This study investigated the use of mutated HIV-1 gp120 as a vaccine immunogen to elicit higher titers of anti-gp120 Ab response effective against the virus. We focused on enhancing titer or potency, but not breadth, of the Ab response by testing recombinant gp120 derived from Env of HIV-1 LAI, a subtype B laboratory-adapted strain in which the principal neutralizing epitopes, located in the V3 loop, are already defined. Two approaches to improve V3 immunogenicity were studied: 1) removal of a single glycan at position 448 by substituting the N448 residue with Q or E, and 2) immune complex formation with the anti-CD4-binding site mAb 654. Each of these strategies has been shown previously to enhance antigenicity (i.e. in vitro
Ab reactivity) of neutralizing V3 epitopes [15
]. The human anti-CD4-binding site mAb 654 was selected for preparing the gp120/mAb complexes because it has a relatively high binding affinity for gp120 and forms a stable immune complex with enhanced V3 reactivity [26
]. No murine anti-CD4-binding site mAbs displaying similar properties are available. Data from this study demonstrate that using the DNA prime/protein boost protocol, immunization with the mutant gp120 did not elicit anti-V3 Abs and failed to stimulate higher titers of anti-gp20 serum Abs than the wild type gp120. The sera also had very weak neutralizing activities comparable to that achieved with the wild type gp120. By contrast, immune complexes made with N448Q or N448E were potent in eliciting higher titers of both neutralizing and non-neutralizing Abs than the wild type complex. Neutralizing activity was directed against V3 and other undefined epitopes, while non-neutralizing Abs were stimulated to higher levels against the inner domain of gp120 including the C2 region. The contribution of these non-neutralizing Abs in protection against HIV-1 remains unclear but these Abs might be important for antiviral activities such as antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated virus inhibition [31
]. The uncomplexed gp120 was not able to induce neutralizing Abs (Supplementary Figure 2C
]), similar to the DNA prime/protein boost data (). These data demonstrate the advantage of combining a select gp120 mutation and immune complex formation for designing a more potent vaccine immunogen that is capable of eliciting higher titers of Ab responses against HIV-1.
Although the DNA prime/protein boost immunization with the N448Q mutant elicited comparable levels of gp120-binding and virus-neutralizing Ab titers as that with the wild type gp120, the mutant elicited higher levels of gp120-specific lymphoproliferation and secretion of Th2-associated cytokines. Significantly, the responses against peptides in the vicinity of residue 448 were significantly enhanced, suggesting that the removal of N448-linked glycan may promote the processing and/or presentation of nearby Th epitopes that result in better induction of Th responses to these epitopes. This finding could not be predicted from our previous in vitro
data which showed that the same glycan removal reduced recognition of epitopes immediately upstream of residue N448 by both human and mouse CD4 T cell lines [15
]. The reasons for this discrepancy are not known, but these data reaffirm the difficulty of extrapolating in vitro
antigenicity of a vaccine to its immunogenicity in vivo
. Enhanced immunogenicity of the gp120 mutants was further demonstrated by the ability of the immune complexes made with N448Q or N448E gp120s to induce higher titers of binding and neutralizing Abs than the wild type complex. It should also be noted that the DNA priming/protein boost protocol induced much higher titers of anti-gp120 Abs than immunization with gp120 protein alone ( vs. Supplementary Figure 2A
), confirming the significant contribution of DNA priming as reported previously [17
]. In these two protocols, animals were immunized three times with either one DNA prime plus two protein boosts or with three injections of proteins only, but the protein only immunization resulted in a very low level of anti-gp120 Abs that was almost comparable to the sham control. Nevertheless, DNA prime/protein boost vaccination, even with the N448Q mutant displaying enhanced V3 reactivity, was not adequate for eliciting Abs against neutralizing V3 epitopes.
In contrast to gp120 alone, immune complexes of gp120 and mAb 654 were highly potent in inducing virus-neutralizing Ab responses. Importantly, using the N448 mutant complexes we were able to further enhance the titers of neutralizing and non-neutralizing anti-gp120 Ab responses beyond those achievable with the wild type counterpart, indicating the synergistic potential of N-glycan removal and immune complex formation in augmenting gp120 immunogenicity. Consistent with our previous reports [21
], DNA priming was not required for these immune complex vaccines, although experiments are currently in progress to evaluate the contribution of DNA priming in further improving the immunogenicity of the immune complex vaccines. The mechanisms by which mAb 654 enhances gp120 immunogenicity are not yet fully understood. The binding of mAb 654 and other anti-CD4-binding site mAbs to gp120 has been shown to cause allosteric changes in the overall gp120 structures that affect exposure of distant Th and B cell epitopes [21
]. Similar to CD4, anti-CD4-binding site mAbs also induce large enthalpic and entropic changes that are not seen upon gp120 interaction with other anti-gp120 mAbs to increase rigidity of the otherwise highly flexible gp120 molecule and increase resistance of the complexed gp120 to degradative enzymes including proteases and endoglycosidases [26
]. To our knowledge this is the first study that demonstrates this effect of anti-CD4 binding site mAbs on specific gp120 epitopes. Hence, Ab reactivity to epitopes in the V3 and C2 regions were specifically enhanced when gp120 was bound to mAb 654. The C2 epitope was also better protected from proteolytic degradation in context of gp120/654 complexes than in uncomplexed gp120. Similar results were observed with V3 in the mutant complex, albeit to a lower extent, indicating that by forming immune complexes, specific gp120 epitopes are recognized better by Abs and also are more resistant to proteolytic degradation. The enhanced antigenicity and stability of these particular Ab epitopes correlated with their increased immunogenicity in vivo
, indicating the importance of these two factors in influencing immunogenicity of Ab epitopes on gp120 and possibly on other antigens.
