Congenital anaemia associated with loss-of-function variants in DNA polymerase epsilon 1.

DNA polymerase epsilon (Pol ε), a component of the core replisome, is involved in DNA replication. Although genetic defects of Pol ε have been reported to cause immunodeficiency syndromes, its role in haematopoiesis remains unknown. Here, we identified compound heterozygous variants (p.[Asp1131fs];[Thr1891del]) in POLE, encoding Pol ε catalytic subunit A (POLE1), in siblings with a syndromic form of severe congenital transfusion-dependent anaemia. In contrast to Diamond-Blackfan anaemia, marked reticulocytopenia or marked erythroid hypoplasia was not found. Their bone marrow aspirates during infancy revealed erythroid dysplasia with strongly positive TP53 in immunostaining. Repetitive examinations demonstrated trilineage myelodysplasia within 2 years from birth. They had short stature and facial dysmorphism. HEK293 cell-based expression experiments and analyses of patient-derived induced pluripotent stem cells (iPSCs) disclosed a reduced mRNA level of Asp1131fs-POLE1 and defective nuclear translocation of Thr1891del-POLE1. Analysis of iPSCs showed compensatory mRNA upregulation of the other replisome components and increase of the TP53 protein, both suggesting dysfunction of the replisome. We created Pole-knockout medaka fish and found that heterozygous fishes were viable, but with decreased RBCs. Our observations expand the phenotypic spectrum of the Pol ε defect in humans, additionally providing unique evidence linking Pol ε to haematopoiesis.

LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition.

During embryonic development, primitive and definitive waves of hematopoiesis take place to provide proper blood cells for each developmental stage, with the possible involvement of epigenetic factors. We previously found that lysine-specific demethylase 1 (LSD1/KDM1A) promotes primitive hematopoietic differentiation by shutting down the gene expression program of hemangioblasts in an Etv2/Etsrp-dependent manner. In the present study, we demonstrated that zebrafish LSD1 also plays important roles in definitive hematopoiesis in the development of hematopoietic stem and progenitor cells. A combination of genetic approaches and imaging analyses allowed us to show that LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition. Analysis of compound mutant lines with Etv2/Etsrp mutant zebrafish revealed that, unlike in primitive hematopoiesis, this function of LSD1 was independent of Etv2/Etsrp. The phenotype of LSD1 mutant zebrafish during the endothelial-to-hematopoietic transition was similar to that of previously reported compound knockout mice of Gfi1/Gfi1b, which forms a complex with LSD1 and represses endothelial genes. Moreover, co-knockdown of zebrafish Gfi1/Gfi1b genes inhibited the development of hematopoietic stem and progenitor cells. We therefore hypothesize that the shutdown of the Gfi1/Gfi1b-target genes during the endothelial-to-hematopoietic transition is one of the key evolutionarily conserved functions of LSD1 in definitive hematopoiesis.

Pathogenic variants in the survival of motor neurons complex gene GEMIN5 cause cerebellar atrophy.

Cerebellar ataxia is a genetically heterogeneous disorder. GEMIN5 encoding an RNA-binding protein of the survival of motor neuron complex, is essential for small nuclear ribonucleoprotein biogenesis, and it was recently reported that biallelic loss-of-function variants cause neurodevelopmental delay, hypotonia, and cerebellar ataxia. Here, whole-exome analysis revealed compound heterozygous GEMIN5 variants in two individuals from our cohort of 162 patients with cerebellar atrophy/hypoplasia. Three novel truncating variants and one previously reported missense variant were identified: c.2196dupA, p.(Arg733Thrfs*6) and c.1831G > A, p.(Val611Met) in individual 1, and c.3913delG, p.(Ala1305Leufs*14) and c.4496dupA, p.(Tyr1499*) in individual 2. Western blotting analysis using lymphoblastoid cell lines derived from both affected individuals showed significantly reduced levels of GEMIN5 protein. Zebrafish model for null variants p.(Arg733Thrfs*6) and p.(Ala1305Leufs*14) exhibited complete lethality at 2 weeks and recapitulated a distinct dysplastic phenotype. The phenotypes of affected individuals and the zebrafish mutant models strongly suggest that biallelic loss-of-function variants in GEMIN5 cause cerebellar atrophy/hypoplasia.

