Linear Epitopes in Vaccinia Virus A27 Are Targets of Protective Antibodies Induced by Vaccination against Smallpox

Abstract

Vaccinia virus (VACV) A27 is a target for viral neutralization and part of the Dryvax smallpox vaccine. A27 is one of the three glycosaminoglycan (GAG) adhesion molecules and binds to heparan sulfate. To understand the function of anti-A27 antibodies, especially their protective capacity and their interaction with A27, we generated and subsequently characterized 7 murine monoclonal antibodies (MAbs), which fell into 4 distinct epitope groups (groups I to IV). The MAbs in three groups (groups I, III, and IV) bound to linear peptides, while the MAbs in group II bound only to VACV lysate and recombinant A27, suggesting that they recognized a conformational and discontinuous epitope. Only group I antibodies neutralized the mature virion in a complement-dependent manner and protected against VACV challenge, while a group II MAb partially protected against VACV challenge but did not neutralize the mature virion. The epitope for group I MAbs was mapped to a region adjacent to the GAG binding site, a finding which suggests that group I MAbs could potentially interfere with the cellular adhesion of A27. We further determined the crystal structure of the neutralizing group I MAb 1G6, as well as the nonneutralizing group IV MAb 8E3, bound to the corresponding linear epitope-containing peptides. Both the light and the heavy chains of the antibodies are important in binding to their antigens. For both antibodies, the L1 loop seems to dominate the overall polar interactions with the antigen, while for MAb 8E3, the light chain generally appears to make more contacts with the antigen.

Importance: Vaccinia virus is a powerful model to study antibody responses upon vaccination, since its use as the smallpox vaccine led to the eradication of one of the world's greatest killers. The immunodominant antigens that elicit the protective antibodies are known, yet for many of these antigens, little information about their precise interaction with antibodies is available. In an attempt to better understand the interplay between the antibodies and their antigens, we generated and functionally characterized a panel of anti-A27 antibodies and studied their interaction with the epitope using X-ray crystallography. We identified one protective antibody that binds adjacent to the heparan sulfate binding site of A27, likely affecting ligand binding. Analysis of the antibody-antigen interaction supports a model in which antibodies that can interfere with the functional activity of the antigen are more likely to confer protection than those that bind at the extremities of the antigen.

FIG 1

Protein ELISA with A27 MAbs. (A) Seven anti-A27 MAbs were tested for their ability to bind purified A27_16–100 protein. All MAbs except MAb 8E3 successfully bound the construct. Anti-D8 MAb Ab12.1 (aD8 Ab12.1) was used as a negative control. (B) Direct comparison of four selected anti-A27 MAbs binding the A27_16–100 protein versus the A27_16–110 protein. All four tested anti-A27 MAbs, including MAb 8E3, bound the longer construct. Anti-L1 MAb M12B9 (aL1 MAb M12B9) was used as a negative control. Dashed lines, the cutoffs for positive results (OD, 1). The experiments were performed three times.

FIG 2

Cross-blocking results for seven A27 MAbs. Anti-A27 antibodies were tested for cross-blocking ability. MAbs 1G6, 12G2, and 8H10 were found to exclusively bind site I and made up the group I MAbs. MAbs 4G5 and 12C3 bound to site II and clustered into another distinct group (group II MAbs). MAbs 8E3 and 6F11 did not cross-react with any other MAbs and bound sites III and IV, respectively, making up group III and IV MAbs, respectively). The experiments were performed twice.

FIG 3

In vitro neutralization assay with anti-A27 MAbs. Anti-A27 antibodies were tested for their ability to neutralize in vitro in a fluorescence-activated cell sorting-based neutralization assay featuring MAbs 1G6, 12G2, 8H10, 6F11, 4G5, 12C3, and 8E3. Anti-L1 MAb M12B9 was used as a positive control. All antibodies were used at a final concentration of 20 μg/ml. Anti-A27 MAbs 1G6, 12G2, and 8H10 (group I) were capable of strong neutralization (>90%) in the presence of complement (bottom) but not in its absence (top). Antibodies 6F11 (group II), 4B5 and 12C3 (group III), and 8E3 (group IV) did not neutralize in the absence of complement and showed weak or no neutralization ability (<20%) in the presence of complement. The positive control, anti-L1 MAb M12B9, strongly neutralized (>95%) the virus in a complement-independent manner. Dashed lines, 50% neutralization. The experiments were performed at least twice.

FIG 4

In vivo protection assays with anti-A27 MAbs. Protection of SCID mice against VACV ACAM2000. In this study, we tested anti-A27 MAbs 1G6 (group I), 6F11 (group II, conformational epitope), and 8E3 (group IV). Anti-H3#41 was used as a positive control. Body weight (A), survival (B), and clinical scores (C) over time are shown for 8 mice per group. Statistical significance was assessed at the final time point. Significance ranges are indicated by asterisks. *, P = 0.05 to 0.01; **, P = 0.01 to 0.005; ***, P = 0.005 to 0.0001; ****, P < 0.0001; ns, not significant. Dashed lines in panel A, initial body weight (100%) and minimum cutoff weight (75% initial body weight). The experiments were performed at least twice.

FIG 5

Determination of linear epitope binding for A27 MAbs. (A) Group I MAbs (MAbs 1G6, 12G2, and 8H10) bound the linear peptide spanning from residues 21 to 40. (B) Group II MAb 6F11 did not a bind a linear epitope; thus, we hypothesized that it bound a conformational, discontinuous epitope instead. (C) Group III MAbs (MAbs 12C3 and 4G5) bound the peptide spanning from residues 81 to 100. Additionally, MAb 12C3 also bound the peptide spanning from residues 21 to 40. (D) Group IV MAb 8E3 bound the peptide spanning from residues 91 to 110. The experiments were performed three times.

FIG 6

Truncation and alanine scan of A27 linear epitopes. (A and C) A truncation assay discovered the epitope of group I MAbs (MAbs 1G6, 12G2, and 8H10) to be residues 31 to 40 of the peptide spanning from residues 21 to 40. The epitope of group IV MAb 8E3 was residues 101 to 110 of the peptide spanning from residues 91 to 110. A low OD indicated that the peptide fragment preincubated with the antibody fully occupied the antibody's binding interface and prevented it from binding to plate-bound full-length peptide. (B and D) An alanine scan of the linear epitopes revealed residues E33, I35, V36, K37, and D39 (the linear epitope from residues 31 to 40) and residues G105, R107, P108, Y109, and (arguably) E110 (the linear epitope from residues 101 to 110) to be key residues for antibody binding. A low OD indicates a decreased ability of the antibody to bind to that particular peptide; thus, the residue in the original peptide replaced by an alanine had a strong impact on the binding ability. When the peptide sequence contained an alanine to begin with, the alanine was replaced by a serine instead. Dashed lines, cutoff for positive results (ODs, 0.5 for the truncation assay and 1.0 for the alanine scan). The experiments were performed three times.

FIG 7

Crystal structures of Fabs 1G6 and 8E3 in complex with A27 peptides. (A) Overview of the Fab-peptide complex of Fabs 1G6 (top row) and 8E3 (bottom row). Green, heavy chain; orange, light chain. (B) Top view onto the antigen binding site of both Fabs, shown as a molecular surface and colored by electrostatic surface potential (red, negative; blue, positive; contoured from −30 to +30 kT/electron). Bound A27 peptides are shown as white sticks, and the 2F_o − F_c electron density is drawn as a blue mesh contoured at 1σ. (C) Polar interactions between Fab residues and the peptide. Blue dotted lines, H bonds and salt bridges.