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Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1β (IL-1β), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarify the roles of inositol-requiring enzyme 1 alpha (IRE1α), an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1β production in RPMs. In RPMs, CTB is incorporated into the ER and induces ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1α-deficient RPMs show a significant impairment of CT- or CTB-induced IL-1β production, indicating that IRE1α is required for CT- or CTB-induced IL-1β production in RPMs. This study demonstrates the critical roles of IRE1α in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages. [Display omitted] • Cholera toxin induces ER stress responses in tissue-resident macrophages • Cholera toxin reaches ER in a ganglioside GM1-dependent manner • IRE1α is required for cholera-toxin-induced IL-1β production in tissue resident macrophages Sasaki et al. find that IRE1α, an endoplasmic reticulum stress sensor, is required for cholera-toxin-induced interleukin-1β production in murine resident peritoneal macrophages. This study demonstrates the critical roles of IRE1α in activation of both the NLRP3 inflammasome and the pyrin inflammasome in tissue-resident macrophages. [ABSTRACT FROM AUTHOR]

Lung CD8+ memory T cells play central roles in protective immunity to respiratory viruses, such as influenza A virus (IAV). Here, we find that alveolar macrophages (AMs) function as antigen-presenting cells that support the expansion of lung CD8+ memory T cells. Intranasal antigen administration to mice subcutaneously immunized with antigen results in a rapid expansion of antigen-specific CD8+ T cells in the lung, which is dependent on antigen cross-presentation by AMs. AMs highly express interleukin-18 (IL-18), which mediates subsequent formation of CD103+CD8+ resident memory T (T RM) cells in the lung. In a mouse model of IAV infection, AMs are required for expansion of virus-specific CD8+ T cells and CD103+CD8+ T RM cells and inhibiting virus replication in the lungs during secondary infection. These results suggest that AMs instruct a rapid expansion of antigen-specific CD8+ T cells in lung, which protect the host from respiratory virus infection. [Display omitted] • Alveolar macrophages (AMs) cross-present antigen to CD8+ T cells in the lung • Antigen cross-presentation by AMs rapidly expands CD8+ T cells in the lung • AMs production of IL-18 mediates CD8+ resident memory T cell formation • AMs prevent influenza virus replication during recurrent infection Host defense against virus infection is provided by virus-specific CD8+ T cells. T. Kawasaki et al. show that antigen cross-presentation by alveolar macrophages induces a rapid expansion of antigen-specific CD8+ T cells and subsequent formation of CD8+ resident memory T cells to protect virus replication in the lungs. [ABSTRACT FROM AUTHOR]

Abstract: The effectiveness of influenza vaccines is still controversial, and the role of adjuvants in such vaccines is briefly reviewed in this paper. Inactivated whole virus vaccines may include components that function as adjuvants, meaning that additive adjuvants are often not required. MF59 and AS03 showed higher adjuvanticity than aluminum salts in several clinical studies. Recent research has suggested that immune cell recruitment is the main mechanism underlying adjuvant actions in general, and that aluminum salts induce this recruitment via inflammation at the injected site. The aspect of how oil-based adjuvants, such as MF59 and AS03, recruit immune cells remains to be clarified. [Copyright &y& Elsevier]

Recent accumulating evidence suggests that the human immune system possesses a variety of innate receptors that recognize, distinguish, and respond to viral infections and to vaccination. These include Toll-like receptors, C-type lectin receptors, RIG-I-like receptors, Nod-like receptors and possibly AIM2-like receptors. However, the precise mechanisms by which these receptors exert their critical roles in the induction of virus-specific adaptive immune responses have not been fully elucidated. In this review, we discuss recent advances in our understanding of the innate immune recognition of viruses and the differential connection to the adaptive immune responses induced by infection or vaccination, with a particular focus on the influenza virus. [ABSTRACT FROM AUTHOR]

During microbial infection and tissue injury, nucleic acids and their metabolites, such as nucleotides, nucleosides and uric acids, can be released from dying host cells and may modify immune responses. These nucleic acids and/or their metabolites are in fact recognized by specific host receptors, such as Toll-like receptors (TLRs), RIG-like receptors (RLRs) and NOD-like receptors (NLRs), purinergic receptors such as P2X and P2Y receptors, and adenosine receptors such as A2A receptors. The resultant responses may vary depending on the balance between immune responses to and metabolism of nucleic acids, thereby contributing not only to the host defense, but also to the homeostatic clearance of host dying cells, or even to deleterious autoimmune diseases. [Copyright &y& Elsevier]

