(= 0) and processed sample (= 120 min) abundances of Man9GlcNAc2 (= 1,905

(= 0) and processed sample (= 120 min) abundances of Man9GlcNAc2 (= 1,905.6) and Man8GlcNAc2 (= 1,743.6) is shown for this IC/and 2 and and ?and2and ?and2B)2B) envelope glycosylation. glycans). This oligomannose (Man5C9GlcNAc2) profile is conserved across primary isolates and geographically divergent clades but is not reflected in the current generation of gp120 antigens used for vaccine trials. In the context of vaccine design, we also note that Man12Man-terminating glycans (Man6C9GlcNAc2) of the type recognized by RSV604 R enantiomer the broadly neutralizing anti-HIV antibody 2G12 are 3-fold more abundant on the native envelope than on the recombinant monomer and are also found on isolates not neutralized by 2G12. The Man12Man residues of gp120 therefore provide a vaccine target that is physically larger and antigenically more conserved than the 2G12 epitope itself. This study revises and extends our understanding of the glycan shield of HIV with implications for AIDS vaccine design. Keywords: HIV, 2G12, gp120, glycosylation, vaccine The surface antigen of HIV, gp120, is covered by an extensive array of N-linked glycans (1, 2). These host-derived carbohydrate structures comprise half the mass of gp120 and shield much of the underlying protein surface (3, 4). Although progress has been made in the structural elucidation of the protein component of gp120 (5, 6), the identity of the glycans that form the immunological shield on the native virus has not been established. A gp120 antigen is likely to be a component of any successful AIDS vaccine (2, 7C9), and this Rabbit polyclonal to Ly-6G glycoprotein has formed the basis RSV604 R enantiomer of extensive phase III clinical trials (10). However, half of this recombinant antigen is of unknown similarity to the native virus. To improve our picture of the antigenic structure of HIV, we therefore RSV604 R enantiomer sought to determine the composition of the glycan shield of gp120 as expressed on virions. The position of N-linked glycans is directly encoded within the viral genome (within the consensus sequence Asn-X-Ser/Thr-X, X Pro), whereas the type(s) of glycans found at a given Asn is determined by the biosynthetic processes of the endoplasmic reticulum (ER) and Golgi apparatus of the infected cell (11, 12) (and and is presented in 1,257.4, 1,419.5, 1,582.5, 1,743.6, and 1,905.6 ([M + Na]+ ions) corresponding to the mannose series Man5C9GlcNAc2 (Fig. 11,460.6, [M + Na]+, <2% of total N-glycans). When interpreted in the context of the biosynthetic pathway (and and 1,403.6 (Fig. 1and and and and = 0) and processed sample (= 120 min) abundances of Man9GlcNAc2 (= 1,905.5) and Man8GlcNAc2 (= 1,743.5) is shown for this ER gp120 glycoform. (= 0) and processed sample (= 120 RSV604 R enantiomer min) abundances of Man9GlcNAc2 (= 1,905.6) and Man8GlcNAc2 (= 1,743.6) is shown for this IC/and 2 and and ?and2and ?and2B)2B) envelope glycosylation. Consideration should therefore be given to the use of glycoprotein immunogens with an antigenic surface resembling that of naturally circulating virus. Materials and Methods PBMC Virus. Human PBMCs were isolated and stimulated as previously described (44). HIV-1JRCSF virus stocks were grown and titered on CD8+-depleted PBMCs (45). Virus production was monitored by p24 ELISA (Aalto Bioreagents). Pseudovirus Preparation. Pseudovirus was generated in HEK 293T or GnT I-deficient HEK 293S cells, as described (46). Briefly, cells were transfected with plasmids (pSVIII) carrying the reporter gene expressing the virus backbone PSG-3 and the functional envelope clone at a ratio of 2:1 using Fugene (Roche) according to the manufacturer’s instructions. Virus was harvested after 3 d. Swainsonine was used at a concentration of 20 M. Envelope Isolation. Virus particles were pelleted by ultracentrifugation (22,000 rpm for 1 h using Optima L-90K preparative ultracentrifuge, SW32 Ti Rotor [Beckman Coulter, Sunnyvale, CA]). Virus pellets were lysed with Nonidet P-40 (1% in PBS with protease inhibitors, 20 min at 4 C). The debris was removed by centrifugation, and the.