Peptide-based vaccine targeting IL17A attenuates experimental spondyloarthritis in HLA-B27 transgenic rats.

OBJECTIVES: Spondyloarthritis (SpA) is known as series of immune-mediated inflammatory disease of the axial and peripheral joints. Human leucocyte antigen (HLA)-B27 is a genetic risk factor for SpA. Recent evidence suggests that the interleukin -17 (IL17) axis strongly contributes to SpA. This study aimed to assess the efficacy of an IL17A peptide-based vaccine on SpA manifestations in model rats. METHODS: HLA-B27/human ß2-microglobulin (hß2M) transgenic rats were immunised with heat-inactivated Mycobacterium tuberculosis (MT) to develop spondylitis and arthritis as an experimental SpA model after immunisation with a keyhole limpet hemocyanin-conjugated IL17A peptide-based vaccine with an alum adjuvant three times. The IL17A antibody titre was assessed using ELISA, and arthritis score and joint thickness were monitored two times a week. Enzyme-linked immunospot (ELISpot) assays for IL4- and interferon-γ-secreting splenocytes were conducted to evaluate IL17A-specific T cell activation. We also evaluated the effect of IL17A vaccine in SpA therapeutic model. RESULTS: The IL17A peptide-based vaccine with alum adjuvant successfully induced antibody production and suppressed the arthritis score and joint thickness. X-ray and histological analyses showed that enthesitis, bone destruction and new bone formation were inhibited by the IL17A vaccine. The ELISpot assay showed that the IL17A peptide-based vaccine did not elicit any IL17A-reactive T cell responses. IL17A vaccine tends to mitigate, but not significant, in SpA treatment model. These data showed that the peptide-based vaccine targeting IL17A alleviated the SpA phenotype in a heat-inactivated MT-induced SpA model in HLA-B27/hß2M transgenic rats. CONCLUSIONS: IL17A peptide-based vaccine may be a therapeutic option for SpA treatment.

Preclinical study of a DNA vaccine targeting SARS-CoV-2.

To fight against the worldwide COVID-19 pandemic, the development of an effective and safe vaccine against SARS-CoV-2 is required. As potential pandemic vaccines, DNA/RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFNγ expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies by a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, and neutralization assays using pseudo-virus, and live SARS-CoV-2. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits. Finally, DNA vaccine protected hamsters from SARS-CoV-2 infection. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic.

AJP001, a novel helper T-cell epitope, induces a humoral immune response with activation of innate immunity when included in a peptide vaccine.

Vaccine design requires well-tailored formulations including a T-cell epitope and adjuvants. We identified a novel cationic peptide, AJP001, which possesses a strong affinity for murine MHC class II alleles (H2-IEd and H2-IAd) and low affinity for H2-IAb. We designed an AJP001 and epitope peptide-conjugated vaccine, AJP001-angiotensin (Ang) II, which was intracutaneously administered to mice three times at 2-week intervals. Indeed, the AJP001-Ang II vaccine induced antibody production against Ang II in BALB/cA mice but not in C57BL/6 mice. To estimate the T-cell-dependent immunogenicity of the AJP001 conjugate vaccine in human cells, naïve human peripheral blood mononuclear cells (PBMCs) were exposed to AJP001-Ang II, and T-cell proliferation was evaluated by analyzing cell division using flow cytometric measurement of carboxyfluorescein succinimidyl ester (CFSE) dye dilution. To activate the immune response, the innate immune system must be activated by adjuvant treatment. Interestingly, treatment with AJP001 induced IL-1ß and IL-18 secretion via NLRP3 inflammasome activation and induced TNF-α and IL-6 production through an NF-κB-dependent pathway in human and mouse macrophages. These results suggest that AJP001 behaves as a T-cell epitope in mice and humans and is a useful tool for the formulation of peptide vaccines without the addition of adjuvants.

The efficacy of intracerebroventricular idursulfase-beta enzyme replacement therapy in mucopolysaccharidosis II murine model: heparan sulfate in cerebrospinal fluid as a clinical biomarker of neuropathology.

Mucopolysaccharidosis II (MPS II) is caused by a deficiency of iduronate-2-sulfatase that results in accumulation of glycosaminoglycans (GAG), including heparan sulfate (HS), which is considered to contribute to neuropathology. We examined the efficacy of intracerebroventricular (ICV) enzyme replacement therapy (ERT) of idursulfase-beta (IDS-ß) and evaluated the usefulness of HS as a biomarker for neuropathology in MPS II mice. We first examined the efficacy of three different doses (3, 10, and 30 µg) of single ICV injections of IDS-ß in MPS II mice. After the single-injection study, its long-term efficacy was elucidated with 30 µg of IDS-ß ICV injections repeated every 4 weeks for 24 weeks. The efficacy was assessed by the HS content in the cerebrospinal fluid (CSF) and the brain of the animals along with histologic examinations and behavioral tests. In the single-injection study, the 30 µg of IDS-ß ICV injection showed significant reductions of HS content in brain and CSF that were maintained for 28 days. Furthermore, HS content in CSF was significantly correlated with HS content in brain. In the long-term repeated-injection study, the HS content in the brain and CSF was also significantly reduced and correlated. The histologic examinations showed a reduction in lysosomal storage. A significant improvement in memory/learning function was observed in open-field and fear-conditioning tests. ICV ERT with 30 µg of IDS-ß produced significant improvements in biochemical, histological, and functional parameters in MPS II mice. Furthermore, we demonstrate for the first time that the HS in the CSF had significant positive correlation with brain tissue HS and GAG levels, suggesting HS in CSF as a useful clinical biomarker for neuropathology.

