Inactivated enterovirus 71 with poly-γ-glutamic acid/Chitosan nano particles (PC NPs) induces high cellular and humoral immune responses in BALB/c mice.

Enterovirus 71 (EV71) is the major causative agent of hand-foot-and-mouth disease (HFMD) and many neurological manifestations. Recently, this virus has become a serious concern because of consecutive epidemics in the Asia-Pacific region. However, no effective vaccine for EV71 has been discovered except two EV71 vaccines which are being used in local communities of China. To develop a safe and efficient EV71 vaccine candidate, we generated inactivated EV71 and evaluated its efficacy with γ-PGA/Chitosan nanoparticles (PC NPs), which are safe, biodegradable and effective as an adjuvant. The subcutaneous administration of inactivated EV71 with PC NPs adjuvant induces higher levels of virus-specific humoral (IgG, IgG1, and IgG2a) and cell-mediated immune responses (IFN-γ and IL-4). Additionally, inactivated EV71 with PC NPs adjuvant induces significantly higher virus neutralizing antibody responses compared to the virus only group, and resulted in a long lasting immunity without any noticeable side effects. Together, our findings demonstrate that PC NPs are safe and effective immunogenic adjuvants which may be promising candidates in the development of more efficacious EV71 vaccines.

Programming of Influenza Vaccine Broadness and Persistence by Mucoadhesive Polymer-Based Adjuvant Systems.

The development of an anti-influenza vaccine with the potential for cross-protection against seasonal drift variants as well as occasionally emerging reassortant viruses is essential. In this study, we successfully generated a novel anti-influenza vaccine system combining conserved matrix protein 2 (sM2) and stalk domain of hemagglutinin (HA2) fusion protein (sM2HA2) and poly-γ-glutamic acid (γ-PGA)-based vaccine adjuvant systems that can act as a mucoadhesive delivery vehicle of sM2HA2 as well as a robust strategy for the incorporation of hydrophobic immunostimulatory 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and QS21. Intranasal coadministration of sM2HA2 and the combination adjuvant γ-PGA/MPL/QS21 (CA-PMQ) was able to induce a high degree of protective mucosal, systemic, and cell-mediated immune responses. The sM2HA2/CA-PMQ immunization was able to prevent disease symptoms, confering complete protection against lethal infection with divergent influenza subtypes (H5N1, H1N1, H5N2, H7N3, and H9N2) that lasted for at least 6 mo. Therefore, our data suggest that mucosal administration of sM2HA2 in combination with CA-PMQ could be a potent strategy for a broad cross-protective influenza vaccine, and CA-PMQ as a mucosal adjuvant could be used for effective mucosal vaccines.

Intranasal administration of poly-gamma glutamate induced antiviral activity and protective immune responses against H1N1 influenza A virus infection.

BACKGROUND: The global outbreak of a novel swine-origin strain of the 2009 H1N1 influenza A virus and the sudden, worldwide increase in oseltamivir-resistant H1N1 influenza A viruses highlight the urgent need for novel antiviral therapy. METHODS: Here, we investigated the antiviral efficacy of poly-gamma glutamate (γ-PGA), a safe and edible biomaterial that is naturally synthesized by Bacillus subtilis, against A/Puerto Rico/8/1934 (PR8) and A/California/04/2009 (CA04) H1N1 influenza A virus infections in C57BL/6 mice. RESULTS: Intranasal administration of γ-PGA for 5 days post-infection improved survival, increased production of antiviral cytokines including interferon-beta (IFN-ß) and interleukin-12 (IL-12), and enhanced activation of natural killer (NK) cells and influenza antigen-specific cytotoxic T lymphocytes (CTL) activity. CONCLUSIONS: These results suggest that γ-PGA protects mice against H1N1 influenza A virus by enhancing antiviral immune responses.

Virulence and transmissibility of H1N2 influenza virus in ferrets imply the continuing threat of triple-reassortant swine viruses.

