In this report, we have described a set of immunogenicity studies on first generation monomeric gp120 mutants that have been engineered toward eliciting b12-like antibodies. The main aims of our experiments were twofold: first, to obtain information on the immunogenicity of the mutants; second, to map serum responses to explore the relationship between antigenicity and immunogenicity. Our findings are being used to improve the design and construction of further mutants.
A key observation from this study is that engineering of gp120 can lead to a targeted reduction in the elicitation of multiple nonneutralizing and weakly neutralizing antibody specificities. The vaccinia-produced GDMR gp120, where residues 473 to 476 were mutated to alanine, mirrored the antigenicity profile previously established (35
) for 293T cell-derived GDMR gp120, and in addition further antigenic properties were defined. Our findings can be summarized as follows: (i) CD4bs antibodies, b6, b3, and F105 that are weakly neutralizing failed to bind, while binding affinity for the broadly neutralizing antibody b12 remained the same as wild-type gp120; (ii) CD4i antibodies failed to bind the GDMR gp120 mutant; (iii) C1-C4/C5 gp120 antibodies bound GDMR gp120 with lowered affinity compared to wild-type gp120; (iv) V3 loop antibodies bound the GDMR gp120 mutant with similar affinities as wild-type gp120; (v) a V2 loop antibody (8.22.2) to a linear epitope bound the GDMR gp120 mutant with similar apparent affinity as wild-type gp120 but the antibody (697-D) to a conformational V2 epitope bound with lower affinity; (vi) a V1/V2/V3 loop-dependent antibody, 4KG5, exhibited lower binding to the GDMR gp120 mutant compared to wild-type gp120. Overall, the difference in antibody binding affinities for GDMR gp120 mutant by antibodies to the variable loops that are conformation dependent suggested a degree of conformational rearrangements in the V1/V2 and V3 variable loops. In addition, the lack of recognition of the GDMR gp120 mutant by CD4i antibodies is likely linked to a change in the orientation of the variable loops.
Mapping of the serum responses to the GDMR gp120 mutant by competition studies showed reduced levels of CD4bs and CD4i antibodies compared to the sera from rabbits immunized with wild-type gp120. These observations are consistent with the antigenicity profiles exhibited by the GDMR gp120 mutant. A large proportion of the total serum antibodies in the GDMR gp120-immunized rabbits were specific to the V3 loop. It has been shown previously that sera raised from humans vaccinated with gp120 contain a large proportion of antibodies directed against the V3 loop (27
). Notably, there was a 20 to 30% increase in the percentage of V3 loop antibodies in the sera from rabbits immunized with GDMR gp120 compared to sera of wild-type gp120-immunized rabbits. This result is consistent with the notion that some rearrangements of the V1/V2 and/or V3 loops in the GDMR gp120 mutant produced increased epitope exposure and improved immunogenicity of V3 loop epitopes. There is also a possibility that the increased levels of V3 loop antibodies to the GDMR gp120 mutant may be a compensatory response due to dampening of the elicitation of CD4bs and CD4i antibodies. The immunogenicity results for the GDMR gp120 mutant are encouraging in that the serum antibody responses correspond fairly well to the antigenic profiles exhibited by the mutant.
The GDMR gp120 immune sera exhibited a somewhat improved neutralizing activity against a few primary viruses compared to sera from wild-type gp120-immunized rabbits. We sought to investigate whether this could be attributed to the elicitation of b12-like antibodies as envisaged for the GDMR gp120 mutant. V3 peptide competition studies showed that most (70 to 90%) of the neutralization, where present, could be attributed to V3 loop antibodies. It would seem that changes in the conformation and/or exposure of the V3 loop as a result of the GDMR mutation increases the titer of a neutralizing antibody response that has some degree of cross-reactivity. Furthermore, the inability of the GDMR gp120 immune sera to neutralize the TCLA viruses HXBc2 and NL4-3 argues against the induction of b12-like antibodies since both these viruses are very sensitive to b12 neutralization and, indeed, to CD4bs antibody neutralization in general (5
Immunization with wild-type gp120 or GDMR gp120 had opposite effects on the relative ability of the resulting sera to neutralize primary and TCLA viruses. Wild-type gp120 immune sera neutralized the TCLA viruses HXBc2 and NL4-3, presumably reflecting the elicitation of antibodies such as CD4bs and CD4i, antibodies that effectively neutralize such viruses. The V3 loop of HXBc2 and NL4-3 is notably different from that of the immunizing JR-FL isolate, and neutralization titers were not significantly affected by the presence of JR-FL V3 peptide. It is therefore highly unlikely that neutralization of the TCLA virus NL4-3 by the wild-type gp120 immune sera was mediated by V3 loop-specific antibodies to linear epitopes. In contrast, the GDMR gp120 immune sera did not neutralize HXBc2 or NL4-3 virus, as opposed to its ability to neutralize some primary viruses. This is consistent with the loss of CD4bs and CD4i antibodies in the corresponding sera. The pattern of these results clearly indicates that neutralization of TCLA virus is not necessarily predictive of neutralization of primary viruses; i.e., one can have primary virus neutralization in the absence of TCLA neutralization. Therefore, the use of TCLA neutralization as a sole prescreen or first stage evaluation of immune sera is probably not advisable.