Mammalian innate immunity possesses a distinct system to recognize aberrant DNA inside the cell. One class of DNA sensors is the Toll-like receptor 9, which is expressed in the specialized immune cells, binds to single-stranded DNA in the endosome to transmit cellular signaling through myeloid differentiation primary response protein 88 (MyD88). Another class of DNA sensors exists in the cytoplasm of most type of cells in the tissue, detecting double-stranded DNA to signal through TANK-binding kinase-1 (TBK1)-mediated type-I interferon production and apoptosis-associated speck-like protein containing a CARD (ASC)-mediated IL-1β secretion. Since DNA sensors have potential to recognize aberrant DNA of both self and nonself origin, their physiological roles in microbial infection, tissue damage, autoimmune diseases, and DNA-based therapeutic applications are being intensively investigated. [Copyright &y& Elsevier]

It has long been known that malaria infection causes host immune modulation by various mechanisms. However, the role of Toll-like receptors (TLRs) in mediating innate immune responses to parasite-derived components during the blood stages of malaria has only recently been described. TLRs might have an important role in pathogenesis during malaria infection, as supported by genetic analyses in mice and humans. Moreover, recent findings revealed that sporozoites can partially differentiate in lymph nodes and that liver stages induce the formation of previously unknown parasite-filled vesicles (merosomes) that could function as immune escape machinery. Elucidation of the mechanisms by which the host innate immune system responds to, and/or is manipulated by, Plasmodium infection will hopefully lead to discoveries of potential targets that will ultimately prevent and/or intervene in malaria infection. [Copyright &y& Elsevier]

DNA in microbes or host cells is normally sequestered from the immune system, and therefore inert, but becomes an active immunostimulatory molecule during infection or tissue damage. Recent evidence suggests that Toll-like receptor (TLR)9, currently the only known immune sensor for DNA, recognizes more diverse elements in its ligand than initially thought, and must cooperate with additional host factors to provoke an optimal innate immune response in the physiological environment. Moreover, the innate immune system possesses a TLR9-independent, as-yet-undefined intracellular recognition machinery of double-stranded DNA that induces type I interferons through distinct signaling pathways. TLR9-dependent and TLR9-independent immune recognition of DNA might play crucial roles in DNA-associated protective immunity and in pathological autoimmunity. [Copyright &y& Elsevier]

Toll-like receptors (TLRs) play a crucial role in innate immune response in mammals. Individual TLRs recognize microbial components that are conserved among pathogens and activate their signaling pathways. Each TLR has its own cascade of signaling pathway for exhibiting its specific responses through selective utilization of TIR domain-containing adaptors. Increasing evidence for roles of TLRs in various diseases provides us new insights for a basis of new therapies. In this review, we discuss the possibilities of therapeutics targeting TLRs in various diseases and explain potential problems associated with such approaches. [Copyright &y& Elsevier]

Dysregulation of the microbiome has been associated with development of complex diseases, such as obesity and diabetes. However, no method has been developed to control disease-associated commensal microbes. We investigated whether immunization with microbial antigens, using CpG oligodeoxynucleotides and/or curdlan as adjuvants, induces systemic antigen-specific IgA and IgG production and affects development of diseases in mice. C57BL/6 mice were given intramuscular injections of antigens (ovalbumin, cholera toxin B-subunit, or pneumococcal surface protein A) combined with CpG oligodeoxynucleotides and/or curdlan. Blood and fecal samples were collected weekly and antigen-specific IgG and IgA titers were measured. Lymph nodes and spleens were collected and analyzed by enzyme-linked immunosorbent assay for antigen-specific splenic T-helper 1 cells, T-helper 17 cells, and memory B cells. Six weeks after primary immunization, mice were given a oral, nasal, or vaginal boost of ovalbumin; intestinal lamina propria, bronchial lavage, and vaginal swab samples were collected and antibodies and cytokines were measured. Some mice were also given oral cholera toxin or intranasal Streptococcus pneumoniae and the severity of diarrhea or pneumonia was analyzed. Gnotobiotic mice were gavaged with fecal material from obese individuals, which had a high abundance of Clostridium ramosum (a commensal microbe associated with obesity and diabetes), and were placed on a high-fat diet 2 weeks after immunization with C ramosum. Intestinal tissues were collected and analyzed by quantitative real-time polymerase chain reaction. Serum and fecal samples from mice given injections of antigens in combination with CpG oligodeoxynucleotides and curdlan for 3 weeks contained antigen-specific IgA and IgG, and splenocytes produced interferon-gamma and interleukin 17A. Lamina propria, bronchial, and vaginal samples contained antigen-specific IgA after the ovalbumin boost. This immunization regimen prevented development of diarrhea after injection of cholera toxin, and inhibited lung colonization by S pneumoniae. In gnotobiotic mice colonized with C ramosum and placed on a high-fat diet, the mice that had been immunized with C ramosum became less obese than the nonimmunized mice. Injection of mice with microbial antigens and adjuvant induces antigen-specific mucosal and systemic immune responses. Immunization with S pneumoniae antigen prevented lung infection by this bacteria, and immunization with C ramosum reduced obesity in mice colonized with this microbe and placed on a high-fat diet. This immunization approach might be used to protect against microbe-associated disorders of intestine. [ABSTRACT FROM AUTHOR]

