Booster vaccination using bivalent DS-5670a/b is safe and immunogenic against SARS-CoV-2 variants in children aged 5-11 years: a phase 2/3, randomized, active-controlled study.

Background: DS-5670 is a messenger ribonucleic acid (mRNA) vaccine platform targeting the receptor-binding domain (RBD) of the spike protein derived from severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Booster vaccination against coronavirus disease 2019 (COVID-19) with monovalent DS-5670a (incorporating mRNA encoding the RBD from the original SARS-CoV-2 strain) or bivalent DS-5670a/b (original and omicron BA.4-5 RBD antigens) is effective and safe in adults. Data from a phase 2/3 active-controlled, non-inferiority, pediatric study evaluating a third booster dose of DS-5670a/b are reported here. Methods: Children aged 5-11 years who had completed the two-dose primary vaccination series with monovalent BNT162b2 (original strain) at least 3 months prior to enrolment were randomly assigned to receive DS-5670a/b (20 µg of mRNA) or bivalent BNT1 62b2 (original/omicron BA.4-5; 10 µg of mRNA) on Day 1. The primary efficacy endpoint was blood neutralization geometric mean titer (GMT) against SARS-CoV-2 (omicron variant BA.5.2.1) and immune response rate (≥ 4-fold increase in post-vaccination circulating anti-SARS-CoV-2 neutralizing activity) on Day 29. Results: Among evaluable participants (DS-5670a/b, n = 74; bivalent BNT162b2, n = 75), the adjusted GMT ratio of DS-5670a/b to bivalent BNT162b2 on Day 29 was 1.636 (95% CI, 1.221, 2.190). Immune response rates were ≥ 89% with both study vaccines; adjusted difference 2.6% (95% CI, -7.8, 13.8). The prespecified non-inferiority margins were exceeded, and the study met the primary endpoint. DS-5670a/b also demonstrated broad neutralization activity across recent omicron sublineages and no cases of COVID-19 between Days 8-29 post-administration were reported. There were no novel safety concerns in the pediatric population at data cut-off. Conclusions: Bivalent DS-5670a/b was non-inferior to bivalent BNT162b2 in terms of immunogenicity, and had a manageable safety profile, when administered as a heterologous booster in children aged 5-11 years. Clinical trial registration: https://jrct.niph.go.jp/, identifier jRCT2031220665.

An mRNA vaccine encoding the SARS-CoV-2 receptor-binding domain protects mice from various Omicron variants.

Here, we assessed the efficacy of a lipid nanoparticle-based mRNA vaccine candidate encoding the receptor-binding domain (LNP-mRNA-RBD) in mice. Mice immunized with LNP-mRNA-RBD based on the ancestral strain (ancestral-type LNP-mRNA-RBD) showed similar cellular responses against the ancestral strain and BA.5, but their neutralizing activity against BA.5 was lower than that against the ancestral strain. The ancestral-type LNP-mRNA-RBD protected mice from the ancestral strain or BA.5 challenge; however, its ability to reduce the viral burdens after BA.5 challenge was limited. In contrast, immunization with bivalent LNP-mRNA-RBD consisting of the ancestral-type and BA.4/5-type LNP-mRNA-RBD or monovalent BA.4/5-type LNP-mRNA-RBD elicited robust cellular responses, as well as high and moderate neutralizing titers against BA.5 and XBB.1.5, respectively. Furthermore, the vaccines containing BA.4/5-type LNP-mRNA-RBD remarkably reduced the viral burdens following BA.5 or XBB.1.5 challenge. Overall, our findings suggest that LNP-mRNA-RBD is effective against SARS-CoV-2 infection.

DS-5670a, a novel mRNA-encapsulated lipid nanoparticle vaccine against severe acute respiratory syndrome coronavirus 2: Results from a phase 2 clinical study.

