Harnessing self-assembling peptide nanofibers toprime robust tumor-specific CD8 T cell responses in mice.

Induction of tumor-specific CD8 + T cell responses is known as a major challenge for cancer vaccine development; here we presented a strategy to improve peptide nanofibers-mounted antitumor immune responses. To this end, peptide nanofibers bearing class I (Kb)-restricted epitope (Epi-Nano) were formulated with polyethylene imine backbone (Epi-Nano-PEI), and characterized using morphological and physicochemicalcharacterizationtechniques. Nanofibers were studied in terms of their uptake by antigen-presenting cells (APCs), antigen cross-presentation capacity, and cytotoxic activity. Furthermore, nanofibers were assessed by their potency to induce NLRP3 inflammasome-related cytokines and factors. Finally, the ability of nanofibers to induce tumor-specific CD8 T cells and tumor protection were investigated in tumor-bearing mice. The formulation of Epi-Nano with PEI led to the formation of short strand nanofibers with a positive surface charge, a low critical aggregation concentration (CAC), and an increased resistancetoproteolytic degradation. Epi-Nano-PEI was significantly taken up more efficiently by antigen-presenting cells (APCs), and was more potent in cross-presentation when compared to Epi-Nano. Moreover, Epi-Nano-PEI, in comparison to Epi-Nano, efficiently up-regulated the expression of NLRP3, caspase-1, IL-1b, IL18 and IL-6. Cell viability analysis showed that formulation of PEI with Epi-Nano not only abolished its cytotoxic activity, but surprisingly induced macrophage proliferation. Furthermore, it demonstrated that Epi-Nano-PEI triggered robust antigen-specific CD8+ T cell responses, and induced maximum antitumor response (tumor growth inhibition and prolonged survival) in tumor-bearing mice that were significantly higher compared to Epi-Nano. Taken together, the formulation of Epi-Nano with PEI is suggested as a promising strategy to improve nanofibers-mounted antitumor immune response.

Genetically engineered fusion of allergen and viral-like particle induces a more effective allergen-specific immune response than a combination of them.

Chimeric virus-like particles (VLPs) were developed as a candidate for allergen-specific immunotherapy. In this study, hepatitis B core antigen (HBcAg) that genetically fused to Chenopodium album polcalcin (Che a 3)-derived peptide was expressed in E. coli BL21, purified, and VLP formation was evaluated using native agarose gel electrophoresis (NAGE) and transmission electron microscopy (TEM). Chimeric HBc VLPs were characterized in terms of their reactivity to IgE, the induction of blocking IgG and allergen-specific IgE, basophil-activating capacity, and Th1-type immune responses. Results from IgE reactivity and basophil activation test showed that chimeric HBc VLPs lack IgE-binding capacity and basophil degranulation activity. Although chimeric HBc VLPs induced the highest level of efficient polcalcin-specific IgG antibody in comparison to those induced by recombinant Che a 3 (rChe a 3) mixed either with HBc VLPs or alum, they triggered the lowest level of polcalcin-specific IgE in mice following immunization. Furthermore, in comparison to the other antigens, chimeric HBc VLPs produced a polcalcin-specific Th1 cell response. Taken together, genetically fusion of allergen derivatives to HBc VLPs, in comparison to a mix of them, may be a more effective way to induce appropriate immune responses in allergen-specific immunotherapy. KEY POINTS: • The insertion of allergen-derived peptide into major insertion region (MIR) of hepatitis B virus core (HBc) antigen resulted in nanoparticles displaying allergen-derived peptide upon its expression in prokaryotic host. • The resultant VLPs (chimeric HBc VLPs) did not exhibit IgE reactivity with allergic patients' sera and were not able to degranulate basophils. • Chimeric HBc VLPs dramatically improved protective IgG antibody response compared with those induced by allergen mixed either with HBc VLPs or alum. • Chimeric HBc VLPs induced Th1 responses that were counterparts of Th2 responses (allergic). • Chimeric HBc VLPs increased IgG2a/ IgG1 ratio and the level of IFN-γ compared to those induced by allergen mixed with either HBc VLPs or alum. Graphical Abstract.

