Design of immunogens that present the crown of the HIV-1 V3 loop in a conformation competent to generate 447-52D-like antibodies

Abstract

gp120 is a subunit of the envelope glycoprotein of HIV-1. The third variable loop region of gp120 (V3 loop) contains multiple immunodominant epitopes and is also functionally important for deciding cell-tropism of the virus. 447-52D is a monoclonal antibody that recognizes the conserved tip of the V3 loop in a beta-turn conformation. This antibody has previously been shown to neutralize diverse strains of the virus. In an attempt to generate an immunogen competent to generate 447-52D-like antibodies, the known epitope of 447-52D was inserted at three different surface loop locations in the small, stable protein Escherichia coli Trx (thioredoxin). At one of the three locations (between residues 74 and 75), the insertion was tolerated, the resulting protein was stable and soluble, and bound 447-52D with an affinity similar to that of intact gp120. Upon immunization, the V3 peptide-inserted Trx scaffold was able to generate anti-V3 antibodies that could compete out 447-52D binding to gp120. Epitope mapping studies demonstrated that these anti-V3 antibodies recognized the same epitope as 447-52D. Although the 447-52D-type antibodies were estimated to be present at concentrations of 50-400 microg/ml of serum, these were not able to effect neutralization of strains like JRFL and BAL but could neutralize the sensitive MN strain. The data suggest that because of the low accessibility of the V3 loop on primary isolates such as JRFL, it will be difficult to elicit a V3-specific, 447-52D-like antibody response to effectively neutralize such isolates.

Engineering, Expression, Purification, and Characterization of Stable Clade A/B Recombinant Soluble Heterotrimeric gp140 Proteins

“…glycosylation sites either to occlude immunodominant but variable regions or to enhance the exposure of conserved neutralization epitopes, respectively (10,17,32,50,64,66); repositioning of conserved epitopes within more variable, but also more immunogenic, regions of the HIV Env (48, 51); design of non-HIV Env scaffold proteins that express epitopes of broadly neutralizing antibodies (11,18,19,39,62,63); molecular evolution-based approaches (24); generation of ancestral or consensus Env sequences (23,31,45,46,52); and others. Thus far, all of these approaches have had a very limited success in the elicitation of broad and potent neutralizing antibody responses against tier 2 HIV-1 viruses.…”

An oligosaccharide-based HIV-1 2G12 mimotope vaccine induces carbohydrate-specific antibodies that fail to neutralize HIV-1 virions

“…A more plausible cause is that conformational f lexibility in the oligomannan arms is still high, and the immune response is ''diluted'' by recognition of improper structures within the chemical space. This complicating factor has been proposed as a reason for the inability to mimic the broad neutralizing characteristics of other HIV-1-directed mAbs (4,10,33).…”

“…A number of rarely occurring human mAbs, isolated from infected individuals, have demonstrated this desirable neutralization profile (1)(2)(3). For several of these mAbs, identification and extensive characterization of the neutralizing epitopes have been realized (4)(5)(6)(7). This structural knowledge has been used to design antigens intended to direct the primary immune response toward the neutralizing epitope (8)(9)(10).…”

Modification of One Epitope-Flanking Amino Acid Allows for the Induction of Friend Retrovirus-Specific CD8+T Cells by Adenovirus-Based Immunization

“…Also in this construct, the epitope-flanking amino acid is modified compared to the native sequence (C ϩ 1: R), evidently allowing for efficient processing. Thioredoxin has been ascribed immunostimulatory properties (5,6), and bacterial Txn has been used before as a peptide carrier for antibody induction (8,24); the high frequency of GagL 85-93 -specific CD8 ϩ T cells in Ad.TxnGagL-im- munized mice indicates that its immunogenicity may be beneficial for T cell induction as well.…”