BACKGROUND: DS-5670a is a vaccine candidate for coronavirus disease 2019 (COVID-19) harnessing a novel modality composed of messenger ribonucleic acid (mRNA) encoding the receptor-binding domain (RBD) from the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encapsulated in lipid nanoparticles. Here, we report the safety, immunogenicity, and pharmacokinetic profile of DS-5670a from a phase 2 clinical trial in healthy adults who were immunologically naïve to SARS-CoV-2. METHODS: The study consisted of an open-label, uncontrolled, dose-escalation part and a double-blind, randomized, uncontrolled, 2-arm, parallel-group part. A total of 80 Japanese participants were assigned to receive intramuscular DS-5670a, containing either 30 or 60 µg of mRNA, as two injections administered 4 weeks apart. Safety was assessed by characterization of treatment-emergent adverse events (TEAEs). Immunogenicity was assessed by neutralization titers against SARS-CoV-2, anti-RBD immunoglobulin (Ig)G levels, and SARS-CoV-2 spike-specific T cell responses. Plasma pharmacokinetic parameters of DS-5670a were also evaluated. RESULTS: Most solicited TEAEs were mild or moderate with both the 30 and 60 µg mRNA doses. Four participants (10 %) in the 60 µg mRNA group developed severe redness at the injection site, but all cases resolved without treatment. There were no serious TEAEs and no TEAEs leading to discontinuation. Humoral immune responses in both dose groups were greater than those observed in human convalescent serum; the 60 µg mRNA dose produced better responses. Neutralization titers were found to be correlated with anti-RBD IgG levels (specifically IgG1). DS-5670a elicited antigen-specific T helper 1-polarized cellular immune responses. CONCLUSIONS: The novel mRNA-based vaccine candidate DS-5670a provided favorable immune responses against SARS-CoV-2 with a clinically acceptable safety profile. Confirmatory trials are currently ongoing to evaluate the safety and immunogenicity of DS-5670a as the primary vaccine and to assess the immunogenicity when administered as a heterologous or homologous booster. TRIAL REGISTRY: https://jrct.niph.go.jp/latest-detail/jRCT2071210086.

Fever Responses Are Enhanced with Advancing Age during Respiratory Syncytial Virus Infection among Children under 24 Months Old.

The most important risk factor for severe respiratory syncytial virus (RSV) infection is considered young age due to the immature immune system. The risk at young age is reported greater for RSV than for other respiratory infectious agents. Based on the strong association between young age and severity of RSV infection due to immature immunity, we aimed to assess whether there were any age-related differences in fever responses, as one clinical aspect of the immune response. In our observational study over two seasons (2014-2015 and 2015-2016), daily body temperatures of children under 3 years old with RSV infection were recorded from the first medical visit during the acute phase to defervescence. The body temperature records were analyzed among 171 children of four age groups (< 6, < 12, < 24 and ≥ 24 months), in terms of fever development, degrees of fever onset, the highest fever during the period, and fever duration. There were 54 patients in the group of < 6 months, 41 in the group of < 12 months, 58 in the group of < 24 months, and 18 in the group of ≥ 24 months. We thus found the correlation between age and fever responses under 24 months old; namely, the more the age advanced, the more frequently high and prolonged fever was experienced. Importantly, infants under 6 months old tend to show the suppressed fever responses. In conclusion, young infants with reduced fever response during RSV infection do not implicate less severity and needs attentive management.

