Non-clinical and clinical characterization of MAU868, a novel human-derived monoclonal neutralizing antibody targeting BK polyomavirus VP1.

Reactivation of BK polyomavirus (BKPyV) can cause significant kidney and bladder disease in immunocompromised patients. There are currently no effective, BKPyV-specific therapies. MAU868 is a novel, human IgG1 monoclonal antibody that binds the major capsid protein VP1 of BKPyV with picomolar affinity, neutralizes infection by the four major BKPyV genotypes (EC50 ranging from 0.009 to 0.093 µg/ml; EC90 ranging from 0.102 to 4.160 µg/ml), and has comparable activity against variants with highly prevalent VP1 polymorphisms. No resistance-associated variants were identified in long-term selection studies, indicating a high in vitro barrier-to-resistance. The high-resolution crystal structure of MAU868 in complex with VP1 pentamer identified three key contact residues in VP1 (Y169, R170, K172). A first-in-human study was conducted to assess the safety, tolerability, and pharmacokinetics of MAU868 following intravenous and subcutaneous administration to healthy adults in a randomized, placebo-controlled, double-blinded, single ascending dose design. MAU868 was safe and well-tolerated. All adverse events were Grade 1 and resolved. The pharmacokinetics of MAU868 was typical of a human IgG, with dose-proportional systemic exposure and an elimination half-life ranging between 23 and 30 days. These results demonstrate the potential of MAU868 as a first-in-class therapeutic agent for the treatment or prevention of BKPyV disease.

Gene Coexpression Analysis Identifies Genes Associated with Chlorophyll Content and Relative Water Content in Pearl Millet.

Pearl millet is a significant crop that is tolerant to abiotic stresses and is a staple food of arid regions. However, its underlying mechanisms of stress tolerance are not fully understood. Plant survival is regulated by the ability to perceive a stress signal and induce appropriate physiological changes. Here, we screened for genes regulating physiological changes such as chlorophyll content (CC) and relative water content (RWC) in response to abiotic stress by using "weighted gene coexpression network analysis" (WGCNA) and clustering changes in physiological traits, i.e., CC and RWC associated with gene expression. Genes' correlations with traits were defined in the form of modules, and different color names were used to denote a particular module. Modules are groups of genes with similar patterns of expression, which also tend to be functionally related and co-regulated. In WGCNA, the dark green module (7082 genes) showed a significant positive correlation with CC, and the black (1393 genes) module was negatively correlated with CC and RWC. Analysis of the module positively correlated with CC highlighted ribosome synthesis and plant hormone signaling as the most significant pathways. Potassium transporter 8 and monothiol glutaredoxin were reported as the topmost hub genes in the dark green module. In Clust analysis, 2987 genes were found to display a correlation with increasing CC and RWC. Furthermore, the pathway analysis of these clusters identified the ribosome and thermogenesis as positive regulators of RWC and CC, respectively. Our study provides novel insights into the molecular mechanisms regulating CC and RWC in pearl millet.

Unexplored nutritive potential of tomato to combat global malnutrition.

Tomato, a widely consumed vegetable crop, offers a real potential to combat human nutritional deficiencies. Tomatoes are rich in micronutrients and other bioactive compounds (including vitamins, carotenoids, and minerals) that are known to be essential or beneficial for human health. This review highlights the current state of the art in the molecular understanding of the nutritional aspects, conventional and molecular breeding efforts, and biofortification studies undertaken to improve the nutritional content and quality of tomato. Transcriptomics and metabolomics studies, which offer a deeper understanding of the molecular regulation of the tomato's nutrients, are discussed. The potential uses of the wastes from the tomato processing industry (i.e., the peels and seed extracts) that are particularly rich in oils and proteins are also discussed. Recent advancements with CRISPR/Cas mediated gene-editing technology provide enormous opportunities to enhance the nutritional content of agricultural produces, including tomatoes. In this regard, genome editing efforts with respect to biofortification in the tomato plant are also discussed. The recent technological advancements and knowledge gaps described herein aim to help explore the unexplored nutritional potential of the tomato.

Role of silicon in elevating resistance against sheath blight and blast diseases in rice (Oryza sativa L.).

