Tetrameric glycoprotein complex gH/gL/gQ1/gQ2 is a promising vaccine candidate for human herpesvirus 6B.

Primary infection of human herpesvirus 6B (HHV-6B) occurs in infants after the decline of maternal immunity and causes exanthema subitum accompanied by a high fever, and it occasionally develops into encephalitis resulting in neurological sequelae. There is no effective prophylaxis for HHV-6B, and its development is urgently needed. The glycoprotein complex gH/gL/gQ1/gQ2 (called 'tetramer of HHV-6B') on the virion surface is a viral ligand for its cellular receptor human CD134, and their interaction is thus essential for virus entry into the cells. Herein we examined the potency of the tetramer as a vaccine candidate against HHV-6B. We designed a soluble form of the tetramer by replacing the transmembrane domain of gH with a cleavable tag, and the tetramer was expressed by a mammalian cell expression system. The expressed recombinant tetramer is capable of binding to hCD134. The tetramer was purified to homogeneity and then administered to mice with aluminum hydrogel adjuvant and/or CpG oligodeoxynucleotide adjuvant. After several immunizations, humoral and cellular immunity for HHV-6B was induced in the mice. These results suggest that the tetramer together with an adjuvant could be a promising candidate HHV-6B vaccine.

A new subset of CD103+CD8alpha+ dendritic cells in the small intestine expresses TLR3, TLR7, and TLR9 and induces Th1 response and CTL activity.

CD103(+) dendritic cells (DCs) are the major conventional DC population in the intestinal lamina propria (LP). Our previous report showed that a small number of cells in the LP could be classified into four subsets based on the difference in CD11c/CD11b expression patterns: CD11c(hi)CD11b(lo) DCs, CD11c(hi)CD11b(hi) DCs, CD11c(int)CD11b(int) macrophages, and CD11c(int)CD11b(hi) eosinophils. The CD11c(hi)CD11b(hi) DCs, which are CD103(+), specifically express TLR5 and induce the differentiation of naive B cells into IgA(+) plasma cells. These DCs also mediate the differentiation of Ag-specific Th17 and Th1 cells in response to flagellin. We found that small intestine CD103(+) DCs of the LP (LPDCs) could be divided into a small subset of CD8α(+) cells and a larger subset of CD8α(-) cells. Flow cytometry analysis revealed that CD103(+)CD8α(+) and CD103(+)CD8α(-) LPDCs were equivalent to CD11c(hi)CD11b(lo) and CD11c(hi)CD11b(hi) subsets, respectively. We analyzed a novel subset of CD8α(+) LPDCs to elucidate their immunological function. CD103(+)CD8α(+) LPDCs expressed TLR3, TLR7, and TLR9 and produced IL-6 and IL-12p40, but not TNF-α, IL-10, or IL-23, following TLR ligand stimulation. CD103(+)CD8α(+) LPDCs did not express the gene encoding retinoic acid-converting enzyme Raldh2 and were not involved in T cell-independent IgA synthesis or Foxp3(+) regulatory T cell induction. Furthermore, CD103(+)CD8α(+) LPDCs induced Ag-specific IgG in serum, a Th1 response, and CTL activity in vivo. Accordingly, CD103(+)CD8α(+) LPDCs exhibit a different function from CD103(+)CD8α(-) LPDCs in active immunity. This is the first analysis, to our knowledge, of CD8α(+) DCs in the LP of the small intestine.

Microfluidics sorting enables the isolation of an intact cellular pair complex of CD8+ T cells and antigen-presenting cells in a cognate antigen recognition-dependent manner.

Adaptive immune responses begin with cognate antigen presentation-dependent specific interaction between T cells and antigen-presenting cells. However, there have been limited reports on the isolation and analysis of these cellular complexes of T cell-antigen-presenting cell (T/APC). In this study, we successfully isolated intact antigen-specific cellular complexes of CD8+ T/APC by utilizing a microfluidics cell sorter. Using ovalbumin (OVA) model antigen and OT-I-derived OVA-specific CD8+ T cells, we analyzed the formation of antigen-specific and antigen-non-specific T/APC cellular complexes and revealed that the antigen-specific T/APC cellular complex was highly stable than the non-specific one, and that the intact antigen-specific T/APC complex can be retrieved as well as enriched using a microfluidics sorter, but not a conventional cell sorter. The single T/APC cellular complex obtained can be further analyzed for the sequences of T cell receptor Vα and Vß genes as well as cognate antigen information simultaneously. These results suggested that this approach can be applied for other antigen and CD8+ T cells of mice and possibly those of humans. We believe that this microfluidics sorting method of the T/APC complex will provide useful information for future T cell immunology research.

