Cationic-nanogel nasal vaccine containing the ectodomain of RSV-small hydrophobic protein induces protective immunity in rodents.

Respiratory syncytial virus (RSV) is a leading cause of upper and lower respiratory tract infection, especially in children and the elderly. Various vaccines containing the major transmembrane surface proteins of RSV (proteins F and G) have been tested; however, they have either afforded inadequate protection or are associated with the risk of vaccine-enhanced disease (VED). Recently, F protein-based maternal immunization and vaccines for elderly patients have shown promising results in phase III clinical trials, however, these vaccines have been administered by injection. Here, we examined the potential of using the ectodomain of small hydrophobic protein (SHe), also an RSV transmembrane surface protein, as a nasal vaccine antigen. A vaccine was formulated using our previously developed cationic cholesteryl-group-bearing pullulan nanogel as the delivery system, and SHe was linked in triplicate to pneumococcal surface protein A as a carrier protein. Nasal immunization of mice and cotton rats induced both SHe-specific serum IgG and mucosal IgA antibodies, preventing viral invasion in both the upper and lower respiratory tracts without inducing VED. Moreover, nasal immunization induced greater protective immunity against RSV in the upper respiratory tract than did systemic immunization, suggesting a critical role for mucosal RSV-specific IgA responses in viral elimination at the airway epithelium. Thus, our nasal vaccine induced effective protection against RSV infection in the airway mucosa and is therefore a promising vaccine candidate for further development.

5'-CMP and 5'-UMP alleviate dexamethasone-induced muscular atrophy in C2C12 myotubes.

Atrogin-1 and muscle RING finger 1 (MuRF1) are ubiquitin ligases specifically expressed during skeletal muscle atrophy and mediate muscle protein degradation. In contrast, PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α), which is a master regulator of mitochondrial biosynthesis, protects skeletal muscle from atrophy. Pyrimidine nucleoside 5'-monophosphates, such as cytidine 5'-monophosphate (5'-CMP) and uridine 5'-monophosphate (5'-UMP), induce PGC-1α expression and promote myotube formation in mouse C2C12 cells. In this study, we determined the effect of 5'-CMP and 5'-UMP on muscular atrophy in C2C12 myotube cells. 5'-UMP decreased Atrogin-1 and MuRF1 mRNA levels that were upregulated by dexamethasone treatment. 5'-CMP and 5'-UMP ameliorated dexamethasone-mediated atrophy in C2C12 myotubes. Furthermore, the combination of 5'-CMP and 5'-UMP further alleviated dexamethasone-mediated atrophy. In addition, cytidine and uridine, the precursors of 5'-CMP and 5'-UMP, markedly ameliorated dexamethasone-mediated atrophy. Considering nucleotide metabolism and absorption, the active metabolites underlying the observed effects of 5'-CMP and 5'-UMP appear to be cytidine and uridine. Our results indicate that 5'-CMP alleviates muscle atrophy by activating PGC-1α and differentiation, and 5'-UMP alleviates muscle atrophy by suppressing the activation of the myolytic system, whereas the combined use of both enhances the muscle atrophy inhibitory effect. 5'-CMP and 5'-UMP may be an effective and safe treatment for muscular atrophy.

Model for predicting age-dependent safety and immunomodulatory effects of STING ligands in non-human primates.

Stimulator of interferon genes (STING) is a cytoplasmic dinucleotide sensor used as an immunomodulatory agent for cancer treatment. The efficacy of the STING ligand (STING-L) against various tumors has been evaluated in mouse models; however, its safety and efficacy in non-human primates have not been reported. We examined the effects of escalating doses of cyclic-di-adenosine monophosphate (c-di-AMP) or cyclic [G (3',5')pA (3',5'p] (3'-3'-cGAMP) administered intramuscularly or intravenously to cynomolgus macaques. Both ligands induced transient local and systemic inflammatory responses and systemic immunomodulatory responses, including the upregulation of interferon-α (IFN-α) and IFN-γ expression and the activation of multiple immunocompetent cell subsets. Better immunological responses were observed in animals that received c-di-AMP compared with those that received 3'-3'-cGAMP. Multi-parameter analysis using a dataset obtained before administering the ligands predicted the efficacy outcome partially. Importantly, the efficacy of these ligands was reduced in older macaques. We propose that 0.5 mg/kg c-di-AMP via intramuscular administration should be the optimal starting point for clinical studies. Our study is the first to demonstrate the age-dependent safety and efficacy of STING-L in non-human primates and supports the potential of STING-L use as a direct immunomodulator in vivo.

