltration of immunosuppressive Treg cells and myeloid-derived suppressor cells. These findings indicate that ATP is an effective vaccine adjuvant and that nanoparticles adjuvanted with ATP were able to elicit robust antitumor cellular immunity, which may provide a promising therapeutic vaccine candidate for the treatment of clinical malignancies, such as cervical cancer.

A therapeutic HPV16 E7 vaccine in combination with active anti-FGF-2 immunization synergistically elicits robust antitumor immunity in mice.

FGF-2 accumulates in many tumor tissues and is closely related to the development of tumor angiogenesis and the immunosuppressive microenvironment. This study aimed to investigate whether active immunization against FGF-2 could modify antitumor immunity and enhance the efficacy of an HPV16 E7-specific therapeutic vaccine. Combined immunization targeting both FGF-2 and E7 significantly suppressed tumor growth, which was accompanied by significantly increased levels of IFN-γ-expressing splenocytes and effector CD8 T cells and decreased levels of immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells(MDSCs) in both the spleen and tumor; in addition, the levels of FGF-2 and neovascularization in tumors were decreased in the mice receiving the combined immunization, and tumor cell apoptosis was promoted. The combination of an HPV16 E7-specific vaccine and active immunization against FGF-2 significantly enhances antitumor immune responses in mice with TC-1 tumors, indicating a promising strategy for tumor immunotherapy.

Modified bacterial outer membrane vesicles induce autoantibodies for tumor therapy.

Using monoclonal antibodies to block tumor angiogenesis has yielded effective antitumor effects. However, this treatment method has long cycles and is very expensive; therefore, its long-term and extensive application is limited. In this study, we developed a nanovaccine using bacterial biomembranes as carriers for antitumor therapy. The whole basic fibroblast growth factor (BFGF) molecule (154 amino acids (aa)) was loaded onto bacterial outer membrane vesicles (OMVs) using gene recombination technology. The strong adjuvant effect of OMVs was used to induce the host to produce anti-BFGF autoantibodies. We proved that persistent anti-BFGF autoantibodies can be induced in mice after only 3 immunizations to antagonize BFGF functions. The effects included multiple tumor suppression functions, including inhibition of tumor angiogenesis, induction of tumor cell apoptosis, reversal of tumor immune barriers, and promotion of tumor-specific cytotoxic T lymphocytes (CTLs), eventually causing tumor regression. We confirmed that bacterial biomembranes can be used as a vaccine delivery system to induce the production of antibodies against autoantigens, which may be used for tumor therapy. This study expands the application fields of bacterial biomembrane systems and provides insight for tumor immunotherapy other than monoclonal antibody technology. STATEMENT OF SIGNIFICANCE: In this study, we proved that bacteria-released outer membrane vesicles (OMVs) modified via genetic engineering can be used as a vaccine carrier to break autoimmune tolerance and induce the body to produce autoantibodies to antagonize pathological molecules and block pathological signaling pathways for tumor therapy. OMVs naturally released by bacteria were used to successfully load the full-length BFGF protein (154 aa). We proved that persistent anti-BFGF autoantibodies can be induced in tumor-bearing mice after only 3 immunizations to effectively inhibit tumors. Furthermore, the production of these antibodies successfully inhibited tumor angiogenesis, promoted tumor cell apoptosis, reversed the tumor immunosuppressive microenvironment, increased the cytotoxic T lymphocyte (CTL) reaction, and eventually inhibited tumor growth.

Development of novel nanoantibiotics using an outer membrane vesicle-based drug efflux mechanism.

Conventionally used antibiotics are present in low concentrations at the infection site and require multiple administrations to sustain a continuous bactericidal effect, which not only increases their systemic toxicity but also results in bacterial drug resistance. In this study, we first identified an interesting drug resistance mechanism mediated by bacterial outer membrane vesicles (OMVs) and then designed novel antibiotic-loaded OMVs using this mechanism. We show that these antibiotic-loaded OMVs can effectively enter and kill pathogenic bacteria in vitro. In a mouse model of intestinal bacterial infection, one low-dose oral administration of antibiotic-loaded OMVs showed that the drug was retained in the intestine for 36 h, and no systemic spread was detected 12 h after drug administration. The antibiotic-loaded OMVs significantly reduced the bacterial load in the small intestine and feces of infected mice. Safety experiments confirmed that the antibiotic-loaded OMVs had excellent biocompatibility. This study extends the application range of OMVs and provides new ideas for the development of antibacterial drugs.

Potential role of the antimicrobial peptide Tachyplesin III against multidrug-resistant P. aeruginosa and A. baumannii coinfection in an animal model.

BACKGROUND: Tachyplesin III, an antimicrobial peptide (AMP), provides protection against multidrug-resistant (MDR) bacterial infections and shows cytotoxicity to mammalian cells. Mixed bacterial infections, of which P. aeruginosa plus A. baumannii is the most common and dangerous combination, are critical contributors to the morbidity and mortality of long-term in-hospital respiratory medicine patients. Therefore, the development of effective therapeutic approaches to mixed bacterial infections is urgently needed. METHODS AND RESULTS: In this study, we demonstrated that compared with individual infections, mixed infections with MDR bacteria P. aeruginosa and A. baumannii cause more serious diseases, with increased pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α) and chemokines (MCP-1/MIP-2) and reduced mouse survival. In vitro treatment with Tachyplesin III enhanced phagocytosis in a mouse alveolar macrophage cell line (MH-S). Strikingly, in vivo, Tachyplesin III demonstrated a potential role against mixed-MDR bacterial coinfection. The bacterial burden in bronchoalveolar lavage fluid (BALF) was significantly reduced in the Tachyplesin III-treated group. In addition, a systemic reduction in pro-inflammatory cytokines and decreased lung injury occurred with Tachyplesin III therapy. CONCLUSION: Therefore, our study demonstrated that Tachyplesin III represents a potential therapeutic treatment against mixed-MDR bacterial infection in vivo, which sheds light on the development of therapeutic strategies against mixed-MDR bacterial infections.

Pretreatment with cathelicidin-BF ameliorates Pseudomonas aeruginosa pneumonia in mice by enhancing NETosis and the autophagy of recruited neutrophils and macrophages.

Although the antimicrobial peptide cathelicidin-BF shows minimal cytotoxicity in mammalian cells and has excellent direct killing effects on multidrug-resistant clinical pathogens such as Pseudomonas aeruginosa, its clinical application is precluded by its high sensitivity to serum proteases. Here, we demonstrate that intravenous administration of cathelicidin-BF after P. aeruginosa infection did not increase the survival rate of mice with acute pneumonia but that pretreatment with cathelicidin-BF ameliorated pneumonia by effectively activating innate immunity. Enhanced neutrophil extracellular trap (NET) activation and release (NETosis) are key processes for capturing and killing bacteria, concomitantly enhanced macrophage clearance activity, including phagocytosis and autophagy, may eliminate NETs early enough to prevent severe tissue damage. Our study not only suggests a possible approach for applying cathelicidin-BF in vivo but also provides a possible defense strategy against multidrug-resistant pathogens, i.e., efficiently activation of innate immunity.