Anti-idiotype isolation of a broad and potent influenza A virus-neutralizing human antibody

Abstract

The VH6-1 class of antibodies includes some of the broadest and most potent antibodies that neutralize influenza A virus. Here, we elicit and isolate anti-idiotype antibodies against germline versions of VH6-1 antibodies, use these to sort human leukocytes, and isolate a new VH6-1-class member, antibody L5A7, which potently neutralized diverse group 1 and group 2 influenza A strains. While its heavy chain derived from the canonical IGHV6-1 heavy chain gene used by the class, L5A7 utilized a light chain gene, IGKV1-9, which had not been previously observed in other VH6-1-class antibodies. The cryo-EM structure of L5A7 in complex with Indonesia 2005 hemagglutinin revealed a nearly identical binding mode to other VH6-1-class members. The structure of L5A7 bound to the isolating anti-idiotype antibody, 28H6E11, revealed a shared surface for binding anti-idiotype and hemagglutinin that included two critical L5A7 regions: an FG motif in the third heavy chain-complementary determining region (CDR H3) and the CDR L1 loop. Surprisingly, the chemistries of L5A7 interactions with hemagglutinin and with anti-idiotype were substantially different. Overall, we demonstrate anti-idiotype-based isolation of a broad and potent influenza A virus-neutralizing antibody, revealing that anti-idiotypic selection of antibodies can involve features other than chemical mimicry of the target antigen.

Keywords: MEDI8852; VH6-1 class; anti-idiotype; broadly neutralizing antibody; cryo-EM; influenza A virus.

Figure 1

Isolation and characterization of anti-idiotype antibodies from mice immunized with the UCAs of broad VH6-1 influenza neutralizing antibodies. (A) Immunization scheme employing UCAs from broad VH6-1 influenza neutralizing antibodies, and work flow involving isolation of monoclonal anti-idiotype antibodies from UCA immunized mice (figure created with Biorender). (B) Surface plasmon resonance binding for anti-idiotype antibodies, isolated from homologous or heterologous immunized mice in (A) with K_D for each interaction shown above graph, or NB for no binding.

Figure 2

Screening of B cells from donor L16 PBMC with anti-idiotype antibodies. (A) Sorting of naïve and memory B cells from L16 PBMC with anti-idiotype antibodies. (B) Frequencies of anti-idiotype+ naïve (IgD+) and memory (lgG+) B cells isolated using the different anti-idiotype candidates. (C) Frequency comparison of anti-idiotype+ naïve (IgD+) and memory (IgG+) B cells. (D) CDR H3 length distributions of all anti-idiotype+ sequences and only sequences containing either IGHV6-1 or FG motif. (E) VH usage and presence of canonical "FG" motif of sequences from sorted B cells. Degree of usage is indicated by intensity of the color with more intense color indicating higher frequency. Highest frequencies are indicated in colored boxes and are shown as percentages of total number of transcripts isolated by the indicated each anti-idiotype. V-gene and FG motif percentages are calculated independently.

Figure 3

Screening of B cells from donor L5 leukopak with anti-idiotype antibodies identifies VH6-1 antibody with FG motif and different kappa chain from other members of the class. (A) Sorting of naïve and memory B cells from leukopak L5 with anti-idiotype antibodies. (B) Frequency of anti-idiotype positive naïve and memory B cells (C) Frequency of members with VH6-1 or FG motif in sequences isolated using anti-idiotype antibodies from leukopak L5. (D) VH-gene distribution of sequences isolated from leukopak L5 using anti-idiotype antibodies. (E) Venn diagram showing shared genetic elements among sequence members isolated using anti-idiotype antibodies. (F) Phylogenetic tree showing distance between L5A7 lineage members and MEDI8852. (G) Sequence features of L5A7 lineage members.

Figure 4

Binding and neutralization characteristics of isolated VH6-1 antibodies. (A) Binding of L5A7 lineage members to influenza HA shown as area under the curve (AUC). (B) Virus micro-neutralization indicated as IC80 titers (µg antibody per ml).

Figure 5

Structure of L5A7.5 bound to Flu HA shows same binding mode as MEDI8852. (A) Overview of L5A7.5 Fab bound to INDO05 HA. (B) Overlay of MEDI8852 and L5A7.5 showing near identical binding modes. (C) Comparison of the FG motifs and (D) CDR H2s between MEDI8852 and L5A7.5 with HA interacting residues shown as sticks, demonstrating the conservation in the binding of the motifs to HA. Identical residues are labeled in black, while others are labeled in the chain color. (E) Sequence alignment of the IGHV6-1 germline and MEDI8852 and L5A7.5 mature heavy chains. The FG motif is highlighted in red, and yellow underline denotes regions on L5A7.5 with significant HA interactions.

Figure 6

L5A7.5 utilizes a different light chain than other MEDI8852 class members, but maintains a similar sequence. (A) Overview of L5A7.5 binding to INDO05 HA. (B) Comparison of CDR L1s of MEDI8852 and L5A7.5 interacting with HA with common residues labeled in black and others in their chain color. (C) Sequence alignment of the light chain germline genes (IGKV1-39, IGKV3-20, IGKV1-9) and their corresponding mature antibodies (MEDI8852, 56.a.09, L5A7.5). CDRs of L5A7.5 are shown above, and the region with significant HA interaction is underlined in yellow.

Figure 7

Cryo-EM structure of L5A7.5 bound to anti-idiotype reveals regions recognized by anti-idiotype to overlap with HA. (A) Overview of L5A7.5 bound to its anti-idiotype antibody, 28H6E11. (B) Binding surface on L5A7.5 which interacts with either the anti-idiotype (top) or HA (bottom), demonstrating the overlap of the two surfaces. (C) Sequence comparison of the interacting residues of L5A7.5 for both HA and 28H6E11. Residues with more than 25Ų buried surface area at the interface are colored.

Figure 8

Comparison of L5A7.5 interactions with anti-idiotype antibody and HA. (A) Macroscopic view of the interface of L5A7.5 in complex with either HA or the 28H6E11 anti-idiotype, showing the differences in secondary structure recognized. While the epitope on HA is composed primarily of the helical regions, the corresponding epitopes on the anti-idiotype are the variable loops of the antibody. (B) Details of the individual interactions of L5A7.5 with HA or 28H6E11 showing the differences in interactions at the FG motif and CDR L1 interactions, while the mode of interaction at the CDR H2 is very similar.