Development of a novel monoclonal antibody-based competitive ELISA for antibody detection against bovine leukemia virus.

Infection with bovine leukemia virus (BLV) leads to enzootic bovine leukosis, the most prevalent neoplastic disease in cattle. Due to the lack of commercially available vaccines, reliable eradication of the disease can be achieved through the testing and elimination of BLV antibody-positive animals. In this study, we developed a novel competitive ELISA (cELISA) to detect antibodies against BLV capsid protein p24. Recombinant p24 protein expressed by Escherichia coli, in combination with the monoclonal antibody 2G11 exhibiting exceptional performance, was used for the establishment of the cELISA. Receiver-operating characteristic curve analysis showed that the sensitivity and specificity of the assay were 98.85 % and 98.13 %, respectively. Furthermore, the established cELISA was specific for detecting BLV-specific antibodies, without cross-reactivity to antisera for six other bovine viruses. Significantly, experimental infection of cattle and sheep with BLV revealed that the cELISA accurately monitors seroconversion. In a performance evaluation, the established cELISA displayed a high agreement with Western blotting and the commercial BLV gp51 cELISA kit in the detection of 242 clinical samples, respectively. In conclusion, the novel p24 cELISA exhibited the potential to be a reliable and efficient diagnostic tool for BLV serological detection with a broad application prospect.

The helicase-like transcription factor redirects the autophagic flux and restricts human T cell leukemia virus type 1 infection.

Retroviruses and their host have coevolved in a delicate balance between viral replication and survival of the infected cell. In this equilibrium, restriction factors expressed by infected cells control different steps of retroviral replication such as entry, uncoating, nuclear import, expression, or budding. Here, we describe a mechanism of restriction against human T cell leukemia virus type 1 (HTLV-1) by the helicase-like transcription factor (HLTF). We show that RNA and protein levels of HLTF are reduced in primary T cells of HTLV-1-infected subjects, suggesting a clinical relevance. We further demonstrate that the viral oncogene Tax represses HLTF transcription via the Enhancer of zeste homolog 2 methyltransferase of the Polycomb repressive complex 2. The Tax protein also directly interacts with HLTF and induces its proteasomal degradation. RNA interference and gene transduction in HTLV-1-infected T cells derived from patients indicate that HLTF is a restriction factor. Restoring the normal levels of HLTF expression induces the dispersal of the Golgi apparatus and overproduction of secretory granules. By synergizing with Tax-mediated NF-κB activation, physiologically relevant levels of HLTF intensify the autophagic flux. Increased vesicular trafficking leads to an enlargement of the lysosomes and the production of large vacuoles containing viral particles. HLTF induction in HTLV-1-infected cells significantly increases the percentage of defective virions. In conclusion, HLTF-mediated activation of the autophagic flux blunts the infectious replication cycle of HTLV-1, revealing an original mode of viral restriction.

Specific DMPK-promoter targeting by CRISPRi reverses myotonic dystrophy type 1-associated defects in patient muscle cells.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disease that originates from an expansion of CTG microsatellites in the 3' untranslated region of the DMPK gene, thus leading to the expression of transcripts containing expanded CUG repeats (CUGexp). The pathophysiology is explained by a toxic RNA gain of function where CUGexp RNAs form nuclear aggregates that sequester and alter the function of MBNL splicing factors, triggering splicing misregulation linked to the DM1 symptoms. There is currently no cure for DM1, and most therapeutic strategies aim at eliminating CUGexp-DMPK transcripts. Here, we investigate a DMPK-promoter silencing strategy using CRISPR interference as a new alternative approach. Different sgRNAs targeting the DMPK promoter are evaluated in DM1 patient muscle cells. The most effective guides allowed us to reduce the level of DMPK transcripts and CUGexp-RNA aggregates up to 80%. The CUGexp-DMPK repression corrects the overall transcriptome, including spliceopathy, and reverses a physiological parameter in DM1 muscle cells. Its action is specific and restricted to the DMPK gene, as confirmed by genome-wide expression analysis. Altogether, our findings highlight DMPK-promoter silencing by CRISPRi as a promising therapeutic approach for DM1.