Age-Specific Profiles of Antibody Responses against Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is one of the most prevalent causative agents of lower respiratory tract infections worldwide, especially in infants around 3 to 4months old. Infants at such a young age have maternally-transferred passive antibodies against RSV but do not have active immune systems efficient enough for the control of RSV infection. In order to elucidate age-specific profiles of immune responses against RSV protection, antibody responses were examined by using blood samples in both acute and convalescent phases obtained from child patients and adult patients. In addition to the serum neutralization activity, antibody responses to the RSV fusion protein (F protein) were dissected by analyzing levels of total IgG, IgG subclasses, the binding stability, and the levels of antibody for the neutralization epitopes. It was suggested that children's antibody responses against RSV are matured over months and years in at least 5 stages based on 1) levels of the neutralization titer and IgG3 for F protein in the convalescent phase, 2) geometric mean ratios of the neutralization titers and levels of IgG1 and IgG2 for F protein in the convalescent phase compared to those levels in the acute phase, 3) the affinity maturation of IgG for F protein and the cross reactivity of IgG for RSV glycoproteins of groups A and B, 4) levels of neutralization epitope-specific IgG, and 5) augmentation of overall antibody responses due to repetitive RSV infection.

Ligand-induced Ordering of the C-terminal Tail Primes STING for Phosphorylation by TBK1.

The innate immune protein Stimulator of interferon genes (STING) promotes the induction of interferon beta (IFN-ß) production via the phosphorylation of its C-terminal tail (CTT) by TANK-binding kinase 1 (TBK1). Potent ligands of STING are, therefore, promising candidates for novel anti-cancer drugs or vaccine adjuvants. However, the intrinsically flexible CTT poses serious problems in in silico drug discovery. Here, we performed molecular dynamics simulations of the STING fragment containing the CTT in ligand-bound and unbound forms and observed that the binding of a potent ligand cyclic GMP-AMP (cGAMP) induced a local structure in the CTT, reminiscent of the known structure of a TBK1 substrate. The subsequent molecular biological experiments confirmed the observed dynamics of the CTT and identified essential residues for the activation of the IFN-ß promoter, leading us to propose a new mechanism of STING activation.

Efficient antigen delivery to the draining lymph nodes is a key component in the immunogenic pathway of the intradermal vaccine.

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.

Superior immunogenicity profile of the new intradermal influenza vaccine compared to the standard subcutaneous vaccine in subjects 65 years and older: A randomized controlled phase III study.

BACKGROUND: Although the elderly are at high risk for influenza, the immunogenicity in the elderly is lower than that in younger adults. We developed the new type of seasonal influenza vaccine with the novel intradermal (ID) injection system. In the previous exploratory phase I/II study of the ID vaccine with a dose of 15 µg HA per strain showed the superior immunogenicity profile to that of the standard subcutaneous (SC) injection vaccine in subjects aged 20 years and older. METHODS: In this multicenter, randomized, double-blind, active controlled study, 900 adults aged 65 years and older were randomized at an equal ratio to either the ID vaccine group or the licensed standard SC vaccine group. Immunogenicity was assessed using serum hemagglutination inhibition (HAI) titers. The co-primary endpoints were the geometrical mean titers (GMT) and the seroconversion rates (SCR) of HAI titers against 3 vaccine strains on Day 21 (21 days after vaccination). To evaluate the early phase immunogenicity, the GMTs and SCRs on Day 7 were also assessed in the same way as the secondary endpoints. RESULTS: The superiority of the ID vaccine in the GMTs and SCRs were demonstrated in all 3 vaccine strains both on Day 7 and Day 21. The frequency of any injection-site reactions was higher in the ID vaccine group, while the severity of injection-site reactions and the frequency of systemic AEs were comparable between the ID and the SC vaccine groups. CONCLUSIONS: A single-dose of the influenza vaccine with the novel ID injection system and a dose of 15 µg HA was suggested as an appropriate regimen for clinical use in influenza prevention and associated disease burden reduction. It was also suggested that the new ID vaccine has the potential to replace the standard influenza vaccine from the view point of immunogenicity and safety. TRIAL REGISTRATION: JAPIC Clinical Trials Information (JapicCTI-142493).

Immunogenicity and safety of the new intradermal influenza vaccine in adults and elderly: A randomized phase 1/2 clinical trial.