The mCHO gp120 mutant was designed to eliminate weakly neutralizing and nonneutralizing antibodies in the CD4bs, CD4i, C1-C4/C5, and V1/V2 loops and part of the V3 loop while still maintaining binding to antibody b12. The mCHO gp120 mutant with seven extra glycosylation moieties still gave a robust serum antibody binding titer by ELISA, similar to the sera from wild-type gp120-immunized rabbits. Mapping sera by competition studies showed that mCHO gp120 elicited lower levels of CD4bs, CD4i, and C1-C4/C5 conformational antibodies than wild-type gp120 as envisaged by its design. This is an interesting observation that shows that antibody responses to three major gp120 core epitopes can be dampened by the presence of only three extra glycans.
Wild-type gp120 immune sera did not elicit detectable levels of conformational V2 loop-specific antibodies as determined by the lack of serum inhibition of biotinylated 697-D antibody and this was also the case with mCHO gp120 immune sera. However there were some antibodies against linear V2 peptides both in the wild-type gp120 and mCHO gp120 immune sera. It is therefore difficult to speculate whether the extra sugar on the V2 loop necessarily masked responses to the V2 loop in the mCHO gp120 mutant since such antibodies were not elicited in great numbers by wild-type gp120 immunization either. However, an improved serum binding response by the mCHO gp120 immune sera was seen to a linear 15-mer peptide in the V2 loop, the C terminus of the K171 residue where an extra sugar was incorporated.
Sera from mCHO gp120-immunized rabbits did not produce antibodies that bind to the crown of the V3 loop as determined by the lack of inhibition of biotinylated 447-52D antibody binding in the presence of mCHO gp120 immune sera compared to sera from wild-type gp120- or GDMR gp120-immunized rabbits. In addition, two out of the three mCHO gp120 immune sera also failed to bind one of the two V3 crown-specific 15-mer peptides (NNTRKSIHIGPGRAF), against which both wild-type gp120 and GDMR gp120 immune sera bound the best. The reasons for this could be either the mutation of the GPGR sequence or the effective masking of the V3 crown by the extra sugar moiety. The lowered levels of V3 crown-specific antibodies in the mCHO gp120 immune sera is in accordance with the lack of recognition of the mutant by V3 crown antibodies 447-52D and 19b in antigenicity studies. However, in a different setup of the competition ELISA, where inhibition of the mutant immune serum binding to wild-type gp120 is detected in the presence of human monoclonal antibodies, mCHO gp120 immune serum binding was effectively inhibited in the presence of monoclonal antibodies to the V3 loop. The results indicate that there is still a significant proportion of V3 loop antibodies in the mCHO gp120 immune sera to parts of the V3 loop other than the crown. This result is consistent with the antigenicity studies that showed the mCHO gp120 mutant bound well to the V3 stem-specific antibody 39F. It is, however, difficult to say conclusively whether the masking of the crown of the V3 loop or the mutation in the GPGR sequence caused the reduction in antibodies (like 447-52D) specific to the crown. In a report by Garrity et al. (15
), a complete block in the antibody response against the V3 loop as a whole was seen only when there were three to four extra sugar attachment motifs and at least two extra glycans on the V3 loop, and in that case the response shifted to the immunodominant V1 loop. Therefore, in order to completely mask the V3 loop, additional glycosylations may be needed. Similarly, to prevent a shift in antibody response to other variable loops, extra sugars may also be required to fully mask the V1/V2 loops. In light of this, we have recently modified the mCHO gp120 mutant further by the incorporation of several additional glycosylation sites in the V2 and V3 loops (36
