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.