BACKGROUND: Recent clinical evidence indicates that an intradermal (ID) delivery of vaccines confers superior immunogenicity as compared to a standard intramuscular or subcutaneous (SC) delivery. METHODS: In this exploratory study, 600 healthy adults were randomized to 6 study groups with subgroups of young adults (20-64 years old) and older adults (65 years and older). The subjects were either injected by a novel ID injection system with a single dose of 6, 9, or 15 µg HA or two doses (21 days apart) of 15 µg HA per strain or injected by an SC injection method with a single or two doses (21 days apart) of 15 µg HA per strain. Immunogenicity was assessed using hemagglutination inhibition (HAI) titer and microneutralization titer on Days 0, 10, 21, and 42. Solicited and unsolicited adverse events were recorded for 7 and 21 days post-vaccination, respectively. RESULTS: In both young adults and older adults groups, the geometric titer (GMT) ratios of HAI in the ID 15 µg HA group were higher than those in the SC 15 µg HA group on both Day 10 and Day 21, while those in the ID 6 and ID 9 µg HA groups were comparable with those in the SC 15 µg HA group. The kinetics of GMTs of HAI suggested that the ID vaccine has the potential to induce the prompt immune response, which is rather hampered in older adults as seen in the SC vaccine groups. The injection-site AEs were generally mild and transient, and did not occur in a dose or dosage-dependent manner. CONCLUSIONS: The results of this study clearly suggest that the immunologic profile of the ID vaccine is better than that of the SC vaccine, while the safety profile of the ID vaccine is similar to that of the SC vaccine. In this exploratory study with almost 100 subjects per each group, single or two-dose administration of the ID vaccine containing 15 µg HA was suggested to be an appropriate regimen in order to prevent influenza and to reduce the associated disease burden. TRIAL REGISTRATION: JAPIC Clinical Trials Information (JapicCTI-132096).

Role of Extrachromosomal Histone H2B on Recognition of DNA Viruses and Cell Damage.

Histones are essential components of chromatin structure, and histone modification plays an important role in various cellular functions including transcription, gene silencing, and immunity. Histones also play distinct roles in extrachromosomal settings. Extrachromosomal histone H2B acts as a cytosolic sensor to detect double-stranded DNA (dsDNA) fragments derived from infectious agents or damaged cells to activate innate and acquired immune responses in various cell types. It also physically interacts with interferon (IFN)-ß promoter stimulator 1 (IPS-1), an essential adaptor molecule that activates innate immunity, through COOH-terminal importin 9-related adaptor organizing histone H2B and IPS-1 (CIAO), resulting in a distinct signaling complex that induces dsDNA-induced type I IFN production. Such a molecular platform acts as a cellular sensor to recognize aberrant dsDNA in cases of viral infection and cell damage. This mechanism may also play roles in autoimmunity, transplantation rejection, gene-mediated vaccines, and other therapeutic applications.

Innate Immune Signaling by, and Genetic Adjuvants for DNA Vaccination.

DNA vaccines can induce both humoral and cellular immune responses. Although some DNA vaccines are already licensed for infectious diseases in animals, they are not licensed for human use because the risk and benefit of DNA vaccines is still controversial. Indeed, in humans, the immunogenicity of DNA vaccines is lower than that of other traditional vaccines. To develop the use of DNA vaccines in the clinic, various approaches are in progress to enhance or improve the immunogenicity of DNA vaccines. Recent studies have shown that immunogenicity of DNA vaccines are regulated by innate immune responses via plasmid DNA recognition through the STING-TBK1 signaling cascade. Similarly, molecules that act as dsDNA sensors that activate innate immune responses through STING-TBK1 have been identified and used as genetic adjuvants to enhance DNA vaccine immunogenicity in mouse models. However, the mechanisms that induce innate immune responses by DNA vaccines are still unclear. In this review, we will discuss innate immune signaling upon DNA vaccination and genetic adjuvants of innate immune signaling molecules.

Demonstration of innate immune responses in the thyroid gland: potential to sense danger and a possible trigger for autoimmune reactions.