Efficient worldwide swine surveillance for influenza A viruses is urgently needed; the emergence of a novel reassortant pandemic H1N1 (pH1N1) virus in 2009 demonstrated that swine can be the direct source of pandemic influenza and that the pandemic potential of viruses prevalent in swine populations must be monitored. We used the ferret model to assess the pathogenicity and transmissibility of predominant Korean triple-reassortant swine (TRSw) H1N2 and H3N2 influenza viruses genetically related to North American strains. Although most of the TRSw viruses were moderately pathogenic, one [A/Swine/Korea/1204/2009; Sw/1204 (H1N2)] was virulent in ferrets, causing death within 10 d of inoculation, and was efficiently transmitted to naive contact ferrets via respiratory droplets. Although molecular analysis did not reveal known virulence markers, the Sw/1204 virus acquired mutations in hemagglutinin (HA) (Asp-225-Gly) and neuraminidase (NA) (Ser-315-Asn) proteins during the single ferret passage. The contact-Sw/1204 virus became more virulent in mice, replicated efficiently in vitro, extensively infected human lung tissues ex vivo, and maintained its ability to replicate and transmit in swine. Reverse-genetics studies further indicated that the HA(225G) and NA(315N) substitutions contributed substantially in altering virulence and transmissibility. These findings support the continuing threat of some field TRSw viruses to human and animal health, reviving concerns on the capacity of pigs to create future pandemic viruses. Apart from warranting continued and enhanced global surveillance, this study also provides evidence on the emerging roles of HA(225G) and NA(315N) as potential virulence markers in mammals.

Postbiotic potential of Bacillus velezensis KMU01 cell-free supernatant for the alleviation of obesity in mice.

Attention toward the preventive effects of postbiotics on metabolic diseases has increased because of greater stability and safety over probiotics. However, studies regarding the bioactive effects of postbiotics, especially from probiotic Bacillus strains, are relatively limited. The anti-obesity effects of the cell-free culture supernatant of Bacillus velezensis KMU01 (CFS-B.vele) were evaluated using high-fat-diet (HFD)-induced mice. HFD-induced mice (n = 8 per group) received equal volumes of (1) CFS-B.vele (114 mg/kg) in PBS, (2) Xenical in PBS, or (3) PBS alone by oral gavage daily for 13 weeks. The results demonstrated that CFS-B.vele changed the gut microbiota and showed anti-obesity effects in HFD-induced obese mice. The elevated Firmicutes/Bacteroidota ratio induced by HFD was decreased in the CFS-B.vele group compared to the other groups (p < 0.05). The CFS-B.vele intervention led to the enrichment of SCFA-producers, such as Roseburia and Eubacterium, in the cecum, suggesting their potential involvement in the amelioration of obesity. Due to these changes, the various obesity-related biomarkers (body weight, fat in tissue, white adipose tissue weight and size, serum LDL-cholesterol level, hepatic lipid accumulation, and adipogenesis/lipogenesis-related gene/protein expression) were improved. Our findings suggest that CFS-B.vele has potential as a novel anti-obesity agent through modulation of the gut microbiota.

Polymer nanomicelles for efficient mucus delivery and antigen-specific high mucosal immunity.

Micelles for mucosal immunity: A mucosal vaccine system based on γ-PGA nanomicelles and viral antigens was synthesized. The intranasal administration of the vaccine system induces a high immune response both in the humoral and cellular immunity (see picture).

Self-fluorescence of chemically crosslinked MRI nanoprobes to enable multimodal imaging of therapeutic cells.

Old chemistry for novel materials: Self-fluorescent high-relaxivity T(2)-weighted magnetic resonance imaging (MRI) contrast agents are produced. They are a novel type of MR/optical dual-modality in vivo imaging nanoprobe using glutaraldehyde crosslinking chemistry, and they are used to label and monitor therapeutic cells both in vitro and in vivo.

Design of Injectable Poly(γ-glutamic acid)/Chondroitin Sulfate Hydrogels with Mineralization Ability.

Biocompatible hydrogels are considered promising agents for application in bone tissue engineering. However, the design of reliable hydrogels with satisfactory injectability, mechanical strength, and a rapid biomineralization rate for bone regeneration remains challenging. Herein, injectable hydrogels are fabricated using hydrazide-modified poly(γ-glutamic acid) and oxidized chondroitin sulfate by combining acylhydrazone bonds and ionic bonding of carboxylic acid groups or sulfate groups with calcium ions (Ca2+). The resulting hydrogels display a fast gelation rate and good self-healing ability due to the acylhydrazone bonds. The introduction of Ca2+ at a moderate concentration enhances the mechanical strength of the hydrogels. The self-healing capacity of hydrogels is improved, with a healing efficiency of 87.5%, because the addition of Ca2+ accelerates the healing process of hydrogels. Moreover, the hydrogels can serve as a robust template for biomineralization. The mineralized hydrogels with increasing Ca2+ concentration exhibit rapid formation and high crystallization of apatite after immersion in simulated body fluid. The hydrogels containing the aldehyde groups possess good bioadhesion to the bone and cartilage tissues. With these superior properties, the developed hydrogels demonstrate potential applicability in bone tissue engineering.

