Intratracheal trimerized nanobody cocktail administration suppresses weight loss and prolongs survival of SARS-CoV-2 infected mice.

BACKGROUND: SARS-CoV-2 Omicron variants are highly resistant to vaccine-induced immunity and human monoclonal antibodies. METHODS: We previously reported that two nanobodies, P17 and P86, potently neutralize SARS-CoV-2 VOCs. In this study, we modified these nanobodies into trimers, called TP17 and TP86 and tested their neutralization activities against Omicron BA.1 and subvariant BA.2 using pseudovirus assays. Next, we used TP17 and TP86 nanobody cocktail to treat ACE2 transgenic mice infected with lethal dose of SARS-CoV-2 strains, original, Delta and Omicron BA.1. RESULTS: Here, we demonstrate that a novel nanobody TP86 potently neutralizes both BA.1 and BA.2 Omicron variants, and that the TP17 and TP86 nanobody cocktail broadly neutralizes in vitro all VOCs as well as original strain. Furthermore, intratracheal administration of this nanobody cocktail suppresses weight loss and prolongs survival of human ACE2 transgenic mice infected with SARS-CoV-2 strains, original, Delta and Omicron BA.1. CONCLUSIONS: Intratracheal trimerized nanobody cocktail administration suppresses weight loss and prolongs survival of SARS-CoV-2 infected mice.

Antibodies are made by the immune system to identify and inactivate infectious agents such as viruses. Alpacas produce a simple type of antibodies called nanobodies. We previously developed two nanobodies named P17 and P86 that inactivate SARS-CoV-2. In this study, we modified these nanobodies to create two nanobodies named TP17 and TP86. The cocktail of these nanobodies inactivated different types of SARS-CoV-2 viruses including Omicron BA.1 and BA.2. The cocktail also prolonged survival of mice infected with lethal doses of SARS-CoV-2.

Pharmacologic characterization of TBP1901, a prodrug form of aglycone curcumin, and CRISPR-Cas9 screen for therapeutic targets of aglycone curcumin.

Curcumin (aglycone curcumin) has antitumor properties in a variety of malignancies via the alteration of multiple cancer-related biological pathways; however, its clinical application has been hampered due to its poor bioavailability. To overcome this limitation, we have developed a synthesized curcumin ß-D-glucuronide sodium salt (TBP1901), a prodrug form of aglycone curcumin. In this study, we aimed to clarify the pharmacologic characteristics of TBP1901. In ß-glucuronidase (GUSB)-proficient mice, both curcumin ß-D-glucuronide and its active metabolite, aglycone curcumin, were detected in the blood after TBP1901 injection, whereas only curcumin ß-D-glucuronide was detected in GUSB-impaired mice, suggesting that GUSB plays a pivotal role in the conversion of TBP1901 into aglycone curcumin in vivo. TBP1901 itself had minimal antitumor effects in vitro, whereas it demonstrated significant antitumor effects in vivo. Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screen disclosed the genes associated with NF-κB signaling pathway and mitochondria were among the highest hit. In vitro, aglycone curcumin inhibited NF-kappa B signaling pathways whereas it caused production of reactive oxygen species (ROS). ROS scavenger, N-acetyl-L-cysteine, partially reversed antitumor effects of aglycone curcumin. In summary, TBP1901 can exert antitumor effects as a prodrug of aglycone curcumin through GUSB-dependent activation.

ILF2 enhances the DNA cytosine deaminase activity of tumor mutator APOBEC3B in multiple myeloma cells.

DNA cytosine deaminase APOBEC3B (A3B) is an endogenous source of mutations in many human cancers, including multiple myeloma. A3B proteins form catalytically inactive high molecular mass (HMM) complexes in nuclei, however, the regulatory mechanisms of A3B deaminase activity in HMM complexes are still unclear. Here, we performed mass spectrometry analysis of A3B-interacting proteins from nuclear extracts of myeloma cell lines and identified 30 putative interacting proteins. These proteins are involved in RNA metabolism, including RNA binding, mRNA splicing, translation, and regulation of gene expression. Except for SAFB, these proteins interact with A3B in an RNA-dependent manner. Most of these interacting proteins are detected in A3B HMM complexes by density gradient sedimentation assays. We focused on two interacting proteins, ILF2 and SAFB. We found that overexpressed ILF2 enhanced the deaminase activity of A3B by 30%, while SAFB did not. Additionally, siRNA-mediated knockdown of ILF2 suppressed A3B deaminase activity by 30% in HEK293T cell lysates. Based on these findings, we conclude that ILF2 can interact with A3B and enhance its deaminase activity in HMM complexes.

The SARS-CoV-2 Lambda variant exhibits enhanced infectivity and immune resistance.