Incorporation of the Tat cell-penetrating peptide into nanofibers improves the respective antitumor immune response.

A major challenge for the development of anticancer vaccines is the induction of a safe and effective immune response, particularly mediated by CD8+ T lymphocytes, in an adjuvant-free manner. In this respect, we present a simple strategy to improve the specific CD8+ T cell responses using KFE8 nanofibers bearing a Class I (Kb)-restricted peptide epitope (called E. nanofibers) without the use of adjuvant. We demonstrate that incorporation of Tat, a cell-penetrating peptide (CPP) of the HIV transactivator protein, into E. nanofibers remarkably enhanced tumor-specific CD8+ T cell responses. E. nanofibers containing 12.5% Tat peptide (E.Tat12.5 nanofiber) increased antigen cross-presentation by bone marrow-derived dendritic cells as compared with E. nanofibers, or E. nanofibers containing 25 or 50% the Tat peptide. Uptake of KFE8.Tat12.5 nanofibers by dendritic cells (DCs) was significantly increased compared with KFE8 nanofiber lacking Tat. Peritoneal and lymph node DCs of mice immunized with E.Tat12.5 nanofibers exhibited increased presentation of the H2kb-epitope (reminiscent for cross-presentation) compared with DCs obtained from E. nanofiber vaccinated mice. Tetrameric and intracellular cytokine staining revealed that vaccination with E.Tat12.5 triggered a robust and specific CD8+ T lymphocyte response, which was more pronounced than in mice vaccinated with E. nanofibers alone. Furthermore, E.Tat12.5 nanofibers were more potent than E. nanofiber to induce antitumor immune response and tumor-infiltrating IFN-γ CD8 T lymphocyte. In terms of cancer vaccine development, we propose that harnessing the nanofiber-based vaccine platform with incorporated Tat peptide could present a simple and promising strategy to induce highly effective antitumor immune response.

Substitution of lysine for isoleucine at the center of the nonpolar face of the antimicrobial peptide, piscidin-1, leads to an increase in the rapidity of bactericidal activity and a reduction in toxicity.

Purpose: Piscidin-1 is an effective antimicrobial peptide (AMP) against a variety of microbes. However, its toxicity has been reported as a limitation for its potential therapeutic applications. The toxicity of piscidin-1 may be related to the long nonpolar face of this AMP. Here, we investigated different piscidin-1 analogs to reach a peptide with the reduced toxicity. Material and methods: In vitro and in vivo antibacterial activity and toxicity of piscidin-1 analogs generated by replacement of isoleucine at the border (I9) or the center (I16) of the nonpolar face of piscidin-1 by alanine or lysine were investigated. Results: The results indicated that among all peptides, piscidin-1 with the highest HPLC retention time (RT) and I16K-piscidin-1 with the lowest RT had the highest and lowest cytotoxicity, respectively. Although I16K-piscidin-1 possessed the same MIC value as the parent peptide (piscidin-1) and other analogs, I16K-piscidin-1 exhibited a higher rapidity of bactericidal action at 5×MIC. The ß-galactosidase leakage and propidium iodide staining assays indicated a higher pore-forming capacity of I16K-piscidin-1 relative to the parent peptide (piscidin-1). Taken together, RT is suggested to have a direct association with the toxicity and an inverse association with the rapidity of bactericidal action and pore-forming capacity. After infection of mice with clinical colistin-resistant Acinetobacter baumannii or clinical methicillin-resistant Staphylococcus aureus strains, treatment with I16K-piscidin-1, but not piscidin-1 and other analogs, resulted in a significantly stronger bactericidal potency. Furthermore, I16K-piscidin-1 exhibited the lowest in vivo toxicity.  Conclusion: Overall, in vitro and in vivo comparison of piscidin-1 and its analogs together documented that replacement of isoleucine at the center of the nonpolar face of piscidin-1(I16) by lysine leads to not only a decrease in toxicity potential but also an increase in bactericidal potential.