Here we describe a novel image-based methodology for assaying HIV neutralization in an automated high-content manner. Our high-content assay allows, for the first time, evaluation of both HIV neutralization and inhibition of cell-cell fusion within the same assay. This is achieved by quantifying the reduction in number of plaques and mean plaque area, respectively. The image analysis pipeline for the APR assay in GHOST(3) cells is based on the freely available CellProfiler software
 and has now been optimized and adapted for automated reading. Automation offers many advantages: it is higher throughput, standardized, and inexpensive compared both to the manual PR assay and the classic virus infection assay performed in peripheral blood mononuclear cells (PBMC). Automated image analysis increases the objectivity of the plaque counting demonstrated by the very low intra-well variation among triplicate wells (data not shown) as well as low background in the negative controls.
A striking feature of the image-based approach is that it allows extraction of additional phenotypic features of viruses, including cell-cell fusion capacity. We observed differences between the eight HIV-1 isolates used in the present study. In fact, based on statistically significant differences in mean plaque area, viruses could be divided into two groups yielding large or small plaques. Large plaques may result from fusion of a higher number of cells than small plaques. It has been suggested that virus ability to mediate cell-cell fusion, i.e. syncytia, depends on the strength of the interaction of viral envelope protein with CD4 and coreceptors
[14, 15]. In addition, Env clustering and mobility in the cell membrane may influence the outcome of syncytia formation
Our results show that a higher concentration of inhibitory reagent was needed to get a reduction in plaque area than that needed for neutralization. This was previously proposed by Yee et al.
 and given the explanation that the interaction of the envelope glycoproteins gp120/gp41 (Env) with cell membrane CD4 may be different during cell-cell fusion than during virus-cell membrane fusion
. It is plausible that the molecular organization and surface density of gp120/gp41 may be different in Env-expressing cells and HIV-1 virions. Indeed, Purtscher et al.
 proposed, similarly to what we show, that elevated concentrations of antibody are necessary to inhibit fusion between infected and target cells, and suggested this to be due to higher levels of gp120/gp41 expressed on the surface of infected cells as compared to the viral envelope. Interestingly, it was recently shown, in an HIV-1-infected humanized mouse model, that infected T cells form contacts with uninfected cells, and when the frequency of these contacts was reduced, plasma viremia was significantly decreased, strongly suggesting a role for cell-cell contacts in systemic viral spread
. Thus, it is conceivable that cell-cell fusion inhibition capacity of an antibody has a role in mitigating pathogenesis and may also be required for an effective HIV vaccine.
Antibody assessment plays a central role in HIV-1 vaccine development where, analogous to other virus infections, virus neutralization is considered particularly important. Accordingly, international comparisons of a wide range of HIV-1 neutralization assays have been performed and showed that no assay alone detects neutralization over the entire spectrum of virus-reagent combinations
[2, 21]. The APR assay has also been standardized and compared with other HIV neutralization assays within the framework of an international collaboration, Neutnet
[2, 3]. As with the other assays, the sensitivity of our assay was dependent on both the neutralizing reagent and the virus. Thus the APR assay can be considered an information-rich alternative to the PBMC and PSV assays.