Identification of microRNA-96-5p as a postoperative, prognostic microRNA predictor in nonviral hepatocellular carcinoma.

AIM: The microRNA (miR) clusters miR-183/96/182 and miR-217/216a/216b are significantly upregulated in nonviral hepatocellular carcinoma (NBNC-HCC). Here, we investigate the impact of each member of these clusters on the clinical outcome of NBNC-HCC and analyze the antitumor effects of miR-96-5p. METHODS: The association between recurrence-free survival of 111 NBNC-HCC patients and the levels of miR-183-5p, miR-96-5p, miR-182-5p, miR-217-5p, miR-216a-5p, and miR-216b-5p in tumor and adjacent tissues was investigated. The impact of miR-96-5p on apoptosis and invasion of a hepatoma cell line, HepG2, was investigated by cell counting, Transwell assay, and flow cytometry, respectively. RESULTS: MicroRNA-183-5p, miR-96-5p, miR-182-5p, miR-217-5p, and miR-216b-5p were significantly upregulated in tumor tissues compared to the adjacent tissues (p = 0.0005, p = 0.0030, p = 0.0002, p = 0.0011, and p = 0.0288, respectively). By multivariate Cox regression analysis, high tumor/adjacent ratios of miR-182-5p (p = 0.007) and miR-217-5p (p = 0.008) were associated with poor recurrence-free survival. In contrast, a low tumor/adjacent ratio of miR-96-5p (p < 0.001) was associated with poor recurrence-free survival. It suggested that further upregulation of miR-96-5p in tumors might have an inhibitory effect on recurrence. Transfection of miR-96-5p mimic significantly induced apoptosis of HepG2 cells, in association with downregulation of Nucleophosmin 1 (NPM1) and a decrease of phosphorylated AKT protein. Interestingly, simultaneous knockdown of the NPM1 and AKT genes induced apoptosis. MicroRNA-96-5p also suppressed proliferation and invasion, which inhibited epithelial-to-mesenchymal transition of HCC cells. CONCLUSION: MicroRNA-96-5p as a tumor suppressor would be valuable to stratify NBNC-HCC patients at high risk of recurrence.

An aggressive systemic mastocytosis preceded by ovarian dysgerminoma.

BACKGROUND: Aggressive systemic mastocytosis (ASM) is a rare malignant disease characterized by disordered mast cell accumulation in various organs. We here describe a female ASM patient with a previous history of ovarian dysgerminoma. METHODS: Molecular cytogenomic analyses were performed to elucidate an etiological link between the ASM and dysgerminoma of the patient. RESULTS: This patient was affected by ovarian dysgerminoma which was treated by chemotherapy and surgical resection. Having subsequently been in complete remission for 2 years, she developed symptoms of ASM. A somatic D816A mutation in the KIT gene was detected in her bone marrow, which facilitated the diagnosis of ASM. Unexpectedly, this KIT D816A variant was also detected in the prior ovarian dysgerminoma sample. Whole-exome sequencing allowed us to identify a somatic nonsense mutation of the TP53 gene in the bone marrow, but not in the dysgerminoma. Microarray analysis of the patient's bone marrow revealed a copy-number-neutral loss of heterozygosity at the TP53 locus, suggestive of the homozygous nonsense mutation in the TP53 gene. In addition, the loss of heterozygosity at the TP53 locus was also detected in the dysgerminoma. CONCLUSIONS: These results indicated that either the mast cells causing the ASM in this case had originated from the preceding ovarian dysgerminoma as a clonal evolution of a residual tumor cell, which acquired the TP53 mutation, or that both tumors developed from a common cancer stem cell carrying the KIT D816A variation.

Inhibitory Effect of a Human MicroRNA, miR-6133-5p, on the Fibrotic Activity of Hepatic Stellate Cells in Culture.