Real-time intravital imaging of pH variation associated with osteoclast activity.

Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption.

BODIPY-based probes for the fluorescence imaging of biomolecules in living cells.

Fluorescence imaging techniques have been widely used to visualize biological molecules and phenomena. In particular, several studies on the development of small-molecule fluorescent probes have been carried out, because their fluorescence properties can be easily tuned by synthetic chemical modification. For this reason, various fluorescent probes have been developed for targeting biological components, such as proteins, peptides, amino acids, and ions, to the interior and exterior of cells. In this review, we cover advances in the development of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-based fluorescent probes for biological studies over the past decade.

In vitro amyloidogenic peptides of galectin-7: possible mechanism of amyloidogenesis of primary localized cutaneous amyloidosis.

Pathogenesis of primary localized cutaneous amyloidosis (PLCA) is unclear, but pathogenic relationship to keratinocyte apoptosis has been implicated. We have previously identified galectin-7, actin, and cytokeratins as the major constituents of PLCA. Determination of the amyloidogenetic potential of these proteins by thioflavin T (ThT) method demonstrated that galectin-7 molecule incubated at pH 2.0 was capable of binding to the dye, but failed to form amyloid fibrils. When a series of galectin-7 fragments containing ß-strand peptides were prepared to compare their amyloidogenesis, Ser(31)-Gln(67) and Arg(120)-Phe(136) were aggregated to form amyloid fibrils at pH 2.0. The rates of aggregation of Ser(31)-Gln(67) and Arg(120)-Phe(136) were dose-dependent with maximal ThT levels after 3 and 48 h, respectively. Their synthetic analogs, Phe(33)-Lys(65) and Leu(121)-Arg(134), which are both putative tryptic peptides, showed comparable amyloidogenesis. The addition of sonicated fibrous form of Ser(31)-Gln(67) or Phe(33)-Lys(65) to monomeric Ser(31)-Gln(67) or Phe(33)-Lys(65) solution, respectively, resulted in an increased rate of aggregation and extension of amyloid fibrils. Amyloidogenic potentials of Ser(31)-Gln(67) and Phe(33)-Lys(65) were inhibited by actin and cytokeratin fragments, whereas those of Arg(120)-Phe(136) and Leu(121)-Arg(134) were enhanced in the presence of Gly(84)-Arg(113), a putative tryptic peptide of galectin-7. Degraded fragments of the galectin-7 molecule produced by limited trypsin digestion, formed amyloid fibrils after incubation at pH 2.0. These results suggest that the tryptic peptides of galectin-7 released at neutral pH, may lead to amyloid fibril formation of PLCA in the intracellular acidified conditions during keratinocyte apoptosis via regulation by the galectin-7 peptide as well as actin and cytokeratins.

Rapid survey of four Asp isomers in disease-related proteins by LC-MS combined with commercial enzymes.

Until relatively recently, it was considered that D-amino acids were excluded from living systems except for the cell wall of microorganisms. However, D-aspartate residues have now been detected in long-lived proteins from various tissues of elderly humans. Formation of D-aspartate in proteins induces aggregation and loss of function, leading to age-related disorders such as cataracts and Alzheimer disease. A recent study used LC-MS to analyze isomers of Asp residues in proteins precisely without complex purification of the proteins. However, to identify the four Asp isomers (Lα, Lß, Dß, and Dα) on the chromatogram, it was necessary to synthesize reference peptides containing the four different Asp isomers as standards. Here, we describe a method for rapidly and comprehensively identifying Asp isomers in proteins using a combination of LC-MS and commercial enzymes without synthesizing reference peptides. The protein sample is treated with trypsin, trypsin plus Asp-N, trypsin plus PIMT, trypsin plus paenidase, and the resulting peptides are applied to LC-MS. Because Asp-N hydrolyzes peptide bonds on the N-terminus of only Lα-Asp residues, it differentiates peptides containing Lα-Asp from those containing the other three isomers. Similarly, PIMT recognizes only peptides containing Lß-Asp residues, and paenidase internally cleaves the C-terminus of Dα-Asp residues. This approach was successfully applied to the analysis of all tryptic peptides in aged lens. The comprehensive quantitative data of Asp isomer formation in age-related proteins obtained via this method might be used as biomarkers of age-related disease.

