The principal observation of this study is that coimmunization with GRFT enhances the IgG response to gp120 in mice, the serum titration curves for the coimmunized mice being shifted ~10-fold to the left. The enhancement of immunogenicity by GRFT was specific for gp120. There was no such effect in mice that received GRFT together with the nonglycosylated Gag protein. Hence it is reasonable to assume that the action of GRFT is mediated by its binding to the gp120 mannose moieties.
The particular mannose-dependent epitope recognized by 2G12, a prototypical cross-neutralizing Ab, is rarely immunogenic during infection, although Abs are readily elicited to other mannose epitopes.22,23
Obviously, blocking mannoses will not facilitate the elicitation of Abs with the rare 2G12 specificity, for which goal other means must be pursued.22–24
Here we focus on the general IgG response to gp120 that is directed to other, mannose-independent epitopes.
The present results are broadly consistent with those of our earlier study in mice, in which we showed that mannose-depleted gp120 transiently elicited ~50-fold higher binding antibody titers than mannosylated gp120.12
However, the enhancement in that study was somewhat stronger than the modest and transient effect we report here. Although there could be advantages to occluding rather than digesting off mannose residues, there are also potential disadvantages. On the one hand, while deglycosylation could accelerate proteolysis of the immunogen, the formation of GRFT-gp120 complexes might protect gp120 and prolong its tissue half-life. On the other hand, the occluding molecule, GRFT, may attract some of the immune response to itself, at the expense of desired Ab responses to gp120. Furthermore, we found that GRFT partly blocks the binding of NAbs b12 and VRC01 to the CD4-binding site (CD4BS) on gp120, suggesting that the lectin would compromise the immunogenicity of this important epitope cluster. An explanation for the partial inhibition of b12 and VRC01 binding could be that GRFT interacts with the N-linked glycan at position 386. This glycosylation site is positioned at the rim of the CD4BS, and its elimination by mutagenesis is known to increase viral sensitivity to b12 neutralization.25–26
We did not attempt to measure NAb responses, because mice are not the best species for induction of NAbs and monomeric gp120 is a poor immunogen for this purpose. Our goal was to investigate influences on the overall immunogenicity of gp120, given that it is such an atypical, extensively glycosylated protein. Although there has been recent speculation that nonneutralizing antibodies to gp120 might play a role in protection from HIV-1 infection,27
we believe that the focus of HIV-1 vaccine research should remain squarely on the induction of NAbs with broad and strong activity. It seems highly unlikely that this goal would ever be achieved by using a monomeric gp120 protein. One alternative approach is the use of gp140 trimers, although that form of the protein is also inadequate for NAb induction at present. We do not yet know whether gp140 mannose moieties adversely influence the immune response to trimers, and hence whether GRFT coimmunization would increase their immunogenicity. However, the proportion of glycans present as unprocessed oligomannose forms is greater on trimers than on monomers when the two forms of Env are expressed in the same cells.28
A higher mannose content of trimers may therefore cause more profound biological effects, but it might also be harder to remove or block.
Whether occluding mannose moieties or removing them by enzyme treatment will be of practical benefit to Env vaccine development remains to be seen. In circumstances in which a complex protein is particularly hard to manufacture in bulk, any strategy that reduces the amount needed for each immunization could be useful. Adjuvants can do that,29,30
and we found that the beneficial effect of mannose depletion was observed only with Alum adjuvant, not with the more complex QS-21.12
However, there may be circumstances, for example, mucosal delivery, in which the use of certain adjuvants is precluded and where other approaches could be considered.
One potential consequence of occluding terminal mannose residues would be to reduce the uptake of gp120 into DCs via MCLRs and hence its presentation as an antigen. If such untoward effects do in fact occur, they must be overridden by proimmunogenic ones, since the net result was an enhancement of Ab reactivity, not a decrease. We can envisage three broad groups of mechanisms by which formation of a GRFT complex could increase the immunogenicity of gp120, but at present we cannot discriminate among them. The first is that GRFT, by binding to mannose moieties, inhibits the mannose-dependent interactions of gp120 with DC-SIGN and other MCLRs that would otherwise trigger the release of anti-inflammatory cytokines such as IL-10 from DCs, or impair the maturation of these cells.10
Enzymatic removal of the mannose moieties from gp120 had a marginally stronger effect on its immunogenicity, and at least some of the consequences of mannose depletion were mimicked by coadministering an anti-IL-10-receptor antibody with unmodified gp120.12
The second possibility is that GRFT binding prolongs the tissue half-life of gp120 by blocking proteolysis or by interfering with mannose-dependent scavenging systems. In principle, increasing the longevity of deposited gp120 could increase its immunogenicity.29
However, mannose-receptor-dependent clearance, which takes place mostly in the liver and spleen,31
may be more relevant when the immunogen is given intravenously than, as here, subcutaneously.