BACKGROUND: We recently identified 39 human microRNAs, which effectively suppress hepatitis B virus (HBV) replication in hepatocytes. Chronic HBV infection often results in active, hepatitis-related liver fibrosis; hence, we assessed whether any of these microRNAs have anti-fibrotic potential and predicted that miR-6133-5p may target several fibrosis-related genes. METHODS: The hepatic stellate cell line LX-2 was transfected with an miR-6133-5p mimic and subsequently treated with Transforming growth factor (TGF)-ß. The mRNA and protein products of the COL1A1 gene, encoding collagen, and the ACTA2 gene, an activation marker of hepatic stellate cells, were quantified. RESULTS: The expression of COL1A1 and ACTA2 was markedly reduced in LX-2 cells treated with miR-6133-5p. Interestingly, phosphorylation of c-Jun N-terminal kinase (JNK) was also significantly decreased by miR-6133-5p treatment. The expression of several predicted target genes of miR-6133-5p, including TGFBR2 (which encodes Transforming Growth Factor Beta Receptor 2) and FGFR1 (which encodes Fibroblast Growth Factor Receptor 1), was also reduced in miR-6133-5p-treated cells. The knockdown of TGFBR2 by the corresponding small interfering RNA greatly suppressed the expression of COL1A1 and ACTA2. Treatment with the JNK inhibitor, SP600125, also suppressed COL1A1 and ACTA2 expression, indicating that TGFBR2 and JNK mediate the anti-fibrotic effect of miR-6133-5p. The downregulation of FGFR1 may result in a decrease of phosphorylated Akt, ERK (extracellular signal-regulated kinase), and JNK. CONCLUSION: miR-6133-5p has a strong anti-fibrotic effect, mediated by inactivation of TGFBR2, Akt, and JNK.

Type I Interferon Signaling Prevents Hepatitis B Virus-Specific T Cell Responses by Reducing Antigen Expression.

Robust virus-specific CD8+ T cell responses are required for the clearance of hepatitis B virus (HBV). However, the factors that determine the magnitude of HBV-specific CD8+ T cell responses are poorly understood. To examine the impact of genetic variations of HBV on HBV-specific CD8+ T cell responses, we introduced three HBV clones (Aa_IND [Aa], C_JPN22 [C22], and D_IND60 [D60]) that express various amounts of HBV antigens into the livers of C57BL/6 (B6) (H-2b) mice and B10.D2 (H-2d) mice. In B6 mice, clone C22 barely induced HBV-specific CD8+ T cell responses and persisted the longest, while clone D60 elicited strong HBV-specific CD8+ T cell responses and was rapidly cleared. These differences between HBV clones largely diminished in H-2d mice. Interestingly, the magnitude of HBV-specific CD8+ T cell responses in B6 mice was associated with the HB core antigen expression level during the early phase of HBV transduction. Surprisingly, robust HBV-specific CD8+ T cell responses to clone C22 were induced in interferon-α/ß receptor-deficient (IFN-αßR-/-) (H-2b) mice. The induction of HBV-specific CD8+ T cell responses to C22 in IFN-αßR-/- mice reflects enhanced HBV antigen expression because the suppression of antigen expression by HBV-specific small interfering RNA (siRNA) attenuated HBV-specific T cell responses in IFN-αßR-/- mice and prolonged HBV expression. Collectively, these results suggest that HBV genetic variation and type I interferon signaling determine the magnitude of HBV-specific CD8+ T cell responses by regulating the initial antigen expression levels.IMPORTANCE Hepatitis B virus (HBV) causes acute and chronic infection, and approximately 240 million people are chronically infected with HBV worldwide. It is generally believed that virus-specific CD8+ T cell responses are required for the clearance of HBV. However, the relative contributions of genetic variation and innate immune responses to the induction of HBV-specific CD8+ T cell responses are not fully understood. In this study, we discovered that different clearance rates between HBV clones after hydrodynamic transduction were associated with the magnitude of HBV-specific CD8+ T cell responses and initial HB core antigen expression. Surprisingly, type I interferon signaling negatively regulated HBV-specific CD8+ T cell responses by reducing early HBV antigen expression. These results show that the magnitude of the HBV-specific CD8+ T cell response is regulated primarily by the initial antigen expression level.

Virological characteristics of hepatitis B genotype G/A2 recombination virus in Japan.

