However, we were able to obtain the highly purified CHO expressed MN-rgp120 immunogen (MNGNE) that was incorporated into the AIDSVAXB/E vaccine of the RV144 trial, to use for comparison. Cell culture supernatants from transiently transfected GnTI- cells expressing MN rgp120 with glycan epitope insertions were normalized Kenpaullone for gp120 concentration and subjected to Endo H glycosidase digests to confirm size and oligomannose content (Fig 4). RV144 trial were enriched for glycans containing high levels of sialic acid, and lacked critical N-linked glycosylation sites required for binding by several families of bN-mAbs. The absence of these epitopes may have contributed to the low level of efficacy achieved in this study. In this report, we describe our efforts to improve the antigenic structure of the rgp120 immunogens used in the vaccine by optimizing glycan-dependent epitopes recognized by multiple bN-mAbs. Our results demonstrated that by shifting the location of one PNGS in A244-rgp120, and by adding two PNGS to MN-rgp120, in conjunction with the production of both proteins in a cell line that favors the incorporation of oligomannose glycans, we could significantly improve the binding by three major families of bN-mAbs. The immunogens described here represent a second generation of gp120-based vaccine immunogens that exhibit potential for use in RV144 follow-up studies. Introduction The RV144 clinical trial has been the only human clinical trial to show that vaccination can provide protection from HIV infection . The RV144 vaccination protocol consisted of immunization with the ALVAC (VCP1521) canarypox virus vector , designed to elicit a robust cell-mediated immune response, followed by co-immunization with the bivalent AIDSVAX B/E gp120 vaccine, designed to Kenpaullone elicit an anti- gp120 antibody response [3C5]. This regimen provided statistically significant protection (Vaccine Efficacy = 31.2%, P = 0.04) over 3.5 years, with up to 60% efficacy within the first year after vaccination . Follow-up analysis revealed that protection correlated with: antibodies to the V2 domain of gp120, high levels of antibody-dependent cellular cytotoxicity (ADCC) , and HIV-1 specific IgG3 antibodies , but not with gp120-specific CD8+ T-cell responses . Together, these studies indicated a role for anti-gp120 antibodies in the modest but significant level of protection afforded by the vaccine. The importance of the antibody response was further supported by additional antibody binding studies [8, 9] and sieve analysis of breakthrough viruses . Such studies associating safety with anti-gp120 antibodies offered a rationale for further development of gp120-centered immunogens. Since the completion of the RV144 TIE1 trial, we have accumulated considerable insight regarding the structure of gp120, as well as of the specificity of neutralizing antibodies against it. The isolation of bN-mAbs from HIV-infected individuals revealed highly conserved protein and glyco-peptide epitopes on gp120 that were unfamiliar when the AIDSVAX/Become vaccine was first developed. Of particular relevance was the recognition of oligomannose terminal glycans targeted by multiple families of bN-mAbs. These glycans are located at conserved N-linked glycosylation sites in the V1/V2 website (N301 and N332), near the apex of the gp120 trimer, and near the stem of the V3 website [11C21], referred to as the high mannose patch . The apparent preference of these bN-mAbs for gp120 within trimeric constructions, as compared to monomeric gp120, suggested a requirement for quaternary structure for bN-mAb binding [18, 19]. However, it is becoming apparent that variations in glycan processing and glycan convenience between monomeric and trimeric gp120 constructions, in part, can account for this preference. While trimeric gp120, the practical unit of gp120 displayed on the surface of virions, is Kenpaullone definitely enriched for oligomannose glycans, recombinant monomeric gp120 displays mainly complex, sialic acid-terminal, glycans [22, 23]. This discrepancy is at least partially explained by incomplete glycan processing in the ER and Golgi Apparatus, thought to be a consequence of steric hindrance to glycosidase enzymes during trimer formation [21, 24]. The AIDSVAXB/E immunogens were produced in a Chinese Hamster Ovary (CHO) cell collection, and consequently possessed a high degree of N-linked glycan sialylation . High sialic acid content Kenpaullone is desired for a majority of biotherapeutics, as its presence in recombinant glycoproteins is known to impart a.