Preparation of pH-Responsive Poly(γ-glutamic acid) Hydrogels by Enzymatic Cross-Linking.

pH-responsive hydrogels are important for oral drug release applications, and they are increasingly demanded to reduce the adverse side effects of drug release and improve drug absorption. In this study, a new type of pH-responsive hydrogel comprised of poly(γ-glutamic acid) modified with tyramine (PGA-Tyr) was developed through enzymatic cross-linking in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The gelation rate, stiffness, swelling behavior, and pore size of the resulting hydrogels were tuned by changing the concentrations of HRP and H2O2 or the degree of substitution (DS) of PGA-Tyr. The pH responsiveness of the hydrogels was evaluated by the swelling ratio in solutions with various pH values, and their pH responsiveness exhibited a good reversibility in pH 2.0 and 7.0 solutions. The degradation rate of the hydrogels in simulated intestinal fluid (SIF) was faster than that in simulated gastric fluid (SGF). Moreover, indomethacin (IM), a hydrophobic drug model, was encapsulated in the hydrogels by rapid in situ gelation, and the pH-dependent drug release of IM-loaded hydrogels was achieved in SGF and SIF. Importantly, when IM was entrapped in pluronic F-127 to form drug micelles, the burst release of the IM-micelle-loaded hydrogels with a high DS of PGA-Tyr was remarkably decreased in SGF, and sustained drug release was presented in SIF. Thus, pH-responsive PGA-based hydrogels have tremendous promise for biomedical applications, especially oral drug delivery.

Bacterial Community of Galchi-Baechu Kimchi Based on Culture-Dependent and - Independent Investigation and Selection of Starter Candidates.

In this study, the bacterial community of galchi-baechu kimchi was determined using culture-based and culture-independent techniques (next generation sequencing:NGS), and showed discrepancies between results. Weissella koreensis and Pediococcus inopinatus were the dominant species according to the NGS results, while Bacillus species and P. inopinatus were dominant in the culture-dependent analysis. To identify safe starter candidates, sixty-five Bacillus strains isolated from galchi-baechu kimchi using culture-dependent methods were evaluated for their antibiotic resistance, presence of toxin genes, and hemolytic activity. Strains were then assessed for salt tolerance and protease and lipase activity. As a result, four strains-B. safensis GN5_10, B. subtilis GN5_19, B. velezensis GN5_25, and B. velezensis GT8-were selected as safe starter candidates for use in fermented foods.

Postbiotics Enhance NK Cell Activation in Stress-Induced Mice through Gut Microbiome Regulation.

Recent studies have revealed that probiotics and their metabolites are present under various conditions; however, the role of probiotic metabolites (i.e., postbiotics in pathological states) is controversial. Natural killer (NK) cells play a key role in innate and adaptive immunity. In this study, we examined NK cell activation influenced by a postbiotics mixture in response to gut microbiome modulation in stress-induced mice. In vivo activation of NK cells increased in the postbiotics mixture treatment group in accordance with Th1/Th2 expression level. Meanwhile, the Red Ginseng treatment group, a reference group, showed very little expression of NK cell activation. Moreover, the postbiotics mixture treatment group in particular changed the gut microbiome composition. Although the exact role of the postbiotics mixture in regulating the immune system of stress-induced mice remains unclear, the postbiotics mixture-induced NK cell activation might have affected gut microbiome modulation.

Bioderived polyelectrolyte nanogels for robust antigen loading and vaccine adjuvant effects.

An easy but robust strategy for the synthesis of bioderived polyelectrolyte nanogels for protein antigen loading and vaccine adjuvant systems that can improve both humoral (Th2) and cellular immunity (Th1) is presented. The synthesized polyelectrolyte nanogels promote the uptake of antigens into antigen-presenting cells and strongly induce ovalbumin-specific INF-γ producing cells, cytotoxic T cell activity, and antibody production.

pH-stimuli-responsive near-infrared optical imaging nanoprobe based on poly(γ-glutamic acid)/poly(ß-amino ester) nanoparticles.

pH-stimuli-responsive near-infrared optical imaging nanoprobes are designed and synthesized in this study in a facile one-step synthesis process based on the use of the biocompatible and biodegradable polymer poly(γ-glutamic acid) (γ-PGA)/poly(ß-amino ester) (PBAE). PBAE has good transfection efficiency and promotes degradation properties under acidic conditions. This pH-responsive degradability can be used for the effective release of encapsulating materials after cellular uptake. As an optical imaging probe, indocyanine green (ICG) is an FDA-approved near-infrared fluorescent dye with a quenching property at a high concentration. In this regard, we focus here on the rapid degradation of PBAE in an acidic environment, in which the nanoparticles are disassembled. This allows the ICG dyes to show enhanced fluorescence signals after being releasing from the particles. We demonstrated this principle in cellular uptake experiments. We expect that the developed pH-stimuli-responsive smart nanoprobes can be applied in intracellular delivery signaling applications.