AIM: We identified four cases of infection with hepatitis B virus genotype G and A2 recombinant (HBV/G/A2) strains, which were initially overlooked by enzyme immunoassay-based genotyping. The patients were all men who have sex with men (MSM) and inhabited several metropolitan areas of Japan, suggesting that the recombinant strains may be circulating among high-risk groups such as MSM. Here, we investigated the genomic structure and virological properties of the HBV/G/A2 strains. METHODS: Complete genome sequences of the isolates were determined and phylogenetically analyzed. Replication efficiency of HBV/G/A2 was investigated by transfecting plasmids containing 1.24-fold viral genome. The in vivo viral kinetics of HBV/G/A2 were investigated using chimeric mice with humanized livers. RESULTS: Phylogenetic analysis revealed that the four strains were almost identical (>99.7% homologous). The preS2/S region of these strains was highly homologous to that of genotype A2 and the remaining region was almost identical to that of genotype G, reflecting inter-genotypic recombination. Interestingly, in all four cases, genotype A was co-infected as a minor population. In vitro analysis revealed that HBV/G/A2 had a low replication rate. Although detectable viremia was not measurable following the inoculation of HBV/G/A2 into chimeric mice, subsequent superinfection of HBV genotype A greatly enhanced HBV/G/A2 replication and viral spread. CONCLUSION: We found that four cases of HBV/G/A2 recombinant among MSM patients in the metropolitan areas of Japan, and HBV/A co-infections are required for its efficient replication. High-risk groups such as MSM should be carefully tested for infection of genotype G-derived variants.

Postexposure prophylactic effect of hepatitis B virus (HBV)-active antiretroviral therapy against HBV infection.

Retrospective study indicates that hepatitis B virus (HBV)-active nucleoside (nucleotide) analogues (NAs) used for antiretroviral therapy reduce the incidence of acute HBV infections in human immunodeficiency virus (HIV)-infected patients. Learning from HIV postexposure prophylaxis (PEP), we explored the possibility of using NAs in PEP following HBV exposure, if preexposure prophylaxis is feasible clinically. Using freshly isolated primary human hepatocytes cultured in vitro, we analyzed the effect of HBV-active tenofovir and lamivudine in primary HBV infection and also the effect of treatment with these NAs after HBV infection. HBV-active NAs applied from 24 h before inoculation could not prevent the secretion of hepatitis B surface antigen into the culture medium, and cessation of the NAs after inoculation allowed the cells to establish an apparent HBV infection. In contrast, hepatitis B immune globulin was able to prevent HBV infection completely. NA treatment before infection, however, can control the spread of HBV infection, as detected by immunohistochemistry. Practically, starting NA treatment within 2 days of primary HBV infection inhibited viral spread effectively, as well as preexposure treatment. We demonstrated that preexposure NA treatment was not able to prevent the acquisition of HBV infection but prevented viral spread by suppressing the production of mature progeny HBV virions. The effect of postexposure treatment within 2 days was similar to the effect of preexposure treatment, suggesting the possibility of HBV PEP using HBV-active NAs in HIV- and HBV-susceptible high-risk groups.

Comprehensive screening for antigens overexpressed on carcinomas via isolation of human mAbs that may be therapeutic.

Although several murine mAbs that have been humanized became useful therapeutic agents against a few malignancies, therapeutic Abs are not yet available for the majority of the human cancers because of our lack of knowledge of which antigens (Ags) can become useful targets. In the present study we established a procedure for comprehensive identification of such Ags through the extensive isolation of human mAbs that may become therapeutic. Using the phage-display Ab library we isolated a large number of human mAbs that bind to the surface of tumor cells. They were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. The Ags recognized by those clones were isolated by immunoprecipitation and identified by MS. We isolated 2,114 mAbs with unique sequences and identified 21 distinct Ags highly expressed on several carcinomas. Of those 2,114 mAbs 356 bound specifically to one of the 21 Ags. After preparing complete IgG(1) Abs the in vitro assay for Ab-dependent cell-mediated cytotoxicity (ADCC) and the in vivo assay in cancer-bearing athymic mice were performed to examine antitumor activity. The mAbs converted to IgG(1) revealed effective ADCC as well as antitumor activity in vivo. Because half of the 21 Ags showed distinct tumor-specific expression pattern and the mAbs isolated showed various characteristics with strong affinity to the Ag, it is likely that some of the Ags detected will become useful targets for the corresponding carcinoma therapy and that several mAbs will become therapeutic agents.

What Do We See in Spectra?: Assignment of High-Intensity Peaks of Cutibacterium and Staphylococcus Spectra of MALDI-TOF Mass Spectrometry by Interspecies Comparative Proteogenomics.

Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is a widely used and reliable technology to identify microbial species and subspecies. The current methodology is based on spectral fingerprinting, analyzing protein peaks, most of which are yet to be characterized. In order to deepen the understanding of these peaks and to develop a more reasonable identification workflow, we applied proteogenomic approaches to assign the high-intensity peaks of MALDI-TOF spectra of two bacterial genera. First, the 3-22 kD proteomes of 5 Cutibacterium strains were profiled by UPLC-MS/MS, and the amino acid sequences were refined by referring to their genome in the public database. Then, the sequences were converted to m/z (x-axis) values based on their molecular masses. When the interspecies comparison of calculated m/z values was well-fitted to the observed peaks, the peak assignments for the five Cutibacterium species were confirmed. Second, the peak assignments for six Staphylococcus species were performed by using the above result for Cutibacterium and referring to ribosomal subunit proteins coded on the S10-spc-alpha operon (the S10-GERMS method), a previous proteomics report by Becher et al., and comprehensive genome analysis. We successfully assigned 13 out of 15 peaks for the Cutibacterium species and 11 out of 13 peaks for the Staphylococcus species. DNA-binding protein HU, the CsbD-like protein, and 50S ribosomal protein L7/L12 were observed in common. The commonality suggests they consist of high-intensity peaks in the MALDI spectra of other bacterial species. Our workflow may lead to the development of a more accurate species identification database of MALDI-TOF mass spectrometry based on genome data.

Common Features and Intra-Species Variation of Cutibacterium modestum Strains, and Emended Description of the Species.

Cutibacterium modestum is a new species coined in 2020 as the fifth species of genus Cutibacterium, which includes Cutibacterium acnes. The species is predicted as a minor but common member of skin microbiome and includes a group tentatively named as "Propionibacterium humerusii". The description of the species has been provided only with a single strain. To establish the characteristics of C. modestum and search for possible disease-related subtypes, we investigated the biochemical characteristics of eight live strains and performed in silico comparison of nine genomes. The common features, which included the morphology of Gram-stain positive short rods, the negativity of phenylalanine arylamidase, and several unique MALDI-TOF MS spectral peaks, were considered useful in laboratory identification. Pairwise comparisons of the genomes by in silico DNA-DNA hybridization showed similarity values of 98.1% or larger, which were far higher than the subspecies cutoff of 79-80%. The 16S rRNA gene sequences of thirteen isolates and genomes were identical. Their recA gene sequences were identical except for two strains, HM-510 (HL037PA2) and Marseille-P5998, which showed unique one-nucleotide polymorphisms. The biochemical features using API kits were slightly different among the isolates but far closer than those of the nearest other species, C. acnes and Cutibacterium namnetense. Spectra of MALDI-TOF mass spectrometry showed slight differences in the presence of m/z 10,512 (10 kD chaperonin GroS) and three other peaks, further clustering the eight isolates into three subtypes. These results indicated that these isolates did not separate to form subspecies-level clusters, but subtyping is possible by using recA gene sequences or MALDI-TOF mass spectrometry spectra. Moreover, this work has confirmed that a group "P. humerusii" is included in C. modestum.

The antiviral effects of human microRNA miR-302c-3p against hepatitis B virus infection.

BACKGROUND: Conventional treatments of chronic hepatitis B virus (HBV) infection rarely achieve a decline of serum hepatitis B surface antigen (HBsAg) levels and eradication of the virus. AIM: To elucidate the antiviral mechanisms of a human microRNA, miR-302c-3p, against HBV replication. METHODS: The antiviral effect of miR-302c-3p was evaluated in vitro and in vivo by transfecting the miR-302c-3p mimic into HBV-infected HepG2-hNTCP-C4 cells and HBV transgenic mice respectively. RESULTS: miR-302c-3p decreased not only HBV replication but also production of HBsAg. Pregenomic RNA and HBsAg mRNA concentrations decreased in the cells treated with miR-302c-3p. Interestingly, the amount of cccDNA was significantly reduced in the miR-302c-3p-treated cells, in association with disappearance of the HBV core protein. An RNA immunoprecipitation assay showed that miR-302c-3p decreased the binding of the HBV polymerase to the pregenomic RNA by hybridising with the ε-loop region. A number of host genes were downregulated in miR-302c-3p-treated cells, including BMPR2 and HNF4A. Knockdown of these two genes by corresponding siRNAs also suppressed HBV replication and HBsAg secretion. The antiviral effect of miR-302c-3p was also observed in HBV transgenic mice. CONCLUSION: miR-302c-3p had anti-HBV activity, in vitro and in vivo, via several mechanisms.