Innate immune sensing of nucleic acids derived from invading pathogens or tumor cells via pattern recognition receptors is crucial for mounting protective immune responses against infectious disease and cancer. Recently, discovery of tremendous amounts of nucleic acid sensors as well as identification of natural and synthetic ligands for these receptors revealed the potential of adjuvants targeting nucleic acid sensing pathways for designing efficacious vaccines. Especially, current data indicated that unique adjuvants targeting TLR9 and stimulator of interferon genes (STING)-dependent cytosolic nucleic acid sensing pathways along with the combinations of already existing adjuvants are promising candidates for this purpose. Here, we review current vaccine adjuvants targeting nucleic acid sensors and their modes of action. [ABSTRACT FROM AUTHOR]

Background: We reported previously that serum levels of IL-33 are significantly increased in patients with allergic rhinitis (AR). However, very little is known about the role of IL-33 for the development of AR. Objective: We thought to develop a novel murine model of ragweed pollen–specific AR and examined the pathologic role for ragweed-induced IL-33 in the development of AR manifestation using IL-33–deficient (il33 −/− ) mice. Methods: Ragweed-immunized and ragweed-challenged mice were examined for early- and late-phase nasal responses. IL-33 protein expression in the nasal epithelial cells of the AR murine model and patients with AR were assessed by using confocal microscopy. Results: After nasal challenge with ragweed pollen, ragweed-immunized wild-type mice manifested early-phase (sneezing) and late-phase (eosinophilic and basophilic accumulation) responses. In contrast, il33 −/− and FcεRI−/− mice did not have both early- and late-phase AR responses. IL-33 protein was constitutively expressed in the nucleus of nasal epithelial cells and was promptly released into nasal fluids in response to nasal exposure to ragweed pollen. In human subjects we revealed constitutive expression of IL-33 protein in the nasal epithelial cells of healthy control subjects and downregulated expression of IL-33 protein in inflamed nasal epithelial cells of patients with AR. IL-33–stimulated mast cells and basophils contributed to the early- and late-phase AR manifestation through increasing histamine release and production of chemoattractants for eosinophils/basophils, respectively. Conclusions: Ragweed pollen–driven endogenous IL-33 contributed to the development of AR responses. IL-33 might present an important therapeutic target for the prevention of AR. [ABSTRACT FROM AUTHOR]

• We propose the revision of the infectious disease vaccine clinical trial guidelines. • New vaccines should be evaluated for the protective efficacy with multiregional clinical trials. • In non-major subjects, immunogenicity should be compared with that of main subjects. The development of vaccines against infectious diseases requires a different approach from that of therapeutics, because vaccines are inoculated into healthy individuals and have a preventive effect by activating the immunity of the inoculated human. In Japan, "The Guideline for Clinical Trials of Vaccines for the Prevention of Infectious Diseases" was published in 2010 before changes occurred in the vaccine development environment in Japan, such as the introductions of foreign vaccines and simultaneous global development. This study aimed to identify current challenges in vaccine development through a questionnaire-based survey of pharmaceutical companies in Japan and by comparing the domestic and international guidelines and surveying review reports of 35 vaccines approved in Japan between April 2010 and December 2020. Identified challenges included the requirement for protective efficacy trials, efficacy evaluation of combination vaccines, development of multiregional and foreign clinical trials, and immunization of older adults and immunocompromised patients. We propose that new vaccines against infectious diseases should be evaluated for the protective efficacy, preferably through multiregional clinical trials. Additionally, differences in the incidence of infectious diseases or in epidemic virus strains between regions may affect the trials, when multiregional clinical trials are conducted, but immunogenicity-based studies can be conducted if a correlation between protective efficacy and immunogenicity has been established. We suggest that licensed combination vaccines can be used as comparators when an antigen is added to a licensed combination vaccine. We also proposed that the efficacy of a vaccine in non-major subjects, such as older adults or immunocompromised patients could be evaluated by comparing immunogenicity in major subjects with the confirmed protective effects of the vaccine. It is expected that these revisions will lead to the rapid advancement of vaccine development, which should contribute to the improvement of public health. [ABSTRACT FROM AUTHOR]