Development of combination adjuvant for efficient T cell and antibody response induction against protein antigen.

Most current clinical vaccines work primarily by inducing the production of neutralizing antibodies against pathogens. Vaccine adjuvants that efficiently induce T cell responses to protein antigens need to be developed. In this study, we developed a new combination adjuvant consisting of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), D35, and an aluminum salt. Among the various combinations tested, the DOTAP/D35/aluminum salt adjuvant induced strong T cell and antibody responses against the model protein antigen with a single immunization. Adjuvant component and model antigen interaction studies in vitro also revealed that the strong mutual interactions among protein antigens and other components were one of the important factors for this efficient immune induction by the novel combination adjuvant. In addition, in vivo imaging of the antigen distribution suggested that the DOTAP component in the combination adjuvant formulation elicited transient antigen accumulation at the draining lymph nodes, possibly by antigen uptake DC migration. These results indicate the potential of the new combination adjuvant as a promising vaccine adjuvant candidate to treat infectious diseases and cancers.

Microfluidic-prepared DOTAP nanoparticles induce strong T-cell responses in mice.

For the induction of antigen-specific T-cell responses by vaccination, an appropriate immune adjuvant is required. Vaccine adjuvants generally provide two functions, namely, immune potentiator and delivery, and many adjuvants that can efficiently induce T-cell responses are known to have the combination of these two functions. In this study, we explored a cationic lipid DOTAP-based adjuvant. We found that the microfluidic preparation of DOTAP nanoparticles induced stronger CD4+ and CD8+ T-cell responses than liposomal DOTAP. The further addition of Type-A CpG D35 in DOTAP nanoparticles increased the induction of T-cell responses, particularly in CD4+ T cells. Further investigations revealed that the size of DOTAP nanoparticles, prepared buffer conditions, and physicochemical interaction with vaccine antigen are important factors for the efficient induction of T-cell responses with a relatively small antigen dose. These results suggested that microfluidic-prepared DOTAP nanoparticles plus D35 are a promising adjuvant for a vaccine that induces therapeutic T-cell responses for treating cancer and infectious diseases.

Comprehensive Screening of Mouse T-Cell Epitopes in Human Herpesvirus 6B Glycoprotein H/L/Q1/Q2 Tetramer Complex.

Human herpesvirus 6 (HHV-6) infects over 90% of people. The HHV-6 subtype, HHV-6B in particular, is often associated with exanthem subitum in early childhood. Exanthem subitum is usually self-limiting and good prognosis disease; however, some infants primarily infected with HHV-6B develop encephalitis/encephalopathy, and half of the patients developed encephalopathy reported to have neurological sequelae. Furthermore, after primary infection, HHV-6B remains in a latent state and sometimes reactivated in immunosuppressed patients, causing life-threatening severe encephalopathy. However, effective immunotherapies or vaccines for controlling HHV-6B infection and reactivation have not yet been established. Recently, we have found that the HHV-6B tetrameric glycoprotein (g) complex, gH/gL/gQ1/gQ2 is a promising vaccine candidate, and currently under preclinical development. To confirm our vaccine candidate protein complex induce detectable T-cell responses, in this study, we comprehensively screened CD4+ and CD8+ T-cell epitopes in the gH/gL/gQ1/gQ2 tetrameric complex protein in mice immunisation model. Both BALB/c and C57BL/6 mice were immunised with the tetrameric complex protein or plasmid DNA encoding gH, gL, gQ1, and gQ2, and then restimulated with 162 20-mer peptides covering the whole gH/gL/gQ1/gQ2 sequences; multiple CD4+ and CD8+ T-cell-stimulating peptides were identified in both BALB/c and C57BL/6 mice. Our study demonstrates that gH/gL/gQ1/gQ2 tetramer-targeted vaccination has potential to induce T-cell responses in two different strains of mice and supports the future development and application of T-cell-inducing vaccine and immunotherapies against HHV-6B.

Lipid nanoparticles of Type-A CpG D35 suppress tumor growth by changing tumor immune-microenvironment and activate CD8 T cells in mice.