A clue about the third possible explanation is provided by the specific and reciprocal enhancing effects of gp120 and GRFT on the respective Ab responses. Thus both the anti-GRFT and the anti-gp120 titers were higher in the mice that received gp120+GRFT than in mice given, respectively, GRFT+Gag or gp120 without GRFT. Although the combination was more immunogenic than either protein individually, the resulting Abs mostly recognized the individual components with few, if any, appearing to be strictly complex specific (). What mechanisms could underlie a mutually enhanced immunogenicity that mostly yields Abs against the individual components of the complex? The size of an immunogen is an important factor.29
Antigen-presenting cells take up soluble proteins inefficiently, but the Alum adjuvant used in our experiments already promotes uptake by creating immunogen complexes of vastly increased size. Nonetheless, the array of epitopes available on Alum–immunogen complexes may differ when gp120 is presented with and without GRFT. An effect of this nature might be particularly important for the smaller protein, GRFT, akin to how conjugating short peptides to carrier proteins increases their immunogenicity. The response to the GRFT moiety may also have benefited from the T-helper cell epitopes present in gp120. We note that all responses to gp120, Gag and GRFT were IgG1-dominated and thus TH
2-polarized. GRFT binds to gp120 with a stoichiometry of 10 to 1 and an overall Kd
In addition, GRFT may be able to multimerize gp120 molecules, thereby creating substantial aggregates that, regardless of any further agglomeration by Alum, present arrays of epitopes that favorably cross-link B cell receptors.29
Furthermore, some of the mannose residues may remain unblocked, and the net effect of aggregation could be to enhance antigen uptake into DCs, particularly for GRFT. Varying the molar ratio of the two components might create different forms of gp120–GRFT complexes and could enable the response to the more relevant component, gp120, to be optimized.
It is also noteworthy that mice immunized with only gp120 produced some Abs that could bind to uncomplexed gp120 but not to gp120–GRFT complexes in an ELISA. GRFT may compete with Ab binding either directly or by steric hindrance. In either case, such blocking effects on the immunogen may hinder the elicitation of certain Abs. Thus, the net enhancement of the anti-gp120 response by GRFT might carry the price of immunosilencing a subset of gp120 epitopes. In this regard, it is problematic that GRFT partly blocked the binding of the NAbs b12 and VRC01, which are directed to important, cross-reactive CD4BS epitopes on gp120. If GRFT complexes of Env proteins more relevant to NAb induction were tested as immunogens, this point should be addressed so that important neutralization epitopes remain unoccluded. For example, it may be advantageous to mutate glycosylation sites adjacent to neutralization epitopes on the edges of the glycan shield, while instead occluding the bulk of the glycans; eliminating the latter would expose peptidic epitopes that are inaccessible to Ab on the native protein.
Does the increase in the anti-GRFT response in the presence of gp120 refute arguments that gp120 might be immunosuppressive, reducing the antibody response to both itself and other coadministered immunogens?2,32
We believe the present data are not definitive on this issue, because the formation of the gp120–GRFT complex may eliminate at least some of the possible immunosuppressive effects of gp120, notably those mediated by its mannose moieties. Differently designed studies would be required to answer this point. It should be noted, however, that the unexpected converse enhancement of the response to GRFT by gp120 may constitute a disadvantage. Thus, the immunogen complexes would rapidly become coated by preexisting Abs during subsequent immunizations, which could further impede B cell responses to gp120.
We conclude that ligating terminal mannose residues on gp120 by the small algal lectin GRFT enhances the immunogenicity of gp120 in a manner that is compatible with, but may go beyond, the blocking of MCLR interactions.