Thus, we sought to test whether we could stain diverse sites on the HIV Env reliably and sensitively using a large panel of publicly available monoclonal antibodies (mAbs). virions with a panel of 85 monoclonal antibodies targeting different regions of Env. A broad range of antibodies yielded robust staining of Env, with V3 antibodies showing the highest quantitative staining. A subset of antibodies tested in parallel on viruses produced in CD4+ T cell lines, HEK293T cells, and primary cells showed that the cellular model of virus production can impact Env detection. Finally, in addition to being able to highlight Env heterogeneity on virions, we show FV can sensitively detect differences in Env conformation when soluble CD4 is added to virions before staining. Keywords: human immunodeficiency virus (HIV), calibrated flow virometry, molecules of equivalent soluble fluorophore (MESF), nanoscale flow cytometry, gp120/gp41, HIV Env, HIV trimer, Env conformation, virion capture, neutralization 1. Introduction Despite decades of research dedicated to studying the human immunodeficiency virus (HIV), an efficacious preventative vaccine remains elusive. One of the prime reasons for this is due to the difficulty that exists with targeting the viral envelope glycoprotein (Env) [1,2,3,4]. Env has been highly studied due to its critical roles in mediating HIV entry through binding and fusion with CD4+ cells (reviewed in [5,6,7]). Early work in this field identified Env as a trimer, consisting of heterodimers of the surface unit, gp120, and the transmembrane subunit, gp41 [5,7]. As the sole viral protein on the HIV surface, Env is a key antigen for anti-HIV immune responses and for the development of an effective HIV cure. Unfortunately, targeting Env has proved challenging due to its heavy glycosylation, high genetic variability, conformational flexibility, and its low abundance on virions [1,8,9,10,11,12,13,14]. Despite this, Env continues to hold promise as a viable target for vaccine strategies, and thus a need to better understand this protein remains. The variability of Env is particularly well documented, with extensive variation among different viral strains and five hypervariable regions of gp120 interspersed among more constant regions of the protein [15,16,17]. While A-966492 individuals mount antiviral responses that produce antibodies targeting the trimer [18,19], these antibodies alone fail at controlling infection [3] because of the trimers glycan shielding and high propensity to accumulate mutations. However, a subset of individuals generate broadly neutral antibodies (bNAbs) with notable potency and breadth [20,21,22,23]. These antibodies have helped bring A-966492 forth a better understanding of Env structure and have been characterized to target distinct epitopes on the trimer, including the following: the CD4 binding site (CD4bs), CD4 inducible epitopes (CD4i), glycan-dependent regions at the first and second variable (V1V2) loop apex and third variable loop (V3), gp41, and the membrane proximal region (MPER) [17,24,25,26]. While the promise of bNAbs in managing infection in vivo is an area of ongoing investigation [25,26,27], these antibodies have proven to be a powerful tool in the laboratory, where they can be used in antibody-based assays such as neutralization and immunoprecipitation assays. Although such antibody-based techniques have been routinely employed in the HIV field with high success, the use of antibody techniques that assess viruses at a single particle level is less common. Flow virometry (FV), or flow cytometry applied to viruses, is an emerging technique that can be used to help bridge the A-966492 gap in this field. Flow virometry was originally coined in 2013 when Arakelyan et al. studied the surface proteins of HIV with flow cytometry using viruses bound to magnetic nanoparticles [28]. Since then, their group has continued to develop this methodology for use on HIV and Dengue virus [29,30,31,32]. While flow cytometry A-966492 has been utilized for decades to enumerate viruses in the marine biology field [33,34,35], visualization of viruses on conventional cytometers often requires labelling with a fluorescent protein or Rabbit Polyclonal to NF1 tag [36,37,38,39,40], since virions typically fall within the range of instrument A-966492 background noise when using light scatter for detection based on their size. More recently, advances in flow cytometry instrumentation have allowed for viruses and vesicles in the 100 nm range to readily be detected on high-sensitivity cytometers [41,42,43,44,45,46]. With these.