Potent neutralization of vaccinia virus by divergent murine antibodies targeting a common site of vulnerability in L1 protein

Abstract

Vaccinia virus (VACV) L1 is an important target for viral neutralization and has been included in multicomponent DNA or protein vaccines against orthopoxviruses. To further understand the protective mechanism of the anti-L1 antibodies, we generated five murine anti-L1 monoclonal antibodies (MAbs), which clustered into 3 distinct epitope groups. While two groups of anti-L1 failed to neutralize, one group of 3 MAbs potently neutralized VACV in an isotype- and complement-independent manner. This is in contrast to neutralizing antibodies against major VACV envelope proteins, such as H3, D8, or A27, which failed to completely neutralize VACV unless the antibodies are of complement-fixing isotypes and complement is present. Compared to nonneutralizing anti-L1 MAbs, the neutralization antibodies bound to the recombinant L1 protein with a significantly higher affinity and also could bind to virions. By using a variety of techniques, including the isolation of neutralization escape mutants, hydrogen/deuterium exchange mass spectrometry, and X-ray crystallography, the epitope of the neutralizing antibodies was mapped to a conformational epitope with Asp35 as the key residue. This epitope is similar to the epitope of 7D11, a previously described potent VACV neutralizing antibody. The epitope was recognized mainly by CDR1 and CDR2 of the heavy chain, which are highly conserved among antibodies recognizing the epitope. These antibodies, however, had divergent light-chain and heavy-chain CDR3 sequences. Our study demonstrates that the conformational L1 epitope with Asp35 is a common site of vulnerability for potent neutralization by a divergent group of antibodies.

Importance: Vaccinia virus, the live vaccine for smallpox, is one of the most successful vaccines in human history, but it presents a level of risk that has become unacceptable for the current population. Studying the immune protection mechanism of smallpox vaccine is important for understanding the basic principle of successful vaccines and the development of next-generation, safer vaccines for highly pathogenic orthopoxviruses. We studied antibody targets in smallpox vaccine by developing potent neutralizing antibodies against vaccinia virus and comprehensively characterizing their epitopes. We found a site in vaccinia virus L1 protein as the target of a group of highly potent murine neutralizing antibodies. The analysis of antibody-antigen complex structure and the sequences of the antibody genes shed light on how these potent neutralizing antibodies are elicited from immunized mice.

FIG 1

Cross-blocking results for five L1 MAbs. Anti-L1 antibodies were tested for cross-blocking ability. 8C8 and 39D4 were found to exclusively bind sites 2 and 3, respectively. M12B9, M2E9, and M7B6 share a mutual binding site, 1.

FIG 2

In vitro neutralization assay with anti-L1 MAbs. Anti-L1 antibodies have been tested for their ability to neutralize in vitro. (A) FACS-based neutralization assay featuring anti-L1 MAbs M12B9, M2E9, M7B6, 39D4, and 8C8. Anti-H3 MAbs (41 and JH4), anti-A27 MAbs (1G6 and 12G2), and anti-D8 MAbs (JE10 and LA5) were used as controls. All antibodies were used at a final concentration of 20 μg/ml. Anti-L1 MAbs 39D4 and 8C8 did not neutralize the virus in the absence or presence of complement. Anti-L1 group I MAbs (M12B9, M7B6, and M2E9) are capable of neutralizing in the absence of complement, whereas control MAbs required the presence of complement to provide neutralization. (B, top) Neutralization levels for two L1 MAbs (M12B9 and M2E9) and one control MAb for A27, H3, and D8 each in the absence of complement. (Bottom) Results in the presence of complement. A 5-fold titration of antibodies was done for all antibodies, starting with a concentration of 20 μg/ml (green bars). Consistent with the FACS results, the 2 anti-L1 MAbs were able to neutralize in a complement-independent manner, whereas anti-H3 MAb 41 was not able to neutralize in the absence of complement. Error bars are based on four data points per sample (averages from duplicates in two independent experiments, normalized).

FIG 3

In vivo protection assays with anti-L1 MAbs. (A and B) Protection of BALB/c mice against lethal intranasal VACV_WR. (A) Body weights. (B) Survival. (C to E) Protection of SCID mice against VACV ACAM2000. Also shown are body weight (C), survival (D), and clinical scores (E) over time. There were 6 to 8 mice per group. Significance ranges were the following: *, P = 0.05 to 0.01; **, P = 0.01 to 0.005; ***, P = 0.005 to 0.0001; ****, P < 0.0001; ns, not significant.

FIG 4

Survival of C57BL/6 mice treated with M12B9 and B126 following a lethal ectromelia virus challenge. Survival of C57BL/6 mice treated with anti-L1 M12B9 MAb at D₋₁, followed by a 1,000-PFU ECTV challenge at D₀. By day 12 of the experiment, all mice of group M12B9 were dead, but B126-treated mice were protected. There were 5 mice per group.

FIG 5

Linear epitope determination of 39D4 MAb. (A) Summary of epitope mapping of VACV anti-L1 MAbs by linear peptide ELISA. All 5 MAbs bind to the full-length L1 protein, but only 39D4 binds an individual peptide (121-140). (B and D) Truncation assay discovered the epitope to be amino acids 123 to 132 of peptide 121-140. A low optical density (OD) indicates that the peptide fragment preincubated with the antibody fully occupies the antibody's episome and prevents it from binding to plate-bound full-length peptide. (C and E) Alanine scan of the linear (lin.) epitope 123-132 revealed N124, K125, I128, and I132 to be key residues for antibody binding. A lowered OD indicates the decreased ability of the antibody to bind to that particular peptide; thus, the alanine-substituted residue in the original peptide has a large effect on the binding ability. Dashed lines indicate a cutoff for positive results (OD of 1.0). w/o, without.

