Functional Human CD141+ Dendritic Cells in Human Immune System Mice.

BACKGROUND: For the purpose of studying functional human dendritic cells (DCs) in a humanized mouse model that mimics the human immune system (HIS), a model referred to as HIS mice was established. METHODS: Human immune system mice were made by engrafting NOD/SCID/IL2Rgammanull (NSG) mice with human hematopoietic stem cells (HSCs) following the transduction of genes encoding human cytokines and human leukocyte antigen (HLA)-A2.1 by adeno-associated virus serotype 9 (AAV9) vectors. RESULTS: Our results indicate that human DC subsets, such as CD141+CD11c+ and CD1c+CD11c+ myeloid DCs, distribute throughout several organs in HIS mice including blood, bone marrow, spleen, and draining lymph nodes. The CD141+CD11c+ and CD1c+CD11c+ human DCs isolated from HIS mice immunized with adenoviruses expressing malaria/human immunodeficiency virus (HIV) epitopes were able to induce the proliferation of malaria/HIV epitopes-specific human CD8+ T cells in vitro. Upregulation of CD1c was also observed in human CD141+ DCs 1 day after immunization with the adenovirus-based vaccines. CONCLUSIONS: Establishment of such a humanized mouse model that mounts functional human DCs enables preclinical assessment of the immunogenicity of human vaccines in vivo.

Co-administration of α-GalCer analog and TLR4 agonist induces robust CD8(+) T-cell responses to PyCS protein and WT-1 antigen and activates memory-like effector NKT cells.

In the present study, the combined adjuvant effect of 7DW8-5, a potent α-GalCer-analog, and monophosphoryl lipid A (MPLA), a TLR4 agonist, on the induction of vaccine-induced CD8(+) T-cell responses and protective immunity was evaluated. Mice were immunized with peptides corresponding to the CD8(+) T-cell epitopes of a malaria antigen, a circumsporozoite protein of Plasmodium yoelii, and a tumor antigen, a Wilms Tumor antigen-1 (WT-1), together with 7DW8-5 and MPLA, as an adjuvant. These immunization regimens were able to induce higher levels of CD8(+) T-cell responses and, ultimately, enhanced levels of protection against malaria and tumor challenges compared to the levels induced by immunization with peptides mixed with 7DW8-5 or MPLA alone. Co-administration of 7DW8-5 and MPLA induces activation of memory-like effector natural killer T (NKT) cells, i.e. CD44(+)CD62L(-)NKT cells. Our study indicates that 7DW8-5 greatly enhances important synergistic pathways associated to memory immune responses when co-administered with MPLA, thus rendering this combination of adjuvants a novel vaccine adjuvant formulation.

Comprehensive survey of transposon mPing insertion sites and transcriptome analysis for identifying candidate genes controlling high protein content of rice.

Rice is the most important crop species in the world, being staple food of more than 80% of people in Asia. About 80% of rice grain is composed of carbohydrates (starch), with its protein content as low as 7-8%. Therefore, increasing the protein content of rice offers way to create a stable protein source that contributes to improving malnutrition and health problems worldwide. We detected two rice lines harboring a significantly higher protein content (namely, HP5-7 and HP7-5) in the EG4 population. The EG4 strain of rice is a unique material in that the transposon mPing has high transpositional activity and high copy numbers under natural conditions. Other research indicated that mPing is abundant in the gene-rich euchromatic regions, suggesting that mPing amplification should create new allelic variants, novel regulatory networks, and phenotypic changes in the EG4 population. Here, we aimed to identify the candidate genes and/or mPing insertion sites causing high protein content by comprehensively identifying the mPing insertion sites and carrying out an RNA-seq-based transcriptome analysis. By utilizing the next-generation sequencing (NGS)-based methods, ca. 570 mPing insertion sites were identified per line in the EG4 population. Our results also indicated that mPing apparently has a preference for inserting itself in the region near a gene, with 38 genes in total found to contain the mPing insertion in the HP lines, of which 21 and 17 genes were specific to HP5-7 and HP7-5, respectively. Transcriptome analysis revealed that most of the genes related to protein synthesis (encoding glutelin, prolamin, and globulin) were up-regulated in HP lines relative to the control line. Interestingly, the differentially expressed gene (DEG) analysis revealed that the expression levels of many genes related to photosynthesis decreased in both HP lines; this suggests the amount of starch may have decreased, indirectly contributing to the increased protein content. The high-protein lines studied here are expected to contribute to the development of high protein-content rice by introducing valuable phenotypic traits such as high and stable yield, disease resistance, and abundant nutrients.