• Hydroxypropyl-β-cyclodextrin (HP-β-CyD) exhibits adjuvant function. • A trial was run using an influenza vaccine adjuvanted with HP-β-CyD (Flu/CyD-vac) • Flu/CyD-vac is safe and sufficiently immunogenic despite a reduced antigen dose. • Hydroxypropyl-β-cyclodextrin is a promising vaccine adjuvant candidate. Hydroxypropyl-β-cyclodextrin (HP-β-CyD), an oligosaccharide used as an excipient in pharmaceutical preparation, was recently reported to function as a vaccine adjuvant to co-administered antigens. In this study, we investigated the safety and immunogenicity of a seasonal influenza vaccine adjuvanted with HP-β-CyD (FluCyD-vac) in healthy adults compared with those of a standard seasonal influenza vaccine (Flu-vac). We conducted a single-blinded randomized phase 1 clinical trial study, and used two quadrivalent split seasonal influenza vaccines: FluCyD-vac containing 9 μg of HA/strain and 20% w/v of HP-β-CyD, and Flu-vac containing 15 μg of hemagglutinin (HA)/strain only. All participants were randomly assigned to receive a single dose of Flu/CyD-vac or Flu-vac at a ratio of 2:1. We assessed solicited and unsolicited adverse events (AEs) and immune responses using hemagglutination inhibition (HI) titers. In addition, we assessed T-cell function in peripheral blood mononuclear cells (PBMCs), after stimulation with HA vaccine strains, using flow cytometry. Among 36 healthy volunteers enrolled in the study (FluCyD-vac, n = 24; Flu-vac, n = 12), FluCyD-vac was well tolerated. Most of the solicited AEs were mild local skin reactions at the injection site. No serious AEs were reported in either group. HI titers 21 days after vaccination with FluCyD-vac were comparable with those of Flu-vac and sufficient to meet international criteria, despite reduced HA antigen doses. When PBMCs were stimulated with the four HA antigens in the vaccine, tumor necrosis factor (TNF)-α-producing CD4+ T cells were enhanced in the FluCyD-vac group. FluCyD-vac was well-tolerated and immunogenic, despite containing 40% less HA antigens than Flu-vac. This study showed that HP-β-CyD is a potentially safe, novel adjuvant for human influenza vaccine. Clinical trial registry: UMIN000028530. [ABSTRACT FROM AUTHOR]

• We identified the current challenges in the development of vaccines and propose revision of the guidelines for the non-clinical studies of vaccines. • The results of repeated-dose toxicity studies can be used to decide whether safety pharmacology studies are required. • The studies to evaluate toxicity due to systemic effects may not be necessary for both intramuscular and subcutaneous administration. • Women of childbearing potential could be included in clinical trials with appropriate pregnancy avoidance prior to the reproductive toxicity studies. The efficacy and safety of vaccines for the prevention of infectious diseases are mostly evaluated based on the induction of an immune response against antigens, and do not necessarily depend on the dose administered. Therefore, there are some specific aspects that need to be considered in the development of vaccines and have been described in "The Guidelines for the non-clinical studies of vaccines for the prevention of infectious disease" in Japan. Recent changes in the vaccine development field, such as the introduction of vaccines developed overseas in Japan and vaccine development on a global scale have increased the need for revision of these guidelines. In this study, we identified the current challenges in the development of vaccines through comparison of Japanese and international guidelines. We conducted a questionnaire-based survey of pharmaceutical industries in Japan, and found issues related to non-clinical studies, such as the necessity of safety pharmacology studies and repeated-dose toxicity studies for each route of administration. We examined international guidelines on these issues as well as review reports by regulatory authorities, and determined that the results of repeated-dose toxicity studies can be used to decide whether safety pharmacology studies are required, and that studies to evaluate toxicity due to systemic effects may not be necessary for both intramuscular and subcutaneous administration. We propose revision of the guidelines for the non-clinical studies of vaccines in Japan taking international harmonizaion into account. We expected that the revised guidelines will promote smooth and rational vaccine development. [ABSTRACT FROM AUTHOR]

Abstract: IgG subclass antibody responses are not fully understood. Alum-adjuvanted H5N1whole virion inactivated vaccine (WIV), a genetically reassortant vaccine seed strain originating from H5N1/A/Vietnam/1194/2004 and PR-8, induced significantly stronger antibody responses in neutralizing antibodies in children. In this report, IgG subclass antibody responses were investigated, and most serum samples were positive for IgG1 antibody before immunization. A significant response (more than 4-fold increase) of IgG1 antibody was observed in 67/193 (34.7%) and that of gG4 antibodies in 42/193(21.8%). Children <4 years of age showed a significant increase in IgG subclass antibodies but those ≥4 years showed lower responses. Alum- adjuvanted H5N1WIV induced an efficient immune response in young children especially <4 years. [Copyright &y& Elsevier]

