Polymeric Caffeic Acid Acts as an Antigen Delivery Carrier for Mucosal Vaccine Formulation by Forming a Complex with an Antigenic Protein.

The development of mucosal vaccines, which can generate antigen-specific immune responses in both the systemic and mucosal compartments, has been recognized as an effective strategy for combating infectious diseases caused by pathogenic microbes. Our recent research has focused on creating a nasal vaccine system in mice using enzymatically polymerized caffeic acid (pCA). However, we do not yet understand the molecular mechanisms by which pCA stimulates antigen-specific mucosal immune responses. In this study, we hypothesized that pCA might activate mucosal immunity at the site of administration based on our previous findings that pCA possesses immune-activating properties. However, contrary to our initial hypothesis, the intranasal administration of pCA did not enhance the expression of various genes involved in mucosal immune responses, including the enhancement of IgA responses. Therefore, we investigated whether pCA forms a complex with antigenic proteins and enhances antigen delivery to mucosal dendritic cells located in the lamina propria beneath the mucosal epithelial layer. Data from gel filtration chromatography indicated that pCA forms a complex with the antigenic protein ovalbumin (OVA). Furthermore, we examined the promotion of OVA delivery to nasal mucosal dendritic cells (mDCs) after the intranasal administration of pCA in combination with OVA and found that OVA uptake by mDCs was increased. Therefore, the data from gel filtration chromatography and flow cytometry imply that pCA enhances antigen-specific antibody production in both mucosal and systemic compartments by serving as an antigen-delivery vehicle.

Development of a concise and reliable method for quantifying the antibody loaded onto lipid nanoparticles modified with Herceptin.

Antibodies are essential components of the immune system with a wide range of molecular targets. They have been recognized as modalities for treating several diseases and more than 130 approved antibody-based therapeutics are available for clinical use. However, limitations remain associated with its efficacy, tissue permeability, and safety, especially in cancer treatment. Nanoparticles, particularly those responsive to external stimuli, have shown promise in improving the efficacy of antibody-based therapeutics and tissue-selective delivery. In this study, we developed a reliable and accurate method for quantifying the amount of antibody loaded onto lipid nanoparticles modified with Herceptin® (Trastuzumab), an antibody-based therapeutic used to treat HER2-positive cancers, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. This method proved to be a suitable alternative to commonly used protein quantification techniques, which are limited by lipid interference present in the samples. Furthermore, the amount of Herceptin modified on the liposomes, measured by this method, was confirmed by Herceptin's antibody-dependent cell-mediated cytotoxicity activity. Our results demonstrate the potential of this method as a critical tool for developing tissue-selective antibody delivery systems, leading to improved efficacy and reduced side effects of antibody-based therapeutics.

Development of a Gene and Nucleic Acid Delivery System for Skeletal Muscle Administration via Limb Perfusion Using Nanobubbles and Ultrasound.

Strategies for gene and nucleic acid delivery to skeletal muscles have been extensively explored to treat Duchenne muscular dystrophy (DMD) and other neuromuscular diseases. Of these, effective intravascular delivery of naked plasmid DNA (pDNA) and nucleic acids into muscles is an attractive approach, given the high capillary density in close contact with myofibers. We developed lipid-based nanobubbles (NBs) using polyethylene-glycol-modified liposomes and an echo-contrast gas and found that these NBs could improve tissue permeability by ultrasound (US)-induced cavitation. Herein, we delivered naked pDNA or antisense phosphorodiamidate morpholino oligomers (PMOs) into the regional hindlimb muscle via limb perfusion using NBs and US exposure. pDNA encoding the luciferase gene was injected with NBs via limb perfusion into normal mice with application of US. High luciferase activity was achieved in a wide area of the limb muscle. DMD model mice were administered PMOs, designed to skip the mutated exon 23 of the dystrophin gene, with NBs via intravenous limb perfusion, followed by US exposure. The number of dystrophin-positive fibers increased in the muscles of mdx mice. Combining NBs and US exposure, which can be widely delivered to the hind limb muscles via the limb vein, could be an effective therapeutic approach for DMD and other neuromuscular disorders.

