In this and previous publications, both polyclonal and monoclonal anti-V3 Abs have been shown to neutralize diverse primary isolates of HIV-1 (8
), although not all viruses are neutralized by these reagents. The neutralizing activities of anti-V3 Abs may be limited due to antigenic variation of the V3 region, lack of V3 exposure on the surfaces of intact virions, and/or the nature of the Ab specificity. For clarification of this issue, a panel of 32 human anti-V3 MAbs were screened for binding to intact virions, for binding to solubilized gp120 from these viruses, and for neutralization. The results suggest that the V3 loop is similarly exposed on solubilized gp120 and on intact virions, that the V3 loop is accessible in all viruses studied, that the strength of binding of anti-V3 MAbs to intact virions correlates strongly with the potency of neutralizing activity, and that the V3 loops of diverse primary isolates display shared epitopes that can be recognized by anti-V3 MAbs with neutralizing activities.
For this study, the viruses that were selected represented a sampling within the spectrum of neutralization sensitivities and resistances. For example, BaL, SF162, and JR-CSF are well established as viruses that are sensitive to neutralization by various MAbs, including anti-V3 MAbs (29
). Other viruses, including ADA, P15, P27, aBL-01, and dBR-07, represent viruses that are relatively resistant to anti-V3 MAbs. The remaining viruses tested fall between these two extremes. The rationale for the use of these viruses was to determine if and how exposure of the V3 loop on the virus surface correlates with neutralization by anti-V3 MAbs. Binding studies revealed that the V3 loop was exposed in all viruses tested, since all could be captured with one or more of the anti-V3 MAbs studied. Each of these viruses, except aBL-01, was also neutralized by at least one MAb, and a highly significant correlation was found between strength of binding to intact virions and potency of neutralization (P
< 0.0001) (Fig. and ).
While all of the viruses examined in this study displayed V3 on their surfaces, data shown above and published previously suggest that the degree of exposure differs with the virus and the conditions studied. Thus, by using saturating conditions in the virus binding assay, in which virions are captured on ELISA plates coated with 10 μg of MAb/ml, we noted large differences in the capture of different viruses. For example, on average, much more BaL was captured by the anti-V3 MAbs than 92US717 (Fig. ). Nonetheless, the V3 epitopes seemed to be comparably recognized when these same MAbs reacted with the solubilized gp120s from these two viruses (Table ). These data lead to the conclusions that the relevant V3 epitopes are present within the gp120s of these viruses but that the V3 loop is more accessible on the BaL virus than it is on the 92US717 virus. This view is supported by data in Fig. showing that the average half-max binding of the anti-V3 MAbs is much lower (i.e., the relative affinity is much higher) for BaL than it is for 92US717. Similarly, each MAb tested neutralized BaL more strongly than 92US717 (Table ). These data reflect the differential exposure of the V3 loop on the intact virions of these two strains, and similar data in the cited tables and figures support the notion that V3 is differentially exposed by different strains of the virus. This concept is extended by data from other studies suggesting that some viruses apparently have cryptic V3 loops (3
), although, from the data presented above, this appears to be the exception rather than the rule.
Sensitivity to anti-V3 Abs may also be affected by the mobility of the V3 loop upon interaction of gp120 with CD4. Thus, Mbah et al. (30
) showed that, upon treatment with soluble CD4, the exposure of the V3 loop on virions of primary isolates is increased, but this occurs to different degrees with different viruses. Consequently, the sensitivity of viruses to anti-V3 Abs may also be affected by the mobility of the V3 loop during the conformational changes that occur in gp120 upon binding to CD4 (31
The relative affinities of anti-V3 MAbs were also found to play a profound role in the neutralization process. A highly significant correlation (P
< 0.0001) was demonstrated between the percent neutralization of psSF162 tested in a luciferase assay and the half-max values for binding of 32 anti-V3 MAbs with this pseudovirus (Fig. ). Similarly, a highly significant correlation (P
< 0.0001) was found when the percent neutralization in a single-round PBMC assay and half-max virion binding values were analyzed for 90 MAb-virus combinations (Fig. ). This extends earlier studies in which primary isolates from clades A to F were tested against seven human anti-V3 MAbs for MAb-virion binding and for neutralization (by the GHOST assay and the conventional PHA-blasted PBMC assay), and significant correlations were again found (P
< 0.0001 and P
< 0.001, respectively) (15
). Earlier studies, which were limited to neutralization of TCLA strains and binding to either monomeric gp120 or V3 peptides, also concluded that anti-V3 MAb affinity correlates with its neutralizing potency and further suggested that the dissociation rate (K−1
) rather than the association rate (K1
) is the principal component which determines neutralizing activity (27
). While Abs against other epitopes of the HIV envelope may not be constrained by the same affinity limitations (41
), experiments in a multitude of systems consistently support the association between affinity and neutralization for anti-V3 Abs.