BACKGROUND: Autoimmune thyroid disease is an archetypal organ-specific autoimmune disorder that is characterized by the production of thyroid autoantibodies and lymphocytic infiltration into the thyroid. However, the underlying mechanisms by which specific thyroid antibodies are produced are largely unknown. Recent studies have shown that innate immune responses affect both the phenotype and the severity of autoimmune reactions. Moreover, it appears that even non-immune cells, including thyroid cells, have an ability to launch such responses. The aim of this study was to conduct a more detailed analysis of innate immune responses of the thyroid upon stimulation with various "non-self" and "self" factors that might contribute to the initiation of autoimmune reactions. METHODS: We used rat thyroid FRTL-5 cells, human thyroid cells, and mice to investigate the effects of various pathogen-associated molecular patterns (PAMPs), danger-associated molecular patterns (DAMPs), and iodide on gene expression and function that were related to innate immune responses. RESULTS: RT-PCR analysis showed that both rat and human thyroid cells expressed mRNAs for Toll-like receptors (TLRs) that sensed PAMPs. Stimulation of thyrocytes with TLR ligands resulted in activation of the interferon-beta (IFN-ß) promoter and the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)-dependent promoter. As a result, pro-inflammatory cytokines, chemokines, and type I interferons were produced. Similar activation was observed when thyroid cells were stimulated with double-stranded DNA, one of the typical DAMPs. In addition to these PAMPs and DAMPs, treatment of thyroid cells with high concentrations of iodide increased mRNA expression of various cytokines. CONCLUSION: We show that thyroid cells express functional sensors for exogenous and endogenous dangers, and that they are capable of launching innate immune responses without the assistance of immune cells. Such responses may relate to the development of thyroiditis, which in turn may trigger autoimmune reactions.

Intracellular inflammatory sensors for foreign invaders and substances of self-origin.

In order to survive, all organisms must recognize and eliminate foreign invaders such as infectious pathogens, chemicals, ultraviolet rays, metabolites and damaged or transformed self-tissues, as well as allogenic organs in cases of transplantation. Recent research in innate immunity has elucidated that there are versatile inflammatory sensors on spatiotemporal 'sentry duty' that recognize substances derived from both 'nonself' and 'self', e.g., Toll-like receptors, retinoic acid-inducible gene-I-like receptors, nucleotide oligomerization domain-like receptors and c-type lectin receptors. Having acquired high-level functions through the development of multiple molecules, higher organisms have established both extracellular and intracellular sensors that can discriminate danger-associated molecular patterns from promiscuous, but biologically similar, molecular patterns. In addition, 'loss-of-function' or 'gain-of-function' mutations in these inflammatory sensors have been linked (at least in part) with the etiology and severity of autoimmune diseases, autoinflammatory diseases and immunocompromised diseases in humans. Further studies focusing on the role of these inflammatory sensors in the development of immune disorders would highlight new avenues for the development of novel diagnostic and therapeutic applications with regard to these diseases.

Recognition of damage-associated molecular patterns related to nucleic acids during inflammation and vaccination.

All mammalian cells are equipped with large numbers of sensors for protection from various sorts of invaders, who, in turn, are equipped with molecules containing pathogen-associated molecular patterns (PAMPs). Once these sensors recognize non-self antigens containing PAMPs, various physiological responses including inflammation are induced to eliminate the pathogens. However, the host sometimes suffers from chronic infection or continuous injuries, resulting in production of self-molecules containing damage-associated molecular patterns (DAMPs). DAMPs are also responsible for the elimination of pathogens, but promiscuous recognition of DAMPs through sensors against PAMPs has been reported. Accumulation of DAMPs leads to massive inflammation and continuous production of DAMPs; that is, a vicious circle leading to the development of autoimmune disease. From a vaccinological point of view, the accurate recognition of both PAMPs and DAMPs is important for vaccine immunogenicity, because vaccine adjuvants are composed of several PAMPs and/or DAMPs, which are also associated with severe adverse events after vaccination. Here, we review as the roles of PAMPs and DAMPs upon infection with pathogens or inflammation, and the sensors responsible for recognizing them, as well as their relationship with the development of autoimmune disease or the immunogenicity of vaccines.