SARS-CoV-2 Lambda, a variant of interest, has spread in some South American countries; however, its virological features and evolutionary traits remain unclear. In this study, we use pseudoviruses and reveal that the spike protein of the Lambda variant is more infectious than that of other variants due to the T76I and L452Q mutations. The RSYLTPGD246-253N mutation, a unique 7-amino acid deletion in the N-terminal domain of the Lambda spike protein, is responsible for evasion from neutralizing antibodies and further augments antibody-mediated enhancement of infection. Although this mutation generates a nascent N-linked glycosylation site, the additional N-linked glycan is dispensable for the virological property conferred by this mutation. Since the Lambda variant has dominantly spread according to the increasing frequency of the isolates harboring the RSYLTPGD246-253N mutation, our data suggest that the RSYLTPGD246-253N mutation is closely associated with the substantial spread of the Lambda variant in South America.

Enhanced fusogenicity and pathogenicity of SARS-CoV-2 Delta P681R mutation.

During the current coronavirus disease 2019 (COVID-19) pandemic, a variety of mutations have accumulated in the viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and, at the time of writing, four variants of concern are considered to be potentially hazardous to human society1. The recently emerged B.1.617.2/Delta variant of concern is closely associated with the COVID-19 surge that occurred in India in the spring of 2021 (ref. 2). However, the virological properties of B.1.617.2/Delta remain unclear. Here we show that the B.1.617.2/Delta variant is highly fusogenic and notably more pathogenic than prototypic SARS-CoV-2 in infected hamsters. The P681R mutation in the spike protein, which is highly conserved in this lineage, facilitates cleavage of the spike protein and enhances viral fusogenicity. Moreover, we demonstrate that the P681R-bearing virus exhibits higher pathogenicity compared with its parental virus. Our data suggest that the P681R mutation is a hallmark of the virological phenotype of the B.1.617.2/Delta variant and is associated with enhanced pathogenicity.

APOBEC3B is preferentially expressed at the G2/M phase of cell cycle.

APOBEC3B (A3B) is a cytosine deaminase that converts cytosine to uracil in single-stranded DNA. Cytosine-to-thymine and cytosine-to-guanine base substitution mutations in trinucleotide motifs (APOBEC mutational signatures) were found in various cancers including lymphoid hematological malignancies such as multiple myeloma and A3B has been shown to be an enzymatic source of mutations in those cancers. Although the importance of A3B is being increasingly recognized, it is unclear how A3B expression is regulated in cancer cells as well as normal cells. To answer these fundamental questions, we analyzed 1276 primary myeloma cells using single-cell RNA-sequencing (scRNA-seq) and found that A3B was preferentially expressed at the G2/M phase, in sharp contrast to the expression patterns of other APOBEC3 genes. Consistently, we demonstrated that A3B protein was preferentially expressed at the G2/M phase in myeloma cells by cell sorting. We also demonstrated that normal blood cells expressing A3B were also enriched in G2/M-phase cells by analyzing scRNA-seq data from 86,493 normal bone marrow mononuclear cells. Furthermore, we revealed that A3B was expressed mainly in plasma cells, CD10+ B cells and erythroid cells, but not in granulocyte-macrophage progenitors. A3B expression profiling in normal blood cells may contribute to understanding the defense mechanism of A3B against viruses, and partially explain the bias of APOBEC mutational signatures in lymphoid but not myeloid malignancies. This study identified the cells and cellular phase in which A3B is highly expressed, which may help reveal the mechanisms behind carcinogenesis and cancer heterogeneity, as well as the biological functions of A3B in normal blood cells.

CAGE-seq reveals that HIV-1 latent infection does not trigger unique cellular responses in a Jurkat T cell model.