Intranasal administration of sodium nitroprusside augments antigen-specific mucosal and systemic antibody production in mice.

The coronavirus disease 2019, i.e., the COVID-19 pandemic, caused by a highly virulent and transmissible pathogen, has profoundly impacted global society. One approach to combat infectious diseases caused by pathogenic microbes is using mucosal vaccines, which can induce antigen-specific immune responses at both the mucosal and systemic sites. Despite its potential, the clinical implementation of mucosal vaccination is hampered by the lack of safe and effective mucosal adjuvants. Therefore, developing safe and effective mucosal adjuvants is essential for the fight against infectious diseases and the widespread clinical use of mucosal vaccines. In this study, we demonstrated the potent mucosal adjuvant effects of intranasal administration of sodium nitroprusside (SNP), a known nitric oxide (NO) donor, in mice. The results showed that intranasal administration of ovalbumin (OVA) in combination with SNP induced the production of OVA-specific immunoglobulin A in the mucosa and increased serum immunoglobulin G1 levels, indicating a T helper-2 (Th2)-type immune response. However, an analog of SNP, sodium ferrocyanide, which does not generate NO, failed to show any adjuvant effects, suggesting the critical role of NO generation in activating an immune response. In addition, SNPs facilitated the delivery of antigens to the lamina propria, where antigen-presenting cells are located, when co-administered with antigens, and also transiently elicited the expression of interleukin-6, interleukin-1ß, granulocyte colony-stimulating factor, C-X-C motif chemokine ligand 1, and C-X-C motif chemokine ligand 2 in nasal tissue. These result suggest that SNP is a dual-functional formulation with antigen delivery capabilities to the lamina propria and the capacity to activate innate immunity. In summary, these results demonstrate the ability of SNP to induce immune responses via an antigen-specific Th2-type response, making it a promising candidate for further development as a mucosal vaccine formulation against infectious diseases.

Refinement and complete solution NMR analysis of the structure of a 6-branched 1,3-ß-D-glucan (OL-2) isolate from Omphalialapidescens.

OL-2 is a water-soluble ß-glucan produced by Omphalia lapidescens. This versatile glucan has potential applications in various industries, including food, cosmetics, and pharmaceuticals. In addition, OL-2 is known for its promising applications as a biomaterial and drug, owing to its reported antitumor and antiseptic properties. Although the biological activities of ß-glucans vary depending on their primary structure, holistic clarification of OL-2 via solution NMR spectroscopy to ascertain its complete and unambiguous structure has not yet been achieved. In this study, a chain of solution NMR techniques, such as correlation spectroscopy, total correlation spectroscopy (TOCSY), nuclear Overhauser effect and exchange spectroscopy, 13C-edited heteronuclear single quantum coherence (HSQC), HSQC-TOCSY, heteronuclear multiple bond correlation, and heteronuclear 2-bond correlation pulse sequences were used to unambiguously assign all 1H and 13C atoms in OL-2. Based on our investigation, OL-2 consists of a 1,3-ß-glucan backbone chain decorated with a single 6-branched ß-glucosyl side unit on every fourth residue.

Potentiation of Antitumor Activity by Antibody Drugs and Mushroom-Derived ß-Glucans in Natural Killer Cell-Mediated Tumoricidal Activities against Non-Hodgkin's B-Cell Lymphoma.

