The development of immunogens that elicit broadly neutralizing antibodies remains a high priority for the development of an effective AIDS vaccine. Among the targets of neutralization is the highly conserved CD4-BS. MAb b12 binds primarily to the surface of gp120 utilized in the initial contact with the CD4 receptor. This site is recessed on the viral spike, is surrounded by both N-linked glycans and immunodominant variable loops, and represents only one among many potential B-cell epitopes within gp160.
One strategy for optimizing the presentation of this site is “minimization,” to remove all immunogenic Env surfaces other than the target site. Such minimization would have utility both to define antibodies to this region in complex antisera and to develop new prototypes as potential immunogens. However, it has been a challenge to isolate the site of b12 binding independent of the rest of the HIV Env, particularly the inner domain. In this report, we show that an expression vector encoding the OD and α5-helix of Env linked to a transmembrane domain retains b12-binding activity and interacts specifically with antisera with broadly neutralizing CD4-BS antibodies. Absorption of one of those antisera selectively removed neutralizing activity against several viruses. These data suggest that it is possible to generate simplified proteins that can eliminate multiple specificities detected by anti-HIV-1 sera and to focus recognition on the CD4-BS.
Previous studies have shown that a soluble outer domain protein, OD1, is recognized by 2G12 and a number of V3 antibodies, but recognition by b12 was hampered by an enhanced off rate (36
). Laboratory-adapted virus strain R3A TA1 was chosen for further modification because it was highly sensitive to b12 neutralization and lacked most of the variable loops. It is likely that similar results can be obtained by removing analogous Env regions in other strains, and while a soluble OD based on R3A TA1 was similar to previously described ODs (36
; and data not shown), here, the addition of a cellular transmembrane domain significantly improved b12 reactivity as determined by flow cytometry. This feature was observed with a variety of membrane anchors and was retained upon modification of the V3 region. It is important to remove the V3 from candidate immunogens because of its strong but strain-specific antigenicity.
The OD-TM described here was generated after attempting to develop secreted versions of OD. Secreted OD formed aggregates when expressed in 293 cells and failed to interact with IgG1 b12, similar to another soluble OD described previously (36
). We reasoned that the loss of reactivity of soluble OD was due in part to the loss of appropriate folding, a result of inner domain removal. We further reasoned that an alternative surface might stabilize OD and that, possibly, a cell membrane might provide a suitable surface. Thus, appropriately anchoring OD to a membrane might stabilize it sufficiently to retain a conformation recognized with high affinity by b12. Other fusion protein approaches had failed to confer this property, although it remains possible that secreted ODs could be generated with heterologous domains using independent mutations.
The reasons for the improved reactivity of membrane-anchored OD are not fully known but include the possibility that membrane anchoring allows protein contacts with the cell surface that stabilize exposed regions that would otherwise interact with the inner domain in the nontruncated Env (Fig. ). In this context, the membrane may also occlude ligand binding to the anchored OD in a manner similar to that of the native viral spike. Finally, we note that cell surface avidity, present in the membrane-anchored context but not the soluble context, should reduce the off rate, thereby stabilizing b12 interactions.
Interestingly, the V3 loop modification mutants tested here showed that both the length and charge of V3 affect b12 binding. This result is somewhat unexpected since the V3 region emanating from the core is distal from both the site of inner domain truncation and the site of b12 recognition. To eliminate the V3 immunodominance, we both truncated V3 and made modifications to reduce its immunogenicity. In some cases, modifications affected both 2G12 and b12 binding, indicating an effect on OD-TM expression, but in others, b12 recognition was specifically enhanced, suggesting that its exposure was increased relative to those of other epitopes on the OD-TM.
The deletion of the β20-β21 hairpin has been suggested to reduce steric hindrance around the CD4-BS, to expand the CD4-binding pocket, and to increase b12 accessibility (2
). Our data showed that the deletion of the β20-β21 hairpin improved b12 and 2G12 binding. These data suggest that the β20-β21 loop is not necessary for b12 binding to the membrane-anchored OD proteins, although the increased binding to 2G12 suggests that this deletion improves overall protein expression.
Both 2G12 and b12 are conformational antibodies (5
). Our results showed that without the inner domain, the outer domain of gp120 itself can form a stable structure that allows those antibodies to become accessible. The optimized ODs developed here specifically recognize b12 but not most other nonpotent neutralizing CD4-BS antibodies. Moreover, OD(GSL)(Δβ20-21)(hCD4-TM) can absorb the HIV-1-neutralizing antibody from the human sample Zeptometrix 1642. Zeptometrix 1642 serum has reasonable breadth and potency, although its activity is lower than that of patient 1 sera described previously (17
). Other HIV-positive patient sera have been well characterized, including sera from patients 1 and 45 (18
). Both samples contain CD4-BS antibodies, but their relationship to b12, b13, and Zeptometrix 1642 is unknown, and they might be expected to differ since these sera show broader reactivity across divergent clades than b12. A possible explanation for the lack of binding of patient 1 serum with the membrane-anchored OD is that it may require an interaction with an additional region, such as the inner domain or bridging sheet, not present in the outer domain. It is also unclear whether the activity of patient 1 sera can be explained by a single antibody or a combination of CD4-BS antibodies directed against diverse epitopes. The identification of other broadly neutralizing MAbs to the CD4-BS from such subjects will be instructive in this regard.
Efforts to use OD-TM as a vaccine immunogen are currently in progress. Structure-based calculations show that the proportion of b12-reactive surface relative to the total exposed surface on the optimized OD is significantly higher than that in core gp120 or OD1 contexts (Table and Fig. ). Because of its transmembrane nature, gene-based vaccination is required to elicit immune responses. Nonetheless, the findings reported here suggest that the membrane-anchored OD represents an alternative form of the Env glycoprotein that can detect neutralizing antibodies directed to the site of CD4 binding and may contribute to the rational design of an AIDS vaccine.
FIG. 7. b12 epitope relative surface area on core gp120, OD1, and membrane-anchored OD. The b12 surface area was calculated in the context of three immunogens: core gp120 (14), OD1 (36), and the membrane-anchored OD developed here, OD(GSL)(Δβ20-21)(hCD4-TM). (more ...)