Excess of blood eosinophils prior to therapy correlates with worse prognosis in mesothelioma.

Background: Only a fraction of patients with malignant pleural mesothelioma (MPM) will respond to chemo- or immunotherapy. For the majority, the condition will irremediably relapse after 13 to 18 months. In this study, we hypothesized that patients' outcome could be correlated to their immune cell profile. Focus was given to peripheral blood eosinophils that, paradoxically, can both promote or inhibit tumor growth depending on the cancer type. Methods: The characteristics of 242 patients with histologically proven MPM were retrospectively collected in three centers. Characteristics included overall survival (OS), progression-free survival (PFS), overall response rate (ORR) and disease control rate (DCR). The mean absolute eosinophil counts (AEC) were determined by averaging AEC data sets of the last month preceding the administration of chemo- or immunotherapy. Results: An optimal cutoff of 220 eosinophils/µL of blood segregated the cohort into two groups with significantly different median OS after chemotherapy (14 and 29 months above and below the threshold, p = 0.0001). The corresponding two-year OS rates were 28% and 55% in the AEC ≥ 220/µL and AEC < 220/µL groups, respectively. Based on shorter median PFS (8 vs 17 months, p < 0.0001) and reduced DCR (55.9% vs 35.2% at 6 months), the response to standard chemotherapy was significantly affected in the AEC ≥ 220/µL subset. Similar conclusions were also drawn from data sets of patients receiving immune checkpoint-based immunotherapy. Conclusion: In conclusion, baseline AEC ≥ 220/µL preceding therapy is associated with worse outcome and quicker relapse in MPM.

Assessment of community efforts to advance network-based prediction of protein-protein interactions.

Comprehensive understanding of the human protein-protein interaction (PPI) network, aka the human interactome, can provide important insights into the molecular mechanisms of complex biological processes and diseases. Despite the remarkable experimental efforts undertaken to date to determine the structure of the human interactome, many PPIs remain unmapped. Computational approaches, especially network-based methods, can facilitate the identification of previously uncharacterized PPIs. Many such methods have been proposed. Yet, a systematic evaluation of existing network-based methods in predicting PPIs is still lacking. Here, we report community efforts initiated by the International Network Medicine Consortium to benchmark the ability of 26 representative network-based methods to predict PPIs across six different interactomes of four different organisms: A. thaliana, C. elegans, S. cerevisiae, and H. sapiens. Through extensive computational and experimental validations, we found that advanced similarity-based methods, which leverage the underlying network characteristics of PPIs, show superior performance over other general link prediction methods in the interactomes we considered.

A proteome-scale map of the SARS-CoV-2-human contactome.

Understanding the mechanisms of coronavirus disease 2019 (COVID-19) disease severity to efficiently design therapies for emerging virus variants remains an urgent challenge of the ongoing pandemic. Infection and immune reactions are mediated by direct contacts between viral molecules and the host proteome, and the vast majority of these virus-host contacts (the 'contactome') have not been identified. Here, we present a systematic contactome map of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with the human host encompassing more than 200 binary virus-host and intraviral protein-protein interactions. We find that host proteins genetically associated with comorbidities of severe illness and long COVID are enriched in SARS-CoV-2 targeted network communities. Evaluating contactome-derived hypotheses, we demonstrate that viral NSP14 activates nuclear factor κB (NF-κB)-dependent transcription, even in the presence of cytokine signaling. Moreover, for several tested host proteins, genetic knock-down substantially reduces viral replication. Additionally, we show for USP25 that this effect is phenocopied by the small-molecule inhibitor AZ1. Our results connect viral proteins to human genetic architecture for COVID-19 severity and offer potential therapeutic targets.

A safe and effective vaccine against bovine leukemia virus.