Screening of microRNAs for a repressor of hepatitis B virus replication.

BACKGROUND: Hepatitis B virus (HBV) infection is a leading cause of persistent liver diseases, cirrhosis and hepatocellular carcinoma (HCC) worldwide. Since deregulation of microRNA (miRNA) expression by HBV infection contributes to enhanced viral replication and pathogenesis, modulation of miRNA activity can be a novel therapeutic approach towards HBV eradication. As the effects of the vast majority of miRNAs on HBV replication have not been empirically investigated, here, we aim to identify novel therapeutic targets that have a strong antiviral effect on HBV. METHODS: HepG2-hNTCP-C4 cells were infected with HBV, and then were individually transfected with the library mimics of 2048 miRNAs. To assess the amount of intracellular and extracellular DNA and HBsAg, qPCR and ELISA were performed respectively. RESULTS: From miRNA library screening, we identified 39 miRNAs as candidate repressors of HBV replication. Among them, 9 miRNAs, including miR-204, strongly decreased both HBV DNA and HBsAg in culture supernatant of HepG2-hNTCP-C4 cells. Furthermore, we also showed that inhibition of Rab22a, one of the targets of miR-204, also suppressed intracellular and extracellular HBV DNA expression in HepG2.2.15.7 cells. CONCLUSIONS: Our findings contribute to the understanding of the roles of miRNAs underlying HBV replication and show the possibility of developing a novel strategy for miRNA-mediated HBV treatment.

Identification of the novel 3' UTR sequences of human IL-21 mRNA as potential targets of miRNAs.

Hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma worldwide. However, the strategy of HBV to escape from the host immune system remains largely unknown. In this study, we examined extracellular vesicles (EVs) secreted from human hepatocytes infected with HBV. EVs includeing exosomes are nano-size vesicles with proteins, mRNAs, and microRNAs (miRNAs), which can be transmitted to different cells. We found that 104 EV associated miRNAs were increased in hepatocytes more than 2-fold by HBV infection. We then selected those that were potentially implicated in immune regulation. Among them, five HBV-induced miRNAs were found to potentially target multiple sequences in the 3'UTR of IL-21, a cytokine that induces anti-viral immunity. Moreover, expression of a reporter gene with the 3' UTR of human IL-21 mRNA was suppressed by the five miRNAs individually. Finally, IL-21 expression in cloned human T cells was down-regulated by the five miRNAs. Collectively, this study identified the novel 3' UTR sequences of human IL-21 mRNA and potential binding sites of HBV-induced EV-miRNAs.

Effects of vaccine-acquired polyclonal anti-HBs antibodies on the prevention of HBV infection of non-vaccine genotypes.

BACKGROUND: In universal hepatitis B (HB) vaccination, single vaccine-derived polyclonal anti-HBs antibodies (anti-HBs) need to inhibit infection of HB viruses (HBV) of non-vaccine genotypes. We experimentally addressed this issue. METHODS: Anti-HBs-positive sera were obtained by vaccination with genotype A- or C-derived HBs antigen (HBsAg, gtA-sera or gtC-sera). Their reactivity to genotype A- and C-derived HBsAg (gtA-Ag and gtC-Ag) was measured by ELISA. The capacity of sera to neutralize HBV was evaluated using an in vitro infection model. RESULTS: Of 135 anti-gtA-Ag-reactive gtA-sera, 134 (99.3%) were anti-gtC-Ag-reactive. All (100%) 120 anti-gtC-Ag-reactive gtC-sera were anti-gtA-Ag-reactive. The reactivity to gtA-Ag was strongly correlated with that to gtC-Ag (gtA-sera, ρ = 0.989; gtC-sera, ρ = 0.953; p < 0.01). In gtA-sera (n = 10), anti-HBs to gtA-Ag were less completely absorbed with gtC-Ag (96.4%) than with gtA-Ag (100%, p < 0.05). Similarly, in gtC-sera (n = 10), anti-HBs to gtC-Ag were less completely absorbed with gtA-Ag (96.0%) than with gtC-Ag (100%, p < 0.01). Thus, 3.6 and 4.0% of anti-HBs in gtA-sera and gtC-sera were vaccine genotype HBsAg-specific, respectively. In the neutralization test, gtA-sera (n = 4) and gtC-sera (n = 3) with anti-HBs titers adjusted to 100 mIU/mL equally inhibited genotype C HBV infection (92.8 vs. 95.4%, p = 0.44). However, at 30 mIU/mL, the gtA-sera less effectively inhibited infection than the gtC-sera (60.2 vs. 90.2%, p < 0.05). CONCLUSIONS: Vaccination with genotype A- or C-derived HBsAg provided polyclonal anti-HBs that sufficiently bound to non-vaccine genotype HBsAg. However, a small portion of anti-HBs were specific to the vaccine genotype HBsAg. High anti-HBs titers would be required to prevent HBV infection of non-vaccine genotypes. UMIN/CTR UMIN000014363.

