The envelope glycoproteins on HIV particles are structurally heterogeneous, making it difficult to equate antigenicity with neutralization sensitivity. Efforts to address this question typically employ immobilized reagents and indirect measures of antibody interactions, which can produce inexact antigenicity profiles. Towards this, we applied a single molecule based approach using fluorescence correlation spectroscopy (FCS) that uses fluctuations in fluorescent signals to measure diffusion and reaction kinetics of fluorescently-labeled anti-envelope monoclonal antibodies (MAbs) as they attach to virions. Our method allows monitoring antibody-virion binding interaction with all reactants continuously in solution. FCS binding profiles of anti-envelope antibodies were determined using different virus types. Anti-gp120 MAbs against the 2G12 or b12 epitopes marking functional envelope structures potently neutralized CCR5-tropic JRFL and BaL pseudotyped viruses and exhibited efficient virion binding in solution. MAbs against various CD4-induced (CD4i) epitopes considered hidden on functional envelope structures exhibited limited binding to these pseudoviruses and were not neutralizing. Anti- gp41 MAb 2F5 neutralized both pseudoviruses despite limited virion binding. Overall our experimental data suggest that virion antigenicity in solution is not the only factor that might determine neutralization sensitivity. A variety of mechanisms may converge to produce the overall neutralizing profile of a given HIV variant, depending on variables such as envelope genotype, coreceptor tropism, and virus production method.
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