5'-CMP and 5'-UMP promote myogenic differentiation and mitochondrial biogenesis by activating myogenin and PGC-1α in a mouse myoblast C2C12 cell line.

Ribonucleotides are basic monomeric building blocks for RNA considered as conditionally essential nutrients. They are normally produced in sufficient quantity, but can become insufficient upon stressful challenges. The administration of pyrimidine nucleotides, such as cytidine-5'-monophosphate (5'-CMP) and uridine-5'-monophosphate (5'-UMP), enables rats to endure prolonged exercise. However, the underlying mechanisms have remained elusive. To investigate these mechanisms, we studied the effect of 5'-CMP and 5'-UMP on muscular differentiation and mitochondrial biogenesis in myoblast C2C12 cells. 5'-CMP and 5'-UMP were found to increase the mRNA levels of myogenin, which is a myogenic regulatory protein expressed during the final differentiation step and fusion of myoblasts into myotubes. 5'-CMP and 5'-UMP also promoted myoblast differentiation into myotube cells. 5'-CMP and 5'-UMP further increased the mRNA levels of PGC-1α which regulates mitochondrial biogenesis and skeletal muscle fiber type. In addition, 5'-CMP and 5'-UMP increased mitochondrial DNA copy number and enhanced mRNA levels of slow-muscle myosin heavy chains. Moreover, cytidine and uridine, nucleosides corresponding to 5'-CMP and 5'-UMP, markedly promoted myotube formation in C2C12 cells. Considering the metabolism and absorption of nucleotides, the active bodies underlying the effects observed with 5'-CMP and 5'-UMP could be cytidine and uridine. In conclusion, our results indicate that 5'-CMP and 5'-UMP can promote myogenic differentiation and mitochondrial biogenesis, as well as increase slow-twitch fiber via the activation of myogenin and PGC-1α. In addition, 5'-CMP and 5'-UMP may be considered as safe and effective agents to enhance muscle growth and improve the endurance in skeletal muscles.

Intranasal vaccination against angiotensin II type 1 receptor and pneumococcal surface protein A attenuates hypertension and pneumococcal infection in rodents.

OBJECTIVES: To combat global increases in the prevalence of lifestyle-related diseases and concomitant infectious diseases, we aimed to develop an innovative intranasal vaccine that simultaneously targets both hypertension and pneumonia, is not given by invasive injection, and offers prolonged therapeutic effect and reduced frequency of administration. METHODS: Angiotensin II type 1 receptor-pneumococcal surface protein A (AT1R-PspA) vaccine, consisting of a cationic nanometer-sized hydrogel incorporating AT1R partial peptide conjugated with PspA and cyclic diguanylate monophosphate adjuvant, was created and given intranasally to spontaneously hypertensive rats (SHRs). Antigen-specific antibodies and blood pressure were examined to evaluate immune responses and the antihypertensive effect of the vaccine. To examine the protective effect of antibodies induced by vaccination on pneumococcal infection, sera obtained from immunized SHRs were incubated with a lethal dose of Streptococcus pneumoniae and then administered to mice. RESULTS: Five doses of AT1R-PspA nasal-vaccine-induced AT1R-specific serum IgG antibody production and attenuated the development of hypertension in SHRs in the long term. Both in-vitro and in-vivo studies revealed that responses to angiotensin II were suppressed in vaccinated rats. Mice passively immunized with sera obtained from AT1R-PspA-vaccinated SHRs were protected from lethal pneumococcal infection. CONCLUSION: Intranasal immunization with AT1R-PspA vaccine has the potential to simultaneously attenuate the development of hypertension and protect from lethal pneumococcal infection.