Previous attempts to develop a vaccine against bovine leukemia virus (BLV) have not been successful because of inadequate or short-lived stimulation of all immunity components. In this study, we designed an approach based on an attenuated BLV provirus by deleting genes dispensable for infectivity but required for efficient replication. The ability of the vaccine to protect from natural BLV infection was investigated in the context of dairy productive conditions in an endemic region. The attenuated vaccine was tested in a farm in which the prevalence rose from 16.7% in young cattle at the beginning of the study to more than 90% in adult individuals. Sterilizing immunity was obtained in 28 out of 29 vaccinated heifers over a period of 48 months, demonstrating the effectiveness of the vaccine. As indicated by the antiviral antibody titers, the humoral response was slightly reduced compared to wild-type infection. After initial post-vaccination bursts, the proviral loads of the attenuated vaccine remained most frequently undetectable. During the first dairy cycle, proviral DNA was not detected by nested-PCR in milk samples from vaccinated cows. During the second dairy cycle, provirus was sporadically detected in milk of two vaccinated cows. Forty-two calves born from vaccinated cows were negative for proviral DNA but had antiviral antibodies in their peripheral blood. The attenuated strain was not transmitted to sentinels, further supporting the safety of the vaccine. Altogether, these data thus demonstrate that the vaccine against BLV is safe and effective in herd conditions characterized by a very high incidence. This cost-effective approach will thus decrease the prevalence of BLV without modification of production practices. After facing a series of challenges pertaining to effectiveness and biosafety, the vaccine is now available for further large-scale delivery. The different challenges and hurdles that were bypassed may be informative for the development of a vaccine against HTLV-1.

HDAC Inhibition with Valproate Improves Direct Cytotoxicity of Monocytes against Mesothelioma Tumor Cells.

The composition of the tumor microenvironment (TME) mediates the outcome of chemo- and immunotherapies in malignant pleural mesothelioma (MPM). Tumor-associated macrophages (TAMs) and monocyte myeloid-derived immunosuppressive cells (M-MDSCs) constitute a major fraction of the TME. As central cells of the innate immune system, monocytes exert well-characterized functions of phagocytosis, cytokine production, and antibody-dependent cell-mediated cytotoxicity (ADCC). The objective of this study was to evaluate the ability of monocytes to exert a direct cytotoxicity by cell-to-cell contact with MPM cells. The experimental model is based on cocultures between human blood-derived monocytes sorted by negative selection and mesothelioma cell lines. Data show (i) that blood-derived human monocytes induce tumor cell death by direct cell-to-cell contact, (ii) that VPA is a pharmacological enhancer of this cytotoxic activity, (iii) that VPA increases monocyte migration and their aggregation with MPM cells, and (iv) that the molecular mechanisms behind VPA modulation of monocytes involve a downregulation of the membrane receptors associated with the M2 phenotype, i.e., CD163, CD206, and CD209. These conclusions, thus, broaden our understanding about the molecular mechanisms involved in immunosurveillance of the tumor microenvironment and open new prospects for further improvement of still unsatisfactory MPM therapies.

Activation of DNA Damage Tolerance Pathways May Improve Immunotherapy of Mesothelioma.

Immunotherapy based on two checkpoint inhibitors (ICI), programmed cell death 1 (PD-1, Nivolumab) and cytotoxic T-lymphocyte 4 (CTLA-4, Ipilimumab), has provided a significant improvement in overall survival for malignant mesothelioma (MM). Despite this major breakthrough, the median overall survival of patients treated with the two ICIs only reached 18.1 months vs. 14 months in standard chemotherapy. With an objective response rate of 40%, only a subset of patients benefits from immunotherapy. A critical step in the success of immunotherapy is the presentation of tumor-derived peptides by the major histocompatibility complex I (MHC-I) of tumor cells. These neoantigens are potentially immunogenic and trigger immune responses orchestrated by cytotoxic cells. In MM, tumor development is nevertheless characterized by a low mutation rate despite major structural chromosomal rearrangements driving oncogenesis (BAP1, NF2, CDKN2AB). In this opinion, we propose to investigate an approach based on the mechanisms of the DNA damage tolerance (DDT) pathways to increase the frequency of non-synonymous mutations. The idea is to transiently activate the error-prone DDT in order to generate neoantigens while preserving a fully competent antitumor immune response.