Identification of the Babesia-responsive leucine-rich repeat domain-containing protein from the hard tick Haemaphysalis longicornis.

Haemaphysalis longicornis is a tick known for transmitting Babesia parasites, including Babesia gibsoni, in East Asian countries. The vector tick must have strategies to control Babesia parasites, while Babesia parasites are also considered to establish an evasive mechanism from the tick's innate immunity. Due to this mutual tolerance, H. longicornis is considered to be a vector of Babesia parasites. Recent studies have shown the important roles of leucine-rich repeat (LRR) domain-containing proteins in innate immunity in many living organisms. Some LRR domain-containing proteins were identified in ticks; however, their functions are still unknown. In this study, a novel LRR domain-containing protein was identified from H. longicornis (HlLRR). HlLRR contains two LRR domains, and the expression levels of mRNA and proteins were upregulated during blood feeding, particularly in the salivary glands and midgut. In addition, recombinant HlLRR (rHlLRR) demonstrated growth inhibition activity against B. gibsoni in vitro without a hemolytic effect at any concentration used. Moreover, the diameters of Babesia merozoites treated with rHlLRR were significantly larger than those of the control group. These results strongly indicate the key roles of HlLRR in the tick's innate immunity against Babesia parasites. Furthermore, HlLRR might be a potential alternative drug to treat babesiosis.

Contrast-enhanced endoscopic ultrasonography can predict a higher malignant potential of gastrointestinal stromal tumors by visualizing large newly formed vessels.

BACKGROUND: The aim of this study was to elucidate the histologic and clinical implications of detection of intratumoral vessels on contrast-enhanced endoscopic ultrasonography (CE-EUS) in gastrointestinal stromal tumors (GISTs). METHODS: Thirteen patients with a GIST, all of whom were referred for surgery, underwent presurgical CE-EUS. The malignant potential, assessed according to the modified Fletcher risk classification system, and the histologic degree of angiogenesis were compared with the presence or absence of intratumoral vessels on CE-EUS. RESULTS: Of the six tumors with intratumoral vessels observed on CE-EUS, five were intermediate- or high-risk GISTs, and the remaining seven negative cases were categorized as very low risk or low risk. The presence of intratumoral vessels on CE-EUS was significantly correlated with a higher-risk classification (p = 0.005). On histologic examination, all GISTs having visualized vessels incorporated vessels of more than 500 µm in diameter. The large intratumoral vessels of the five intermediate- or high-risk GISTs lacked elastic fibers, suggesting that they were neovascular in nature. These higher-risk tumors were also found, by immunohistochemical analysis, to have high expression of vascular endothelial growth factor. CONCLUSIONS: Intratumoral vessels observed in GISTs on CE-EUS are correlated with a higher degree of angiogenesis, resulting in higher malignant potential.

[Frontiers in Live Bone Imaging Researches. Development of fluorescent pH probe for imaging osteoclast activation].

For the clinical diagnosis of osteoporosis, X-ray CT and biochemical bone metabolism markers, however, there is no method to monitor osteoclast activity in the living system. We have developed the fluorescent probe to monitor osteoclast activity, by the combination of pH sensitive fluorescent dye with bisphosphonate which is delivered to the bone surface by its high affinity to hydroxyl apatite, which is named BAp-E. In vivo mouse imaging of activated osteoclast was successfully demonstrated using two photon excitation laser microscope.

Enhanced Cycle Stability of LiNiO2 in a Highly Concentrated Ionic Liquid Electrolyte.

LiNiO2 (LNO) is a promising positive material for next-generation vehicle batteries because of its high theoretical capacity and lower cost compared to the Co analogues. However, its unstable performance such as Ni dissolution results in capacity fade and poor cycle life, impeding its practical application. Since hydrogen fluoride (HF), the hydrolysis product of LiPF6, is highly reactive with LNO positive electrodes, exploring LiPF6-free electrolytes is attractive to improve cycle stability and eliminate parasitic reactions. Herein, a series of ionic liquids (ILs) with Li[FSA] ([FSA]- = bis(fluorosulfonyl)amide) salts are investigated as electrolytes compatible with the LNO positive electrode. The use of IL electrolytes enhances cycle performance, achieving a high capacity retention of 73.1% in Li/LNO cells after 500 cycles with a high Li salt concentration. Further characterizations confirm that the cathode electrolyte interphase formed on the LNO positive electrode in the highly Li-salt concentrated ILs suppresses Ni dissolution, structural degradation, and side reactions. Meanwhile, the above electrolyte is capable of effectively alleviating Al corrosion at high potentials. This work highlights the role of electrolytes and contributes to addressing the stability concerns of positive electrode components at high voltages.

