Hum. and we investigated the molecular details of those binding interactions. The IgG versions of PG9 and PG16 recognize the soluble trimeric gp140 form less efficiently than the corresponding monomeric gp140 form. In contrast, the Fab versions of PG9 and PG16 acknowledged the monomeric and trimeric gp140 forms with identical binding kinetics and with binding affinities similar to the high binding affinity of the anti-V3 antibody 447D to its epitope. Our data also indicate that, depending on the Env backbone, the interactions of PG9 and PG16 with gp140 may be facilitated by the presence of the gp41 ectodomain and are independent of the proper enzymatic cleavage of gp140 into gp120 and gp41. The identification of soluble Env proteins that express the PG9 and PG16 epitopes and the detailed characterization of the molecular interactions between these two antibodies and their ligands provide important and novel information that will assist in improving the engineering of future Env immunogens. INTRODUCTION It is currently widely accepted that an effective vaccine against human immunodeficiency computer virus (HIV) must elicit broad antiviral neutralizing-antibody (NAb) responses: antibodies that can prevent contamination by diverse circulating primary HIV-1 isolates (31, 37). Such broad anti-HIV neutralizing-antibody responses have not AR-M 1000390 hydrochloride yet been achieved by immunization (1, 3, 8, 11, AR-M 1000390 hydrochloride 13, 17, 21, 25, 26, 35, 40, 42, 58, 63, 66). Initially, it was thought that such antiviral responses are extremely rare, even in the context of natural HIV-1 contamination, and therefore, their elicitation by vaccination would be extremely difficult, if not impossible. However, recent evidence suggests that approximately a third of those infected with HIV-1 develop broad and potent neutralizing-antibody responses (16, 20, 50, 53, 60). Such responses typically develop within the first 2 to 3 3 years of contamination and as early as the first 12 months of contamination (39). The neutralizing-antibody response against HIV-1 exclusively targets the viral envelope glycoprotein (Env), which is the only virus-encoded protein around the surfaces of viral particles. Env is initially expressed as a AR-M 1000390 hydrochloride 160-kDa precursor protein (gp160), which is usually cleaved posttranslationally into two noncovalently associated subunits: the extracellular IRA1 subunit, gp120, and the transmembrane subunit, gp41. This cleavage is performed by furin-like cellular proteases. Around the surfaces of infectious virions, the functional Env is expressed as a trimer of gp120-gp41 heterodimers. gp120 is responsible for binding to the CD4 and CCR5/CXCR4 cell surface proteins, while gp41 mediates fusion of the virion and host cell membranes. The earliest Env-based immunogens that aimed at the elicitation of anti-HIV NAbs were based on the gp120 subunit alone and derivatives of that protein (2, 4, 22, 26C28, 33, 38, 41, 57, 59). This type of immunogen elicited binding antibodies that were largely nonneutralizing or primarily displayed neutralizing activity against the computer virus from which the Env immunogen was derived, but not heterologous primary isolates. The elicitation of nonneutralizing antibodies by such immunogens is due to the exposure on soluble gp120 immunogens of epitopes that are normally occluded within the functional HIV Env trimer. The elicitation of strain-specific NAbs is due primarily to the natural immunodominance of variable epitopes (i.e., epitopes that are not conserved among diverse HIV isolates) on soluble gp120 proteins but also to improper presentation of more conserved neutralization epitopes (i.e., epitopes that are present on diverse isolates) (see reviews in recommendations 32 and 45). Second-generation soluble HIV Env immunogens were based on the entire extracellular a part of gp160, i.e., not only the gp120 subunit, but also the extracellular part of the gp41 subunit. These constructs are commonly referred to as gp140s and can be trimeric. Soluble trimeric gp140 Env immunogens were shown by several groups to elicit antibody responses with broader neutralizing activities than those elicited by soluble monomeric gp120 immunogens (1, 13, 66). Overall, however, gp140s elicit NAbs with much narrower breadth than those we wish to elicit by vaccination and the antibodies generated by approximately a third of those infected with HIV-1 (16, 20, 50, 53, 60). It was hoped that this trimeric nature of soluble gp140 constructs would better present epitopes that are also present around the virion-associated gp160 Env trimer. However, even in the context of soluble gp140 trimers, the presentation of conserved neutralization epitopes differs from that around the native virion-associated gp160 Env trimers. This is particularly true for gp140 constructs on which the gp120-gp41 cleavage site was.