Two classes of broadly neutralizing antibodies within a single lineage directed to the high-mannose patch of HIV envelope

Abstract

The high-mannose patch of human immunodeficiency virus (HIV) envelope (Env) elicits broadly neutralizing antibodies (bnAbs) during natural infection relatively frequently, and consequently, this region has become a major target of vaccine design. However, it has also become clear that antibody recognition of the region is complex due, at least in part, to variability in neighboring loops and glycans critical to the epitopes. bnAbs against this region have some shared features and some distinguishing features that are crucial to understand in order to design optimal immunogens that can induce different classes of bnAbs against this region. Here, we compare two branches of a single antibody lineage, in which all members recognize the high-mannose patch. One branch (prototype bnAb PGT128) has a 6-amino-acid insertion in CDRH2 that is crucial for broad neutralization. Antibodies in this branch appear to favor a glycan site at N332 on gp120, and somatic hypermutation is required to accommodate the neighboring V1 loop glycans and glycan heterogeneity. The other branch (prototype bnAb PGT130) lacks the CDRH2 insertion. Antibodies in this branch are noticeably effective at neutralizing viruses with an alternate N334 glycan site but are less able to accommodate glycan heterogeneity. We identify a new somatic variant within this branch that is predominantly dependent on N334. The crystal structure of PGT130 offers insight into differences from PGT128. We conclude that different immunogens may be required to elicit bnAbs that have the optimal characteristics of the two branches of the lineage described.

Importance: Development of an HIV vaccine is of vital importance for prevention of new infections, and it is thought that elicitation of HIV bnAbs will be an important component of an effective vaccine. Increasingly, bnAbs that bind to the cluster of high-mannose glycans on the HIV envelope glycoprotein, gp120, are being highlighted as important templates for vaccine design. In particular, bnAbs from IAVI donor 36 (PGT125 to PGT131) have been shown to be extremely broad and potent. Combination of these bnAbs enhanced neutralization breadth considerably, suggesting that an optimal immunogen should elicit several antibodies from this family. Here we study the evolution of this antibody family to inform immunogen design. We identify two classes of bnAbs that differ in their recognition of the high-mannose patch and show that different immunogens may be required to elicit these different classes.

FIG 1

Importance of indels for neutralization breadth and potency by bnAbs from donor 36. Neutralization breadth and potency of mutated and chimeric antibodies were determined on a 6-pseudovirus panel previously shown to be predictive of breadth and potency on a larger panel (21). (A) PGT128, (B) PGT128+del (5-residue CDRL1 deletion restored), (C) PGT128-insert (6-residue CDRH2 insertion removed), (D) PGT128-insert+del (6-residue CDRH2 insert removed and 5-residue CDRL1 deletion restored), (E) PGT130, and (F) PGT130+insert (PGT128 6-residue CDRH2 insert introduced into PGT130 CDRH2 and G32C permitting disulfide formation).

FIG 2

Neutralization breadth and potency of PGT125 to PGT131 variants identified by next-generation sequencing and phylogenetic analysis. (A) Simplified antibody tree representing analysis of next-generation sequencing data using the ImmuniTree algorithm (for full analysis, see Fig. S1a and S1b in the supplemental material). The mutation level is listed for heavy (H) and light (L) chains individually and based on nucleotide changes from the germ line (gl) over the V and J segments. (B) Percent breadth and potency on the 6-virus panel of predicted precursor bnAbs of PGT128 and PGT130 and a new somatic variant (9H 46L). Values are reported as IC₅₀ (antibody concentrations required to inhibit HIV activity by 50%) and measured in micrograms per milliliter. The cells in the table are colored as follows; red, IC₅₀ of <0.01 μg/ml; orange, IC₅₀ of 0.01 to 0.1 μg/ml; yellow, IC₅₀ of 0.1 to 1 μg/ml; green, IC₅₀ of >1 μg/ml.