Analysis of Nucleotide Sequence of Tax, miRNA and LTR of Bovine Leukemia Virus in Cattle with Different Levels of Persistent Lymphocytosis in Russia.

Bovine Leukemia Virus (BLV) is the etiological agent of enzootic bovine leucosis (EBL), a lymphoproliferative disease of the bovine species. In BLV-infected cells, the long terminal repeat (LTR), the viral Tax protein and viral miRNAs promote viral and cell proliferation as well as tumorigenesis. Although their respective roles are decisive in BLV biology, little is known about the genetic sequence variation of these parts of the BLV genome and their impact on disease outcome. Therefore, the objective of this study was to assess the relationship between disease progression and sequence variation of the BLV Tax, miRNA and LTR regions in infected animals displaying either low or high levels of persistent lymphocytosis (PL). A statistically significant association was observed between the A(+187)C polymorphism in the downstream activator sequence (DAS) region in LTR (p-value = 0.00737) and high lymphocytosis. Our study also showed that the mutation A(-4)G in the CAP site occurred in 70% of isolates with low PL and was not found in the high PL group. Conversely, the mutations G(-133)A/C in CRE2 (46.7%), C(+160)T in DAS (30%) and A(310)del in BLV-mir-B4-5p, A(357)G in BLV-mir-B4-3p, A(462)G in BLV-mir-B5-5p, and GA(497-498)AG in BLV-mir-B5-3p (26.5%) were often seen in isolates with high PL and did not occur in the low PL group. In conclusion, we found several significant polymorphisms among BLV genomic sequences in Russia that would explain a progression towards higher or lower lymphoproliferation. The data presented in this article enabled the classification between two different genotypes; however, clear association between genotypes and the PL development was not found.

MyD88 Mediates Colitis- and RANKL-Induced Microfold Cell Differentiation.

Intestinal microfold (M) cells are critical for sampling antigens in the gut and initiating the intestinal mucosal immune response. In this study, we found that the oral administration of dextran sulfate sodium (DSS) and Salmonella infection induced colitis. In the process, the expression levels of M cell differentiation-related genes were synchronized with the kinetics of pro-inflammatory cytokines. Compared to wild-type (WT) mice, MyD88-/- mice exhibited significantly lower expression levels of M cell differentiation-related genes. However, DSS induced colitis in MyD88-/- mice but failed to promote the transcription of M cell differentiation related genes. Furthermore, the receptor activator of the Nuclear Factor-κB ligand (RANKL) upregulated the transcription of M cell differentiation related genes in murine intestinal organoids prepared from both WT and MyD88-/- mice. Meanwhile, fewer changes in M cell differentiation related genes were found in MyD88-/- mice as compared to WT mice. Hence, we concluded that myeloid differentiation factor 88 (MyD88) is an essential molecule for colitis- and RANKL-related differentiation of M cells.

Non-cytotoxic 1,2,3-triazole tethered fused heterocyclic ring derivatives display Tax protein inhibition and impair HTLV-1 infected cells.

Human T cell lymphotropic virus type 1 (HTLV-1) is a human retrovirus that infects approximately 10-20 million people worldwide and causes an aggressive neoplasia (adult T-cell leukemia/lymphoma - ATL). Therapeutic approaches for the treatment of ATL have variable effectiveness and poor prognosis, thus requiring strategies to identify novel compounds with activity on infected cells. In this sense, we initially screened a small series of 25 1,2,3-triazole derivatives to discover cell proliferation inhibitors and apoptosis inducers in HTLV-1-infected T-cell line (MT-2) for further assessment of their effect on viral tax activity through inducible-tax reporter cell line (Jurkat LTR-GFP). Eight promising compounds (02, 05, 06, 13, 15, 21, 22 and 25) with activity ≥70% were initially selected, based on a suitable cell-based assay using resazurin reduction method, and evaluated towards cell cycle, apoptosis and Tax/GFP expression analyses through flow cytometry. Compound 02 induced S phase cell cycle arrest and compounds 05, 06, 22 and 25 promoted apoptosis. Remarkably, compounds 22 and 25 also reduced GFP expression in an inducible-tax reporter cell, which suggests an effect on Tax viral protein. More importantly, compounds 02, 22 and 25 were not cytotoxic in human hepatoma cell line (Huh-7). Therefore, the discovery of 3 active and non-cytotoxic compounds against HTLV-1-infected cells can potentially contribute, as an initial promising strategy, to the development process of new drugs against ATL.

