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

A groundbreaking third-generation lentiviral vector system has been developed to enhance flow virometry-based analysis of HIV-1 Env and other viral envelope proteins. This innovative system addresses the challenge of characterizing non-genetic diversity in HIV and other viruses, which can impact antibody recognition and the formation of persistent virus fractions that remain resistant to broadly neutralizing antibodies.

The researchers successfully integrated fluorescent proteins into the HIV-1 Gag polyprotein, specifically between the matrix- and capsid-encoding regions, using viral protease cleavage sites. This resulted in the creation of packaging constructs pLentiFlow-eGFP and pLentiFlow-mCherry. The LVPs generated using these constructs retained infectivity similar to those produced by the parental vector system, although with reduced particle production.

Through optimization, the researchers established a reliable single-particle analysis concentration range for these LVPs using p24-based detection on a CytoFLEX nano platform. Comprehensive controls confirmed that background signals from the assessed sources accounted for no more than 3% of events within a broad eGFP+ gate, and verified that the detected fluorescent particles were predominantly membrane-enclosed.

To demonstrate the system's versatility, the researchers pseudotyped the LVPs with vesicular stomatitis virus glycoprotein, severe acute respiratory syndrome coronavirus 2 spike, or HIV-1 Env. The researchers then successfully detected envelope-specific antibody binding using 8G5F11, CR3022, or PGT128 antibodies, respectively. This proof of concept highlights the system's ability to analyze different viral surface proteins on a common particle backbone.

In conclusion, this third-generation lentiviral vector system for flow virometry combines intrinsic particle labeling with high biosafety and flexible pseudotyping capabilities. By providing a robust platform for investigating envelope protein-associated non-genetic diversity and its implications for antibody recognition in HIV and other viruses, this system represents a significant advancement in the field.