In the current study, we directly compared WT trimers to two CD4 binding-defective trimer variants to determine the impact of the high-affinity in vivo interaction between Env and CD4 on vaccine-elicited immune responses. To our knowledge, this is the first comparative analysis of CD4 binding-competent and CD4 binding-defective Env immunogens analyzed in nonhuman primates, a highly relevant model for human vaccine testing. Furthermore, the novel design of the 423/425/431 trimer variant, which eliminated CD4 binding in an indirect manner by exploiting the two-step binding mechanism of the gp120-CD4 interaction, is the first example of such an Env immunogen and its analysis in nonhuman primates.
This study follows directly on our previous report in which we investigated the neutralizing Ab response elicited by soluble HIV-1 Env trimers in macaques, WT rabbits, and rabbits transgenic for human CD4 (15
). In that analysis, we demonstrated that the elicitation of CoRbs-directed Abs requires the presence of high-affinity primate CD4, suggesting that some fraction of CD4-binding-competent Env immunogens interacts with primate CD4 in vivo
, affecting the quality of the elicited B-cell response. The elicitation of CoRbs-directed Abs also was observed in humans immunized with purified monomeric gp120 in the Vaxgen phase III clinical trial (15
), likely a result of gp120 interactions with human CD4. Here, we show that CoRbs-directed Abs were elicited in all animals immunized with WT trimers but not in the animals immunized with CD4-binding-defective trimer variants. These results definitively confirm that the CoRbs on gp120 is not efficiently recognized by the naïve B-cell repertoire unless it is presented in complex with host CD4. We also demonstrated that both the direct disruption of the initial interaction between the gp120 outer domain and CD4 and the disruption of the conformational change required for stabilizing the Env-CD4 interaction completely abolished the elicitation of CoRbs-directed Abs.
The role of different types of CoRbs-directed Abs in virus neutralization and protection against infection remains something of an enigma. The detection of CoRbs Abs prior to autologous virus neutralization in HIV-1-infected patients suggests that CoRbs-directed Abs generally are nonneutralizing (16
). This is supported by data from the phase III Vaxgen clinical trial where no protection was observed (30
) despite high titers of CoRbs-directed Abs (15
). Furthermore, the access of two intact Ab molecules to the conserved CoRbs on the functional virus spike was shown to be sterically restricted (23
), while Ab subdomains derived from these same two Abs can access this region and inhibit viral entry (6
). These data argue against a role of CoRbs-directed Abs for virus neutralization. However, CoRbs-directed Abs recently were shown to contribute to the neutralization of some primary viruses in vitro
, at least when present at high titers (10
). It is possible that some yet-to-be-defined class of CoRbs-directed Abs exists that can contribute to virus neutralization during natural infection. Such specificities were suggested in two recent mapping studies (17
). Well-characterized Env immunogens, such as the 423/425/431 trimers described here, which selectively lack the capacity to elicit CoRbs-directed Abs but retain the ability to elicit other classes of Abs, will be important for future investigations aimed at determining the role of CoRbs-directed Abs in virus neutralization and in vivo
In addition to the formation of the CoRbs epitope, conformational changes induced by CD4 ligation may affect the exposure of other Env epitopes, altering the Env-specific B-cell repertoire and neutralizing Ab responses. Recent electron tomographic analyses of HIV-1 suggested dramatic conformational changes in the quarternary structure of the liganded forms of the trimer compared to the native, unliganded form (27
). Other reports show the stabilization and/or exposure of the V3 region on gp120 when it is in complex with CD4 (19
). Increased breadth of V3-mediated neutralization also was reported against a panel of HIV-1 reference strains in the presence of sCD4 using both vaccine-elicited sera and human V3-specific MAbs (46
). An increase in the exposure of the V3 region on the soluble gp140-F trimers also may occur upon CD4 binding, as suggested by an increased proportion of V3-specific compared to total Env-specific Abs in rabbits transgenic for human CD4 compared to that of WT rabbits, as shown in our previous studies (15
). However, whether CD4 binding alters the exposure of other potential neutralizing epitopes on Env besides V3 remains unknown.