ß-glucans are polysaccharides that activate innate immunity. We herein investigated whether P-glucans promote the immunological effects of antibody drugs against malignant tumor cells using human peripheral blood mononuclear cells (PBMCs). Rituximab bound to CD20-specific lymphoma and exhibited cytotoxic activity in the presence of human mononuclear cells, but not neutrophils. The addition of Sparassis crispa (cauliflower mushroom)-derived ß-glucan (SCG) and granulocyte macrophage colony-stimulating factor (GM-CSF) to co-cultures of PBMCs and Raji lymphoma cells further promoted antibody-dependent cell-mediated cytotoxicity (ADCC). The GM-CSF treatment increased ß-glucan receptor expression on adherent cells in PBMCs. A co-stimulation with GM-CSF and SCG of PBMCs induced an increase in the number of spreading cells and the activation of natural killer (NK) cells. The enhancement in ADCC was abolished by the removal of NK cells, indicating that SCG and GM-CSF increased ADCC against lymphoma by activating ß-glucan receptor-expressing cells in PBMCs and enhancing NK cell activity. The synergistic mechanisms of action of mushroom-derived ß-glucans and biopharmaceuticals, including recombinant cytokines and antibodies, in the treatment of malignant tumor cells provide important insights into the clinical efficacy of ß-glucans from mushrooms.

Involvement of splenic marginal zone macrophages in the recognition of systemically administered phosphatidylserine-coated liposomes in mice.

Autoimmune diseases present a significant clinical problem, highlighting the need for the development of novel or improved therapeutic methods. One of the factors that causes autoimmune diseases is a defect in the clearance of apoptotic cells by phagocytes. Thus, improved apoptotic cell processing has been considered as a strategy to treat autoimmune diseases. However, therapeutic strategies focusing on apoptotic cell clearance have not been approved till date. We have reported that liposomes composed of phosphatidylserine (PS liposomes) exhibit anti-inflammatory or immunosuppressive effects in macrophages. A PS liposome display PS on its surface, which plays a crucial role in the phagocytosis of apoptotic cells by marginal zone macrophages (MZMs), a key player in the clearance of apoptotic cells, by recognizing PS exposed on the surface of apoptotic cells. Therefore, we hypothesized that PS liposomes could be used as "antigen delivery vesicles" to act as a substitute for apoptotic cells in the treatment of autoimmune diseases. In this study, we showed that systemically administered PS liposomes accumulated in the marginal zone of the spleen due to recognition of surface-displayed PS by MZMs because it was observed that liposomes without PS did not accumulate in the marginal zone. In conclusion, PS liposomes may be useful vehicles to function as active agents and/or antigens against autoimmune diseases.

Role of Interleukin-6 in the Antigen-Specific Mucosal Immunoglobulin A Responses Induced by CpG Oligodeoxynucleotide-Loaded Cationic Liposomes.

An advantage of mucosal vaccines over conventional parenteral vaccines is that they can induce protective immune responses not only at mucosal surfaces but also in systemic compartments. Despite this advantage, few live attenuated or inactivated mucosal vaccines have been developed and applied clinically. We recently showed that the intranasal immunization of ovalbumin (OVA) with class B synthetic oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motif (CpG ODN)-loaded cationic liposomes synergistically exerted both antigen-specific mucosal immunoglobulin A (IgA) and systemic immunoglobulin G (IgG) responses in mice. However, the mechanism underlying the mucosal adjuvant activity of CpG ODN-loaded liposomes remains unknown. In the present study, we showed that the intranasal administration of CpG ODN-loaded cationic liposomes elicited interleukin (IL)-6 release in nasal tissues. Additionally, pre-treatment with an anti-IL-6 receptor (IL-6R) antibody attenuated antigen-specific nasal IgA production but not serum IgG responses. Furthermore, the intranasal administration of OVA and CpG ODN-loaded cationic liposomes increased the number of IgA+/CD138+ plasma cells and IgA+/B220+ B cells in the nasal passages. This increase was markedly suppressed by pre-treatment with anti-IL-6R blocking antibody. In conclusion, IL-6 released by CpG ODN-loaded cationic liposomes at the site of administration may play a role in the induction of antigen-specific IgA responses by promoting differentiation into IgA+ plasma cells for IgA secretion from B cells.