Novel strategies to improve DNA vaccine immunogenicity.

DNA vaccines can induce both humoral and cellular immune responses in animals. Some DNA vaccines are already licensed for infectious diseases such as West Nile virus encephalitis in horses. When used in humans, however, DNA vaccines suffer from lower immunogenicity profiles. Although the reasons for this are poorly understood, various hypotheses have been proposed. This review aims to provide better understanding of the molecular and immunological mechanisms by which DNA vaccines work and how such knowledge can be used to bring about improvements in their efficacy. Recent studies have provided evidence that the 'adjuvant effect' of plasmid DNA is mediated by its double-stranded structure. This structure activates stimulator of interferon genes/TANK-binding kinase 1 (STING/TBK1)- dependent innate immune signaling pathways in the absence of Toll-like receptors. Indeed, type-I interferons (IFNs), induced in vivo via the STING/TBK1 pathway, were found to be crucial for both direct- and indirect-antigen presentation via distinct cell types (i.e. dendritic cells (DC) and muscle cells, respectively). Importantly, incorporation of TBK1 into a DNA vaccine was found to enhance the antigen-specific humoral immune responses targeting the Plasmodium falciparum serine repeat antigen (SERA), a candidate vaccine antigen expressed in the blood-stages of human malaria parasites. Thus, the results of these studies may offer new ways to develop DNA vaccines, as well as delivering novel vaccine adjuvants against infectious diseases.

Fragments of genomic DNA released by injured cells activate innate immunity and suppress endocrine function in the thyroid.

Activation of innate and acquired immune responses, which can be induced by infection, inflammation, or tissue injury, may impact the development of autoimmunity. Although stimulation of cells by double-stranded DNA (dsDNA) has been shown to activate immune responses, the role of self-genomic DNA fragments released in the context of sterile cellular injury is not well understood. Using cultured thyroid cells, we show that cell injury prompts the release of genomic DNA into the cytosol, which is associated with the production of type I interferons, inflammatory cytokines, and chemokines. Molecules necessary for antigen processing and presentation to lymphocytes are also induced in thyroid cells by injury. dsDNA strongly suppressed the expression of sodium/iodide symporter and radioiodine uptake. To identify molecules responsible for sensing cytosolic dsDNA, we directly identified the cellular proteins that bound a dsDNA Sepharose column by mass spectrometry. Our analysis identified histone H2B, which was previously demonstrated to be an essential factor that mediates the activation of innate immunity induced by dsDNA. Knockdown of histone H2B using specific small interfering RNA abolished cell injury-induced innate immune activation and increased sodium/iodide symporter expression. These results indicate that genomic DNA fragments released by cell injury are recognized by extrachromosomal histone H2B, which results in the activation of genes involved in both innate and acquired immune responses in thyroid cells and suppression of thyroid function. These results suggest that sterile thyroid injury, in the absence of infection, may be sufficient to trigger autoimmune reaction and to induce thyroid dysfunction.

NLRP4 negatively regulates autophagic processes through an association with beclin1.