Effects of Naturally Occurring Mutations in Bovine Leukemia Virus 5'-LTR and Tax Gene on Viral Transcriptional Activity.

Bovine leukemia virus (BLV) is a deltaretrovirus infecting bovine B cells and causing enzootic bovine leucosis (EBL). The long terminal repeat (LTR) plays an indispensable role in viral gene expression. The BLV Tax protein acts as the main transactivator of LTR-driven transcription of BLV viral genes. The aim of this study was to analyze mutations in the BLV LTR region and tax gene to determine their association with transcriptional activity. LTRs were obtained from one hundred and six BLV isolates and analyzed for their genetic variability. Fifteen variants were selected and characterized based on mutations in LTR regulatory elements, and further used for in vitro transcription assays. Reporter vectors containing the luciferase gene under the control of each variant BLV promoter sequence, in addition to variant Tax expression vectors, were constructed. Both types of plasmids were used for cotransfection of HeLa cells and the level of luciferase activity was measured as a proxy of transcriptional activity. Marked differences in LTR promoter activity and Tax transactivation activity were observed amongst BLV variants. These results demonstrate that mutations in both the BLV LTR and tax gene can affect the promoter activity, which may have important consequences on proviral load, viral fitness, and transmissibility in BLV-infected cattle.

bta-miR-23a Regulates the Myogenic Differentiation of Fetal Bovine Skeletal Muscle-Derived Progenitor Cells by Targeting MDFIC Gene.

miR-23a, a member of the miR-23a/24-2/27a cluster, has been demonstrated to play pivotal roles in many cellular activities. However, the mechanisms of how bta-miR-23a controls the myogenic differentiation (MD) of PDGFRα- bovine progenitor cells (bPCs) remain poorly understood. In the present work, bta-miR-23a expression was increased during the MD of PDGFRα- bPCs. Moreover, bta-miR-23a overexpression significantly promoted the MD of PDGFRα- bPCs. Luciferase reporter assays showed that the 3'-UTR region of MDFIC (MyoD family inhibitor domain containing) could be a promising target of bta-miR-23a, which resulted in its post-transcriptional down-regulation. Additionally, the knockdown of MDFIC by siRNA facilitated the MD of PDGFRα- bPCs, while the overexpression of MDFIC inhibited the activating effect of bta-miR-23a during MD. Of note, MDFIC might function through the interaction between MyoG transcription factor and MEF2C promoter. This study reveals that bta-miR-23a can promote the MD of PDGFRα- bPCs through post-transcriptional downregulation of MDFIC.

TGFα Promotes Chemoresistance of Malignant Pleural Mesothelioma.

Background: There is no standard chemotherapy for refractory or relapsing malignant pleural mesothelioma (MPM). Our previous reports nevertheless indicated that a combination of an anthracycline (doxorubicin) and a lysine deacetylase inhibitor (valproic acid, VPA) synergize to induce the apoptosis of MPM cells and reduce tumor growth in mouse models. A Phase I/II clinical trial indicated that this regimen is a promising therapeutic option for a proportion of MPM patients. Methods: The transcriptomes of mesothelioma cells were compared after Illumina HiSeq 4000 sequencing. The expression of differentially expressed genes was inhibited by RNA interference. Apoptosis was determined by cell cycle analysis and Annexin V/7-AAD labeling. Protein expression was assessed by immunoblotting. Preclinical efficacy was evaluated in BALB/c and NOD-SCID mice. Results: To understand the mechanisms involved in chemoresistance, the transcriptomes of two MPM cell lines displaying different responses to VPA-doxorubicin were compared. Among the differentially expressed genes, transforming growth factor alpha (TGFα) was associated with resistance to this regimen. The silencing of TGFα by RNA interference correlated with a significant increase in apoptosis, whereas the overexpression of TGFα desensitized MPM cells to the apoptosis induced by VPA and doxorubicin. The multi-targeted inhibition of histone deacetylase (HDAC), HER2 and TGFα receptor (epidermal growth factor receptor/EGFR) improved treatment efficacy in vitro and reduced tumor growth in two MPM mouse models. Finally, TGFα expression but not EGFR correlated with patient survival. Conclusions: Our data show that TGFα but not its receptor EGFR is a key factor in resistance to MPM chemotherapy. This observation may contribute to casting light on the promising but still controversial role of EGFR signaling in MPM therapy.