Functional Annotation Genome Unravels Potential Probiotic Bacillus velezensis Strain KMU01 from Traditional Korean Fermented Kimchi.

Bacillus velezensis strain KMU01 showing γ-glutamyltransferase activity as a probiotic candidate was isolated from kimchi. However, the genetic information on strain KMU01 was not clear. Therefore, the current investigation was undertaken to prove the probiotic traits of B. velezensis strain KMU01 through genomic analysis. Genomic analysis revealed that strain KMU01 did not encode enterotoxin genes and acquired antibiotic resistance genes. Strain KMU01 genome possessed survivability traits under extreme conditions such as in the presence of gastric acid, as well as several probiotic traits such as intestinal epithelium adhesion and the production of thiamine and essential amino acids. Potential genes for human health enhancement such as those for γ-glutamyltransferase, nattokinase, and bacteriocin production were also identified in the genome. As a starter candidate for food fermentation, the genome of KMU01 encoded for protease, amylase, and lipase genes. The complete genomic sequence of KMU01 will contribute to our understanding of the genetic basis of probiotic properties and allow for the assessment of the effectiveness of this strain as a starter or probiotic for use in the food industry.

Functional Genomic Insights into Probiotic Bacillus siamensis Strain B28 from Traditional Korean Fermented Kimchi.

Bacillus siamensis strain B28 was previously isolated from traditional Korean fermented kimchi and inhibited expression of the microphthalmia-associated transcription factor and ß-catenin in human embryonic kidney 293 cells. Here, we determined the complete genome sequence of strain B28 and compared it with other strains to elucidate its potential probiotic properties. Strain B28 does not contain antibiotic resistance-, hemolysin- or enterotoxin-encoding genes. The genome includes genes related to survival in extreme conditions, adhesion in the gut, and synthesis of the bacteriocin. Considering the potential for enhancement of human health, the strain B28 genome encodes genes related to production of eight essential amino acids, γ-aminobutyric acid, branched-chain fatty acids, γ-glutamyltransferase, and subtilisin. There are genes for the synthesis of uracil, lipoteichoic acid, glutathione, and several reactive oxygen species-scavenging enzymes. Experimentally, strain B28 exhibited sensitivity to eight antibiotics and antibacterial activity against seven foodborne pathogens. B. siamensis B28 is a safe strain with potential for development as a probiotic.

Injectable poly(γ-glutamic acid)-based biodegradable hydrogels with tunable gelation rate and mechanical strength.

Polypeptide-based hydrogels have potential applications in polymer therapeutics and regenerative medicine. However, designing reliable polypeptide-based hydrogels with a rapid injection time and controllable stiffness for clinical applications remains a challenge. Herein, a class of injectable poly(γ-glutamic acid) (PGA)-based hydrogels were constructed using furfurylamine and tyramine-modified PGA (PGA-Fa-Tyr) and the crosslinker dimaleimide poly(ethylene glycol) (MAL-PEG-MAL), through a facile strategy combining enzymatic crosslinking and Diels-Alder (DA) reaction. The injectable hydrogels could be quickly gelatinized and the gelation time, ranging from 10 to 95 s, could be controlled by varying the hydrogen peroxide (H2O2) concentration. Compared with hydrogels formed by single enzymatic crosslinking, the compressive stress and strain of the injectable hydrogels were remarkably enhanced because of the occurrence of the subsequent DA reaction in the hydrogels, suggesting the DA network imparted an outstanding toughening effect on the hydrogels. Furthermore, the mechanical strength, swelling ratio, pore size, and degradation behavior of the injectable hydrogels could be easily controlled by changing the molar ratios of H2O2/Tyr or furan/maleimide. More importantly, injectable hydrogels encapsulating bovine serum albumin exhibited sustained release behavior. Thus, the developed hydrogels hold great potential for applications in biomedical fields, such as tissue engineering and cell/drug delivery.

Human papillomavirus type 16 E6-specific antitumor immunity is induced by oral administration of HPV16 E6-expressing Lactobacillus casei in C57BL/6 mice.