Synthesis of unsymmetrical dialkoxydiarylsilanes and diarylsilanediols from tetraalkoxysilane having a dioxasilepane unit.

The tetraalkoxysilane carrying a stable seven-membered dioxasilepane moiety and two trifluoroethoxy groups undergoes reliable iterative substitution of the two trifluoroethoxy groups by sequential treatment with different aryl Grignard reagents while keeping the seven-membered structure intact. The process results in the synthesis of unsymmetrical dialkoxydiarylsilanes and eventually diarylsilanediols after proper hydrolysis.

Post-embolization syndrome-like symptoms due to shedding of necrotic material of hepatocellular carcinoma into the bile duct following transcatheter arterial chemoembolization: an instructive case.

Fever, abdominal pain, and liver dysfunction are almost inevitable complications of transcatheter arterial chemo embolization (TACE) for hepatocellular carcinoma, but these symptoms may also be due to bile duct obstruction caused by shedding of necrotic tumor material into the bile duct. A 68-year-old man presented with persistent fever, liver dysfunction, and abdominal pain after TACE. Computed tomography revealed stone-like hyperdensities in the bile duct. Endoscopic retrograde cholangiopancreatography revealed these structures to be necrotic material from hepatocellular carcinoma. We believe this is an instructive case of an often overlooked situation.

Gene expression profiles in rice gametes and zygotes: identification of gamete-enriched genes and up- or down-regulated genes in zygotes after fertilization.

In angiosperms, fertilization and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries; therefore, these processes are poorly elucidated. In this study, microarray-based transcriptome analyses were conducted on rice sperm cells, egg cells, and zygotes isolated from flowers to identify candidate genes involved in gametic and/or early zygotic development. Cell type-specific transcriptomes were obtained, and up- or down-regulated genes in zygotes after fertilization were identified, in addition to genes enriched in male and female gametes. A total of 325 putatively up-regulated and 94 putatively down-regulated genes in zygotes were obtained. Interestingly, several genes encoding homeobox proteins or transcription factors were identified as highly up-regulated genes after fertilization, and the gene ontology for up-regulated genes was highly enriched in functions related to chromatin/DNA organization and assembly. Because a gene encoding methyltransferase 1 was identified as a highly up-regulated gene in zygotes after fertilization, the effect of an inhibitor of this enzyme on zygote development was monitored. The inhibitor appeared partially to affect polarity or division asymmetry in rice zygotes, but it did not block normal embryo generation.

Multiple ferritins are vital to successful blood feeding and reproduction of the hard tick Haemaphysalis longicornis.

Ticks are obligate hematophagous parasites and important vectors of diseases. The large amount of blood they consume contains great quantities of iron, an essential but also toxic element. The function of ferritin, an iron storage protein, and iron metabolism in ticks need to be further elucidated. Here, we investigated the function a newly identified secreted ferritin from the hard tick Haemaphysalis longicornis (HlFER2), together with the previously identified intracellular ferritin (HlFER1). Recombinant ferritins, expressed in Escherichia coli, were used for anti-serum preparation and were also assayed for iron-binding activity. RT-PCR and western blot analyses of different organs and developmental stages of the tick during blood feeding were performed. The localization of ferritins in different organs was demonstrated through an indirect immunofluorescent antibody test. RNA interference (RNAi) was performed to evaluate the importance of ferritin in blood feeding and reproduction of ticks. The midgut was also examined after RNAi using light and transmission electron microscopy. RT-PCR showed differences in gene expression in some organs and developmental stages. Interestingly, only HlFER2 was detected in the ovary during oviposition and in the egg despite the low mRNA transcript. RNAi induced a reduction in post-blood meal body weight, high mortality and decreased fecundity. The expression of vitellogenin genes was affected by silencing of ferritin. Abnormalities in digestive cells, including disrupted microvilli, and alteration of digestive activity were also observed. Taken altogether, our results show that the iron storage and protective functions of ferritin are crucial to successful blood feeding and reproduction of H. longicornis.

