ABSTRACT
Protein minimization is an attractive approach for designing vaccines against rapidly evolving pathogens such as human immunodeficiency virus, type 1 (HIV-1), because it can help in focusing the immune response toward conserved conformational epitopes present on complex targets. The outer domain (OD) of HIV-1 gp120 contains epitopes for a large number of neutralizing antibodies and therefore is a primary target for structure-based vaccine design. We have previously designed a bacterially expressed outer-domain immunogen (ODEC) that bound CD4-binding site (CD4bs) ligands with 3-12 µm affinity and elicited a modest neutralizing antibody response in rabbits. In this study, we have optimized ODEC using consensus sequence design, cyclic permutation, and structure-guided mutations to generate a number of variants with improved yields, biophysical properties, stabilities, and affinities (KD of 10-50 nm) for various CD4bs targeting broadly neutralizing antibodies, including the germline-reverted version of the broadly neutralizing antibody VRC01. In contrast to ODEC, the optimized immunogens elicited high anti-gp120 titers in rabbits as early as 6 weeks post-immunization, before any gp120 boost was given. Following two gp120 boosts, sera collected at week 22 showed cross-clade neutralization of tier 1 HIV-1 viruses. Using a number of different prime/boost combinations, we have identified a cyclically permuted OD fragment as the best priming immunogen, and a trimeric, cyclically permuted gp120 as the most suitable boosting molecule among the tested immunogens. This study also provides insights into some of the biophysical correlates of improved immunogenicity.
Subject(s)
AIDS Vaccines/immunology , CD4 Antigens/immunology , HIV Envelope Protein gp120/chemistry , HIV Infections/immunology , HIV-1/chemistry , AIDS Vaccines/chemistry , AIDS Vaccines/therapeutic use , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Binding Sites , Broadly Neutralizing Antibodies , CD4 Antigens/chemistry , Crystallography, X-Ray , Epitopes/chemistry , Epitopes/immunology , HIV Antibodies/chemistry , HIV Antibodies/immunology , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , HIV Infections/genetics , HIV-1/immunology , HIV-1/pathogenicity , Humans , Ligands , Protein Binding , RabbitsABSTRACT
The gp120 subunit of the HIV-1 envelope (Env) protein is heavily glycosylated at â¼25 glycosylation sites, of which â¼7-8 are located in the V1/V2 and V3 variable loops and the others in the remaining core gp120 region. Glycans partially shield Env from recognition by the host immune system and also are believed to be indispensable for proper folding of gp120 and for viral infectivity. Previous attempts to alter glycosylation sites in Env typically involved mutating the glycosylated asparagine residues to structurally similar glutamines or alanines. Here, we confirmed that such mutations at multiple glycosylation sites greatly diminish viral infectivity and result in significantly reduced binding to both neutralizing and non-neutralizing antibodies. Therefore, using an alternative approach, we combined evolutionary information with structure-guided design and yeast surface display to produce properly cleaved HIV-1 Env variants that lack all 15 core gp120 glycans, yet retain conformational integrity and multiple-cycle viral infectivity and bind to several broadly neutralizing antibodies (bNAbs), including trimer-specific antibodies and a germline-reverted version of the bNAb VRC01. Our observations demonstrate that core gp120 glycans are not essential for folding, and hence their likely primary role is enabling immune evasion. We also show that our glycan removal approach is not strain restricted. Glycan-deficient Env derivatives can be used as priming immunogens because they should engage and activate a more divergent set of germlines than fully glycosylated Env. In conclusion, these results clarify the role of core gp120 glycosylation and illustrate a general method for designing glycan-free folded protein derivatives.
Subject(s)
HIV Envelope Protein gp120/metabolism , HIV-1/metabolism , Immune Evasion , Models, Molecular , Protein Processing, Post-Translational , Amino Acid Substitution , Antibodies, Neutralizing/metabolism , Antibodies, Viral , Antibody Specificity , Asparagine/metabolism , Glycosylation , HIV Envelope Protein gp120/antagonists & inhibitors , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp41/antagonists & inhibitors , HIV Envelope Protein gp41/chemistry , HIV Envelope Protein gp41/metabolism , HIV-1/immunology , HIV-1/pathogenicity , Humans , Mutagenesis, Site-Directed , Mutation , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Conformation , Protein Engineering , Protein Folding , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
Zinc-α2-glycoprotein (ZAG) is an adipokine with an MHC class I-like protein fold. Even though zinc causes ZAG to precipitate from plasma during protein purification, no zinc binding has been identified to date. Using mass spectrometry, we demonstrated that ZAG contains one strongly bound zinc ion, predicted to lie close to the α1 and α2 helical groove. UV, CD and fluorescence spectroscopies detected weak zinc binding to holo-ZAG, which can bind up to 15 zinc ions. Zinc binding to 11-(dansylamino) undecanoic acid was enhanced by holo-ZAG. Zinc binding may be important for ZAG binding to fatty acids and the ß-adrenergic receptor.