A possible mechanism of horseback riding on dynamic trunk alignment.

The study aimed to clarify the regularity of the motions of horse's back, rider's pelvis and spine associated with improvement of rider's dynamic trunk alignment. The study used a crossover design, with exercise using the horseback riding simulator (simulator hereafter) as the control condition. The experiments were conducted at Tokyo University of Agriculture Bio-therapy Center. The sample consisted of 20 healthy volunteers age 20-23 years. Participants performed 15-min sessions of horseback riding with a Hokkaido Pony and exercise using the simulator in experiments separated by ≥2 weeks. Surface electromyography (EMG) after horseback riding revealed decreased activity in the erector spinae. Exploratory data analysis of acceleration and angular velocity inferred associations between acceleration (Rider's neck/longitudinal axis [Y hereafter]) and angular velocity (Horse saddle/Y) as well as angular velocity (Rider's pelvis/Y) and angular velocity (Horse saddle/Y). Acceleration (Rider's neck/Y) tended to be associated with angular velocity (Rider's pelvis/Y). Surface EMG following exercise revealed decreased activity in the rectus abdominis and erector spinae after the simulator exercise. Horseback riding improved the rider's dynamic trunk alignment with a clear underlying mechanism, which was not observed with the simulator.

Co-localization of a CD1d-binding glycolipid with an adenovirus-based malaria vaccine for a potent adjuvant effect.

A CD1d-binding, invariant (i) natural killer T (NKT)-cell stimulatory glycolipid, α-Galactosylceramide (αGalCer), has been shown to act as an adjuvant. We previously identified a fluorinated phenyl ring-modified αGalCer analog, 7DW8-5, displaying a higher binding affinity for CD1d molecule and more potent adjuvant activity than αGalCer. In the present study, 7DW8-5 co-administered intramuscularly (i.m.) with a recombinant adenovirus expressing a Plasmodium yoelii circumsporozoite protein (PyCSP), AdPyCS, has led to a co-localization of 7DW8-5 and a PyCSP in draining lymph nodes (dLNs), particularly in dendritic cells (DCs). This occurrence initiates a cascade of events, such as the recruitment of DCs to dLNs and their activation and maturation, and the enhancement of the ability of DCs to prime CD8+ T cells induced by AdPyCS and ultimately leading to a potent adjuvant effect and protection against malaria.

Human CD8+ T cells mediate protective immunity induced by a human malaria vaccine in human immune system mice.

A number of studies have shown that CD8+ T cells mediate protective anti-malaria immunity in a mouse model. However, whether human CD8+ T cells play a role in protection against malaria remains unknown. We recently established human immune system (HIS) mice harboring functional human CD8+ T cells (HIS-CD8 mice) by transduction with HLA-A∗0201 and certain human cytokines using recombinant adeno-associated virus-based gene transfer technologies. These HIS-CD8 mice mount a potent, antigen-specific HLA-A∗0201-restricted human CD8+ T-cell response upon immunization with a recombinant adenovirus expressing a human malaria antigen, the Plasmodium falciparum circumsporozoite protein (PfCSP), termed AdPfCSP. In the present study, we challenged AdPfCSP-immunized HIS-CD8 mice with transgenic Plasmodium berghei sporozoites expressing full-length PfCSP and found that AdPfCSP-immunized (but not naïve) mice were protected against subsequent malaria challenge. The level of the HLA-A∗0201-restricted, PfCSP-specific human CD8+ T-cell response was closely correlated with the level of malaria protection. Furthermore, depletion of human CD8+ T cells from AdPfCSP-immunized HIS-CD8 mice almost completely abolished the anti-malaria immune response. Taken together, our data show that human CD8+ T cells mediate protective anti-malaria immunity in vivo.

A full-length Plasmodium falciparum recombinant circumsporozoite protein expressed by Pseudomonas fluorescens platform as a malaria vaccine candidate.

The circumsporozoite protein (CSP) of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP)-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP) production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS) showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime/boost strategy, ultimately acting as an effective vaccine candidate for the mitigation of P. falciparum-induced malaria.