Rice blast caused by Magnaporthe oryzae and sheath blight caused by Rhizoctonia solani, are the two major diseases of rice that cause enormous losses in rice production worldwide. Identification and utilization of broad-spectrum resistance resources have been considered sustainable and effective strategies. However, the majority of the resistance genes and QTLs identified have often been found to be race-specific, and their resistance is frequently broken down due to continuous exposure to the pathogen. Therefore, integrated approaches to improve plant resistance against such devastating pathogen have great importance. Silicon (Si), a beneficial element for plant growth, has shown to provide a prophylactic effect against many pathogens. The application of Si helps the plants to combat the disease-causing pathogens, either through its deposition in different parts of the plant or through modulation/induction of specific defense genes by yet an unknown mechanism. Some reports have shown that Si imparts resistance to rice blast and sheath blight. The present review summarizes the mechanism of Si transport and deposition and its effect on rice growth and development. A special emphasis has been given to explore the existing evidence showing Si mediated blast and sheath blight resistance and the mechanism involved in resistance. This review will help to understand the prophylactic effects of Si against sheath blight and blast disease at the mechanical, physiological, and genetic levels. The information provided here will help develop a strategy to explore Si derived benefits for sustainable rice production.

Identification and characterization of a spotted-leaf mutant spl40 with enhanced bacterial blight resistance in rice.

BACKGROUND: Spotted leaf mutants show typical necrotic lesions that appear spontaneously in the absence of any pathogen attack. These mutants are often characterized to exhibit programmed cell death (PCD) and activation of plant defense responses resulting in enhanced disease resistance to multiple pathogens. Here, we reported a novel spotted-leaf mutant, spl40 that showed enhanced disease resistance response. RESULTS: Initially lesions appeared at leaf tips during seedling stage and gradually covered the whole leaf at the tillering stage. The lesion development was light-dependent. spl40 showed obvious cell death at and around the lesion, and burst of reactive oxygen species (ROS) was accompanied by disturbed ROS scavenging system. Photosynthetic capacity was compromised as evidenced by significant reductions in chlorophyll content, important photosynthesis parameters and downregulated expression of photosynthesis-related genes which ultimately led to poor performance of major agronomic traits. spl40 exhibited enhanced resistance to 14 out of 16 races of bacterial blight pathogen of rice, caused by Xanthomonas oryzae pv. oryzae, most probably though activation of SA and JA signaling pathways, owing to upregulated expression of SA and JA signaling genes, though the exact mechanism remain to be elucidated. The spotted-leaf phenotype was controlled by a novel single recessive nuclear gene. Genetic mapping combined with high throughput sequencing analysis identified Os05G0312000 as the most probable candidate gene. Sequencing of ORF revealed a single SNP change from C to T that resulted in non-synonymous change in amino acid residue from leucine to phenylalanine. Interestingly, the complementation plants did not display lesions before heading but showed lesions at the heading stage and the transgenic T1 progenies could be classified into 3 categories based on their lesion intensity, indicating the complex genetic nature of the spl40 mutation. CONCLUSION: The results obtained here clearly show that genes related to defense and PCD were upregulated in accordance with enhanced disease resistance and occurrence of PCD, whereas the photosynthetic capacity and overall ROS homeostasis was compromised in spl40. Our data suggest that a novel spotted-leaf mutant, spl40, would help to elucidate the mechanism behind lesion development involving programmed cell death and associated defense responses.

Human Memory B Cells Harbor Diverse Cross-Neutralizing Antibodies against BK and JC Polyomaviruses.

Human polyomaviruses cause a common childhood infection worldwide and typically elicit a neutralizing antibody and cellular immune response, while establishing a dormant infection in the kidney with minimal clinical manifestations. However, viral reactivation can cause severe pathology in immunocompromised individuals. We developed a high-throughput, functional antibody screen to examine the humoral response to BK polyomavirus. This approach enabled the isolation of antibodies from all peripheral B cell subsets and revealed the anti-BK virus antibody repertoire as clonally complex with respect to immunoglobulin sequences and isotypes (both IgM and IgG), including a high frequency of monoclonal antibodies that broadly neutralize BK virus subtypes and the related JC polyomavirus. Cryo-electron microscopy of a broadly neutralizing IgG single-chain variable fragment complexed with BK virus-like particles revealed the quaternary nature of a conserved viral epitope at the junction between capsid pentamers. These features unravel a potent modality for inhibiting polyomavirus infection in kidney transplant recipients and other immunocompromised patients.

Identification of a Novel Semi-Dominant Spotted-Leaf Mutant with Enhanced Resistance to Xanthomonas oryzae pv. oryzae in Rice.