To investigate the impact of in vivo CD4 binding on the Ab response elicited by the three trimer variants, we used several complementary approaches. Striking differences were observed at the level of antibody subspecificities, in particular the capacity of the WT trimers, but not the 368 and 423/425/431 trimers, to elicit CoRbs-directed Abs, and the capacity of WT and 423/425/431, but not the 368 trimers, to elicit either binding or weakly neutralizing CD4bs Abs. For HxBc2, it was possible to examine only one animal immunized with the 368 trimers in adsorption studies (F73, the only animal in this group that displayed weak HxBc2 neutralization). No evidence of CD4bs neutralizing Abs was detected in this animal. Although not formally shown, the absence of CD4bs-directed neutralizing Abs in all animals immunized with 368 trimers may explain the overall poor HxBc2 neutralization seen in this group. In contrast, both animals inoculated with 423/425/431 trimers generated CD4bs-directed Abs that contributed to the neutralization of both HxBc2 and MW965. Furthermore, all animals in the WT and 423/425/431 trimer-inoculated groups elicited CD4bs-directed binding Abs as detected by a b12 cross-competition assay. These results suggest that optimally designed CD4-binding-defective immunogens provide a means to minimize the potential occlusion of broadly neutralizing epitopes caused by in vivo ligation to high-affinity primate CD4. This may be an important consideration as Env immunogens progress from preclinical small-animal studies into human trials. No apparent advantage of the 423/425/431 trimers over the WT trimers in their capacity to stimulate neutralizing Abs against the CD4bs was detected in our studies, but further modifications to the 423/425/431 trimers may improve the performance of this immunogen.
Another observation was that the animals inoculated with WT trimers exhibited higher ID50 neutralizing titers against HxBc2 and MW965 after the fifth immunization than after the second immunization, while no increase in neutralizing titers was observed between the second and the fifth inoculation in the 423/425/431 trimer group. These data suggest that some undefined Ab specificity, or specificities, were selectively boosted in the animals inoculated with WT trimers. The boost effect suggests that some element(s) of the soluble trimers is selectively exposed upon CD4 binding, perhaps the V3 region. In that regard, we did not detect differences in the frequencies of memory B cells specific for the V123 regions elicited by WT and the CD4-binding-defective soluble trimers by the differential B-cell ELISpot assay. However, this analysis would not reveal qualitative differences, for example, potential differences in antibody affinity elicited by the immunogens. Since the boost effect was present only in the WT trimer-inoculated animals, it also is possible that this represents some class of CoRbs-directed Abs that is not detected by the HIV-2 cross-neutralization assay, since no boost effect was observed between the second and fifth inoculations in this assay (Fig. ).
One interpretation of the neutralization data is that the WT trimers are superior to the 423/425/431 trimers, as they elicit higher neutralizing titers against a subset of the viruses tested. However, because these viruses are sensitive to neutralizing specificities that are not effective against many primary circulating isolates (i.e., V3-directed responses), these responses may not reflect those required to accomplish broad neutralization. Therefore, our interpretation of the data generated in this study is that the WT trimers are not markedly superior to the 423/425/431 trimers in terms of vaccine-relevant broadly protective responses.
A potential consequence of in vivo
CD4 binding during immunization is that the ligation of Env to cell surface-expressed CD4 stimulates signaling events that negatively impact T-cell function. CD4 plays an important role as an accessory molecule to activate T-cell proliferation in response to cognate interactions between the T-cell receptor and major histocompatibility complex class II-restricted antigen presentation. Studies using full-length membrane-bound, but not soluble, HIV-1 Env trimers showed that CD4-dependent Env interactions suppressed CD4+
T-cell activation and proliferation in vitro
), suggesting that the elimination of Env interaction with CD4 in the context of vaccination is beneficial to better elicit functional T-cell help and more potent neutralizing Ab responses. In the present study, we investigated the potential effects of CD4 ligation by a comprehensive analysis of Env-specific immune responses elicited by WT and CD4 binding-defective trimers. We show that the titers of circulating Env-specific binding Abs were similar between the three groups, as were the frequencies of Env-specific memory B cells and the magnitude and functionality of Env-specific CD4+
T cells. These results suggest that the disruption of the Env-CD4 interaction is not critical for the induction of potent immune responses against Env. Although not addressed here, our data also argue against a role for Env-CD4 interactions in promoting immune dysfunction during HIV-1 infection. However, due to the multitude of differences between natural infection and active Env protein vaccination, cautious extrapolation between the two systems is advised.
It remains to be determined if in vivo CD4 interactions limit the elicitation of antibodies directed against the CD4 binding site as a result of potential occlusion effects in an animal system both possessing and lacking primate CD4 (i.e., huCD4 transgenic small animals). In this regard, our data demonstrate that modifications to the CD4 binding site should be avoided or approached with care to not disrupt the antigenic surface of the CD4bs, as observed for the 368D/R mutation. To circumvent potential CD4 occlusion effects and to limit the elicitation of nonneutralizing CoRbs-directed responses, rationally designed CD4 binding-defective Env immunogens that retain the capacity to stimulate Ab responses against the CD4bs may remain worthy of further pursuit.