Development of an Antibody Delivery Method for Cancer Treatment by Combining Ultrasound with Therapeutic Antibody-Modified Nanobubbles Using Fc-Binding Polypeptide.

A key challenge in treating solid tumors is that the tumor microenvironment often inhibits the penetration of therapeutic antibodies into the tumor, leading to reduced therapeutic efficiency. It has been reported that the combination of ultrasound-responsive micro/nanobubble and therapeutic ultrasound (TUS) enhances the tissue permeability and increases the efficiency of delivery of macromolecular drugs to target tissues. In this study, to facilitate efficient therapeutic antibody delivery to tumors using this combination system, we developed therapeutic antibody-modified nanobubble (NBs) using an Fc-binding polypeptide that can quickly load antibodies to nanocarriers; since the polypeptide was derived from Protein G. TUS exposure to this Herceptin®-modified NBs (Her-NBs) was followed by evaluation of the antibody's own ADCC activity, resulting the retained activity. Moreover, the utility of combining therapeutic antibody-modified NBs and TUS exposure as an antibody delivery system for cancer therapy was assessed in vivo. The Her-NBs + TUS group had a higher inhibitory effect than the Herceptin and Her-NBs groups. Overall, these results suggest that the combination of therapeutic antibody-modified NBs and TUS exposure can enable efficient antibody drug delivery to tumors, while retaining the original antibody activity. Hence, this system has the potential to maximize the therapeutic effects in antibody therapy for solid cancers.

Role of interleukin-6 in antigen-specific mucosal immunoglobulin A induction by cationic liposomes.

The COVID-19 pandemic, caused by a highly virulent and transmissible pathogen, has proven to be devastating to society. Mucosal vaccines that can induce antigen-specific immune responses in both the systemic and mucosal compartments are considered an effective measure to overcome infectious diseases caused by pathogenic microbes. We have recently developed a nasal vaccine system using cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane and cholesteryl 3ß-N-(dimethylaminoethyl)carbamate in mice. However, the comprehensive molecular mechanism(s), especially the host soluble mediator involved in this process, by which cationic liposomes promote antigen-specific mucosal immune responses, remain to be elucidated. Herein, we show that intranasal administration of cationic liposomes elicited interleukin-6 (IL-6) expression at the site of administration. Additionally, both nasal passages and splenocytes from mice nasally immunized with cationic liposomes plus ovalbumin (OVA) were polarized to produce IL-6 when re-stimulated with OVA in vitro. Furthermore, pretreatment with anti-IL-6R antibody, which blocks the biological activities of IL-6, attenuated the production of OVA-specific nasal immunoglobulin A (IgA) but not OVA-specific serum immunoglobulin G (IgG) responses. In this study, we demonstrated that IL-6, exerted by nasally administered cationic liposomes, plays a crucial role in antigen-specific IgA induction.

Surface Phenotype Changes and Increased Response to Oxidative Stress in CD4+CD25high T Cells.

Conversion of CD4+CD25+FOXP3+ T regulatory cells (Tregs) from the immature (CD45RA+) to mature (CD45RO+) phenotype has been shown during development and allergic reactions. The relative frequencies of these Treg phenotypes and their responses to oxidative stress during development and allergic inflammation were analysed in samples from paediatric and adult subjects. The FOXP3lowCD45RA+ population was dominant in early childhood, while the percentage of FOXP3highCD45RO+ cells began increasing in the first year of life. These phenotypic changes were observed in subjects with and without asthma. Further, there was a significant increase in phosphorylated ERK1/2 (pERK1/2) protein in hydrogen peroxide (H2O2)-treated CD4+CD25high cells in adults with asthma compared with those without asthma. Increased pERK1/2 levels corresponded with increased Ca2+ response to T cell receptor stimulation. mRNA expression of peroxiredoxins declined in Tregs from adults with asthma. Finally, CD4+CD25high cells from paediatric subjects were more sensitive to oxidative stress than those from adults in vitro. The differential Treg sensitivity to oxidative stress observed in children and adults was likely dependent on phenotypic CD45 isoform switching. Increased sensitivity of Treg cells from adults with asthma to H2O2 resulted from a reduction of peroxiredoxin-2, -3, -4 and increased pERK1/2 via impaired Ca2+ response in these cells.

