These include the substitution I559P, introduction of the N-glycosylation site at N332 (in Env from viral strain BG505 only, as the other strains we used already contain Asn at position 332), as well as the disulfide-forming substitutions A501C and T605C. depend on Env conformation and N-linked glycan. Based on antibody recognition and sensitivity to glycosidases, yeast glycosylation patterns partially mimic high mannose-type N-glycosylation in mammalian cells. However, yeast-displayed Env is not recognized by some anti-Env antibodies sensitive to quaternary structure, suggesting either that Citraconic acid the displayed protein exists in a monomeric state or that for these antibodies, yeast glycosylation in certain regions hinders recognition or access. Consistent with studies in other systems, reconstructed predicted unmutated precursors to anti-Env antibodies exhibit little affinity for the yeast-displayed envelope protein. == Introduction == Despite the success of anti-retroviral drugs in treating HIV-infected individuals and slowing the spread of infection, these drugs remain unaffordable by much of the world and are subject to side effects and development of resistance. The use of a vaccine against HIV is generally viewed as the most effective long term approach for controlling or eradicating the worldwide AIDS pandemic [1]. To date, Citraconic acid however, intensive efforts to develop an effective AIDS vaccine (more than 200 clinical trials [2]) have failed to yield an effective immunogen, aside from a single trial showing only a modest protective effect [3]. The potential feasibility of effective vaccination is suggested by the observation that some infected individuals develop antibodies that can neutralize a broad array of virus strainsin vitroand can prevent virus transmission in animal models. In addition, an attenuated form of the related simian SIV virus provides protection against infection [4] and passive immunization using broadly neutralizing antibodies (bnAbs) derived from infected humans is now an active area of research [5]. All known broadly neutralizing antibody responses against HIV are targeted to the viral envelope glycoprotein (Env) that is initially translated as a 160 kDa product (gp160), then cleaved by cellular furin proteases into two chains, the gp120 and gp41 glycoproteins. The difficulty of developing a protective vaccine appears to arise, in part, from the high rate of mutagenesis of the HIV virus, the masking function of the viral glycan shield, the sequestered nature of conserved structures involved in cell binding and entry, and the very low affinity of germline antibodies for neutralizing epitopes on Env. In view of these factors, there has been intensive investigation of the sequences and structural features mediating interactions between Env and known neutralizing antibodies, resulting in identification of several targets of bnAbs. These include the CD4 receptor binding site [6,7], certain glycans on the Env surface [8], sites on gp41 that are likely to be involved in membrane fusion with target cells [9], and sites on the V2 and V3 loops [10] Rabbit Polyclonal to DP-1 and V1 and V2 loops [11] of gp120 that appear to depend on a particular quaternary structure of the envelope protein. In cases where it has been possible to identify germline precursors of broadly neutralizing antibodies, the precursors display little [12] or no [1315] affinity for Env. This has led to an interest in designing immunogens that more efficiently engage germline B cell receptors. However, the current level of structural characterization of Env-antibody complexes and the existing capabilities of procedures for rational protein engineering, may not be sufficient to allow design of an immunogen that can elicit the desired protective responses. An alternative (or complement) to the rational design of more effective immunogens is the use of random mutagenic or gene shuffling approaches followed by screening for variant forms of Env with a desired set of antigenic/immunogenic properties. This approach has previously been applied to in several systems: 1) Random libraries of short peptides comprising potential epitopes from Env have been expressed in bacterial cells and displayed on phage particles [16,17]; 2) Mutated versions of a scaffolded form of gp120 lacking variable loop regions have been displayed on the surface of yeast cells in order to identify variant forms with enhanced binding to precursor forms of anti-CD4 binding site antibodies [1821]; 3) Mutated forms of gp140 have been displayed on yeast cells in a screen for optimized expression, stability, and binding of mature anti-CD4 binding site antibodies [22]; and 4) Variant forms of gp120 have been expressed in cultured mammalian cells [23] and used to screen for variants with enhanced binding to Citraconic acid precursor forms of anti-variable loop bnAbs [24]. Previous screening in the yeast display system for variant forms of Env with enhanced affinity for bnAb precursors used scaffolded versions of HIV Env lacking variable loops. While these efforts.