Increased neutralization of HIV-1 isolates by antibody combinations could be explained by at least two mechanisms. First, the antibodies may act on the same virion particles. An interaction of antibodies against nonoverlapping epitopes might lead to cooperative binding and enhanced neutralization. In addition, increased affinity for envelope spikes as a result of cooperative binding might also result in a broadening of the range of HIV-1 isolates recognized. Second, the antibodies may act on different virion particles. The recognition of a broader range of HIV-1 quasispecies by an antibody combination may then result in increased neutralization of heterogeneous HIV-1 isolates containing multiple quasispecies. An HIV-1 isolate, for example, might contain quasispecies which are neutralized by antibody 1 but resistant to antibody 2 and vice versa. A combination of these two neutralizing antibodies may then achieve a significantly greater reduction in virus growth than that predicted from assays performed with the two antibodies alone. Whereas both mechanisms may explain enhanced neutralization by a broadening of the response, only the first mechanism would be influenced by changes in antibody affinity due to binding cooperativity of antibody combinations. In addition to this, other mechanisms not directly related to antibody binding may apply.
In the primary isolate neutralization assays with constant antibody ratio design, we calculated CIs in a range of 0.6 to 0.8. Chou, Talalay, and Hayball have previously determined that a CI of 0.3 to 0.7 indicates synergism, 0.7 to 0.85 indicates moderate synergism, 0.85 to 0.9 indicates slight synergism, and 0.9 to 1.1 indicates additivity (9
). The synergism observed in neutralization of HIV-1JR-CSF
by combinations of b12, 2G12, and 2F5 (CI = 0.6 to 0.8) is similar to that reported for these antibodies previously in a study by Li et al. In the latter study, the CIs reported were in the range 0.6 to 1.0 for all three possible double combinations for neutralization of an SHIV containing the envelope glycoprotein of the T-cell-line-adapted strain HIV-1IIIB
). Mascola et al. examined the neutralization of SHIV89.6PD
by using 2G12 and 2F5 (24
). Some neutralization enhancement by the 2F5-2G12 combination was observed, although the neutralization of this virus by MAb 2G12 was very weak. The enhancement at the ID90
was about fourfold, which is in the range we have observed for neutralization of HIV-1 JR-CSF, 89.6, and SF162. In another study, neutralization synergy of the same order of magnitude was reported by Mascola and colleagues for neutralization of a subset of clade B HIV-1 primary isolates by combinations of MAb 2F5 and 2G12 (25
). Our data are also in agreement with a study by Potts et al. in which neutralization synergies between anti-CD4 and anti-V3 loop MAbs for T-cell-line-adapted viruses were assessed (42
). Although CIs suggesting intermediate to strong synergy were calculated, it was found that there was only a limited (two- to fourfold) increase in neutralization potency in most cases (42
). Laal et al. also reported apparently significant CIs, while neutralization only increased two- to fourfold (21
). Similarly, dose reduction indices calculated with the mathematical model in our study often appeared overly optimistic and suggested greater dose reductions than were observed (Tables , , and ). We carried out neutralization using different target cells (H9, U87, PHA-activated PBMC), reporter systems (p24 ELISA, β-galactosidase, and luciferase expression), and viruses (T-cell-line-adapted viruses, primary viruses, and recombinant viruses) in this study to make certain that the effects observed were reproducible under different neutralization assay conditions. The good agreement between the results in the various neutralization assay formats indicates that this is indeed the case.