FIG 3

Precursor bnAbs show distinct patterns of N332 and N334 neutralization. (A) Overall percent breadth and potency of precursor bnAbs against a cross-clade panel of 54 pseudoviruses. (B) Percent breadth and potency of precursor bnAbs separated for N332 and N334 viruses. (C) Cumulative frequency distribution of IC₅₀s by precursor bnAbs compared to fully mutated bnAbs. Antibodies in the PGT128 and PGT130 branch are shown in black and green, respectively. (D) Ability of predicted precursor bnAbs to neutralize N332A/N334A and N334-shifted mutant viruses. The cells in the tables in panel D are IC₅₀ values and are colored as follows: red, <0.01 μg/ml; orange, 0.01 to 0.1 μg/ml; yellow, 0.1 to 1 μg/ml; green, >1 μg/ml.

FIG 4

bnAbs in donor 36 evolved to avoid V1 loop glycans in some virus strains. V1 loop glycans were removed by site-directed mutagenesis, and the effect on neutralization was measured. Wild-type (WT) viruses are shown as solid lines, and glycan mutant viruses are shown as dashed lines. (A) JRCSF and N135A variant, (B) SF162 and N136A variant, (C) 92RW020 and N131A variant, (D) JR-FL and N134A variant, (E) BG505 N332 and N137A variant, (F) BG505 and N137A variant, (G) 92TH021 and N151A variant, (H) CNE8 and N141A and N149A variants.

FIG 5

Increased somatic hypermutation of donor 36 bnAbs allows better accommodation of viral gp120 glycan heterogeneity. BJOX015000.11.5 pseudoviruses were prepared in the presence of glycosidase inhibitors kifunensine (Kif) (endoplasmic reticulum [ER]-mannosidase I inhibitor), swainsonine (Golgi-α-mannosidase II inhibitor) and in a GnT1-deficient cell line (HEK-293S), and the effect on neutralization was determined for 74H 3L (A), PGT130 (B), 95H 71L (C), PGT128 (D), and 9H 46L (E).

FIG 6

Crystal structure of PGT130 Fab highlights differing modes of binding by the antibody classes. (A) The crystal structure of the PGT130 Fab variable region is represented as a gray ribbon. The CDR loops are individually labeled and colored, with small red circles indicating parts of the CDR H3 loop that are disordered in the structure. The two N-linked glycosylation sites are also labeled and shown in green ball-and-stick representations. To the right, the combining site of PGT130 Fab is displayed in more detail. Residues that are important for binding gp120, as determined by alanine-scanning mutagenesis, are labeled, and their side chains are shown as ball-and-sticks. The side chain of Trp^H100E is not shown because it is disordered in the crystal structure. (B) The crystal structures of the PGT130 (gray ribbon) and PGT128 (thin blue ribbon) (PDB ID or accession no. 3TV3) variable regions are shown superposed. CDR loops with significant differences between the structures are labeled. (C) The CDR loops of PGT128 are shown as individual ribbons at its extended epitope on the SOSIP trimer extending across portions of the V3 loop (yellow surface) and the V1 loop (gray surface). The V1 component of the epitope is modeled here based on superposing the PGT128-gp120 outer domain structure (PDB ID 3TYG) on the BG505 SOSIP trimer structure (PDB ID 4NCO) only on gp120. Glycans are shown as colored spheres. Insertions/deletions on the CDR loops are colored red. (D) The glycan shift from position N332 to N334 is modeled in relation to the PGT128 CDR H2 loop colored as in panel C. (E) Alanine-scanning mutagenesis of the PGT130 paratope. Amino acids at positions known to be important for PGT128 neutralization were mutated to alanine in PGT130, and the effect on neutralization of JR-FL and JR-CSF was determined. The fold increase in the neutralization IC₅₀ compared to WT IgG is depicted in the table with different colors as follows: green, 3- to 5-fold; yellow, 6- to 20-fold; orange, 21- to 100-fold; red, >100-fold. The residues mutated in PGT130 and PGT128 are shown in red and black in the variant column, respectively. N/A, not applicable, as that particular residue not present at that position in the somatic variant; n.d., not determined.