• BK-SE36/CpG is a candidate blood-stage malaria vaccine against P. falciparum. • In healthy Japanese adults, BK-SE36/CpG has an acceptable safety profile. • No indication of autoimmune condition was observed in BK-SE36/CpG vaccinees. • Two dosing, 21-days apart, of full-dose BK-SE36/CpG was highly immunogenic. BK-SE36 is blood-stage malaria vaccine candidate that is undergoing clinical trials. Here, the safety and immunogenicity of BK-SE36 with a novel adjuvant, CpG-ODN(K3) (thus, BK-SE36/CpG) was assessed in a phase 1a trial in Japan. An investigator-initiated, randomised, single-blind, placebo-controlled, dose-escalation study was conducted at Osaka University Hospital with 26 healthy malaria naïve Japanese male adults. The trial was conducted in two stages: Stage/Group 1, half-dose (n = 7 for BK-SE36/CpG and n = 3 for control) and Stage/Group 2, full-dose (n = 11 for BK-SE36/CpG and n = 5 for control). There were two intramuscular vaccinations 21 days apart for both half-dose (0.5 ml: 50 µg SE36 + 500 µg aluminum + 500 µg K3) and full-dose (1.0 ml: 100 µg SE36 + 1000 µg aluminum + 1000 µg K3). A one-year follow-up was done to monitor changes in autoimmune markers and vaccine-induced antibody response. BK-SE36/CpG was well tolerated. Vaccination site reactions were similar to those observed with BK-SE36. During the trial and follow-up period, no subject had clinical evidence of autoimmune disease. The full-dose group had significantly higher titres than the half-dose group (Student's t -test, p = 0.002) at 21 days post-second vaccination. Antibody titres remained above baseline values during 12 months of follow-up. The vaccine induced antibody was mostly composed of IgG1 and IgM, and recognised epitopes close to the polyserine region located in the middle of SE36. BK-SE36/CpG has an acceptable safety profile. Use of CpG-ODN(K3) greatly enhanced immunogenicity in malaria naïve Japanese adults when compared to BK-SE36 alone. The utility of BK-SE36/CpG is currently under evaluation in a malaria endemic setting in West Africa. Trial Registration. JMACCT Clinical Trial Registry JMA-IIA00109. [ABSTRACT FROM AUTHOR]

Vaccines are administered to healthy humans, including infants, so the safety and efficacy must be very high. Therefore, evaluating vaccine safety in preclinical and clinical studies, according to World Health Organization guidelines, is crucial for vaccine development and clinical use. A change in the route of administration is considered to alter a vaccine’s immunogenicity. Several adjuvants have also been developed and approved for use in vaccines. However, the addition of adjuvants to vaccines may cause unwanted immune responses, including facial nerve paralysis and narcolepsy. Therefore, a more accurate and comprehensive strategy must be used to develope next-generation vaccines for ensuring vaccine safety. Previously, we have developed a system with which to evaluate vaccine safety in rats using a systematic vaccinological approach and 20 marker genes. In this study, we developed a safety evaluation system for nasally administered influenza vaccines and adjuvanted influenza vaccines using these marker genes. Expression of these genes increased dose-dependent manner when mice were intranasally administered the toxicity reference vaccine. When the adjuvant CpG K3 or a CpG-K3-combined influenza vaccine was administered intranasally, marker gene expression increased in a CpG-K3-dose-dependent way. A histopathological analysis indicated that marker gene expression correlated with vaccine- or adjuvant-induced phenotypic changes in the lung and nasal mucosa. We believe that the marker genes expression analyses will be useful in preclinical testing, adjuvant development, and selecting the appropriate dose of adjuvant in nasal administration vaccines. [ABSTRACT FROM AUTHOR]

Intranasal vaccination with inactivated influenza viral antigens is an attractive and valid alternative to currently available influenza (flu) vaccines; many of which seem to need efficient and safe adjuvant, however. In this study, we examined whether hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used pharmaceutical excipient to improve solubility and drug delivery, can act as a mucosal adjuvant for intranasal flu vaccines. We found that intranasal immunization of mice with hemagglutinin split- as well as inactivated whole-virion influenza vaccine with HP-β-CD resulted in secretion of antigen-specific IgA and IgGs in the airway mucosa and the serum as well. As a result, both HP-β-CD adjuvanted-flu intranasal vaccine protected mice against lethal challenge with influenza virus, equivalent to those induced by experimental cholera toxin-adjuvanted ones. Of note, intranasal use of HP-β-CD as an adjuvant induced significantly lower antigen-specific IgE responses than that induced by aluminum salt adjuvant. These results suggest that HP-β-CD may be a potent mucosal adjuvant for seasonal and pandemic influenza vaccine. [ABSTRACT FROM AUTHOR]