Although more than 20 putative members have been assigned to the nucleotide-binding and oligomerization domain-like receptor (NLR) family, their physiological and biological roles, with the exception of the inflammasome, are not fully understood. In this article, we show that NLR members, such as NLRC4, NLRP3, NLRP4, and NLRP10 interact with Beclin1, an important regulator of autophagy, through their neuronal apoptosis inhibitory protein, MHC class II transcription activator, incompatibility locus protein from Podospora anserina, and telomerase-associated protein domain. Among such NLRs, NLRP4 had a strong affinity to the Beclin1 evolutionally conserved domain. Compromising NLRP4 via RNA interference resulted in upregulation of the autophagic process under physiological conditions and upon invasive bacterial infections, leading to enhancement of the autophagic bactericidal process of group A streptococcus. NLRP4 recruited to the subplasma membrane phagosomes containing group A streptococcus and transiently dissociated from Beclin1, suggesting that NLRP4 senses bacterial infection and permits the initiation of Beclin1-mediated autophagic responses. In addition to a role as a negative regulator of the autophagic process, NLRP4 physically associates with the class C vacuolar protein-sorting complex, thereby negatively regulating maturation of the autophagosome and endosome. Collectively, these results provide novel evidence that NLRP4, and possibly other members of the NLR family, plays a crucial role in biogenesis of the autophagosome and its maturation by the association with regulatory molecules, such as Beclin1 and the class C vacuolar protein-sorting complex.

Modulation of intracellular signaling using protein-transduction technology.

Protein-transduction technology is one of the most promising therapeutic tools for the control of intracellular events. A number of studies have demonstrated that minimal and efficient protein-transduction domains (PTDs) can act as a peptide vector to transfer bioactive cargo molecules from outside to inside the cell. PTD-mediated transduction has the ability to cross the blood-brain barrier, with transduction taking place in most tissues and cell types in vivo. Thus, recombinant proteins fused to or conjugated with PTDs have the potential to be harnessed as supplementary and/or intervention agents directly modulating cell signaling and/or metabolism, or to be applied to vaccine antigens/adjuvants that are efficiently delivered to the optimal site of action to enhance vaccine immunogenicity. This review introduces the mechanism of action, recent applications, and future perspectives of protein-transduction technology as an alternative therapeutic in the post-genome era.

C/EBP{delta} and STAT-1 are required for TLR8 transcriptional activity.

Toll-like receptor 8 (TLR8), which is expressed primarily in myeloid cells, plays a central role in initiating immune responses to viral single-stranded RNA. Despite the great interest in the field of TLR8 research, very little is known in terms of TLR8 biology and its transcriptional regulation. Here, we describe the isolation of the hTLR8 promoter and the characterization of the molecular mechanisms involved in its regulation. Reporter gene analysis and ChIP assays demonstrated that the hTLR8 regulation of the basal transcription is regulated via three C/EBP cis-acting elements that required C/EBPδ and C/EBPß activity. In addition, we observed that R848 stimulation increases TLR8 transcriptional activity via an enhanced binding of C/EBPδ, and not C/EBPß, to its responsive sites within the TLR8 promoter. Moreover, we showed that IFN-γ also increased TLR8 transcription activity via the binding of STAT1 transcription factor to IFN-γ activated sequence elements on the TLR8 promoter and enhanced TLR8 functionality. These results shed new light on the mechanisms involved during TLR8-mediated innate immune response.

Immunogenicity of whole-parasite vaccines against Plasmodium falciparum involves malarial hemozoin and host TLR9.

Although whole-parasite vaccine strategies for malaria infection have regained attention, their immunological mechanisms of action remain unclear. We find that immunization of mice with a crude blood stage extract of the malaria parasite Plasmodium falciparum elicits parasite antigen-specific immune responses via Toll-like receptor (TLR) 9 and that the malarial heme-detoxification byproduct, hemozoin (HZ), but not malarial DNA, produces a potent adjuvant effect. Malarial and synthetic (s)HZ bound TLR9 directly to induce conformational changes in the receptor. The adjuvant effect of sHZ depended on its method of synthesis and particle size. Although natural HZ acts as a TLR9 ligand, the adjuvant effects of synthetic HZ are independent of TLR9 or the NLRP3-inflammasome but are dependent on MyD88. The adjuvant function of sHZ was further validated in a canine antiallergen vaccine model. Thus, HZ can influence adaptive immune responses to malaria infection and may have therapeutic value in vaccine adjuvant development.