Usefulness of contrast-enhanced endoscopic sonography for discriminating mural nodules from mucous clots in intraductal papillary mucinous neoplasms: a single-center prospective study.

OBJECTIVES: The aim of this study was to evaluate the ability of contrast-enhanced endoscopic sonography for discrimination of mural nodules from mucous clots in intraductal papillary mucinous neoplasms of the pancreas. METHODS: Contrast-enhanced endoscopic sonography was performed in 17 consecutive patients who had an intraductal papillary mucinous neoplasm with mural lesions. To perform contrast-enhanced endoscopic sonography, we used a second-generation sonographic contrast agent. After reconstitution with 2 mL of sterile water for injection, 0.7 mL of the agent was administered through a peripheral vein. From 10 to 30 seconds after injection of the contrast agent, the presence or absence of vascularity in mural lesions was assessed. All cases were referred to surgery, and diagnoses were finally obtained by pathologic examination. Diagnoses of mural nodules versus mucous clots based on the sonographic results were compared with tumor histopathologic findings. RESULTS: Pathologic findings revealed 12 cases with mural nodules and 5 cases without. Contrast-enhanced endoscopic sonography depicted vascularity in all 12 cases with pathologically confirmed mural nodules, whereas all 4 cases without vascularity had mucous clots. Only 1 case without a pathologically confirmed mural nodule was overestimated by contrast-enhanced endoscopic sonography as having a mural nodule. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of contrast-enhanced endoscopic sonography for mural nodule detection were 100%, 80%, 92%, 100%, and 94%, respectively. CONCLUSIONS: Evaluation of vascularity by contrast-enhanced endoscopic sonography could be useful for distinguishing mural nodules from mucous clots in intraductal papillary mucinous neoplasms. Contrast-enhanced endoscopic sonography could be a necessary option to determine surgical indications for intraductal papillary mucinous neoplasms when mural lesions are observed.

Inhibitory effect of cyclophilin A from the hard tick Haemaphysalis longicornis on the growth of Babesia bovis and Babesia bigemina.

Haemaphysalis longicornis is known as one of the most important ticks transmitting Babesia parasites in East Asian countries, including Babesia ovata and Babesia gibsoni, as well as Theileria parasites. H. longicornis is not the natural vector of Babesia bovis and Babesia bigemina. Vector ticks and transmitted parasites are thought to have established unique host-parasite interaction for their survival, meaning that vector ticks may have defensive molecules for the growth control of parasites in their bodies. However, the precise adaptation mechanism of tick-Babesia parasites is still unknown. Recently, cyclophilin A (CyPA) was reported to be important for the development of Babesia parasites in ticks. To reveal a part of their adaptation mechanism, the current study was conducted. An injection of B. bovis-infected RBCs into adult female H. longicornis ticks was found to upregulate the expression profiles of the gene and protein of CyPA in H. longicornis (HlCyPA). In addition, recombinant HlCyPA (rHlCyPA) purified from Escherichia coli exhibited significant inhibitory growth effects on B. bovis and B. bigemina cultivated in vitro, without any hemolytic effect on bovine RBCs at all concentrations used. In conclusion, our results suggest that HlCyPA might play an important role in the growth regulation of Babesia parasites in H. longicornis ticks, during natural acquisition from an infected host. Furthermore, rHlCyPA may be a potential alternative chemotherapeutic agent against babesiosis.

Radiotherapy for mucosa-associated lymphoid tissue (MALT) lymphoma of the esophagus: a case report with a diagnostic and therapeutic discussion.