A study by Vijh-Warrier et al. suggested a correlation between virus heterogeneity and synergy (55
). In that study, a combination of chimpanzee antibodies against the V2 and V3 loop, for example, appeared to neutralize HIV-1IIIB
synergistically (CI at ID90
of 0.5), whereas the same antibodies were suggested to be only additive against HIV-1HxB2
, a molecular clone of IIIB (CI at ID90
of 0.8 to 1.0) (55
). In a study by Thali et al. (51
), using molecular HIV-1 clones, cooperativity in neutralization by antibodies against the CD4 binding site and V3 loop were examined in HIV-1 envelope glycoprotein complementation assays. An enhancement of neutralization using neutralizing antibody pairs was found only sporadically. The effects observed were also weak (twofold or less) and could not be predicted by antibody binding to envelope glycoprotein expressed on the surface of COS cells (51
The studies above suggest that the estimation of synergy is difficult and its magnitude may be overestimated when using the mathematical model. An alternative approach to assess synergy is to vary the concentration of one neutralizing antibody in the test while adding a second neutralizing antibody at a fixed concentration at or just below its neutralization threshold (variable antibody ratio design). The level of occupancy of the second antibody on HIV-1 envelope spikes is thus expected to be fixed, and a smaller number of parameters are varied in the assay. Significant changes in the ID90 that might indicate synergy are assessed more easily compared to results from assays based on the classical approach to determine synergy. By using this alternative strategy, a moderate two- to fourfold enhancement of primary isolate neutralization by b12, 2G12, and 2F5 combinations became apparent.
The absence of neutralization enhancement by antibody combinations in assays using the molecular clone HIV-1HxB2
suggested that virus heterogeneity may play a role in the moderate neutralization enhancement observed with HIV-1 isolates JR-CSF, 89.6, and SF162. To address this question we assessed neutralization with the molecularly cloned envelopes of HIV-1JR-CSF
in envelope complementation assays. We found a similar enhancement of neutralization by the b12, 2G12, and 2F5 combination for the cloned envelope compared to results from assays using HIV-1 grown by several passages through PBMC. Therefore, heterogeneity as a result of the presence of HIV-1 quasispecies is not an apparent explanation for the neutralization synergy observed. Primary isolates may differ in this aspect from T-cell-line-adapted viruses. Using the molecular clone HIV-1HxB2
, we did not observe any enhancement of neutralization by antibody combinations in both combination assay formats used. This is in agreement with the trend observed by Vijh-Warrier et al. (55
As discussed above, increased neutralization by antibody combinations could be the result of cooperative binding of antibodies to envelope spikes. Binding of b12 and 2G12 to envelope glycoprotein expressed on the surface of cells was therefore assessed in flow cytometry studies. Similar studies using MAbs 2F5 and 4E10 are difficult, as these antibodies bind poorly to envelope expressed on the surface of cells (46
). No cooperativity of binding between b12 and 2G12 was apparent for binding to HxB2 as well as 89.6 envelope. For HIV-1HxB2
this is expected, since neutralization by b12 and 2G12 is additive in both assay formats discussed above. However, there is an apparent conflict between the b12-2G12 neutralization data and the envelope-binding assay (Fig. , Tables and ). Whereas b12 and 2G12 enhance each other approximately fourfold in neutralization, they do not appear to affect each other's binding to 89.6 envelope expressed on recombinant vaccinia virus-infected cells. This may indicate that the enhanced neutralization observed is not directly related to binding. It should be noted, however, that neutralization curves are typically steep and small increases in binding therefore may result in strong increases in neutralization. A two- to fourfold increase in neutralization may therefore be due to an increase in binding which would be difficult to assess in this type of assay.
A good correlation between antibody binding and neutralization has been observed in studies with T-cell-line-adapted strains of HIV-1, such as the HxB2 molecular clone of HIV-1 used above (37
). As it is presently unclear whether such analyses extend to primary isolates of HIV-1, we analyzed antibody binding to BHK cells expressing the envelope glycoprotein of a recombinant primary isolate. It should be noted, however, that recombinant envelope expressed on such cells may be present in molecular forms distinct from those present on the primary virion. Thus, recombinant vaccinia virus-expressing cells may express unprocessed gp160 as well as mature envelope spikes on their surface. Studies in which binding of Fab b12 to the recombinant 89.6-expressing BHK cells was compared to binding of the nonneutralizing CD4 binding site antibody Fab b6 (which binds gp160 strongly but binds mature envelope poorly [34
]), however, showed that Fab b12 bound ~10 times more strongly than Fab b6 (data not shown). This suggests that the majority of HIV-189.6
envelope on the surface of these BHK cells is present as mature envelope.