Polymeric Caffeic Acid Acts as a Nasal Vaccine Formulation against Streptococcus pneumoniae Infections in Mice.

Infectious diseases are the second leading cause of death worldwide, highlighting the importance of the development of a novel and improved strategy for fighting pathogenic microbes. Streptococcus pneumoniae is a highly pathogenic bacteria that causes pneumonia with high mortality rates, especially in children and elderly individuals. To solve these issues, a mucosal vaccine system would be the best solution for the prevention and treatment of these diseases. We have recently reported that enzymatically polymerized caffeic acid (pCA) acts as a mucosal adjuvant when co-administered with antigenic proteins via the nasal route. Moreover, the sources of caffeic acid and horseradish peroxidase are ingredients found commonly in coffee beans and horseradish, respectively. In this study, we aimed to develop a pneumococcal nasal vaccine comprising pneumococcal surface protein A (PspA) and pCA as the mucosal adjuvant. Intranasal immunization with PspA and pCA induced the production of PspA-specific antibody responses in the mucosal and systemic compartments. Furthermore, the protective effects were tested in a murine model of S. pneumoniae infection. Intranasal vaccination conferred antigen-dependent protective immunity against a lethal infection of S. pneumoniae. In conclusion, pCA is useful as a serotype-independent universal nasal pneumococcal vaccine formulation.

Enzymatically polymerised polyphenols prepared from various precursors potentiate antigen-specific immune responses in both mucosal and systemic compartments in mice.

Despite significant modern medicine progress, having an infectious disease is a major risk factor for humans. Mucosal vaccination is now widely considered as the most promising strategy to defeat infectious diseases; however, only live-attenuated and inactivated mucosal vaccines are used in the clinical field. To date, no subunit mucosal vaccine was approved mainly because of the lack of safe and effective methodologies to either activate or initiate host mucosal immune responses. We have recently elucidated that intranasal administration of enzymatically polymerised caffeic acid potentiates antigen-specific mucosal and systemic antibody responses in mice. However, our earlier study has not confirmed whether these effects are specific to the polymer synthesised from caffeic acid. Here, we show that enzymatically polymerised polyphenols (EPPs) from various phenolic compounds possess mucosal adjuvant activities when administered nasally with an antigen to mice. Potentiation of antigen-specific immune responses by all EPPs tested in this study showed no clear difference among the precursors used. We found that intranasal administration of ovalbumin as the antigen, in combination with all enzymatically polymerised polyphenols used in this study, induced ovalbumin-specific mucosal IgA in the nasal cavity, bronchoalveolar lavage fluid, vaginal fluids, and systemic IgG, especially IgG1, in sera. Our results demonstrate that the mucosal adjuvant activities of polyphenols are not limited to polymerised caffeic acid but are broadly observable across the studied polyphenols. These properties of polyphenols may be advantageous for the development of safe and effective nasal vaccine systems to prevent and/or treat various infectious diseases.

Essential Role of Host Double-Stranded DNA Released from Dying Cells by Cationic Liposomes for Mucosal Adjuvanticity.

Infectious disease remains a substantial cause of death. To overcome this issue, mucosal vaccine systems are considered to be a promising strategy. Yet, none are approved for clinical use, except for live-attenuated mucosal vaccines, mainly owing to the lack of effective and safe systems to induce antigen-specific immune responses in the mucosal compartment. We have reported that intranasal vaccination of an antigenic protein, with cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane and 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl], induced antigen-specific mucosal and systemic antibody responses in mice. However, precise molecular mechanism(s) underlying the mucosal adjuvant effects of cationic liposomes remain to be uncovered. Here, we show that a host double-stranded DNA (dsDNA), released at the site of cationic liposome injection, plays an essential role for the mucosal adjuvanticity of the cationic liposome. Namely, we found that nasal administration of the cationic liposomes induced localized cell death, at the site of injection, resulting in extracellular leakage of host dsDNA. Additionally, in vivo DNase I treatment markedly impaired OVA-specific mucosal and systemic antibody production exerted by cationic liposomes. Our report reveals that host dsDNA, released from local dying cells, acts as a damage-associated molecular pattern that mediates the mucosal adjuvant activity of cationic liposomes.