The increased levels of antibodies to a 15-mer V2 loop peptide in the mCHO gp120 immune sera in contrast to wild-type gp120 immune sera raised some concern about the possible generation of neo-epitopes within the mCHO gp120 mutant. Therefore, the mCHO gp120 immune sera were tested for the presence of antibodies to such putative epitopes by using serum antibody binding ELISAs. Immune sera from mCHO gp120 bound to itself somewhat better than to wild-type gp120 and also to gp120 with a deletion of the V1/V2 loop, indicating that some of the serum antibodies from mCHO gp120-immunized rabbits recognized epitopes that were not well exposed on wild-type gp120. Mapping of mCHO gp120 immune sera using linear peptides spanning the whole gp120 sequence (115, 15-mer, overlapping peptides) detected improved recognition by all three rabbit sera from mCHO gp120 immunization of chiefly two 15-mer peptides, one in the C terminus of the V2 loop and other in the conserved C4 region of gp120, in contrast to wild-type gp120 immune sera. Therefore, clearly the mCHO gp120 mutant elicits some neo-epitope antibodies, but the levels are not overwhelming, and, more importantly, most serum antibodies raised against this mutant recognized wild-type gp120 as well as immune sera from wild-type gp120-immunized rabbits.
The mCHO gp120 immune sera exhibited a loss of neutralization efficiency compared to wild-type gp120. This is probably due to the lowered levels of weakly neutralizing CD4bs and CD4i antibodies and to reduced levels of V3 crown-specific antibodies that are an important fraction of the neutralizing antibody repertoire in the immune sera from monomeric gp120. The sera from rabbits immunized with wild-type gp120 elicited CD4bs antibodies as detected by competition assays with bio-b12, bio-b6, or bio-F105. However, the wild-type gp120 immune sera (rabbits 7262, 7263, and 7264) failed to neutralize antibody b12-sensitive viruses such as HIV-1 strains JR-FL, JR-CSF, or ADA to any significant degree. This suggests that b12-like antibodies are not elicited following wild-type gp120 immunization. We had hoped that, unlike wild-type gp120, the engineered gp120s designed to expose the b12 epitope would elicit b12-like antibodies. In the case of the mCHO gp120, the failure to elicit b12-like antibodies might be due to the lowered exposure of the b12 epitope as determined by the reduced affinity for b12 antibody binding. As indicated above, we have sought to address this problem by the engineering of mutants with improved affinity for b12 (36
) that may prove superior in eliciting b12-like antibodies.
In most studies, where a number of potential HIV-1 antigens are being tested for immunogenicity in small animal models, testing for serum titers and testing for neutralizing antibody response are the regular assays of choice (3
). Serum mapping studies aimed at determining the specificities of antibodies raised by an antigen are not examined to any great extent. Competition assays between monoclonal antibodies and polyclonal sera do not always give direct answers regarding the different types of antibodies present in polyclonal sera. This is also true when monoclonal antibodies are competed against other monoclonal antibodies, where decreases in binding can be observed when one antibody binds to gp120 even though the second antibody does not bind to precisely the same epitope (30
). Mapping polyclonal immune serum is not straightforward but vital and provides answers as to the success of a strategy to modify HIV-1 Env to elicit neutralizing antibodies.
To summarize, we have immunized with engineered monomeric gp120 that favorably presents the conserved epitope for the broadly neutralizing antibody b12 while lowering the exposure of the weakly neutralizing and nonneutralizing immunodominant epitopes. Two strategies were used, one where residues were changed on the gp120 core such that a selection of weakly neutralizing antibodies fail to bind (GDMR gp120 mutant). The second was to mask immunodominant loops, the CD4-induced site and the nonneutralizing face of gp120 with carbohydrate moieties (mCHO gp120 mutant). One significant finding in this report is that through design, it is possible to reduce elicitation of unwanted antibodies to a multiplicity of epitopes. However, we have not yet been able to focus the response to the desired b12 epitope, and this will likely require further engineering of gp120.