In a recent study an attempt was made to demonstrate and explain synergy by assessing b12, 2G12, and 2F5 binding to recombinant monomeric gp120 and multimeric gp160 by using surface plasmon resonance (57
). It was found that MAb 2G12 binding to gp160 interfered with the binding of MAb b12. This is in contrast to observations with monomeric gp120 (29
) and our observations on b12 and 2G12 binding to envelope spikes on infected cells. In the study mentioned above, MAb 2G12 furthermore enhanced 2F5 binding to oligomeric gp160 (57
). A number of studies, however, have suggested that unprocessed gp160 may have a different conformation from that of mature envelope spikes on the surface of the virus and infected cells (15
), and the study may therefore have little predictive value for binding of antibodies to envelope spikes.
The observations in our study have significance for the development of a humoral component of a vaccine against HIV-1. The results should be interpreted with the appropriate caution, however. It should be noted that although the observed synergy results in neutralization of HIV-1 isolates at decreased concentration of individual neutralizing antibodies, the total neutralizing antibody concentration increases in most cases. The dose of MAb b12 required to neutralize HIV-1SF162
, for example, may be reduced 5-fold by combining it with MAb 2F5; the total antibody concentration (b12 and 2F5 combined) required to neutralize HIV-1SF162
by the cocktail, however, was about 40-fold increased (compared to neutralization by b12 alone) (Table ). Similarly, HIV-1JR-CSF
is neutralized by the quadruple (b12, 2F5, 2G12, and 4E10) combination when each of the components is present at reduced concentrations, with individual dose reductions ranging from 5- (b12) to 38-fold (4E10). The total antibody concentration required to neutralize HIV-1JR-CSF
by the cocktail, however, is increased by about eightfold (compared to that for neutralization by b12 alone) (Table ). There are only very limited data on this issue from in vivo studies. Single antibodies and antibody combinations have been compared in passive antibody transfer, SHIV challenge studies by Mascola et al. (24
), but the data are in agreement with the above discussion. MAbs 2G12 and 2F5, for example, displayed moderate neutralization synergy against the isolate tested (SHIV89.6PD
). Whereas 2G12 protected 2 out of 4 animals at a plasma concentration of ~200 μg/ml, 2 out of 5 animals were protected by the 2F5-2G12 antibody cocktail, with a (higher) combined concentration of ~400 μg/ml (26
It has been of concern for HIV-1 vaccine design that neutralizing antibody concentrations required to protect against HIV-1 infection are high and exceed levels which can likely be reached and sustained by vaccination (26
). Neutralization synergy of antibody combinations therefore may seem promising. As discussed, however, neutralization synergy may not lead to reduction of the total HIV-1-specific antibody concentration required for neutralization. It can be argued that it may be easier to induce intermediate antibody titers against multiple epitopes by a vaccine than to induce high titers against a single epitope, even though the effective antibody concentration in the multiple-epitope vaccine may be higher. This information, however, is presently unavailable and is therefore an important question to address to further develop knowledge-based approaches to vaccine design.
In summary, our data suggest that neutralization enhancement may be observed in HIV-1 neutralization assays with combinations of broadly neutralizing antibodies. Studies on primary isolates (89.6, SF162, and JR-CSF) showed an enhancement of neutralization which was relatively weak between antibody pairs, with a maximum enhancement of two- to fourfold. A significantly greater enhancement was observed by triple and quadruple antibody combinations which, depending on the isolate tested, increased neutralization titers by up to about 10-fold. This observation encourages enthusiasm for the development of a humoral component of a vaccine against HIV-1, as individual antibodies might be able to provide sterile protection or benefit at lower levels than suggested by passive transfer studies using single antibodies or double antibody combinations (24
). The possible implications of neutralization synergy for vaccine development, however, are presently unclear and require further investigation.