Prevention of Asthma Exacerbation in a Mouse Model by Simultaneous Inhibition of NF-κB and STAT6 Activation Using a Chimeric Decoy Strategy.

Transactivation of inflammatory and immune mediators in asthma is tightly regulated by nuclear factor κB (NF-κB) and signal transducer and activator of transcription 6 (STAT6). Therefore, we investigated the efficacy of simultaneous inhibition of NF-κB and STAT6 using a chimeric decoy strategy to prevent asthma exacerbation. The effects of decoy oligodeoxynucleotides were evaluated using an ovalbumin-induced mouse asthma model. Ovalbumin-sensitized mice received intratracheal administration of decoy oligodeoxynucleotides 3 days before ovalbumin challenge. Fluorescent-dye-labeled decoy oligodeoxynucleotides could be detected in lymphocytes and macrophages in the lung, and activation of NF-κB and STAT6 was inhibited by chimeric decoy oligodeoxynucleotide transfer. Consequently, treatment with chimeric or NF-κB decoy oligodeoxynucleotides protected against methacholine-induced airway hyperresponsiveness, whereas the effect of chimeric decoy oligodeoxynucleotides was significantly greater than that of NF-κB decoy oligodeoxynucleotides. Treatment with chimeric decoy oligodeoxynucleotides suppressed airway inflammation through inhibition of overexpression of interleukin-4 (IL-4), IL-5, and IL-13 and inflammatory infiltrates. Histamine levels in the lung were reduced via suppression of mast cell accumulation. A significant reduction in mucin secretion was observed due to suppression of MUC5AC gene expression. Interestingly, the inhibitory effects on IL-5, IL-13, and histamine secretion were achieved by transfer of chimeric decoy oligodeoxynucleotides only. This novel therapeutic approach could be useful to treat patients with various types of asthma.

Accurate estimation of compression in simultaneous masking enables the simulation of hearing impairment for normal-hearing listeners.

This chapter presents a unified gammachirp framework for -estimating cochlear compression and synthesizing sounds with inverse compression that -cancels the compression of a normal-hearing (NH) listener to simulate the -experience of a hearing-impaired (HI) listener. The compressive gammachirp (cGC) filter was -fitted to notched-noise masking data to derive level-dependent -filter shapes and the cochlear compression function (e.g., Patterson et al., J Acoust Soc Am 114:1529-1542, 2003). The procedure is based on the analysis/synthesis technique of Irino and Patterson (IEEE Trans Audio Speech Lang Process 14:2222-2232, 2006) using a dynamic cGC filterbank (dcGC-FB). The level dependency of the dcGC-FB can be reversed to produce inverse compression and resynthesize sounds in a form that cancels the compression applied by the -auditory system of the NH listener. The chapter shows that the estimation of compression in simultaneous masking is improved if the notched-noise procedure for the derivation of auditory filter shape includes noise bands with different levels. Since both the estimation and resynthesis are performed within the gammachirp framework, it is possible for a specific NH listener to experience the loss of a -specific HI listener.

Hepatocyte growth factor stimulated angiogenesis without inflammation: differential actions between hepatocyte growth factor, vascular endothelial growth factor and basic fibroblast growth factor.

Based on the potent angiogenic effects of hepatocyte growth factor (HGF), therapeutic angiogenesis using human HGF plasmid DNA increased tissue perfusion and reduced symptoms in patients with critical limb ischemia (CLI) in randomized placebo-controlled clinical trials. To explore further the potent angiogenic activity of HGF, the present study compared the effects of HGF, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) on angiogenesis and vascular inflammation. All of HGF, VEGF and bFGF significantly induced the formation of capillary blood vessel and granulation tissue in the rat paper disc model as an in vivo animal model of angiogenesis. However, although HGF, bFGF and VEGF significantly increased the growth of vascular endothelial cells, bFGF alone, but not HGF or VEGF, significantly increased the growth of vascular smooth muscle cells (VSMCs) in the in vitro proliferation assay. In addition, bFGF, but not HGF or VEGF, significantly activated an essential transcription factor for inflammation, NFκB, and gene expression of its downstream inflammation-related cytokines (IL-8 and MCP-1) in VSMCs, accompanied by an increase in the vascular permeability in the rat paper disc model. Thus, the present results indicated that HGF induced angiogenesis without vascular inflammation, different from bFGF and VEGF. These different properties between HGF, VEGF and bFGF might affect the efficiency of therapeutic angiogenesis.

Folate-linked lipid-based nanoparticles deliver a NFkappaB decoy into activated murine macrophage-like RAW264.7 cells.

Activated macrophages are the key effector cells in rheumatoid arthritis (RA) and secrete multiple mediators of inflammation including proinflammatory cytokines. We investigated delivery of a nuclear factor kappa B (NFkappaB) decoy by folate-linked lipid-based nanoparticles (NP-F) into murine macrophages. The expression of folate receptor (FR) in RAW264.7 cells activated by lipopolysaccaride was confirmed by strong expression of FR mRNA, and association of FITC-labeled folate-BSA conjugate. When transfected via NP-F, the NFkappaB decoy was strongly detected in the cytoplasm, and an inhibitory effect on the translocation of NFkappaB into the nucleus was observed at 0.03 microM of the decoy, suggesting that NP-F effectively delivered the NFkappaB decoy into the cytoplasm. This information is of value for the design of NFkappaB decoy carrier systems targeting FR in activated macrophages in gene therapy for autoimmune diseases such as RA.