In summary, our data indicate that neutralization sensitivity to anti-V3 Abs appears to be affected by the presence or absence of the relevant epitope(s) on the virion envelope, the exposure of the V3 loop on the intact virion, the mobility of the V3 loop during the conformational change induced by CD4, and the affinity of the Ab. Additional factors influencing neutralization sensitivity are the density and/or number of Env oligomers on the surface of a given virus. All of these parameters contribute to the shape of the neutralization curve and the ultimate outcome of Ab-virus interactions. These multiple parameters, and most probably several others, contribute to the complex equation that determines neutralization sensitivity or resistance. Thus, viruses such as BaL, SF162, and JR-CSF, which according to the data shown in Fig. have a very low threshold for resisting Ab-mediated neutralization, appear to have well-exposed V3 loops for which the anti-V3 MAbs have high affinities (Fig. ); in contrast, viruses such as BR07, ADA, and aBL01 have higher thresholds as a consequence, at least in part, of a poorly exposed V3 loop and low-affinity interactions with anti-V3 MAbs. The existence of a threshold was suggested previously in a study showing that anti-V3 MAb neutralization of TCLA HIV-1 was incremental rather than all or nothing and that each MAb binding an Env oligomer reduced infectivity (46
). Recently, Franti et al. also described a threshold effect when studying the interaction between JF-CSF and the anti-gp120 MAbs b12, 447-52D, and 2G12 (M. Franti, S. Frost, M. Guyader, K. Delgado, D. R. Burton, and P. Poignard, Abstr. AIDS Vaccine 2003, abstr. 118, 2003). The data included in Fig. , however, indicate for the first time that the levels of these thresholds will vary with each individual strain of virus.
The affinities of the anti-V3 MAbs tested here for psSF162 and 13 clade B viruses grown in PBMCs also varied by more than 3 orders of magnitude (Fig. , , and ); similarly, profound differences were described previously in the affinities of several of these MAbs for the V3-FP and V3 peptide of JR-CSF (15
). Clearly, HIV-infected subjects make a broad range of anti-V3 Abs, both with respect to affinity and specificity (57
). With the single, but important, exception of MAb 447, which was selected with the V3 peptide of MN, the most avid anti-V3 MAbs were selected with V3-FPs, which maintain the native conformation of the V3 loop (22
). These data have important implications for vaccine design, as they establish the fact that the human Ab repertoire includes anti-V3 Abs with broad and potent cross-neutralizing activities and they identify the characteristics of the types of anti-V3 Abs which will have the greatest neutralizing efficacies and therefore the highest probabilities of blocking a virus inoculum. These characteristics include broad immunochemical cross-reactivity (Table ) (35
), broad neutralizing activity (Table ) (15
), and a high affinity for gp120 and intact virus particles (Table ) (15
). Antigens used to select MAbs with these characteristics, such as V3-FP, provide a template for the design of immunogens that will focus the immune response on the V3 loop and should induce high-affinity, broadly reactive Abs to conformational epitopes on V3. The apparent requirement for conformational aspects of V3 revealed by our studies may also provide an explanation for the disappointing results with previously used V3 immunogens (1
) which lacked the appropriate conformational aspects of the V3 loop.