In this study we have produced A32-rgp120 complexes with stably expressed coreceptor binding sites and have compared these complexes as immunogens with uncomplexed rgp120. Our study is important in that we determined (i) that stable enhanced expression of the coreceptor binding site is not sufficient for induction of broadly reactive NA in the absence of CD4, (ii) oil adjuvant formulation did not prevent A32-rgp120 broadly neutralizing antigen expression, and (iii) rgp120BaL alone was the best Env immunogen tested.
Fouts et al. have reported that cross-linked CD4-rgp120IIIB
complexes, unlike uncross-linked rgp120 or rgp140, induced antibodies that neutralized a spectrum of HIV-1 primary isolates from multiple HIV-1 subtypes (9
). Soluble CD4 in cross-linked CD4-rgp120 complexes could be important as a component for the ability to induce cross-reactive NA in multiple ways. Human CD4 induces anti-CD4 antibodies that themselves neutralize HIV-1. Indeed, absorption of anti-CD4 antibodies removed some but not all of the cross-subtype neutralizing activity induced by CD4-rgp120 complexes (5
). Second, CD4, when complexed to rgp120, can be part of a new neutralizing determinant in the complex and/or induce new neutralizing determinants on rgp120 following induction of conformational changes in rgp120 by CD4 (5
We chose to study MAb A32-rgp120 complexes for several reasons: (i) MAb A32 reacts with many HIV-1 isolates of multiple subtypes, (ii) MAb A32 is as potent an inducer of the CD4i coreceptor binding site as is sCD4, and (iii) MAb A32 does not bind to the CD4BS, thus leaving it open for induction of anti-CD4BS antibodies (26
While A32-rgp120 complexes with rgp12089.6
induced slightly more neutralizing breadth than rgp12089.6
alone, comparative studies with A32-rgp120BaL
showed that A32-rgp120BaL
complexes were less immunogenic for broadly reactive NA than uncomplexed rgp120BaL
(Tables and ). Thus, our carefully characterized A32-rgp120 complexes provide the first data demonstrating that exposure of the coreceptor binding site alone is not sufficient for induction of broadly reactive NA. Immunofluorescence studies on live HIV-infected cells have shown limited accessibility of the coreceptor binding site during fusion (8
). In contrast, the fusion protein CD4-17b Fab is a potent bivalent inhibitor of most HIV-1 primary isolates (7
). Clearly envelope constructs more native than A32-rgp120 monomers, such as A32-bound gp140 or gp160 trimers, need to be tested as immunogens.
DeVico et al. have shown that expression of CD4-induced neutralizing determinants defined by MAbs raised against CD4-rgp120 complexes vary in their expression on native gp120s, being constitutively expressed on some gp120s and less so on others (5
). In this regard, rgp12089.6
had constitutive low-level expression of MAb 17b (7
) binding that increased after MAb A32 binding while rgp120BaL
had no constitutive MAb 17b binding. However, rgp120BaL
induced antibodies that neutralized 64% (9 of 14) of subtype B isolates tested while rgp12089.6
was less effective, which could be due to constitutive expression of as-yet undefined conserved neutralizing determinants on rgp120BaL
. These latter data give credence to the strategy of testing multiple HIV-1 primary isolate Envs to select a best Env or best polyvalent Envs for induction of broadly reactive NA.
Generally, most analyses of immune sera from animals and humans have shown little neutralizing activity for HIV-1 primary isolates (13
). However, recent analysis of immune sera raised against either polyvalent gp120 (4
), gp120 subunit peptides (15
), or oligomeric gp160 (25
) have demonstrated the ability to induce antibodies that neutralized select HIV-1 primary isolates. Clearly, the majority of HIV-1 primary isolates neutralized by anti-gp120 immune sera in our study are more easily neutralizable than others, and the breadth of neutralization elicited by the best immunogen, rgp120BaL
, is not sufficient to anticipate widespread utility as a vaccine. It is important to note that AT-2-inactivated HIV-1 virions (21
) have been grown in vitro in T-cell lines and are readily neutralized in syncytium from without inhibition assays. Thus, the syncytium inhibition assay used in our study is useful to demonstrate the presence of inhibiting activities, but the assay appears to be more sensitive than the luciferase-base multiple-round infection assay, the single-round infection cytoplasmic p24 staining assay, and the tissue culture supernatant p24 production in PBMC assay.
Fouts et al. also compared CD4-rgp120IIIB
complexes and found no difference in immunogenicity (9
). However, we have recently shown in a direct comparison of rgp120ADA
and purified trimeric rgp120ADA
as immunogens, that trimeric rgp140ADA
was superior to monomeric rgp120 for NA induction (M. Kim, H.-X. Liao, D. Montefiori, E. Reinherz, and B. Haynes, unpublished observations). We have begun to express and purify rgp140BaL
trimers for comparison in immunogenicity studies with rgp120BaL
as well to determine if A32-rgp140 trimer complexes have an enhanced capacity for inducing NA over uncomplexed rgp140 trimers.
A critical issue for us in this study was to determine the role that adjuvants might play in the ability of conformational epitopes on constrained envelope complexes to be immunogenic. While not an exhaustive study, we found no significant difference in the breadth of NA induced with oil (mineral oil, squalene) versus aqueous (CT and LPS) adjuvants. It was important to rule out that the adjuvant formulation was not preventing immunogenicity. Our data suggest that induction of broadly reactive NA was not prevented because of adjuvant interference with immunogenicity. However, in this regard, Van Cott et al. have shown that mineral oil adjuvants such as CFA and IFA can induce decreased recognition of conformational epitopes to some degree on rgp140 oligomers (25
). This issue will remain a concern in future studies of other immunogens, such as Env trimers.
Finally, it is critical to note that each incremental improvement in induction of breadth of NA by experimental immunogens is important. The rgp120 human Phase III trial was not successful, suggesting that rgp120s may not be viable vaccine candidates in and of themselves (12
). However, that rgp120BaL
alone can induce antibodies that neutralize ~60% of subtype B HIV-1 isolates provides a beach head from which to work to improve the immunogenicity. Clearly, most HIV-1 isolates neutralized by anti-rgp120BaL
sera were the isolates known to be easily neutralized (e.g., IIIB, BaL, SF162, 89.6), while the more difficult to neutralize HIV isolates (e.g., BG11681, 6101, US1) were not neutralized. Thus, rgp120BaL
in and of itself is not likely to solve the HIV-1 vaccine NA immunogen problem, but as a present best Env it can serve as a starting point for design of more native Env immunogens. These strategies include formulation of trimeric rgp140BaL
immunogens and production of constrained gp140 trimer complexes. In this regard, Fouts et al. have described the production of a single-chain polypeptide analogue of the CD4-rgp120 complex utilizing the gene for rgp120BaL
Taken together, our data suggest that HIV-1 BaL rgp120 is a promising R5 envelope that can serve as a starting point for development of an HIV-1 immunogen for induction of broadly reactive NA against subtype B HIV-1 primary isolates.