Ablation of non-coding RNAs affects bovine leukemia virus B lymphocyte proliferation and abrogates oncogenesis.

Viruses have developed different strategies to escape from immune response. Among these, viral non-coding RNAs are invisible to the immune system and may affect the fate of the host cell. Bovine leukemia virus (BLV) encodes both short (miRNAs) and long (antisense AS1 and AS2) non-coding RNAs. To elucidate the mechanisms associated with BLV non-coding RNAs, we performed phenotypic and transcriptomic analyzes in a reverse genetics system. RNA sequencing of B-lymphocytes revealed that cell proliferation is the most significant mechanism associated with ablation of the viral non-coding RNAs. To assess the biological relevance of this observation, we determined the cell kinetic parameters in vivo using intravenous injection of BrdU and CFSE. Fitting the data to a mathematical model provided the rates of cell proliferation and death. Our data show that deletion of miRNAs correlates with reduced proliferation of the infected cell and lack of pathogenesis.

ORF Capture-Seq as a versatile method for targeted identification of full-length isoforms.

Most human protein-coding genes are expressed as multiple isoforms, which greatly expands the functional repertoire of the encoded proteome. While at least one reliable open reading frame (ORF) model has been assigned for every coding gene, the majority of alternative isoforms remains uncharacterized due to (i) vast differences of overall levels between different isoforms expressed from common genes, and (ii) the difficulty of obtaining full-length transcript sequences. Here, we present ORF Capture-Seq (OCS), a flexible method that addresses both challenges for targeted full-length isoform sequencing applications using collections of cloned ORFs as probes. As a proof-of-concept, we show that an OCS pipeline focused on genes coding for transcription factors increases isoform detection by an order of magnitude when compared to unenriched samples. In short, OCS enables rapid discovery of isoforms from custom-selected genes and will accelerate mapping of the human transcriptome.

BLV: lessons on vaccine development.

Vaccination against retroviruses is a challenge because of their ability to stably integrate into the host genome, undergo long-term latency in a proportion of infected cells and thereby escape immune response. Since clearance of the virus is almost impossible once infection is established, the primary goal is to achieve sterilizing immunity. Besides efficacy, safety is the major issue since vaccination has been associated with increased infection or reversion to pathogenicity. In this review, we discuss the different issues that we faced during the development of an efficient vaccine against bovine leukemia virus (BLV). We summarize the historical failures of inactivated vaccines, the efficacy and safety of a live-attenuated vaccine and the economical constraints of further industrial development.

Bta-miR-24-3p Controls the Myogenic Differentiation and Proliferation of Fetal, Bovine, Skeletal Muscle-Derived Progenitor Cells by Targeting ACVR1B.

MicroRNAs modulate a variety of cellular events, including skeletal muscle development, but the molecular basis of their functions in fetal bovine skeletal muscle development is poorly understood. In this study, we report that bta-miR-24-3p promotes the myogenic differentiation of fetal bovine PDGFRα- progenitor cells. The expression of bta-miR-24-3p increased during myogenic differentiation. Overexpression of bta-miR-24-3p significantly promoted myogenic differentiation, but inhibited proliferation. A dual-luciferase assay identified ACVR1B as a direct target of bta-miR-24-3p. Similarly, knocking down ACVR1B by RNA interference also significantly inhibited proliferation and promoted the differentiation of bovine PDGFRα- progenitor cells. Thus, our study provides a mechanism in which bta-miR-24-3p regulates myogenesis by inhibiting ACVR1B expression.