Validation of cross-genotype neutralization by hepatitis B virus-specific monoclonal antibodies by in vitro and in vivo infection.

Vaccines based on hepatitis B virus (HBV) genotype A have been used worldwide for immunoprophylaxis and are thought to prevent infections by non-A HBV strains effectively, whereas, vaccines generated from genotype C have been used in several Asian countries, including Japan and Korea, where HBV genotype C is prevalent. However, acute hepatitis B caused by HBV genotype A infection has been increasing in Japan and little is known about the efficacy of immunization with genotype C-based vaccines against non-C infection. We have isolated human monoclonal antibodies (mAbs) from individuals who were immunized with the genotype C-based vaccine. In this study, the efficacies of these two mAbs, HB0116 and HB0478, were analyzed using in vivo and in vitro models of HBV infection. Intravenous inoculation of HBV genotype C into chimeric mice with human hepatocytes resulted in the establishment of HBV infection after five weeks, whereas preincubation of the inocula with HB0116 or HB0478 protected chimeric mice from genotype C infection completely. Interestingly, both HB0116 and HB0478 were found to block completely genotype A infection. Moreover, infection by a genotype C strain with an immune escape substitution of amino acid 145 in the hepatitis B surface protein was also completely inhibited by incubation with HB0478. Finally, in vitro analysis of dose dependency revealed that the amounts of HB0478 required for complete protection against genotype C and genotype A infection were 5.5 mIU and 55 mIU, respectively. These results suggested that genotype C-based vaccines have ability to induce cross-genotype immunity against HBV infection.

Antibody-dependent cell-mediated cytotoxicity is induced by a single-chain Fv-protein III fusion in the presence of a rabbit anti-protein III polyclonal antibody.

The use of phage-displayed antibody libraries has enabled the isolation of several thousand cancer-specific monoclonal antibodies. To further select for clones among these antibodies which have therapeutic potential for cancer, several types of in vitro anti-tumor assay, such as an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, are required. The cytotoxic activities of effector cells are triggered by the binding of the Fc portion of IgG to its receptor, necessitating the conversion of a candidate clone with a single-chain variable fragment (scFv) form into a human IgG form. In the laboratory however, this conversion process is expensive and involves laborious steps such as the cloning of mammalian cells that contain an IgG expression vector, the subsequent production of protein, and affinity purification. In our current study, we show that an original fusion of scFv and protein III, a coat protein of the M13 bacteriophage, can induce ADCC activity towards its target cells in the presence of a rabbit anti-protein III polyclonal antibody. Our modified assay method thus enables the more rapid selection of potentially therapeutic clones from phage-displayed antibody libraries.

Single chain variable fragment antibodies against Shiga toxins isolated from a human antibody phage display library.

Shiga toxins (Stxs) are involved in the pathogenesis of hemolytic-uremic syndrome and other severe systemic complications following enterohemorrhagic Escherichia coli infection in humans. Passive immunotherapies using monoclonal antibodies have been shown to be effective for neutralizing the toxic effects of Stxs. However, animal-derived monoclonal antibodies are sometimes immunogenic and their production is both laborious and expensive. We here report the isolation of single-chain variable fragment antibodies against Stxs by screening a phage display library constructed from a naïve human repertoire. An antibody among the selected clones designated B22 bound to the binding subunits of both Stx-1 and Stx-2, and strongly neutralized the cytotoxicity of Stx-1. This is the first example of a monovalent antibody showing Stx-neutralizing activity. The B22 antibody is also completely naturally occurring in human, which reduces the possibility of adverse immunological effects, and can be easily produced using bacterial protein synthesis systems.