Type-A CpG oligodeoxynucleotides (ODNs), which have a natural phosphodiester backbone, is one of the highest IFN-α inducer from plasmacytoid dendritic cells (pDC) via Toll-like receptor 9 (TLR9)-dependent signaling. However, the in vivo application of Type-A CpG has been limited because the rapid degradation in vivo results in relatively weak biological effect compared to other Type-B, -C, and -P CpG ODNs, which have nuclease-resistant phosphorothioate backbones. To overcome this limitation, we developed lipid nanoparticles formulation containing a Type-A CpG ODN, D35 (D35LNP). When tested in a mouse tumor model, intratumoral and intravenous D35LNP administration significantly suppressed tumor growth in a CD8 T cell-dependent manner, whereas original D35 showed no efficacy. Tumor suppression was associated with Th1-related gene induction and activation of CD8 T cells in the tumor. The combination of D35LNP and an anti-PD-1 antibody increased the therapeutic efficacy. Importantly, the therapeutic schedule and dose of intravenous D35LNP did not induce apparent liver toxicity. These results suggested that D35LNP is a safe and effective immunostimulatory drug formulation for cancer immunotherapy.

Quantifying the relative immune cell activation from whole tissue/organ-derived differentially expressed gene data.

Evaluation of immune responses in individual immune cell types is important for the development of new medicines. Here, we propose a computational method designated ICEPOP (Immune CEll POPulation) to estimate individual immune cell type responses from bulk tissue and organ samples. The relative gene responses are scored for each cell type by using the data from differentially expressed genes derived from control- vs drug-treated sample pairs, and the data from public databases including ImmGen and IRIS, which contain gene expression profiles of a variety of immune cells. By ICEPOP, we analysed cell responses induced by vaccine-adjuvants in the mouse spleen, and extended the analyses to human peripheral blood mononuclear cells and gut biopsy samples focusing on human papilloma virus vaccination and inflammatory bowel disease treatment with Infliximab. In both mouse and human datasets, our method reliably quantified the responding immune cell types and provided insightful information, demonstrating that our method is useful to evaluate immune responses from bulk sample-derived gene expression data. ICEPOP is available as an interactive web site ( https://vdynamics.shinyapps.io/icepop/ ) and Python package ( https://github.com/ewijaya/icepop ).

Development of a Novel Virus-Like Particle Vaccine Platform That Mimics the Immature Form of Alphavirus.

Virus-like particles (VLPs) are noninfectious multiprotein structures that are engineered to self-assemble from viral structural proteins. Here, we developed a novel VLP-based vaccine platform utilizing VLPs from the chikungunya virus. We identified two regions within the envelope protein, a structural component of chikungunya, where foreign antigens can be inserted without compromising VLP structure. Our VLP displays 480 copious copies of an inserted antigen on the VLP surface in a highly symmetric manner and is thus capable of inducing strong immune responses against any inserted antigen. Furthermore, by mimicking the structure of the immature form of the virus, we altered our VLP's in vivo dynamics and enhanced its immunogenicity. We used the circumsporozoite protein (CSP) of the Plasmodium falciparum malaria parasite as an antigen and demonstrated that our VLP-based vaccine elicits strong immune responses against CSP in animals. The sera from immunized monkeys protected mice from malaria infection. Likewise, mice vaccinated with P. yoelii CSP-containing VLPs were protected from an infectious sporozoite challenge. Hence, our uniquely engineered VLP platform can serve as a blueprint for the development of vaccines against other pathogens and diseases.

Advax, a Delta Inulin Microparticle, Potentiates In-built Adjuvant Property of Co-administered Vaccines.

Advax, a delta inulin-derived microparticle, has been developed as an adjuvant for several vaccines. However, its immunological characteristics and potential mechanism of action are yet to be elucidated. Here, we show that Advax behaves as a type-2 adjuvant when combined with influenza split vaccine, a T helper (Th)2-type antigen, but behaves as a type-1 adjuvant when combined with influenza inactivated whole virion (WV), a Th1-type antigen. In addition, an adjuvant effect was not observed when Advax-adjuvanted WV vaccine was used to immunize toll-like receptor (TLR) 7 knockout mice which are unable to respond to RNA contained in WV antigen. Similarly, no adjuvant effect was seen when Advax was combined with endotoxin-free ovalbumin, a neutral Th0-type antigen. An adjuvant effect was also not seen in tumor necrosis factor (TNF)-α knockout mice, and the adjuvant effect required the presences of dendritic cells (DCs) and phagocytic macrophages. Therefore, unlike other adjuvants, Advax potentiates the intrinsic or in-built adjuvant property of co-administered antigens. Hence, Advax is a unique class of adjuvant which can potentiate the intrinsic adjuvant feature of the vaccine antigens through a yet to be determined mechanism.

Optimization of physiological properties of hydroxyapatite as a vaccine adjuvant.

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-1ß 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-1ß production via NLRP3 inflammasome is dispensable for the adjuvanticity of HAps in mice.

RNA is an Adjuvanticity Mediator for the Lipid-Based Mucosal Adjuvant, Endocine.

