Nanofibers of PVF2−TrFE/PEDOT−PSS were fabricated at room temperature using electrospinning with the thinnest fiber having a diameter of ∼15 nm. This process for generating PVF2−TrFE/PEDOT−PSS composite nanofibers is cheap, fast, and reliable. The presence of conducting PEDOT−PSS assisted in the fabrication of PVF2−TrFE nanofibers at low polymer concentrations in N,N,-dimethylformamide and energy dispersive X-ray spectroscopy confirmed the presence of PEDOT−PSS in the nanofibers. As a fiber mat, they were electrically conducting and used in the fabrication of a Schottky diode, and the diode parameters were calculated assuming the standard thermionic emission model of a Schottky junction. Being a composite, these nanofibers are promising candidates for use in a variety of applications that can take advantage of the ferroelectric and/or conducting properties of each individual component. In addition, the large aspect ratio and even larger surface to volume ratio of the fibers makes them ideal candidates in the fabrication of miniaturized, low power consumption devices, and supersensitive sensors.
BackgroundThe current live vaccinia virus vaccine used in the prevention of smallpox is contraindicated for millions of immune-compromised individuals. Although vaccination with the current smallpox vaccine produces protective immunity, it might result in mild to serious health complications for some vaccinees. Thus, there is a critical need for the production of a safe virus-free vaccine against smallpox that is available to everyone. For that reason, we investigated the impact of imiquimod and resiquimod (Toll-like receptors agonists), and the codon-usage optimization of the vaccinia virus A27L gene in the enhancement of the immune response, with intent of producing a safe, virus-free DNA vaccine coding for the A27 vaccinia virus protein.MethodsWe analyzed the cellular-immune response by measuring the IFN-γ production of splenocytes by ELISPOT, the humoral-immune responses measuring total IgG and IgG2a/IgG1 ratios by ELISA, and the TH1 and TH2 cytokine profiles by ELISA, in mice immunized with our vaccine formulation.ResultsThe proposed vaccine formulation enhanced the A27L vaccine-mediated production of IFN-γ on mouse spleens, and increased the humoral immunity with a TH1-biased response. Also, our vaccine induced a TH1 cytokine milieu, which is important against viral infections.ConclusionThese results support the efforts to find a new mechanism to enhance an immune response against smallpox, through the implementation of a safe, virus-free DNA vaccination platform.
Smallpox is a disease caused by Variola virus (VARV). Although eradicated by WHO in 1980, the threat of using VARV on a bioterror attack has increased. The current smallpox vaccine ACAM2000, which consists of live vaccinia virus (VACV), causes complications in individuals with a compromised immune system or with previously reported skin diseases. Thus, a safer and efficacious vaccine needs to be developed. Previously, we reported that our virus-free DNA vaccine formulation, a pVAX1 plasmid encoding codon-optimized VACV A27L gene (pA27LOPT) with and without Imiquimod adjuvant, stimulates A27L-specific production of IFN-γ and increases humoral immunity 7 days post-vaccination. Here, we investigated the immune response of our novel vaccine by measuring the frequency of splenocytes producing IFN-γ by ELISPOT, the TH1 and TH2 cytokine profiles, and humoral immune responses two weeks post-vaccination, when animals were challenged with VACV. In all assays, the A27-based DNA vaccine conferred protective immune responses. Specifically, two weeks after vaccination, mice were challenged intranasally with vaccinia virus, and viral titers in mouse lungs and ovaries were significantly lower in groups immunized with pA27LOPT and pA27LOPT + Imiquimod. These results demonstrate that our vaccine formulation decreases viral replication and dissemination in a virus-free DNA vaccine platform, and provides an alternative towards a safer an efficacious vaccine.
Immunogenicity of DNA vaccines can be efficiently improved by adding adjuvants into their formulations. In this regard, the application of nano- and microparticles as vaccines adjuvants, or delivery systems, provides a powerful tool in designing modern vaccines. In the present study, we examined the role of “Supramolecular Hacky Sacks” (SHS) particles, made via the hierarchical self-assembly of a guanosine derivative, as a novel immunomodulator for DNA plasmid preparations. These plasmids code for the proteins HIV-1 Gag (pGag), the wild-type vaccinia virus Western Reserve A27 (pA27L), or a codon-optimized version of the latter (pOD1A27Lopt), which is also linked to the sequence of the outer domain-1 (OD1) from HIV-1 gp120 protein. We evaluated the enhancement of the immune responses generated by our DNA plasmid formulations in a murine model through ELISpot and ELISA assays. The SHS particles increased the frequencies of IFN-γ-producing cells in mice independently immunized with pGag and pA27L plasmids. Moreover, the addition of SHS to pGag and pA27L DNA plasmid formulations enhanced the production of IFN-γ (Th1-type) over IL-4 (Th2-type) cellular immune responses. Furthermore, pGag and pA27L plasmids formulated with SHS, triggered the production of antigen-specific IgG in mice, especially the IgG2a isotype. However, no improvement of either of those adaptive immune responses was observed in mice receiving pOD1A27Lopt+SHS. Here, we demonstrated that SHS particles have the ability to improve both arms of adaptive immunity of plasmid coding “wild-type” antigens without additional strategies to boost their immunogenicity. To the best of our knowledge, this is the first report of SHS guanosine-based particles as DNA plasmid adjuvants.
Poxviruses are complex dsDNA viruses with over 200 genes, many of them with unknown role in the stimulation of immune responses. Among these, the vaccinia virus (VACV) L3L ORF encodes an essential protein for the transcription of the VACV early genes. To the best of our knowledge, the immune response elicited by L3 has not been characterized. In this regard, our data describes a DNA L3-coding plasmid (pL3L) that stimulates both, humoral- and cell-mediated immune responses in a mouse model. Cell-mediated immune responses were measured by IFN-γ and IL-4 ELISPOT assays. We performed CD8+ cells depletion and flow cytometry analysis to account for the contribution of cytotoxic T lymphocytes in the IFN-γ production. Moreover, results from ELISPOT were confirmed by measuring the concentration of IL-4 and IFN-γ in supernatant of antigen-stimulated splenocytes by cytokine ELISA. Additionally, dominant antigenic regions of L3 protein were identified by epitope mapping analysis. Humoral immune responses were assessed by ELISA. Specifically, the production of total IgG, IgG1 (TH-2) and IgG2a (TH-1) were determined one week after the final immunization. Our ELISPOT data shows pL3L-immunized animals to produce significantly higher frequencies of IFN-γ Spot-Forming Cells (SFC) versus controls. IL-4 levels remained unchanged in all three groups, demonstrating the increase in antigen-specific IFN-γ releasing cells. Flow cytometry assay results showed that CD8+ T cells are a major contributor to the production of IFN-γ. Moreover, our formulation enhances the production of total IgG, predominantly IgG2a isotype. Immunization with pL3L promotes a robust cytotoxic immune response, crucial against viral pathogens. In addition, our vaccine candidate promotes an increase in IgG levels, especially IgG2a (TH-1 type). Our data encourages further studies of L3 as a novel antigen in vaccine development against poxviruses.
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