Biology subjects

Ghanam, J.

Publications and source records attributed to Ghanam, J..

3 recordsLinked to original sources

bioRxiv2026

A Third-generation Lentiviral Vector System for Flow Virometry-based Analysis of HIV-1 Env and Other Viral Envelope Proteins

Individual human immunodeficiency virus (HIV) particles can differ in their envelope protein (Env) despite being genetically identical. This non-genetic diversity can influence antibody recognition and has been implicated in the formation of persistent virus fractions that escape complete inhibition by broadly neutralizing antibodies. Investigating non-genetic diversity in HIV and other viruses requires approaches capable of characterizing antibody binding to individual viral particles. Flow virometry provides a promising tool for single-particle analysis, but a system combining high biosafety with specific particle labeling and versatility is lacking. Here, we developed a third-generation lentiviral vector system specifically designed for flow virometry. Fluorescent proteins were incorporated into the HIV-1 Gag polyprotein between the matrix- and capsid-encoding regions, flanked by viral protease cleavage sites, creating the packaging constructs pLentiFlow-eGFP and pLentiFlow-mCherry. Lentiviral particles (LVPs) based on pLentiFlow vectors were fluorescent and retained infectivity comparable to LVPs generated with the parental vector system despite reduced particle production. We optimized LVP detection on a CytoFLEX nano and defined a linear p24-based concentration range for reliable single-particle analysis. Using complementary controls, we demonstrated that background from the assessed sources accounted for no more than approximately 3% of events within a broad eGFP+ gate and confirmed that the detected fluorescent particles were predominantly membrane-enclosed. As a proof of concept, LVPs were pseudotyped with vesicular stomatitis virus glycoprotein, severe acute respiratory syndrome coronavirus 2 spike, or HIV-1 Env. Envelope-specific antibody binding was detected using 8G5F11, CR3022 or PGT128, respectively, demonstrating that different viral surface proteins can be analyzed on a common particle backbone. Together, these results establish a third-generation lentiviral vector system for flow virometry that combines intrinsic particle labeling with high biosafety and flexible pseudotyping. The system provides a platform for investigating envelope protein-associated non-genetic diversity and its consequences for antibody recognition in HIV and other viruses.

microbiology↗

bioRxiv2026

Dual Immunoliposome Targeting of PD-L1 and CSF1R affects T-Cell readouts in tumor-conditioned co-cultures: An In Vitro Study in Glioblastoma and Medulloblastoma

Glioblastoma and medulloblastoma are characterized by an immunosuppressive tumor microenvironment, in which tumor-associated macrophages may impair T-cell function, in part through the expression of PD-L1 and CSF1R. Single-target therapies have shown inconsistent results in brain tumors, likely due to the complex interplay involving tumor-associated macrophages and immunosuppression. We developed two immunoliposomes functionalized with antibodies against PD-L1 and CSF1R to co-target these receptors on M2-like tumor-associated macrophages. TAM2Ms were generated by polarizing THP-1 monocytes with glioblastoma (A172, U87MG) and medulloblastoma (DAOY, ONS-76) tumor-conditioned medium together with IL-4 and IL-13. In co-culture with activated Jurkat T cells under tumor-conditioned medium-influenced conditions, combined PD-L1 and CSF1R immunoliposomes enhanced T-cell proliferation, reduced apoptosis, and improved migration toward tumor spheroids compared to single-target liposomal treatments or free antibodies. These benefits varied between glioblastoma and medulloblastoma in vitro models, reflecting distinct TAM2M phenotypes and microenvironment contexts. Dual immunoliposome targeting of PD-L1 and CSF1R may offer a promising strategy for reprogramming tumor-associated macrophages in brain tumor immunotherapy and warrants further evaluation in primary human macrophages and in vivo models to clarify efficacy and mechanisms.

neuroscience↗

bioRxiv2025

Orphan Non-Coding RNAs Drive Tumorigenesis and Enable Pre-Diagnostic Detection in HPV-Negative Head and Neck Cancer

HPV-negative head and neck squamous cell carcinoma (HNSCC) lacks reliable biomarkers for early detection and molecular stratification. We recently described orphan non-coding RNAs (oncRNAs) as a class of cancer-emergent small RNAs that arise from cryptic promoters largely silent in normal tissues. Here, we define the comprehensive landscape of oncRNAs in HNSCC. Using TCGA and CPTAC-3 data, we demonstrate that these transcripts are not only tumor-specific but provide digital fingerprints that accurately stratify tumors by anatomical site. Moving from association to function, we investigated whether these emergent transcripts actively drive tumorigenesis. We focused on oncRNA64585, a transcript highly enriched in oral cavity tumors and associated with tumor progression. Its depletion significantly impaired proliferation in vitro and suppressed tumor growth in syngeneic murine models. Mechanistically, transcriptomic and chromatin accessibility profiling revealed that oncRNA64585 regulates transcriptional programs governing cell cycle progression and apoptosis through interaction with the Polycomb Repressive Complex 2 (PRC2). Finally, we demonstrate the clinical utility of these signatures: oncRNA profiles in plasma predicted oral cavity cancer with >90% accuracy up to four years prior to clinical diagnosis. These findings establish oncRNAs as active drivers of HNSCC biology and potent tools for early detection and therapeutic targeting.

cancer biology↗