Mucosa-associated lymphoid tissue (MALT) lymphoma is increasing common in various sites; however, MALT lymphoma in the esophagus is still rare, so its diagnostic features have not yet been well recognized and optimal treatment has not been properly discussed. Though radiotherapy is widely preferred for gastric and orbital MALT lymphoma, surgery has been the most frequently reported treatment for esophageal MALT lymphoma. This raises the question: why not radiotherapy for esophageal MALT lymphoma instead of surgery? The only reported case of definitive radiotherapy for esophageal MALT lymphoma lacks follow-up data. Three years ago (2007), we treated a 59-year-old male patient with a large esophageal submucosal tumor, diagnosed as MALT lymphoma, with 36 Gy of solo external beam radiotherapy. The tumor was 15 cm in craniocaudal length, homogeneously weakly contrast-enhanced on X-ray computed tomography (CT), homogeneously hypoechoic and clearly demarcated from the surrounding adventitia, and had a concave pattern between the folds. During and after radiotherapy, no treatment-related complications occurred except for transient Grade 2 leukocytopenia. The tumor showed remarkable reduction and histological negativity in the next month. Over the follow-up period, no recurrence was observed in semiannual PET/CT/MRI studies. Taking the current observation with the well known effectiveness of radiotherapy for MALT lymphoma in various other sites, we recommend considering radiotherapy as a reasonable less-invasive treatment for this rare entity.

Anti-babesial activity of a potent peptide fragment derived from longicin of Haemaphysalis longicornis.

Babesiosis is one of the most important tick-borne diseases affecting livestock that can cause major economic losses worldwide particularly in the tropics. Control relies on controlling both the protozoan parasite and the tick vector. Antiprotozoal drugs are most commonly used for treatment, but problems on emergence of resistant strains and food residues are encountered. Longicin, a defensin-like peptide identified from the hard tick, Haemapysalis longicornis, as well as one of its synthetic partial analogs (P4), were previously reported to exert antimicrobial, fungicidal, and parasiticidal activity. Both longicin and P4 showed babesiacidal activity, in vitro and in vivo. Here, peptide fragments of P4 were studied for in vitro activity against bovine Babesia parasites. One of the peptide fragments, antimicrobial peptide 1 (AMP1), reduced the parasitemia of Babesia bigemina. No peptide had significant effect on Babesia bovis. The sequence of AMP1 corresponded to the longicin sequence which is associated with antiparasitic activity. Although AMP1 caused reduction in parasitemia of B. bigemina, the difference in morphology of the parasite compared with the control group was not statistically significant. However, the percentage occurrence of piroplasms decreased, whereas the abnormal pycnotic form increased. The results demonstrated that this shorter peptide retained the anti-babesial activity of the parent peptide, exerting an antiparasitic effect against a bovine Babesia species. Therefore, this short peptide can be considered for chemical synthesis as an alternative therapeutic agent for babesiosis.

Immunogenicity and protective efficacy of SARS-CoV-2 recombinant S-protein vaccine S-268019-b in cynomolgus monkeys.

The vaccine S-268019-b is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-protein vaccine consisting of full-length recombinant SARS-CoV-2 S-protein (S-910823) as antigen, mixed with the squalene-based adjuvant A-910823. The current study evaluated the immunogenicity of S-268019-b using various doses of S-910823 and its vaccine efficacy against SARS-CoV-2 challenge in cynomolgus monkeys. The different doses of S-910823 combined with A-910823 were intramuscularly administered twice at a 3-week interval. Two weeks after the second dosing, dose-dependent humoral immune responses were observed with neutralizing antibody titers being comparable to that of human convalescent plasma. Pseudoviruses harboring S proteins from Beta and Gamma SARS-CoV-2 variants displayed approximately 3- to 4-fold reduced sensitivity to neutralizing antibodies induced after two vaccine doses compared with that against ancestral viruses, whereas neutralizing antibody titers were reduced >14-fold against the Omicron variant. Cellular immunity was also induced with a relative Th1 polarized response. No adverse clinical signs or weight loss associated with the vaccine were observed, suggesting safety of the vaccine in cynomolgus monkeys. Immunization with 10 µg of S-910823 with A-910823 demonstrated protective efficacy against SARS-CoV-2 challenge according to genomic and subgenomic viral RNA transcript levels in nasopharyngeal, throat, and rectal swab specimens. Pathological analysis revealed no detectable vaccine-dependent enhancement of disease in the lungs of challenged vaccinated monkeys. The current findings provide fundamental information regarding vaccine doses for human trials and support the development of S-268019-b as a safe and effective vaccine for controlling the current pandemic, as well as general protection against SARS-CoV-2 moving forward.