Although adjuvants are a “must-have” component of successful vaccines, there are very few adjuvants licensed for use in humans, there is therefore an urgent need to develop new and safer adjuvants. Synthetic hemozoin (sHZ), a chemical analog of hemozoin which is produced by the malaria parasite, exhibits a potent adjuvant effect which enhances antigen-specific immune responses to vaccines. The potency of sHZ adjuvanticity is not limited to malaria specific vaccines, it has also been demonstrated to be effective in influenza and dog allergy models. While the synthesis of uniformly sized sHZ with consistent characteristics has proven difficult, we have recently successfully optimized the manufacture of sHZ product with an optimal adjuvant effect. Here, we summarize recent developments on the adjuvant properties of optimized sHZ adjuvant, including its good laboratory practice (GLP) non-clinical safety profile in animals. These studies ensure the safety of optimized sHZ product to be readily used as vaccine adjuvant beforehand in veterinary medicine. [ABSTRACT FROM AUTHOR]

Background It has been clinically demonstrated that intradermal (ID) vaccines have a potential to confer a superior immunogenic profile compared to intramuscular (IM) or subcutaneous (SC) vaccines. In terms of distribution of a vaccine antigen depending on the administration routes, at least two independent immunogenic pathways of the vaccines have been proposed: (1) the antigen recognition by the immune cells present at the vaccine-administered site and (2) the antigen recognition by the lymph node (LN)-resident immune cells through the lymphatic flow from the vaccine-administered site after the antigen is directly delivered into the draining LNs. Objective In order to clarify the key components for the immunogenic pathway of the ID vaccine, the correlation between the kinetics of the antigen distribution to the draining LNs and antibody responses to the antigen were evaluated. Methods We compared the antibody responses in the groups with by surgical removal of the administration site immediately after the ID administration, and by surgical removal of the draining LNs before the ID administration. Results The results suggested that the efficient and direct antigen delivery to the draining LNs plays an important role in the antibody responses to the ID vaccine. Indeed, it was confirmed that the direct administration into the draining LNs with the antigen elicited comparable levels of the antibody responses with the ID vaccine. At the cellular level, it was shown that the LN-resident immune cells such as B cells, dendritic cells, and macrophages including medullary macrophages and subcapsular sinus macrophages interacting with the antigens following the ID administration. Finally, we demonstrated by immunofluorescence analysis that the lymphatic vessels are more diffusely distributed in the dermis as compared with the subcutaneous area and muscle. Conclusion The results of the present study suggested that the skin is an optimal tissue to facilitate the vaccine antigen access to the draining LNs, which is an important immunogenic pathway of the ID vaccine. Further elucidation of regulatory mechanisms underlying such an immunogenic pathway of the ID vaccine would provide us with elements for the development of novel adjuvants and devices to enhance the immunogenicity of the ID vaccines. [ABSTRACT FROM AUTHOR]

Various particles such as Alum or silica are known to act as an adjuvant if co-administered with vaccine antigens. Several reports have demonstrated that the adjuvanticity is strongly affected by the physicochemical properties of particles such as the size, shape and surface charge, although the required properties and its relationship to the adjuvanticity are still controversial. Hydroxyapatite particle (HAp) composed of calcium phosphate has been shown to work as adjuvant in mice. However, the properties of HAp required for the adjuvanticity have not been fully characterized yet. In this study, we examined the role of size or shape of HAps in the antibody responses after immunization with antigen. HAps whose diameter ranging between 100 and 400 nm provided significantly higher antibody responses than smaller or larger ones. By comparison between sphere and rod shaped HAps, rod shaped HAps induced stronger inflammasome-dependent IL-1β production than the sphere shaped ones in vitro . However, sphere- and rod-shaped HAp elicited comparable antibody response in WT mice. Vice versa, Nlrp3 −/− , Asc −/− or Caspase1 −/− mice provided comparable level of antibody responses to HAp adjuvanted vaccination. Collectively, our results demonstrated that the size rather than shape is a more critical property, and IL-1β production via NLRP3 inflammasome is dispensable for the adjuvanticity of HAps in mice. [ABSTRACT FROM AUTHOR]

RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, includingAGO2and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells. [ABSTRACT FROM AUTHOR]

