A role of BPTF in viral oncogenicity delineated through studies of heritable Kaposi sarcoma.

Kaposi sarcoma (KS), caused by Herpesvirus-8 (HHV-8; KSHV), shows sporadic, endemic, and epidemic forms. While familial clustering of KS was previously recorded, the molecular basis of hereditary predilection to KS remains largely unknown. We demonstrate through genetic studies that a dominantly inherited missense mutation in BPTF segregates with a phenotype of classical KS in multiple immunocompetent individuals in two families. Using an rKSHV.219-infected CRISPR/cas9-model, we show that BPTFI2012T mutant cells exhibit higher latent-to-lytic ratio, decreased virion production, increased LANA staining, and latent phenotype in viral transcriptomics. RNA-sequencing demonstrated that KSHV infection dysregulated oncogenic-like response and P53 pathways, MAPK cascade, and blood vessel development pathways, consistent with KS. BPTFI2012T also enriched pathways of viral genome regulation and replication, immune response, and chemotaxis, including downregulation of IFI16, SHFL HLAs, TGFB1, and HSPA5, all previously associated with KSHV infection and tumorigenesis. Many of the differentially expressed genes are regulated by Rel-NF-κB, which regulates immune processes, cell survival, and proliferation and is pivotal to oncogenesis. We thus demonstrate BPTF mutation-mediated monogenic hereditary predilection of KSHV virus-induced oncogenesis, and suggest BPTF as a drug target.

Functional binding of PD1 ligands predicts response to anti-PD1 treatment in patients with cancer.

Accurate predictive biomarkers of response to immune checkpoint inhibitors (ICIs) are required for better stratifying patients with cancer to ICI treatments. Here, we present a new concept for a bioassay to predict the response to anti-PD1 therapies, which is based on measuring the binding functionality of PDL1 and PDL2 to their receptor, PD1. In detail, we developed a cell-based reporting system, called the immuno-checkpoint artificial reporter with overexpression of PD1 (IcAR-PD1) and evaluated the functionality of PDL1 and PDL2 binding in tumor cell lines, patient-derived xenografts, and fixed-tissue tumor samples obtained from patients with cancer. In a retrospective clinical study, we found that the functionality of PDL1 and PDL2 predicts response to anti-PD1 and that the functionality of PDL1 binding is a more effective predictor than PDL1 protein expression alone. Our findings suggest that assessing the functionality of ligand binding is superior to staining of protein expression for predicting response to ICIs.

Clinical and Molecular Characterization of a Rare Case of BNT162b2 mRNA COVID-19 Vaccine-Associated Myositis.

Initial clinical trials and surveillance data have shown that the most commonly administered BNT162b2 COVID-19 mRNA vaccine is effective and safe. However, several cases of mRNA vaccine-induced mild to moderate adverse events were recently reported. Here, we report a rare case of myositis after injection of the first dose of BNT162b2 COVID-19 mRNA vaccine into the left deltoid muscle of a 34-year-old, previously healthy woman who presented progressive proximal muscle weakness, progressive dysphagia, and dyspnea with respiratory failure. One month after vaccination, BNT162b2 vaccine mRNA expression was detected in a tissue biopsy of the right deltoid and quadriceps muscles. We propose this case as a rare example of COVID-19 mRNA vaccine-induced myositis. This study comprehensively characterizes the clinical and molecular features of BNT162b2 mRNA vaccine-associated myositis in which the patient was severely affected.

[PRIMARY GASTRIC PLASMACYTOMA IN A BREAST CANCER MALE PATIENT].

INTRODUCTION: Plasmacytoma is a malignant tumor of the plasma cells. Extra-medullary plasmacytoma is rare and with an even lower incidence appears as a primary tumor of the stomach. Initial onset of the disease in the upper gastrointestinal tract is reported in the literature as just second to primary plasmacytomas of the head and neck system. The presenting symptoms are related to the organ involved and systemic symptoms can be weight loss, pain, bleeding and even fever. As this is a rare disease, there is no standard treatment and patients undergo endoscopic resection or chemotherapy with or without additional radiation. The prognosis of the disease depends on the possible future diagnosis of multiple myeloma which can be up to 50% within only a few years. We hereby report a case of a male patient with a past locally advanced breast cancer who was on prolonged adjuvant hormonal treatment. He developed a new symptom of melena and underwent a thorough evaluation including imaging and repeated biopsies from a large gastric lesion. The results were inconclusive mainly because of the differential diagnosis between breast cancer metastases and a new second primary malignancy. In view of a clinical deterioration and lack of diagnosis, an operation of radical gastrectomy was eventually performed only to surprisingly diagnose a rare hematologic disease of the stomach - gastric plasmacytoma. This diagnosis is rare in itself, especially having his previous male breast cancer and maternal multiple myeloma. The diagnostic procedure in this case had also provided the full treatment for his illness.