Polymeric Caffeic Acid Is a Safer Mucosal Adjuvant That Augments Antigen-Specific Mucosal and Systemic Immune Responses in Mice.

Infections remain a major threat to human lives. To overcome the threat caused by pathogens, mucosal vaccines are considered a promising strategy. However, no inactivated and/or subunit mucosal vaccine has been approved for human use, largely because of the lack of a safe and effective mucosal adjuvant. Here, we show that enzymatically synthesized polymeric caffeic acid (pCA) can act as a potent mucosal adjuvant in mice. Intranasal administration of ovalbumin (OVA) in combination with pCA resulted in the induction of OVA-specific mucosal IgA and serum IgG, especially IgG1. Importantly, pCA was synthesized from caffeic acid and horseradish peroxidase from coffee beans and horseradish, respectively, which are commonly consumed. Therefore, pCA is believed to be a highly safe material. In fact, administration of pCA did not show distinct toxicity in mice. These data indicate that pCA has merit for use as a mucosal adjuvant for nasal vaccine formulations.

Intranasal administration of cationic liposomes enhanced granulocyte-macrophage colony-stimulating factor expression and this expression is dispensable for mucosal adjuvant activity.

OBJECTIVE: Infectious diseases remain a threat to human life. Vaccination against pathogenic microbes is a primary method of treatment as well as prevention of infectious diseases. Particularly mucosal vaccination is a promising approach to fight against most infectious diseases, because mucosal surfaces are a major point of entry for most pathogens. We recently developed an effective mucosal adjuvant of cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl] (DC-chol) (DOTAP/DC-chol liposomes). However, the mechanism(s) underlying the mucosal adjuvant effects exerted by the cationic liposomes have been unclear. In this study, we investigated the role of granulocyte-macrophage colony-stimulating factor (GM-CSF), which was reported to act as a mucosal adjuvant, on the mucosal adjuvant activities of DOTAP/DC-chol liposomes when administered intranasally to mice. RESULTS: Here, we show that, although intranasal vaccination with cationic liposomes in combination with antigenic protein elicited GM-CSF expression at the site of administration, blocking GM-CSF function by using an anti-GM-CSF neutralizing antibody did not alter antigen-specific antibody production induced by DOTAP/DC-chol liposomes, indicating that GM-CSF may not contribute to the mucosal adjuvant activity of the cationic liposomes when administered intranasally.

Nasal vaccination with pneumococcal surface protein A in combination with cationic liposomes consisting of DOTAP and DC-chol confers antigen-mediated protective immunity against Streptococcus pneumoniae infections in mice.

Infectious diseases are the second leading cause of death worldwide, suggesting that there is still a need for the development of new and improved strategies for combating pathogens effectively. Streptococcus pneumoniae is the most virulent bacteria causing pneumonia with high mortality, especially in children and the elderly. Because of the emergence of antibiotic resistance in S. pneumoniae, employing a serotype-independent mucosal vaccine would be the best approach to prevent and treat the diseases caused by S. pneumoniae. In this study, we have developed a pneumococcal nasal vaccine, consisting of pneumococcal surface protein A (PspA) and cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and cholesteryl 3ß-N-(dimethylaminoethyl)-carbamate (DC-chol) (DOTAP/DC-chol liposome). The efficiency of this cationic liposome-based PspA nasal vaccine was examined in a murine model of S. pneumoniae infection. Intranasal vaccination with PspA and DOTAP/DC-chol liposomes conferred protective immunity against lethal inhalation of S. pneumoniae, improving the survival rate of infected mice. Moreover, intranasal immunization with PspA and DOTAP/DC-chol liposomes not only induced the production of PspA-specific IgA and IgG by both mucosal and systemic compartments but also elicited PspA-specific Th17 responses, which play a pivotal role in controlling S. pneumoniae infection by host innate immune response. We further demonstrated that DOTAP/DC-chol liposomes enhanced PspA uptake by nasal dendritic cells (DCs), which might be a mechanism for the induction of protective immune responses to S. pneumoniae infection. These results show that DOTAP/DC-chol liposome would be an efficient mucosal vaccine system for a serotype-independent universal nasal vaccine against pneumococcal infection.