An increase in the appearance of nonvaccine serotypes in both children and adults with invasive pneumococcal disease (IPD) after introduction of pneumococcal conjugate vaccine represents a limitation of this vaccine. In this study, we generated three recombinant pneumococcal surface protein A (PspA) proteins comprising PspA families 1 and 2, and we examined the reactivity of antisera raised in mice immunized with a PspA fusion protein in combination with CpG oligonucleotides plus aluminum hydroxide gel. The protective effects of immunization with PspA fusion proteins against pneumococcal challenge by strains with five different PspA clades were also examined in mice. Flow cytometry demonstrated that PspA3+2-induced antiserum showed the greatest binding of PspA-specific IgG to all five challenge strains with different clades. PspA2+4- or PspA2+5-induced antiserum showed the lowest binding of PspA-specific IgG to clade 3. Immunization with PspA3+2 afforded significant protection against pneumococcal challenge by five strains with different clades in mice, but immunization with PspA2+4 or PspA2+5 failed to protect mice from pneumococcal challenge by strains with clades 1 and 3. The binding of PspA-specific IgG in antisera raised by three PspA fusion proteins was examined in 68 clinical isolates from adult patients with IPD. Immunization of mice with PspA3+2-induced antiserum with a high binding capacity for clinical isolates expressing clades 1–4, but not clade 5. Our results suggest that the PspA3+2 vaccine has an advantage over the PspA2+4 or PspA2+5 vaccine in terms of a broad range of cross-reactivity with clinical isolates and cross-protection against pneumococcal challenge in mice. [ABSTRACT FROM AUTHOR]

Because vaccination is an effective means to protect humans from influenza viruses, extensive efforts have been made to develop not only new vaccines, but also for new adjuvants to enhance the efficacy of existing inactivated vaccines. Here, we examined the adjuvanticity of synthetic hemozoin, a synthetic version of the malarial by-product hemozoin, on the vaccine efficacy of inactivated whole influenza viruses in a mouse model. We found that mice immunized twice with hemozoin-adjuvanted inactivated A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1) virus elicited higher virus-specific antibody responses than did mice immunized with non-adjuvanted counterparts. Furthermore, mice immunized with hemozoin-adjuvanted inactivated viruses were better protected from lethal challenge with influenza viruses than were mice immunized with non-adjuvanted inactivated vaccines. Our results show that hemozoin improves the immunogenicity of inactivated influenza viruses, and is thus a promising adjuvant for inactivated whole virion influenza vaccines. [ABSTRACT FROM AUTHOR]

Highlights: [•] sHZ enhanced the immunogenicity of trivalent SV. [•] SV/sHZ did not show pyrogenicity after immunization. [•] sHZ enhanced the protective efficacy of trivalent SV against influenza B virus infection. [Copyright &y& Elsevier]

Abstract: Alum-adjuvanted H5 whole virion inactivated vaccine (WIV) was licensed for adults in Japan but induced marked febrile reactions with significantly stronger antibody responses in children. In this study, the mechanisms behind the different responses were investigated. Lymphocytes were obtained from 25 healthy subjects who were not immunized with H5 vaccine, to examine the innate immune impact of the various vaccine formulations, analyzing the cytokine production profile stimulated with alum adjuvant alone, alum-adjuvanted H5 WIIV, plain H5 WIV, and H5 split vaccine. Alum adjuvant did not induce cytokine production, but H5 split induced IFN-γ and TNF-α. H5 WIV induced IL-6, IL-17, TNF-α, MCP-1, IFN-γ, and IFN-α. An extremely low level of IL-1β was produced in response to H5 WIV, and alum-adjuvanted H5 WIV enhanced IL-1β production, with similar levels of other cytokines stimulated with H5 WIV. Enhanced production of cytokines induced by alum-adjuvanted H5 WIV may be related to the higher incidence of febrile reactions with stronger immune responses in children but it should be further investigated why efficient immune responses with febrile illness were observed only in young children. [Copyright &y& Elsevier]

Abstract: A devastating disease spread by mosquitoes with high-efficiency, malaria imposes an enormous burden for which no licensed vaccine currently exists. Although the genome complexity of the parasite has made vaccine development tenuous, an effective malaria vaccine would be a valuable tool for control, elimination and eventual eradication. The Plasmodium serine repeat antigen 5 (SERA5) is an abundant asexual blood stage antigen that does not show any antigenic variation and exhibits limited polymorphism, making it a suitable vaccine candidate. Identified by comparing the IgG status of people in endemic areas with protective immunity and those with malaria symptoms, the vaccine potential of the N-terminal domain of Plasmodium falciparum SERA5 is also strongly supported by experimental data and immune responses both measured in vitro and in animal challenge models. The current understanding of SERA5 will be presented, particularly in relation to its path towards clinical development. The review highlights lessons learned and sorts out issues upon which further research efforts are needed. [Copyright &y& Elsevier]