Identification of Tumor Antigens in the HLA Peptidome of Patient-derived Xenograft Tumors in Mouse.

Personalized cancer immunotherapy targeting patient-specific cancer/testis antigens (CTA) and neoantigens may benefit from large-scale tumor human leukocyte antigen (HLA) peptidome (immunopeptidome) analysis, which aims to accurately identify antigens presented by tumor cells. Although significant efforts have been invested in analyzing the HLA peptidomes of fresh tumors, it is often impossible to obtain sufficient volumes of tumor tissues for comprehensive HLA peptidome characterization. This work attempted to overcome some of these obstacles by using patient-derived xenograft tumors (PDX) in mice as the tissue sources for HLA peptidome analysis. PDX tumors provide a proxy for the expansion of the patient tumor by re-grafting them through several passages to immune-compromised mice. The HLA peptidomes of human biopsies were compared with those derived from PDX tumors. Larger HLA peptidomes were obtained from the significantly larger PDX tumors as compared with the patient biopsies. The HLA peptidomes of different PDX tumors derived from the same source tumor biopsy were very reproducible, even following subsequent passages to new naïve mice. Many CTA-derived HLA peptides were discovered, as well as several potential neoantigens/variant sequences. Taken together, the use of PDX tumors for HLA peptidome analysis serves as a highly expandable and stable source of reproducible and authentic peptidomes, opening up new opportunities for defining large HLA peptidomes when only small tumor biopsies are available. This approach provides a large source for tumor antigens identification, potentially useful for personalized immunotherapy.

A Vaxfectin(®)-adjuvanted HSV-2 plasmid DNA vaccine is effective for prophylactic and therapeutic use in the guinea pig model of genital herpes.

Here we describe studies in the guinea pig model of genital herpes to evaluate a novel plasmid DNA (pDNA) vaccine encoding the HSV-2 glycoprotein D and UL46 and UL47 genes encoding tegument proteins VP11/12 and VP 13/14 (gD2/UL46/UL47), formulated with a cationic lipid-based adjuvant Vaxfectin(®). Prophylactic immunization with Vaxfectin(®)-gD2/UL46/UL47 significantly reduced viral replication in the genital tract, provided complete protection against both primary and recurrent genital skin disease following intravaginal HSV-2 challenge, and significantly reduced latent HSV-2 DNA in the dorsal root ganglia compared to controls. We also examined the impact of therapeutic immunization of HSV-2 infected animals. Here, Vaxfectin(®)-gD2/UL46/UL47 immunization significantly reduced both the frequency of recurrent disease and viral shedding into the genital tract compared to controls. This novel adjuvanted pDNA vaccine has demonstrated both prophylactic and therapeutic efficacy in the guinea pig model of genital herpes and warrants further development.

Vaxfectin-adjuvanted plasmid DNA vaccine improves protection and immunogenicity in a murine model of genital herpes infection.

