The luciferase activity in the lysate was measured by the Bright-Glo method (Promega) on a Victor3 1420 plate-reading luminometer (Perkin Elmer, Wellesley, MA). We hypothesize that genetic, post-translational or conformational heterogeneity of the Env protein and of other targets for inhibitors can yield apparent synergy and increased cooperativity between inhibitors. == INTRODUCTION == The functional human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein complex (Env) is a trimer of hetero-dimers that each consists of the outer gp120 subunit attached non-covalently to the transmembrane glycoprotein, gp41. The docking of gp120 onto the primary receptor, CD4, triggers conformational changes that allow interactions with the CCR5 or CXCR4 co-receptor; these interactions in turn activate a refolding of the Env complex that unleashes the fusogenic potential of gp41, ultimately allowing the viral core to enter the cytoplasm of the target cell (Doms and Peiper, 1997;Pantophlet and Burton, 2006). Neutralizing antibodies (NAbs) interfere with this process at different stages by binding to different epitopes; some recognize gp120 and impede receptor interactions; others interact with gp41 and interfere with later stages of entry (Klasse and Sattentau, 2002;Ugolini et al., 1997;Zwick and Burton, 2007). The induction of broadly active and potent NAbs is a crucial but elusive requirement for an effective vaccine to prevent HIV-1 infection. The access to epitopes on the native Env complex is restricted, as is the immunogenicity of the few epitopes that bind broadly active NAbs (Burton et al., 2004;Karlsson Hedestam et al., 2008;Klasse et al., 2011;Pantophlet and Burton, 2006;Poignard et al., 1996b;Poignard et al., 2001;Zwick and Burton, 2007). Env-vaccine design aims to induce protective levels of NAbs against these neutralization epitopes. But how well do NAbs act in combination? Do they have stronger or weaker effect when combined than when acting individually? Various small organic molecules, as well as peptides and proteins, can also inhibit HIV-1 entry, again by acting at different stages of the entry process. Some such compounds are used in therapy or may become components of microbicides or oral prevention regimens to block sexual transmission (Grant et al., 2010;Klasse et al., 2008;Lederman et al., 2006;Veazey et al., 2005). The use of inhibitor combinations has long been standard for treating HIV-1 infection, but it may also be advantageous for prevention. For example, more than one inhibitor may be needed in a microbicide to counteract HIV-1 sequence diversity, while any enhanced potency of a combination may allow lower amounts of each drug to be used, reducing cost and improving safety (Grant et al., 2008;Ketas et al., 2007b). The quantitative analysis of combinatorial effects is therefore important both in prevention and therapy. Synergy is a special case of combined effects (Berenbaum, 1977;Greco et al., 1995;Loewe, 1953). Its potential occurrence between NAbs and other entry inhibitors merits a rigorous, quantitative investigation. Synergy can be defined as a greater potency of combined inhibitors than would be predicted from their individual effects (Loewe, 1953); weaker than predicted potency is HAMNO called antagonism; when the combined potency is neither enhanced nor reduced, it is categorized as additivity. The method most commonly used to quantify synergy in the inhibition of HIV-1 replication analyzes the inhibitor-concentration dependence after a linear transformation of the data (Chou and Talalay, 1981,1984). Here, we compare that method with a new, nonlinear approach. What types of infectivity-inhibition assays are suitable for synergy analyses? Valid synergy assessments require proportionality between the infectious dose ENO2 and the resulting propagation of the virus, which can only be guaranteed in certain titration zones of single-cycle replication assays; the distortions inherent in multi-cycle replication can create artifactual, or obliterate authentic, synergy (Ferguson et al., 2001). Moreover, PBMC assays based on production of the HIV-1 p24 Gag antigen lack precision (Choudhry et al., 2006;Heredia et al., 2007a;Heredia et al., 2007b;Ketas et al., 2007). Despite that problem, PBMC or T-cell-line assays with a p24 read-out have been used extensively HAMNO in studies of synergy involving NAbs and other inhibitors (Dorr et al., 2005;Eron et al., 1992;Gantlett et al., 2007;Johnson et al., 1989;Johnson et al., 1990;Johnson et al., 1992;Kennedy et al., 1991;Laal et al., 1994;Li et al., 1997;Mascola et al., 1997;McKeating et al., 1992;Nakata et al., 2008;Strizki et al., 2005;Tremblay et al., 1999;Tremblay et al., 2005a;Tremblay et al., 2005b;Tremblay et al., 2002;Tremblay et al., 2000;Vermeire et al., 2004;Xu et al., 2001;Zwick et al., 2001). Here, we explored HAMNO whether data obtained from a PBMC assay are amenable to synergy analysis. Cooperativity differs from synergy in that it can occur for each individual ligand; it typically arises HAMNO when ligands interact with multimeric proteins (Hill, 1913;Koshland et al., 1966;Monod et al., 1965). At least some NAbs may show negative coperativity in binding to the trimeric HIV-1 Env complex (Gustchina et al., 2010). However, cooperativity as measured by slope coefficients is subject to other molecular influences,.