Crystal Structure of PG16 and Chimeric Dissection with Somatically Related PG9: Structure-Function Analysis of Two Quaternary-Specific Antibodies That Effectively Neutralize HIV-1

Abstract

HIV-1 resists neutralization by most antibodies. Two somatically related human antibodies, PG9 and PG16, however, each neutralize 70 to 80% of circulating HIV-1 isolates. Here we present the structure of the antigen-binding fragment of PG16 in monoclinic and orthorhombic lattices at 2.4 and 4.0 Å, respectively, and use a combination of structural analysis, paratope dissection, and neutralization assessment to determine the functional relevance of three unusual PG9/PG16 features: N-linked glycosylation, extensive affinity maturation, and a heavy chain-third complementarity-determining region (CDR H3) that is one of the longest observed in human antibodies. Glycosylation extended off the side of the light chain variable domain and was not required for neutralization. The CDR H3 formed an axe-shaped subdomain, which comprised 42% of the CDR surface, with the axe head looming ϳ20 Å above the other combining loops. Comprehensive sets of chimeric swaps between PG9 and PG16 of light chain, heavy chain, and CDR H3 were employed to decipher structure-function relationships. Chimeric swaps generally complemented functionally, with differences in PG9/PG16 neutralization related primarily to residue differences in CDR H3. Meanwhile, chimeric reversions to genomic V genes showed isolate-dependent effects, with affinity maturation playing a significant role in augmenting neutralization breadth (P ‫؍‬ 0.036) and potency (P < 0.0001). The structural and functional details of extraordinary CDR H3 and extensive affinity maturation provide insights into the neutralization mechanism of and the elicitation pathway for broadly neutralizing antibodies like PG9 and PG16.To create antibodies capable of effectively neutralizing human immunodeficiency virus type 1 (HIV-1), the adaptive humoral response is driven to exceptional lengths (reviewed in reference 8). Indeed, the response often fails, and sera from individuals infected with HIV-1 typically display limited neutralization breadth (59). After several years of infection, however, antibodies capable of neutralizing diverse viral strains develop in 15 to 25% of infected individuals (3,16,32,33,49,53). Details of the adaptive changes that allow for effective recognition are of direct vaccine relevance, and clues from rare neutralizing antibodies have been eagerly sought.Two broadly neutralizing antibodies, PG9 and PG16, were recently identified with single cell-sequencing techniques after direct microneutralization assessment of secreted antibody from individually plated, stimulated B cells (58). These antibodies are somatically related and appear to be derived from the same recombination of heavy and light chains. They both recognize a site on HIV-1 gp120 composed of elements from the second and third variable regions (V2 and V3). Despite the vaunted diversity of the HIV-1 gp120 envelope and the even higher sequence variability in the V2 and V3 regions (26), neutralization assays indicate that the recognized epitope is conserved in 70 to 80% of circulating viral isolates (58).To inves...

Broad Neutralizing Antibodies to HIV Env and Other Complex Viral Antigens from Vaccinated Cows

“…The substitution of aromatic residues, mainly Trp or tyrosine (Tyr), in MPER binding antibodies reduces the neutralization activity of these antibodies [74,84]. Also, the importance of Cys and aromatic residues such as Trp is shown in neutralization activity of CD4bs BrNAbs antibodies [85,86], highlighting the significant function of these residues in either directly in the antigen binding interaction or involvement in shaping the required structure for epitope binding.…”

“…These CDRH3 regions help the antibodies to penetrate the glycan shield of Env trimer then interact with the V1/V2 and/or V3 region of gp120 [73]. Also, CDRH3 is an important component in gp41-reactive antibodies such as 2F5 (CDRH3 of 22 residues) and 4E10 (CDRH3 of 18 residues) which performs the additional activity causing a hydrophobic interaction with the membrane [74][75][76][77][78][79][80] and forming a loop to contact the highly conserved hydrophobic residues on gp41 [52,[81][82][83]. …”

Point Mutations Associated with HIV-1 Drug Resistance, Evasion of the Immune Response and AIDS Pathogenesis

“…(Corti et al, 2010;Euler et al, 2011;Ho et al, 1991;Y. Li et al, 2011;Pancera et al, 2010;Posner et al, 1991;Walker et al, 2009; Glycans 2G12…”

An Introduction to the Current State of HIV Vaccine Research

“…the conserved CD4-binding site [10], the CD4-induced antigenic site that becomes accessible after Env interacts with the CD4 receptor [11], the semi-conserved V3 loop [12], the membrane-proximal external region of the gp41 protein [13] and the glycan antigenic site [14]. In subsequent years, many additional bnMabs directed to Env epitopes were isolated from HIV-1 infected persons [15][16][17] and had their structures elucidated by X-ray crystallography [18][19][20][21][22]. These bnMabs were then used as templates to reconstruct one of the epitopes that such Mabs are able to recognize using structure-based design technology.…”