The herpes simplex type 2 (HSV-2) envelope glycoprotein (gD2) was evaluated as a potential antigen candidate for a plasmid DNA (pDNA)-based HSV-2 vaccine. The pDNA was formulated with Vaxfectin, a cationic lipid-based adjuvant, and tested in a murine HSV-2 lethal challenge model. gD2 was expressed as full-length (FL) and secreted (S) gD2 forms. A 0.1 µg pDNA dose was tested to distinguish treatment conditions for survival and a 100 µg pDNA dose was tested to distinguish treatment conditions for reduction in vaginal and latent HSV-2 copies. Vaxfectin-formulated gD2 pDNA significantly increased serum IgG titres and survival for both FL gD2 and S gD2 compared with gD2 pDNA alone. Mice immunized with FL gD2 formulated with Vaxfectin showed reduction in vaginal and dorsal root ganglia (DRG) HSV-2 copies. The stringency of this protection was further evaluated by testing Vaxfectin-formulated FL gD2 pDNA at a high 500 LD(50) inoculum. At this high viral challenge, the 0.1 µg dose of FL gD2 Vaxfectin-formulated pDNA yielded 80 % survival compared with no survival for FL gD2 pDNA alone. Vaxfectin-formulated FL gD2 pDNA, administered at a 100 µg pDNA dose, significantly reduced HSV-2 DNA copy number, compared with FL gD2 DNA alone. In addition, 40 % of mice vaccinated with adjuvanted FL pDNA had no detectable HSV-2 viral genomes in the DRG, whereas all mice vaccinated with gD2 pDNA alone were positive for HSV-2 viral genomes. These results show the potential contribution of Vaxfectin-gD2 pDNA to a future multivalent HSV-2 vaccine.

Analysis of biomarkers after intramuscular injection of Vaxfectin-formulated hCMV gB plasmid DNA.

Cationic lipids have been used as delivery systems to enhance the performance of vaccines and immunotherapeutics. However, little is known about the effect of administration of cationic lipid-formulated vaccines on gene expression. This study used DNA microarrays (39,000 transcripts) to characterize early changes in gene expression patterns in mouse muscle 1 and 2 days after intramuscular (i.m.) injection of a hCMV gB plasmid DNA (pDNA) vaccine formulated with the cationic lipid system Vaxfectin; gene expression profiles were compared to those obtained after i.m. injection of pDNA in PBS. Analysis of the DNA microarray data indicated that approximately 1% of the represented transcripts were modulated at least 2-fold compared to the PBS samples at both time points. Functional analysis of the modulated genes revealed that transcripts involved in antigen processing and presentation, apoptosis and the Toll-like receptor pathway were significantly enriched. In addition, confirmation of local and systemic modulation of subsets of biomarkers was achieved using Real-Time PCR and Cytometric Bead Assays. Time course and magnitude of cellular infiltration (F4/80+ and CD11b+ cells) to the injection site was changed in response to formulation of hCMVgB pDNA with Vaxfectin. Since the expression level of the pDNA-encoded transgene in the muscle was not affected by formulation Vaxfectin mechanism of action is expected to rely primarily on modulation of immune pathways and not on an increase in transfection of the antigen-encoding pDNA. Taken together, these data help explain the Vaxfectin-dependent robust enhancement of immune responses.

Repression of HMGA2 gene expression by human cytomegalovirus involves the IE2 86-kilodalton protein and is necessary for efficient viral replication and inhibition of cyclin A transcription.

Human cytomegalovirus (HCMV) infection results in dysregulation of several cell cycle genes, including inhibition of cyclin A transcription. In this work, we examine the effect of the HCMV infection on expression of the high-mobility group A2 (HMGA2) gene, which encodes an architectural transcription factor that is involved in cyclin A promoter activation. We find that expression of HMGA2 RNA is repressed in infected cells. To determine whether repression of HMGA2 is directly related to the inhibition of cyclin A expression and impacts on the progression of the infection, we constructed an HCMV recombinant that expressed HMGA2. In cells infected with the recombinant virus, cyclin A mRNA and protein are induced, and there is a significant delay in viral early gene expression and DNA replication. To determine the mechanism of HMGA2 repression, we used recombinant viruses that expressed either no IE1 72-kDa protein (CR208) or greatly reduced levels of IE2 86-kDa (IE2 86) protein (IE2 86DeltaSX-EGFP). At a high multiplicity of infection, the IE1 deletion mutant is comparable to the wild type with respect to inhibition of HMGA2. In contrast, the IE2 86DeltaSX-EGFP mutant does not significantly repress HMGA2 expression, suggesting that IE2 86 is involved in the regulation of this gene. Cyclin A expression is also induced in cells infected with this mutant virus. Since HMGA2 is important for cell proliferation and differentiation, particularly during embryogenesis, it is possible that the repression of HMGA2 expression during fetal development could contribute to the specific birth defects in HCMV-infected neonates.