Contrary to V3 and C2, the CD4-binding site and the V2 loop were occluded in the gp120/654 complexes from Ab recognition, leading to failure of animals immunized with these complexes to generate Abs against these regions ( and [22
]). Most of anti-CD4 binding site Abs made during HIV infection by slow and rapid progressors have poor or no neutralizing activity and can interfere with gp120 antigen processing that result in suppressed helper CD4 T cell responses [26
]. However, unique neutralizing epitopes such as those recognized by broadly reactive and potent neutralizing mAbs b12 and VRC01 are present in the CD4 binding site [39
]. These epitopes are also blocked in the gp120/654 complexes (unpublished data). Similarly, Abs to V2 that have the potential to neutralize viruses [43
] or mediate other anti-viral functions would not be induced by the gp120/654 complexes. Therefore, further improvements of the current prototypic complexes are needed. A number of anti-gp120 mAbs that enhance the antigenicity of epitopes in the CD4 binding site and the V2 loop have been identified ([45
] and Hioe et al., unpublished data ) and can be utilized to produce additional immune complexes to enhance Ab responses against these epitopes. In conjunction with the complexes studied here, a cocktail of immune complex vaccines may then be employed to address the difficult challenge of increasing the breath of anti-viral Ab responses elicited by immunization. Moreover, we demonstrated that a broader neutralizing anti-V3 Ab response effective against heterologous HIV-1 isolates could be induced by immune complexes made of gp120 JRFL, rather than gp120 LAI [21
]. Because gp120 LAI has an unusual V3 sequence due to a two amino acid insertion and is antigenically distinct from gp120s of other HIV-1 isolates, the Abs generated in response to gp120 LAI are extremely specific for LAI and its homologous virus strains ( and [22
]), and thus, constructing immune complexes with gp120s that better represent the majority of HIV-1 circulating isolates would be advantageous.
Immune complexes have been used as vaccines to augment immune responses to hepatitis B antigens in humans [46
] as well as to infectious bursal disease virus, equine herpesvirus 1 and porcine parvovirus in animals [48
]. Enhanced immunogenicity of immune complex vaccines has been attributed mainly to their Fc-mediated activity, as the Fc interaction with FcRs or complement receptors on dendritic cells and other professional antigen presenting cells (APCs) facilitates targeting and uptake of antigen by APCs and mediates their activation, resulting in enhanced antigen presentation to helper CD4 T cells and development of a more effective T cell-dependent Ab response [51
]. The specific involvement of the Fc fragment in influencing the immunogenicity of gp120/654 complexes was not evaluated in this study. Human mAb 654 (IgG1 subtype) was utilized to make the immune complexes and the Fc fragment of human IgG1 might not engage murine FcRs efficiently. The association constant of human IgG1 Fc for mouse splenic macrophages has been reported to be ~30x lower than that to human peripheral monocytes, and human IgG also displays distinct binding modes for mouse vs. human Fc receptors [54
]. Moreover, we have previously observed that an adjuvant is critical for immunization of mice with the gp120/654 complex, since administration of the complex in PBS induces much lower levels of neutralizing Abs [21
], indicating that the adjuvant effect of Fc-FcR interaction may not play a substantial role in augmenting immunogenicity of gp120/654 complexes in mice. Rather, we postulate that in this in vivo
model the enhanced immunogenicity of the complexes is mediated mainly by Fab-mediated activity that alters gp120 conformation and modulates exposure and stability of specific Ab epitope. Further studies to directly test this hypothesis are needed, but in support of the idea, we have demonstrated that this activity is not displayed by all anti-gp120 mAbs but it is unique to anti-CD4 binding site mAbs and is determined by the specificity of mAbs used to form the complexes [21
In summary, the capacity of HIV-1 gp120 to induce potent neutralizing anti-gp120 Ab responses was enhanced by a combination of select N-glycan removal and immune complex formation with a specific anti-gp120 mAb. Further improvements of this vaccine platform are needed to increase the breadth of the Ab responses in order to confer protection against a broader array of HIV-1 isolates.