Nasal vaccination has the potential to elicit systemic and mucosal immunity against pathogens. However, split and subunit vaccines lack potency at stimulating mucosal immunity, and an adjuvant is indispensable for eliciting potent mucosal immune response to nasal vaccines. Endocine, a lipid-based mucosal adjuvant, potentiates both systemic and mucosal immune responses. Although Endocine has shown efficacy and tolerability in animal and clinical studies, its mechanism of action remains unknown. It has been reported recently that endogenous danger signals are essential for the effects of some adjuvants such as alum or MF59. However, the contribution of danger signals to the adjuvanticity of Endocine has not been explored. Here, we show that RNA is likely to be an important mediator for the adjuvanticity of Endocine. Administration of Endocine generated nucleic acids release, and activated dendritic cells (DCs) in draining lymph nodes in vivo. These results suggest the possibility that Endocine indirectly activates DCs via damage-associated molecular patterns. Moreover, the adjuvanticity of Endocine disappeared in mice lacking TANK-binding kinase 1 (Tbk1), which is a downstream molecule of nucleic acid sensing signal pathway. Furthermore, co-administration of RNase A reduced the adjuvanticity of Endocine. These data suggest that RNA is important for the adjuvanticity of Endocine.

Development of Nonaggregating Poly-A Tailed Immunostimulatory A/D Type CpG Oligodeoxynucleotides Applicable for Clinical Use.

Immunostimulatory CpG ODNs have been developed and utilized as TLR9-dependent innate immune activators and vaccine adjuvants. Four different types of immunostimulatory CpG ODNs (A/D, B/K, C, and P type) have been reported. A/D type ODNs are characterized by high IFN-α production but intrinsically form aggregates, hindering its good manufacturing practice grade preparation. In this study, we developed several D35-derived ODNs (a commonly used A/D type ODN), which were modified with the addition of a phosphorothioate polynucleotide tail (such as dAs40), and examined their physical properties, solubility in saline, immunostimulatory activity on human PBMCs, and vaccine adjuvant potential in monkeys. We found that two modified ODNs including D35-dAs40 and D35core-dAs40 were immunostimulatory, similar to original D35 in human PBMCs, resulting in high IFN-α secretion in a dose-dependent manner. Physical property analysis by dynamic light scattering revealed that both D35-dAs40 and D35core-dAs40 did not form aggregates in saline, which is currently impossible for the original D35. Furthermore, D35-dAs40 and D35core-dAs40 worked as better vaccine adjuvant in monkeys. These results suggested that D35-dAs40 and D35core-dAs40 are two promising prototypes of nonaggregating A/D type ODN with advantages of ease of drug preparation for clinical applications as vaccine adjuvants or IFN-α inducing immunomodifiers.

Olfactory plays a key role in spatiotemporal pathogenesis of cerebral malaria.

Cerebral malaria is a complication of Plasmodium falciparum infection characterized by sudden coma, death, or neurodisability. Studies using a mouse model of experimental cerebral malaria (ECM) have indicated that blood-brain barrier disruption and CD8 T cell recruitment contribute to disease, but the spatiotemporal mechanisms are poorly understood. We show by ultra-high-field MRI and multiphoton microscopy that the olfactory bulb is physically and functionally damaged (loss of smell) by Plasmodium parasites during ECM. The trabecular small capillaries comprising the olfactory bulb show parasite accumulation and cell occlusion followed by microbleeding, events associated with high fever and cytokine storm. Specifically, the olfactory upregulates chemokine CCL21, and loss or functional blockade of its receptors CCR7 and CXCR3 results in decreased CD8 T cell activation and recruitment, respectively, as well as prolonged survival. Thus, early detection of olfaction loss and blockade of pathological cell recruitment may offer potential therapeutic strategies for ECM.

Critical role for chicken Rad17 and Rad9 in the cellular response to DNA damage and stalled DNA replication.

The Rad17-replication factor C (Rad17-RFC) and Rad9-Rad1-Hus1 complexes are thought to function in the early phase of cell-cycle checkpoint control as sensors for genome damage and genome replication errors. However, genetic analysis of the functions of these complexes in vertebrates is complicated by the lethality of these gene disruptions in embryonic mouse cells. We disrupted the Rad17 and Rad9 loci by gene targeting in the chicken B lymphocyte line DT40. Rad17-/- and Rad9-/- DT40 cells are viable, and are highly sensitive to UV irradiation, alkylating agents, and DNA replication inhibitors, such as hydroxyurea. We further found that Rad17-/- and Rad9-/- but not ATM-/- cells are defective in S-phase DNA damage checkpoint controls and in the cellular response to stalled DNA replication. These results indicate a critical role for chicken Rad17 and Rad9 in the cellular response to stalled DNA replication and DNA damage.