This finding suggests a relative enrichment of binding specific for conformational epitopes within the cross-subtype-reactive antibody population. of neutralizing both subtype E and subtype B TCLA HIV-1 isolates. However, neither immunogen induced antibodies capable of neutralizing primary HIV-1 isolates. Antibody induced by rgp120CM235 preferentially bound natively folded gp120 and retained strong cross-reactivity against multiple gp120 strains within subtype E as well as subtype B. In contrast, antibody responses to rgp120SF2 were directed predominantly to linear epitopes poorly exposed on native gp120 and had more limited cross-recognition of divergent gp120. Fine epitope mapping revealed differences in antibody specificities. While both rgp120CM235 and rgp120SF2 DZNep induced antibodies to regions within C1, V1/V2, V3, and C5, unique responses were induced by rgp120CM235 to multiple epitopes within C2 and by rgp120SF2 to multiple epitopes within C3, V4, and C4. These data demonstrate that strain and/or phenotypic differences of HIV-1 subunit gp120 immunogens can substantially alter antibody binding specificities and subsequent HIV-1 neutralizing capacity. Most human immunodeficiency virus type 1 (HIV-1) subunit vaccine candidates are based on genes from prototype T-cell line-adapted (TCLA) subtype B viruses. Examples are gp120 and gp160 immunogens based on HIV-1 strain IIIB, MN, or SF2. Since the HIV-1 epidemic in southeast Asia is largely caused by subtype E viruses (35, 43, 56C58), it may be important to evaluate vaccines expressing antigens from subtype E for use in this region. Subtype E HIV-1 is antigenically distinct from subtype B; sera (39, 40) and neutralizing monoclonal antibodies (MAbs) (48, 78) derived from subtype B-infected donors preferentially neutralize viruses from the same subtype, though other studies have not identified such an association between HIV-1 serum neutralization serotype and genetic subtype (29, 33). HIV-1 sera from subtype B- and E-infected individuals bind preferentially to HIV-1 gp120 and gp160 from subtypes B and E, respectively (39, 80). However, while gp120 from subtype B and subtype E may be distinct antigenically, it remains to be determined whether as immunogens they are capable of inducing cross-subtype functional immune responses. An example of discordance between HIV-1 gp120 antigenic and immunogenic properties was demonstrated by the ability of column-immobilized gp120 to remove primary isolate-neutralizing antibody activity from HIV-1 serum and its inability to elicit such antibodies in animals (70). Previous subunit HIV-1 envelope vaccines using monomeric forms of gp120 or gp160 are immunogenic in small animals, primates, and humans, but the antibody responses, though capable of neutralizing TCLA HIV-1 isolates, have limited neutralizing activity against primary HIV-1 isolates (4, 25, 30, 41, 42, 67, 85); however, recent studies using a resting cell assay obtained significant neutralization of several X4-using primary HIV-1 isolates by sera from individuals immunized with monomeric recombinant HIV-1SF2 gp120 (rgp120SF2) (10, 88). These results may be attributable to the inefficiency of these monomeric gp120 vaccines to elicit antibodies specific for conserved, discontinuous epitopes, since the majority of antibodies are focused primarily to linear epitopes poorly accessible on cell surface expressed gp120-gp41 (81). Monomeric gp120 or gp160 vaccines based on TCLA isolates, therefore, may lack structural properties critical for the ability to induce broadly reactive and neutralizing antibody. These structural properties may be related to the adaptation of the HIV-1 envelope strain, since TCLA and primary isolates have been demonstrated to have significant phenotypic differences with respect to coreceptor usage (1, 14, 15, 18) and susceptibility to antibody- or serum-mediated neutralization (2, 7, 13, 45, 63, 65). Immunization with monomeric gp120 from strains MN and SF2 protected chimpanzees against homologous and heterologous primary isolate HIV-1SF2 challenge (5, 17), and a vaccine containing rgp120SF2 protected rhesus macaques against challenge with the closely related SHIVSF13 (26). However, several individuals enrolled in clinical trials of candidate monomeric gp120 subunit vaccines became HIV-1 infected despite receiving the full vaccination regimen (12, 31, 44), indicating that these vaccines are less than 100% effective. There are several potently neutralizing MAbs which map to regions accessible on monomeric gp120 or gp41 (8, 11, 21, 23, 52, 53, 60, 75, 77, 78). The neutralizing epitopes, present on monomeric gp120, are not currently immunogenic when presented in the context of a vaccine. The majority of the broadly anti-gp120 neutralizing MAbs are directed to conformational epitopes that have been particularly difficult to elicit with monomeric HIV-1 subunit vaccines. Studies designed to DZNep correlate antibody binding and neutralizing capacity have shown poor correlation ARFIP2 with binding to monomeric gp120 and superior correlation with binding to oligomeric forms of HIV-1 envelope (19, 45, 64), though this correlation is not DZNep complete for all antibodies (20)..