The Dual Activity Responsible for the Elongation and Branching of ß-(1,3)-Glucan in the Fungal Cell Wall.

ß-(1,3)-Glucan, the major fungal cell wall component, ramifies through ß-(1,6)-glycosidic linkages, which facilitates its binding with other cell wall components contributing to proper cell wall assembly. Using Saccharomyces cerevisiae as a model, we developed a protocol to quantify ß-(1,6)-branching on ß-(1,3)-glucan. Permeabilized S. cerevisiae and radiolabeled substrate UDP-(14C)glucose allowed us to determine branching kinetics. A screening aimed at identifying deletion mutants with reduced branching among them revealed only two, the bgl2Δ and gas1Δ mutants, showing 15% and 70% reductions in the branching, respectively, compared to the wild-type strain. Interestingly, a recombinant Gas1p introduced ß-(1,6)-branching on the ß-(1,3)-oligomers following its ß-(1,3)-elongase activity. Sequential elongation and branching activity of Gas1p occurred on linear ß-(1,3)-oligomers as well as Bgl2p-catalyzed products [short ß-(1,3)-oligomers linked by a linear ß-(1,6)-linkage]. The double S. cerevisiae gas1Δ bgl2Δ mutant showed a drastically sick phenotype. An ScGas1p ortholog, Gel4p from Aspergillus fumigatus, also showed dual ß-(1,3)-glucan elongating and branching activity. Both ScGas1p and A. fumigatus Gel4p sequences are endowed with a carbohydrate binding module (CBM), CBM43, which was required for the dual ß-(1,3)-glucan elongating and branching activity. Our report unravels the ß-(1,3)-glucan branching mechanism, a phenomenon occurring during construction of the cell wall which is essential for fungal life.IMPORTANCE The fungal cell wall is essential for growth, morphogenesis, protection, and survival. In spite of being essential, cell wall biogenesis, especially the core ß-(1,3)-glucan ramification, is poorly understood; the ramified ß-(1,3)-glucan interconnects other cell wall components. Once linear ß-(1,3)-glucan is synthesized by plasma membrane-bound glucan synthase, the subsequent event is its branching event in the cell wall space. Using Saccharomyces cerevisiae as a model, we identified GH72 and GH17 family glycosyltransferases, Gas1p and Bgl2p, respectively, involved in the ß-(1,3)-glucan branching. The sick phenotype of the double Scgas1Δ bgl2Δ mutant suggested that ß-(1,3)-glucan branching is essential. In addition to ScGas1p, GH72 family ScGas2p and Aspergillus fumigatus Gel4p, having CBM43 in their sequences, showed dual ß-(1,3)-glucan elongating and branching activity. Our report identifies the fungal cell wall ß-(1,3)-glucan branching mechanism. The essentiality of ß-(1,3)-glucan branching suggests that enzymes involved in the glucan branching could be exploited as antifungal targets.

Attachment of class B CpG ODN onto DOTAP/DC-chol liposome in nasal vaccine formulations augments antigen-specific immune responses in mice.