Preparation of chondroitin sulfate libraries containing disulfated disaccharide units and inhibition of thrombin by these chondroitin sulfates.

Chondroitin sulfate (CS) containing GlcA-GalNAc(4,6-SO(4)) (E unit) and CS containing GlcA(2SO(4))-GalNAc(6SO(4)) (D unit) have been implicated in various physiological functions. However, it has been poorly understood how the structure and contents of disulfated disaccharide units in CS contribute to these functions. We prepared CS libraries containing E unit or D unit in various proportions by in vitro enzymatic reactions using recombinant GalNAc 4-sulfate 6-O-sulfotransferase and uronosyl 2-O-sulfotransferase, and examined their inhibitory activity toward thrombin. The in vitro sulfated CSs containing disulfated disaccharide units showed concentration-dependent direct inhibition of thrombin when the proportion of E unit or D unit in the CSs was above 15-17%. The CSs containing both E unit and D unit exhibited higher inhibitory activity toward thrombin than the CSs containing either E unit or D unit alone, if the proportion of the total disulfated disaccharide units of these CSs was comparable. The thrombin-catalyzed degradation of fibrinogen, a physiological substrate for thrombin, was also inhibited by the CS containing both E unit and D unit. These observations indicate that the enzymatically prepared CS libraries containing various amounts of disulfated disaccharide units appear to be useful for elucidating the physiological function of disulfated disaccharide units in CS.

Polyphosphate kinase (PPK2), a potent, polyphosphate-driven generator of GTP.

An enzyme that uses inorganic polyphosphate (poly P) as a donor to convert GDP to GTP has been purified 1,300-fold to homogeneity from lysates of Pseudomonas aeruginosa PAOM5. Poly P chains of 30-50 residues are optimal; those of 15-700 residues can also serve. GDP is preferred over ADP among nucleoside diphosphate acceptors. This nucleoside diphosphate kinase (NDK) activity resides in the same protein isolated for its synthesis of poly P from GTP and designated PPK2 in an accompanying report. The reaction that synthesizes poly P and the reaction that utilizes poly P differ in their kinetic features. Especially notable is the catalytic potency of the NDK activity, which is 75-fold greater than that of poly P synthesis. PPK2 appears in the stationary phase of growth and reaches NDK levels of 5-10% that of the classic NDK; both kinase activities may figure in the generation of the guanosine precursors in the synthesis of alginate, an exopolysaccharide essential for the virulence of P. aeruginosa.

A polyphosphate kinase (PPK2) widely conserved in bacteria.

Synthesis of inorganic polyphosphate (poly P) from the terminal phosphate of ATP is catalyzed reversibly by poly P kinase (PPK, now designated PPK1) initially isolated from Escherichia coli. PPK1 is highly conserved in many bacteria, including some of the major pathogens such as Pseudomonas aeruginosa. In a null mutant of P. aeruginosa lacking ppk1, we have discovered a previously uncharacterized PPK activity (designated PPK2) distinguished from PPK1 by the following: synthesis of poly P from GTP or ATP, a preference for Mn2+ over Mg2+, and a stimulation by poly P. The reverse reaction, a poly P-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the forward reaction, poly P synthesis from GTP. The gene encoding PPK2 (ppk2) was identified from the amino acid sequence of the protein purified near 1,000-fold, to homogeneity. The 5'-end is 177 bp upstream of the annotated genome sequence of a "conserved hypothetical protein"; ppk2 (1,074 bp) encodes a protein of 357 aa with a molecular mass of 40.8 kDa. Sequences homologous to PPK2 are present in two other proteins in P. aeruginosa, in two Archaea, and in 32 other bacteria (almost all with PPK1 as well); these include rhizobia, cyanobacteria, Streptomyces, and several pathogenic species. Distinctive features of the poly P-driven nucleoside diphosphate kinase activity and structural aspects of PPK2 are among the subjects of an accompanying report.