Abstract: Effective pneumococcal vaccines are required for preventing secondary bacterial pneumonia, a life-threatening condition, during epidemics of influenza. We examined whether nasal administration of a low dose of pneumococcal surface protein A (PspA) plus polyinosinic–polycytidylic acid (poly(I:C)) could protect against a fatal secondary pneumococcal pneumonia after influenza A virus infection in mice. PspA-specific IgG but not IgA level was higher in the airways and blood of mice nasally administered a low dose of PspA plus poly(I:C) than in mice nasally administered PspA alone or poly(I:C) alone. Binding of PspA-specific IgG increased C3 deposition on the bacterial surface. The survival rate during secondary infection was higher in mice immunized with PspA plus poly(I:C) than in mice immunized with poly(I:C) alone. The significant reduction in bacterial density in the lung and blood was associated with increased survival of immunized mice with secondary pneumonia. Passive transfer of sera from mice immunized with PspA plus poly(I:C) increased the survival of mice infected with secondary pneumonia. Our data suggest that an intranasal PspA vaccine has promising protective effects against secondary pneumonia after influenza and that PspA-specific IgG plays a critical role in this protection. [Copyright &y& Elsevier]

Abstract: An effective malaria vaccine is a public health priority. Proteins expressed during the blood-stage of the parasite life cycle have been proposed as good vaccine candidates. No such blood-stage vaccine, however, is available against Plasmodium falciparum, the deadliest Plasmodium species. We show here that P. falciparum serine repeat antigen 5 (SERA5) is a potential vaccine immunogen. We have constructed a new recombinant molecule of SERA5, namely SE36, based on previously reported SE47′ molecule by removing the serine repeats. Epidemiological study in the holo-endemic population of Solomon Islands shows highly significant correlation of sero-conversion and malaria protective immunity against this antigen. Animal experiments using non-human primates, and a human phase 1a clinical trial assessed SE36 vaccine immunogenicity. Vaccination of squirrel monkeys with SE36 protein and aluminum hydroxyl gel (SE36/AHG) conferred protection against high parasitemia and boosted serum anti-SE36 IgG after P. falciparum parasite challenge. SE36/AHG was highly immunogenic in chimpanzees, where serum anti-SE36 IgG titers last more than one year. Phase 1a clinical trial (current controlled trials, ISRCTN78679862) demonstrated the safety and immunogenicity of SE36/AHG with 30 healthy adults and 10 placebo controls. Three subcutaneous administrations of 50 and 100μg dose of SE36/AHG were well-tolerated, with no severe adverse events; and resulted in 100% sero-conversion in both dose arms. The current research results for SE36/AHG provide initial clinical validation for future trials and suggest clues/strategies for further vaccine development. [ABSTRACT FROM AUTHOR]

Abstract: To develop an effective nasal vaccine for Streptococcus pneumoniae, the effects of a panel of Toll-like receptor (TLR) agonists in combination with pneumococcal surface protein A (PspA) on induction of PspA-specific antibodies and bacterial clearance were compared in mice. Mice were nasally immunized with 10μg of TLR agonist (TLR 2–4 and 9) and 2.5μg of PspA once per week for 3 weeks. Significantly increased levels of PspA-specific immunoglobulin G (IgG) and IgA in the airways and PspA-specific IgG in plasma were found in mice administered PspA plus each TLR agonist, compared with mice administered PspA alone. In a sub-lethal pneumonia model using a serotype 3 pneumococcal strain, bacterial density in the lungs of mice was significantly reduced in mice administered PspA plus each TLR agonist, compared with mice administered either PspA alone or phosphate-buffered saline alone 3h after bacterial challenge. Similarly, enhanced bacterial clearance was found in the nasopharynx of mice administered PspA plus each TLR agonist 1 day after infection with a serotype 19F strain. Our data suggest that PspA-specific antibody induced by nasal immunization with PspA plus TLR agonist is capable of reducing the bacterial load in both the nasopharynx and lungs after challenge with pneumococci with different serotypes. Despite the skewed Th1/Th2 immune responses, the effects of nasal immunization with PspA plus each TLR agonist on bacterial clearances from the lungs 3h after infection and from nasopharynx 1 day after infection in mice were equivalent. [Copyright &y& Elsevier]

Unmethylated CpG motifs present in bacterial DNA rapidly trigger an innate immune response characterized by the activation of Ig- and cytokine-secreting cells. Synthetic oligonucleotides (ODNs) containing CpG motifs mimic this activity, triggering monocytes to proliferate, secrete and/or differentiate. Analysis of hundreds of novel ODNs led to the identification of two structurally distinct classes of CpG motif that differentially activate human monocytes. ODNs of the “K”-type interact with Toll-like receptor 9 and induce monocytes to proliferate and secrete IL-6. In contrast, “D”-type ODNs trigger monocytes to differentiate into mature dendritic cells. [Copyright &y& Elsevier]