The cure for HIV-1 is currently stalled by our inability to specifically identify and target latently infected cells. HIV-1 viral RNA/DNA or viral proteins are recognized by cellular mechanisms and induce interferon responses in virus producing cells, but changes in latently infected cells remain unknown. HIVGKO contains a GFP reporter under the HIV-1 promoter and an mKO2 reporter under the internal EF1α promoter. This viral construct enables direct identification of HIV-1 both productively and latently infected cells. In this study we aim to identify specific cellular transcriptional responses triggered by HIV-1 entry and integration using Cap Analysis of Gene Expression (CAGE).We deep sequenced CAGE tags in uninfected, latently and productively infected cells and compared their differentially expressed transcription start site (TSS) profiles. Virus producing cells had differentially expressed TSSs related to T-cell activation and apoptosis when compared to uninfected cells or latently infected cells. Surprisingly, latently infected cells had only 33 differentially expressed TSSs compared to uninfected cells. Among these, SPP1 and APOE were down-regulated in latently infected cells. SPP1 or APOE knockdown in Jurkat T cells increased susceptibility to HIVGKO infection, suggesting that they have anti-viral properties. Components of the PI3K/mTOR pathway, MLST8, 4EBP and RPS6, were significant TSSs in productively infected cells, and S6K phosphorylation was increased compared to latently infected cells, suggesting that mTOR pathway activity plays a role in establishing the latent reservoir. These findings indicate that HIV-1 entry and integration do not trigger unique transcriptional responses when infection becomes latent.Importance: Latent HIV-1 infection is established as early as the first viral exposure and remains the most important barrier in obtaining the cure for HIV-1 infection. Here, we used CAGE to compare the transcriptional landscape of latently infected cells with that of non-infected or productively infected cells. We found that latently infected cells and non-infected cells show quite similar transcriptional profiles. Our data suggest that T-cells cannot recognize incoming viral components nor the integrated HIV-1 genome when infection remains latent. These findings should guide future research into widening our approaches to identify and target latent HIV-1 infected cells.

APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and in various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of specifically A3B in cancer cells. To easily and comprehensively investigate A3B function in myeloma cells, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3×FLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced by known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeted therapeutics, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage triggers A3B expression through ATM, ATR and DNA-PK signaling.

[Essential thrombocythemia accompanied by adrenal infarction].

A 64-year-old man presented to the emergency department with sudden-onset upper abdominal pain and pain in the left chest area. His platelet count was 121.7×104/µl. Computed tomography (CT) showed bilateral adrenal swelling and inflammation of the adjacent tissue. Diffusion-weighted magnetic resonance imaging (MRI) showed hyperintensity in the bilateral adrenal glands. The patient was diagnosed with bilateral adrenal infarction. A bone marrow biopsy yielded a diagnosis of essential thrombocythemia, and a positive JAK2 V617F mutation was detected. He presented with recurrent adrenal infarction and developed aortic mural thrombosis and splenic infarction. We administered aspirin and performed cytoreductive therapy with hydroxyurea and anagrelide; however, the patient then went into heart failure resulting from coronary artery stenosis. We then added prasugrel to the list of medicines administered to manage his condition. Bilateral adrenal infarction is a very rare thrombotic event of essential thrombocythemia. CT and MRI were useful for making the diagnosis; however, we also had to rule out acute coronary syndrome or intestinal ischemia. Our patient presented with strong thrombotic diathesis, which prompted us to use dual antiplatelet therapy; however, further studies are needed to confirm the efficacy and safety of this treatment.

Successful engraftment after cord blood transplantation from an HLA-homozygous donor (homo-to-hetero cord blood transplantation) in a primary myelofibrosis patient with broad HLA antibodies.

BACKGROUND: Donor-specific human leukocyte antigen (HLA) antibodies are a significant risk factor for graft failure in cord blood transplantation (CBT). Although there are several treatments to decrease HLA antibodies, such as platelet transfusion, plasma exchange, rituximab, and bortezomib, their effectiveness has not been established. STUDY DESIGN AND METHODS: We herein report the case of a primary myelofibrosis (PMF) patient with broad HLA antibodies who underwent CBT from an HLA-homozygous donor in which the alleles were matched only in the host-versus-graft direction (homo-to-hetero CBT). RESULTS: The cord blood was killer cell immunoglobulin-like receptor (KIR) ligand matched. She received a reduced-intensity conditioning regimen. We used tacrolimus and mycophenolate mofetil as prophylaxis against graft-versus-host disease (GVHD). The neutrophils engrafted on Day 31. A chimerism analysis with fluorescence in situ hybridization of peripheral blood cells showed 99.9% donor type on Day 33. She developed only mild acute skin GVHD and chronic skin GVHD. CONCLUSION: This case indicates the usefulness of homo-to-hetero CBT in a patient with broad HLA antibodies with a strong mean fluorescence intensity, which is a significant risk factor for graft failure. Further studies are necessary to determine the risk of GVHD and to elucidate the association between KIR ligand incompatibility and graft failure in homo-to-hetero CBT.

Substantial Improvement in a Nerve Conduction Study of Lymphoma-associated Demyelinating Neuropathy Treated by Intravenous Immunoglobulin and Chemotherapy.

A 64-year-old woman with lymphoma-associated demyelinating neuropathy was treated by 6 cycles of R-CHOP with intravenous immunoglobulin in the first 2 cycles. We noted substantial improvement in the findings of a nerve conduction study (NCS) after the first cycle, followed by more protracted improvement during the second to sixth cycles. The improvement of the neurological symptoms paralleled the findings of the NCS. Our case provides important information for understanding the etiology and optimization of treatments for lymphoma-associated demyelinating neuropathy.