Many spotted-leaf mutants show enhanced disease resistance to multiple pathogen attacks; however, the mechanisms are largely unknown. Here, we reported a novel semi-dominant spotted-leaf mutant 24 (spl24) obtained from an ethyl methane sulfonate (EMS)-induced IR64 mutant bank. spl24 developed tiny brown lesions on the leaf tip and spread down gradually to the leaf base as well as the sheath at the early heading stage. The performances of major agronomic traits such as the plant height, panicle length, number of panicles/plant, and 1000-grain weight were significantly altered in spl24 when compared to the wild-type IR64. Furthermore, spl24 exhibited a premature senescing phenotype with degeneration of nuclear acids, significantly reduced soluble protein content, increased level of malonaldehyde (MDA), and lowered activities of reactive oxygen species (ROS) scavenging enzymes. Disease evaluation indicated that spl24 showed enhanced resistance to multiple races of Xanthomonas oryzae pv. oryzae, the causal pathogen of bacterial leaf blight in rice, with elevated expression of pathogenesis-related genes, salicylic acid (SA) signaling pathway-associated genes revealed by real-time quantitative PCR and high-throughput RNA sequencing analysis. Genetic analysis and gene mapping indicated that the lesion mimic phenotype was controlled by a novel semi-dominant nuclear gene. The mutation, tentatively termed as OsSPL24, was in a 110 kb region flanked by markers Indel-33 and Indel-12 in chromosome 11. Together, our data suggest that spl24 is a novel lesion mimic mutant with enhanced innate immunity and would facilitate the isolation and functional characterization of the target gene.

A substitution mutation in OsPELOTA confers bacterial blight resistance by activating the salicylic acid pathway.

We previously reported a spotted-leaf mutant pelota (originally termed HM47) in rice displaying arrested growth and enhanced resistance to multiple races of Xanthomonas oryzae pv. oryzae. Here, we report the map-based cloning of the causal gene OsPELOTA (originally termed splHM47 ). We identified a single base substitution from T to A at position 556 in the coding sequence of OsPELOTA, effectively mutating phenylalanine to isoleucine at position 186 in the translated protein sequence. Both functional complementation and over-expression could rescue the spotted-leaf phenotype. OsPELOTA, a paralogue to eukaryotic release factor 1 (eRF1), shows high sequence similarity to Drosophila Pelota and also localizes to the endoplasmic reticulum and plasma membrane. OsPELOTA is constitutively expressed in roots, leaves, sheaths, stems, and panicles. Elevated levels of salicylic acid and decreased level of jasmonate were detected in the pelota mutant. RNA-seq analysis confirmed that genes responding to salicylic acid were upregulated in the mutant. Our results indicate that the rice PELOTA protein is involved in bacterial leaf blight resistance by activating the salicylic acid metabolic pathway.

Correlation of BK Virus Neutralizing Serostatus With the Incidence of BK Viremia in Kidney Transplant Recipients.

BACKGROUND: BK virus (BKV)-associated nephropathy is the second leading cause of graft loss in kidney transplant recipients. Due to the high prevalence of persistent infection with BKV in the general population, it is possible that either the transplant recipient or donor may act as the source of virus resulting in viruria and viremia. Although several studies suggest a correlation between donor-recipient serostatus and the development of BK viremia, specific risk factors for BKV-related complications in the transplant setting remain to be established. METHODS: We retrospectively determined the pretransplant BKV neutralizing serostatus of 116 donors (D)-recipient (R) pairs using infectious BKV neutralization assays with representatives from the 4 major viral serotypes. The neutralizing serostatus of donors and recipients was then correlated with the incidence of BK viremia during the first year posttransplantation. RESULTS: There were no significant differences in baseline demographics or transplant data among the 4 neutralizing serostatus groups, with the exception of calculated panel-reactive antibody which was lowest in the D+/R- group. Recipients of kidneys from donors with significant serum neutralizing activity (D+) had elevated risk for BK viremia, regardless of recipient serostatus (D+ versus D-: odd ratio, 5.0; 95% confidence interval, 1.9-12.7]; P = 0.0008). Furthermore, donor-recipient pairs with D+/R- neutralizing serostatus had the greatest risk for BK viremia (odds ratio, 4.9; 95% confidence interval, 1.7-14.6; P = 0.004). CONCLUSIONS: Donor neutralizing serostatus correlates significantly with incidence of posttransplant BK viremia. Determination of donor-recipient neutralizing serostatus may be useful in assessing the risk of BKV infection in kidney transplant recipients.