FIG 6

Epitope mapping of anti-L1 M12B9 MAb using DXMS. Deuterium exchange data show differences in deuteration levels in the presence compared to the absence of MAb binding at four time points (10 s, 30 s, 100 s, and 1,000 s). Slower deuterium exchange is marked in blue, and faster exchange is marked in red. Residues 25 to 34 and 113 to 131 (red boxes) show the most marked slowing, indicating regions likely incorporating epitope binding sites.

FIG 7

Identification of Asp35 as a key epitope residue for group I MAbs. (A) Part of the L1 sequence of two VACV mutants that are resistant to neutralization by M12B9. The two mutants were plaque purified from VACV that had been mutagenized with ethyl methanesulfonate and escaped the neutralization by M12B9. The L1 coding sequence of the mutants was determined. Shown in the dashed box is a single-nucleotide substitution compared to the wild-type sequence. (B) VACV mutants with D35N or D35Y substitution in L1 were resistant to neutralization by all group I anti-L1 MAbs. The abilities of the anti-L1 MAbs to neutralize wild-type or mutant VACV were determined with the plaque reduction assay. The labels below the x axis indicate the viruses that were used for neutralization. (C) Amino acid comparison of L1 and its ectromelia ortholog (EVM072) shows four differences, one of which is residue 35 (D35 in vaccinia versus N35 in ectromelia).

FIG 8

M12B9-MAb targets the same epitope as 7D11-MAb. (A) Superimposed structures of L1 bound to M12B9 or 7D11-Fab domains. Models were superimposed on L1 using the secondary-structure matching algorithm (SSM) within Coot 0.7 (root mean square deviations [RMSD] of 0.53 Å for L1 using PDB code 2I9L). For simplicity, only L1 of the M12B9 complex is shown in gray throughout. (B) M12B9 footprint on L1 surface colored by contacting CDR, with H1, H2, and H3 shown in green, cyan, and blue, respectively. D35 occupies a central position in the epitope. The associated table lists contact residues. Boldface black labels highlight residues chosen for site-directed mutagenesis. (C, D, E, and F) Detailed contacts of M12B9/L1 and comparison to 7D11/L1. CDRs of M12B9 are shown in orange superimposed on 7D11 (gray), while L1 is gray with side-chain orientation drawn as thin lines. Detailed MAb/L1 interactions are shown for H1 (C), H2 (D), and H3 (E). (F) Framework residue R74 forms a salt bridge with D62 of L1. Yellow dashed lines indicate hydrogen bonds or salt bridges (for L1/M12B9-Fab). Note how H2 in the L1/7D11 interface forms almost identical contacts with L1 residues.

FIG 9

Sequence alignment of 4 anti-L1 MAbs. (A) Heavy-chain sequence alignment of germ line IGHV1S26*01 versus 7D11, M12B9, M7B6, and M2E9. Identical amino acids are marked by a dot, green boxes are marked CDR1 through CDR3, and dashes indicate where the germ line sequence ends. Residues in CDR1 and CDR2 are highly conserved between all 4 MAbs and the germ line, whereas residues in CDR3 show high diversity. 7D11 has 12 heavy-chain residues contacting L1 in the crystal structure (T30, R31, W33, Y50, N52, S54, T55, G56, Y57 T58, D102, and Y104), while M12B9 has four additional heavy-chain residues contacting L1 in the structure (F32, E59, Q101, and N103), highlighted in red. Many of the critical CDR1 and CDR2 residues (30, 32, 50, 52, 54, 55, 57, and 58) are either conserved in the germ line or have conservative substitutions (black arrows). Only residues 31, 33, and 59 have nonconservative substitutions compared to the germ line sequence (red arrows). (B) Light-chain sequence alignment of 7D11 versus M12B9, M7B6, and M2E9. Identical amino acids are marked by a dot, green boxes mark CDR1 through CDR3, and gaps (insertions) are marked by a tilde symbol. Whereas M12B9 and M2E9 show quite similar sequences, overall all 4 MAbs show a highly diverse CDR sequence. Light chains originate from different germ lines; thus, no comparison to a mutual germ line was possible.

FIG 10

Real-time binding curves using biolayer interferometry. Curves show binding of analytes M12B9-Fab, 8C8-Fab, and 39D4-IgG in solution to wild-type antigen L1 (residues 1 to 184) and indicated mutants immobilized on Ni-NTA biosensors. Association (600 s) and dissociation (900 s) steps are represented. Curves are colored according to their specific analyte (Fab or IgG) concentration (middle right, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, and 200 nM). The affinity constant (K_D) is reported as an average from duplicate assays with standard errors.

FIG 11

Immunofluorescence analysis with anti-L1 MAbs. BHK cells were infected with a GFP-expressing VACV at an MOI of 0.5 PFU/cell for 8 h. The cells were fixed, permeabilized, and stained with the indicated anti-L1 MAb, followed by DAPI and goat anti-mouse IgG coupled to Cy3. The DAPI and antibody staining are shown separately in the left and middle panels, respectively. They are shown together with GFP fluorescence in the right panel. F, viral factory. For virion images, sucrose gradient-purified VACV virions were absorbed to glass coverslips coated with fibronectin and fixed with paraformaldehyde. The virions then were stained with DAPI and the indicated anti-L1 MAb. DAPI and antibody staining are shown separately in the left and middle panels, respectively. They are shown together in the right panel. The white arrows point to individual virion particles.