These results provide some insights regarding the qualities of sera that mediate effective ADCC against primary HIV-1-infected CD4+
T cells. In the present study, high levels of ADCC activity were not associated with plasma viral load or CD4+
T cell count. In contrast to some prior reports, we were unable to detect greater ADCC activity in sera from LTNPs/ECs under our experimental conditions (2
). Further, we found no association with neutralization of the virus used in the ADCC assay or with breadth of neutralization. Using single MAbs we were also unable to detect a clear association with affinity or specificity. MAbs mediated very modest or low ADCC activity compared to sera from HIV-1-infected patients. The low ADCC activity of some sera and of most MAbs was associated with lower binding of HIV-1-specific antibodies to the surface of infected cells. Thus, under our experimental conditions, total binding of Env-specific IgG, especially IgG1, tightly correlated with ADCC activity. These results suggest that the total amount of IgG bound to an HIV-1-infected cell is an important determinant of the level of ADCC activity.
The finding that ADCC activity is simply associated with total IgG binding may be surprising at first inspection. However, these results are potentially consistent with those of a recent a study by Sun et al. that showed a correlation between NK cell-mediated ADCC and titers of SIVmac251
gp140 binding antibody in the first 14 weeks of rhesus macaque SIV infection (44
). Although it is unclear exactly how many antibody molecules must bind to an infected cell to trigger degranulation of an NK cell, most estimates predicted that this number was relatively small. If this were the case, one might expect that binding by only a few IgG molecules of an MAb or from patient sera would mediate high levels of ADCC. If surface Env trimers were uniform, one might also expect that high levels of ADCC would be achieved by combining MAbs with different specificities. However, this was not observed even in combinations of 6 broadly neutralizing MAbs with specificities that are widely spaced on the Env trimer. Given the size of IgG molecules relative to the Env trimer, it seems unlikely that additional specificities would result in greater IgG binding. Although modification of the Fc to enhance FcγRIII binding increased ADCC activity, it did not attain the level of complex sera. This result suggests that an important limiting factor is not FcR binding but, rather, is at the level of antigen-specific binding on the target cell. We consistently observed the highest ADCC activity using sera from infected patients, and this was associated with greater HIV-specific IgG1 binding than MAb binding. Consistent with this, among patient sera, greater ADCC activity was closely associated with higher IgG1 binding to HIV-infected cells. One likely explanation for the higher ADCC activity with sera than monoclonal antibodies is that Env is not homogeneous and sera containing multiple specificities are better able to recognize various surface conformations, including nonfunctional spikes. These Env forms would include Env with different levels of glycosylation, triggered Env spikes in which the conformation is altered after receptor binding, shed gp120 bound to CD4 on target cell surfaces, and residual gp41 stumps remaining after shedding of gp120.
One might expect that if Env-specific binding is required for ADCC, neutralization might correlate with ADCC, as neutralization is primarily determined by occupancy of the functional Env trimer by antibody (35
). However, we did not observe a correlation between ADCC and neutralization. One possible explanation is that major variations in ADCC are associated with various levels of binding, nonneutralizing antibodies (11
). Another possible reason for our inability to detect a correlation between ADCC and neutralization may reflect the fact that some ADCC, measured in vitro
, is potentially mediated by Abs recognizing non-Env epitopes (38
), although our results using an env
-deletion mutant show that ADCC responses are predominantly directed against Env.
In contrast to some prior work, we also did not observe an association between viral load and the ADCC activity of sera (2
). One prior study observed considerably greater ADCC activity of sera from LTNPs/ECs than progressors (26
). That prior study used very different experimental conditions, including protein-coated cell line targets and chromium release. Likewise, in other studies, no correlation between ADCC and disease progression was observed (7
). At present, the precise reason for the disparate results remains unclear.
The results of the present study also have some implications for ADCC mediated in passive-transfer studies. Passive-transfer studies examining the relative contributions of ADCC, ADCVI, or phagocytosis in protection of rhesus macaques from SIV infection have used monoclonal antibodies alone or in combination (17
). However, under our in vitro
conditions, the level of ADCC mediated by MAbs against primary HIV-1-infected cells was only a fraction of that mediated by most sera from chronically infected patients. Although this result is in need of confirmation in vivo
, it suggests that compared to passive transfer of IgG from complex sera, use of MAbs may underestimate the contribution of ADCC to sterilizing immunity.
A further understanding of the parameters that dictate ADCC-mediated killing of infected cells may have implications for efforts to stimulate ADCC activity in vaccinees. Specifically, it will be important to further define the relative importance of individual specificities versus the ability to recognize structural variants on the cell surface. Isolation of additional antibodies capable of recognizing a broad array of HIV-1 Envs may uncover specificities that are particularly good targets for ADCC. Alternatively, the high levels of ADCC observed using sera may not be mediated by antibodies specific for conserved regions and may be mediated only by the ability to recognize structural variants. If this is the case, then immunization with multiple Env variants may be required to achieve these levels. It is encouraging that some sera from RV144 mediated ADCC in the range of those from chronically infected patients in assays using HIV-1-infected cell lines or primary CD4+ T cells (A. Smalls-Mantey and G. Ferrari, unpublished observations). A further understanding of the basis of ADCC-mediated protection through passive-transfer studies in either macaques or vaccinated humans should yield important information for improving ADCC activity and possibly enhancing vaccine potency.