Given that local cell-mediated immunity (CMI) against the human papillomavirus type 16 E6 (HPV16 E6) protein is important for eradication of HPV16 E6-expressing cancer cells in the cervical mucosa, the HPV16 E6 protein may be a target for the mucosal immunotherapy of cervical cancer. Here, we expressed the HPV16 E6 antigen on Lactobacillus casei (L. casei) and investigated E6-specific CMI following oral administration of the L. casei-PgsA-E6 to mice. Surface expression of HPV16 E6 antigens was confirmed and mice were orally inoculated with the L. casei-PgsA or the L. casei-PgsA-E6. Compared to the L. casei-PgsA-treated mice, significantly higher levels of serum IgG and mucosal IgA were observed in L. casei-PgsA-E6-immunized mice; these differences were significantly enhanced after boost. Consistent with this, systemic and local CMI were significantly increased after the boost, as shown by increased counts of IFN-gamma-secreting cells in splenocytes, mesenteric lymph nodes (MLN), and vaginal samples. Furthermore, in the TC-1 tumor model, animals receiving the orally administered L. casei-PgsA-E6 showed reduced tumor size and increased survival rate versus mice receiving control (L. casei-PgsA) immunization. We also found that L. casei-PgsA-E6-induced antitumor effect was decreased by in vivo depletion of CD4(+) or CD8(+) T cells. Collectively, these results indicate that the oral administration of lactobacilli bearing the surface-displayed E6 protein induces T cell-mediated cellular immunity and antitumor effects in mice.

Isolation and genetic characterization of H5N2 influenza viruses from pigs in Korea.

Due to dual susceptibility to both human and avian influenza A viruses, pigs are believed to be effective intermediate hosts for the spread and production of new viruses with pandemic potential. In early 2008, two swine H5N2 viruses were isolated from our routine swine surveillance in Korea. The sequencing and phylogenetic analysis of surface proteins revealed that the Sw/Korea/C12/08 and Sw/Korea/C13/08 viruses were derived from avian influenza viruses of the Eurasian lineage. However, although the Sw/Korea/C12/08 isolate is an entirely avian-like virus, the Sw/Korea/C13/08 isolate is an avian-swine-like reassortant with the PB2, PA, NP, and M genes coming from a 2006 Korean swine H3N1-like virus. The molecular characterization of the two viruses indicated an absence of significant mutations that could be associated with virulence or binding affinity. However, animal experiments showed that the reassortant Sw/Korea/C13/08 virus was more adapted and was more readily transmitted than the purely avian-like virus in a swine experimental model but not in ferrets. Furthermore, seroprevalence in swine sera from 2006 to 2008 suggested that avian H5 viruses have been infecting swine since 2006. Although there are no known potential clinical implications of the avian-swine reassortant virus for pathogenicity in pigs or other species, including humans, at present, the efficient transmissibility of the swine-adapted H5N2 virus could facilitate virus spread and could be a potential model for pandemic, highly pathogenic avian influenza (e.g., H5N1 and H7N7) virus outbreaks or a pandemic strain itself.

New biological functions and applications of high-molecular-mass poly-gamma-glutamic acid.

Ultra-high-molecular-weight poly-gamma-glutamic acid (gamma-PGA) is a most promising biodegradable polymer that is produced by Bacillus subtilis (chungkookjang). Attractive properties of gamma-PGA are that it is water soluble, anionic, biodegradable, and edible. Development of gamma-PGA has pursued in terms of cosmetics/skin care, bone care, nanoparticle for drug delivery system, hydrogel, and so on. Very recently, our research has shown that gamma-PGA can be used as an immune-stimulating agent, especially at high molecular weight. This review presents the synthesis and production of high-molecular-weight gamma-PGA and its various applications in industrial fields.

Synthesis of an amphiphilic poly(gamma-glutamic acid)-cholesterol conjugate and its application as an artificial chaperone.

A poly(gamma-glutamic acid) (gammaPGA)-cholesterol conjugate was synthesized and the properties of an aqueous solution were evaluated. The conjugate showed amphiphilic nature derived from the hydrophilic gammaPGA backbone and the hydrophobic cholesterol side chain. The conjugate spontaneously formed nanoparticles in the aqueous solution of the low concentration, and the high concentration resulted in the formation of the physical gel. By utilizing the self-aggregating properties of the conjugate in water, an artificial chaperone was developed. The complex of protein with the nanoparticles of the conjugate was formed and the protein was released upon the dissociation of the nanoparticles by the addition of beta-cyclodextrin. For denatured carbonic anhydrase, the activity was recovered in the artificial chaperone of the nanoparticle conjugate.