A CpG-Ficoll Nanoparticle Adjuvant for Anthrax Protective Antigen Enhances Immunogenicity and Provides Single-Immunization Protection against Inhaled Anthrax in Monkeys.

Nanoparticulate delivery systems for vaccine adjuvants, designed to enhance targeting of secondary lymphoid organs and activation of APCs, have shown substantial promise for enhanced immunopotentiation. We investigated the adjuvant activity of synthetic oligonucleotides containing CpG-rich motifs linked to the sucrose polymer Ficoll, forming soluble 50-nm particles (DV230-Ficoll), each containing >100 molecules of the TLR9 ligand, DV230. DV230-Ficoll was evaluated as an adjuvant for a candidate vaccine for anthrax using recombinant protective Ag (rPA) from Bacillus anthracis. A single immunization with rPA plus DV230-Ficoll induced 10-fold higher titers of toxin-neutralizing Abs in cynomolgus monkeys at 2 wk compared with animals immunized with equivalent amounts of monomeric DV230. Monkeys immunized either once or twice with rPA plus DV230-Ficoll were completely protected from challenge with 200 LD50 aerosolized anthrax spores. In mice, DV230-Ficoll was more potent than DV230 for the induction of innate immune responses at the injection site and draining lymph nodes. DV230-Ficoll was preferentially colocalized with rPA in key APC populations and induced greater maturation marker expression (CD69 and CD86) on these cells and stronger germinal center B and T cell responses, relative to DV230. DV230-Ficoll was also preferentially retained at the injection site and draining lymph nodes and produced fewer systemic inflammatory responses. These findings support the development of DV230-Ficoll as an adjuvant platform, particularly for vaccines such as for anthrax, for which rapid induction of protective immunity and memory with a single injection is very important.

CpG-containing immunostimulatory DNA sequences elicit TNF-alpha-dependent toxicity in rodents but not in humans.

CpG-containing immunostimulatory DNA sequences (ISS), which signal through TLR9, are being developed as a therapy for allergic indications and have proven to be safe and well tolerated in humans when administrated via the pulmonary route. In contrast, ISS inhalation has unexplained toxicity in rodents, which express TLR9 in monocyte/macrophage lineage cells as well as in plasmacytoid DCs (pDCs) and B cells, the principal TLR9-expressing cells in humans. We therefore investigated the mechanisms underlying this rodent-specific toxicity and its implications for humans. Mice responded to intranasally administered 1018 ISS, a representative B class ISS, with strictly TLR9-dependent toxicity, including lung inflammation and weight loss, that was fully reversible and pDC and B cell independent. Knockout mouse experiments demonstrated that ISS-induced toxicity was critically dependent on TNF-alpha, with IFN-alpha required for TNF-alpha induction. In contrast, human PBMCs, human alveolar macrophages, and airway-derived cells from Ascaris suum-allergic cynomolgus monkeys did not produce appreciable TNF-alpha in vitro in response to ISS stimulation. Moreover, sputum of allergic humans exposed to inhaled ISS demonstrated induction of IFN-inducible genes but minimal TNF-alpha induction. These data demonstrate that ISS induce rodent-specific TNF-alpha-dependent toxicity that is absent in humans and reflective of differential TLR9 expression patterns in rodents versus humans.

In vitro methotrexate as a practical approach to selective allodepletion.

Graft-versus-host disease (GVHD) is a major cause of transplant-related morbidity and mortality in recipients of allogeneic hematopoietic stem cell transplantation. As GVHD is mediated predominantly by alloreactive donor T cells, selective allodepletion from the graft may alleviate GVHD, whereas potentially maintaining other advantages conferred by donor T cells, such as graft survival, antiviral immunity, and graft-versus-leukemia effect. In this study, we evaluated the ability of methotrexate, a clinically approved antimetabolite drug, to deplete alloreactive T cells in HLA-mismatched mixed lymphocyte reactions (MLR). We observed that methotrexate could inhibit the proliferation of alloreactive T cells in primary in vitro MLR. On reexposure of methotrexate-treated cells to the same allostimulus, a significant reduction in the alloreactive immune response was observed, whereas responses to third-party allostimuli and viral antigens were preserved. Thus, our results provide preclinical evidence that in vitro methotrexate treatment results in specific allodepletion and may be used as an effective agent for preventing GVHD.