BACKGROUND: To overcome infectious diseases, the development of mucosal vaccines would be an effective strategy, since mucosal surfaces are the entry site for most pathogens. In general, protein antigens show inherently poor immunogenicity when administered by the mucosal route. Therefore, co-administration of an appropriate mucosal adjuvant is required to exert immune responses toward pathogen-derived antigens effectively. However, the development of a safe and effective mucosal adjuvant system is still challenging. Although, recent studies reported that oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motifs (CpG ODNs) act as potent mucosal adjuvants and are useful in the formulation of nasal vaccines, there are some disadvantages. For instance, the administration of phosphorothioate (PS)-modified CpG ODNs can induce adverse systemic effects, such as splenomegaly, in a dose-dependent manner. Therefore, a reduced dose of CpG ODN might be crucial when used as vaccine adjuvant for clinical purposes. Therefore, we prepared a CpG ODN-loaded cationic liposome, and evaluated its mucosal adjuvant activity. RESULTS: We prepared a CpG ODN-loaded DOTAP/DC-chol liposome that was stable during our experiments, by mixing CpG ODNs and liposomes at an N/P ratio of 4. Further, we demonstrated that the attachment of class B CpG ODN to the DOTAP/DC-chol liposomes synergistically enhanced antigen-specific IgA production in the nasal area than that induced by CpG ODN and DOTAP/DC-chol liposomes alone. The endpoint titers were more than tenfolds higher than that induced by either single CpG ODN or single DOTAP/DC-chol liposomes. Additionally, although serum IgG1 responses (indicated as a Th2 response) remained unchanged for DOTAP/DC-chol liposomes and CpG ODN-loaded DOTAP/DC-chol liposomes, the CpG ODN-loaded DOTAP/DC-chol liposomes synergistically induced the production of serum IgG2a (indicated as a Th1 response) than that by the individual liposomes. CONCLUSIONS: We conclude that the advantage of using DOTAP/DC-chol liposome harboring CpG ODN is it induces both antigen-specific mucosal IgA responses and balanced Th1/Th2 responses. Therefore, such a combination enables us to resolve the adverse effects of using CpG ODNs (as a mucosal adjuvant) by reducing the overall dose of CpG ODNs. Further, the biodegradable and essentially non-antigenic nature of the liposomes makes it superior than the other existing mucosal adjuvants.

Intranasal Immunization with DOTAP Cationic Liposomes Combined with DC-Cholesterol Induces Potent Antigen-Specific Mucosal and Systemic Immune Responses in Mice.

Despite the progress made by modern medicine, infectious diseases remain one of the most important threats to human health. Vaccination against pathogens is one of the primary methods used to prevent and treat infectious diseases that cause illness and death. Vaccines administered by the mucosal route are potentially a promising strategy to combat infectious diseases since mucosal surfaces are a major route of entry for most pathogens. However, this route of vaccination is not widely used in the clinic due to the lack of a safe and effective mucosal adjuvant. Therefore, the development of safe and effective mucosal adjuvants is key to preventing infectious diseases by enabling the use of mucosal vaccines in the clinic. In this study, we show that intranasal administration of a cationic liposome composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl] (DC-chol) (DOTAP/DC-chol liposome) has a potent mucosal adjuvant effect in mice. Intranasal vaccination with ovalbumin (OVA) in combination with DOTAP/DC-chol liposomes induced the production of OVA-specific IgA in nasal tissues and increased serum IgG1 levels, suggesting that the cationic DOTAP/DC-chol liposome leads to the induction of a Th2 immune response. Additionally, nasal-associated lymphoid tissue and splenocytes from mice treated with OVA plus DOTAP/DC-chol liposome showed high levels of IL-4 expression. DOTAP/DC-chol liposomes also enhanced OVA uptake by CD11c+ dendritic cells in nasal-associated lymphoid tissue. These data demonstrate that DOTAP/DC-chol liposomes elicit immune responses via an antigen-specific Th2 reaction. These results suggest that cationic liposomes merit further development as a mucosal adjuvant for vaccination against infectious diseases.