To further clarify the identities of the oligomeric forms, 5 g of purified unfractionated or fractionated gp145 was cross linked with 5 mM ethylene glycol bis(succinimidylsuccinate) (EGS) and resolved by SDS-PAGE (36). and larger multimers elicited similar levels of GF1 cross-subtype binding and neutralizing Heptasaccharide Glc4Xyl3 antibodies to tier 1 and some tier 2 viruses. Immunized rabbit sera did not neutralize the highly resistant CO6980v0c22 pseudovirus but did inhibit the homologous infectious molecular clone in a peripheral blood mononuclear cell (PBMC) assay. This Env is currently in good manufacturing practice (GMP) production to be made available for use as a clinical research tool and further evaluation as a candidate vaccine. IMPORTANCE At present, the product pipeline for HIV vaccines is insufficient and is limited by inadequate capacity to produce large quantities of vaccine to standards required for human clinical trials. Such products are required to evaluate critical questions of vaccine formulation, route, dosing, and schedule, as well as to establish vaccine efficacy. The gp145 Env protein presented in this study forms physical trimers, binds to many of the well-characterized broad neutralizing MAbs that target conserved Env epitopes, and induce cross-subtype neutralizing antibodies as measured in both cell line and primary cell assays. This subtype C Env gp145 protein is currently undergoing good manufacturing practice production for use as a reagent for preclinical studies and Heptasaccharide Glc4Xyl3 for human clinical research. This product will serve as a reagent for comparative studies and may represent a next-generation candidate HIV immunogen. INTRODUCTION Human immunodeficiency virus (HIV) vaccine candidates capable of eliciting broad, protective humoral immune responses would significantly advance prevention strategies to control the AIDS pandemic. The diversity of circulating HIV type 1 (HIV-1) envelope (Env) sequences and structures has complicated HIV-1 immunogen design and has impaired the development of a globally efficacious vaccine. Env diversity of up to 20% within a subtype and up to 35% between subtypes has been reported (1), leading vaccine developers to focus on common antigenic features most frequently represented on infectious viral particles. The role of the subtype in cross-protective responses has long been a subject of debate, but numerous studies have indicated that the HIV-1 subtype can play a role in the nature of the functional antibody responses elicited by both vaccination and infection (2,C4). Subtype C accounts for >50% of the current global infections (5, 6) and for the majority of incident cases in southern Africa. Subtype C has also been shown to exhibit greater kinetics, magnitude, and breadth of neutralizing antibody (NAb) development than other subtypes during the course of natural infection (3, 7, 8). Early NAb responses in humans to subtype C infection are predominantly strain specific, targeting the V1V2 and C3 regions of gp120, and can be quite potent (8,C11). Subtype C Env proteins from the acute infection phase also appear to have certain genetic features in Heptasaccharide Glc4Xyl3 common, such as shorter gp120 variable regions and fewer potential N-linked glycosylation sites, indicating selective pressure at transmission, where infection may be established by a single infectious particle (3, 5, 6, 12, 13). Subtype C envelopes derived from the acute infection phase are therefore of considerable interest for vaccine design. The development of broadly reactive NAbs occurs after 2 to 3 3 years in only 10 to 30% of infected individuals (14,C18) and can be mediated by a single or a small number of antibody specificities (19). Some of these broadly reactive antibodies target quaternary neutralizing epitopes (QNEs) that exist in the context of trimeric Env (10). Monoclonal antibodies (MAbs) that show preferential binding to the Env quaternary structure, such as the PG9, PG16, PGT141-145, and CH01-04 MAbs, recognize epitopes and glycosylation motifs in V1, V2, and V3 (20,C22) and have particularly potent antiviral activity. Animal studies have demonstrated that protection is achieved with relatively low titers of HIV-specific antibodies if the antibodies are targeted to the native Env trimer on the virion surface (14, 23). NAbs targeting QNEs, if elicited via vaccination, may prevent infection in humans if present at sufficient titers prior to exposure to HIV. The development of HIV Env trimers to elicit these functional antibodies.