{
  "abstract": "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.",
  "abstract_shown": true,
  "affiliations": [
    "Institute for the Research on HIV and AIDS-associated Diseases, University Hospital Essen, University of Duisburg-Essen, Essen, Germany"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 11,
  "authors": [
    {
      "name": "P. I. Busskamp Castro"
    },
    {
      "name": "J. Parreuter"
    },
    {
      "name": "C. Brands"
    },
    {
      "name": "A.-M. Perkovic"
    },
    {
      "name": "B. van Bilsen"
    },
    {
      "name": "B. Giebel"
    },
    {
      "name": "J. Bohne"
    },
    {
      "name": "T. Tertel"
    },
    {
      "name": "J. Ghanam"
    },
    {
      "name": "B. K. Thakur"
    },
    {
      "affs": [
        0
      ],
      "name": "C. B. Karsten"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/e08av0cb1gg50.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.10.11.755930",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.10.11.755930v1.full",
  "id": "e08av0cb1gg50",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.10.11.755930v1",
  "language": null,
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "microbiology"
    ],
    [
      "openalex",
      "2406|HIV Research and Treatment"
    ]
  ],
  "openalex": "W7221506972",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.10.11.755930v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-11",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/e08av0cb1gg50.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:27:24Z",
      "native_id": "10.64898/2026.10.11.755930",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.10.11.755930v1"
    }
  ],
  "subjects": [
    {
      "field": "biology",
      "origin": "biorxiv:microbiology",
      "subfield": "microbiology"
    }
  ],
  "title": "A Third-generation Lentiviral Vector System for Flow Virometry-based Analysis of HIV-1 Env and Other Viral Envelope Proteins",
  "title_text": "A Third-generation Lentiviral Vector System for Flow Virometry-based Analysis of HIV-1 Env and Other Viral Envelope Proteins",
  "type": "preprint",
  "updated_at": "2026-10-30T12:27:24Z",
  "url": "https://research.draconitic.com/paper/e08av0cb1gg50",
  "venue": "bioRxiv",
  "versions": []
}