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. 2014 Sep:464-465:264-273.
doi: 10.1016/j.virol.2014.06.039. Epub 2014 Aug 9.

A highly specific monoclonal antibody against monkeypox virus detects the heparin binding domain of A27

Laura J Hughes  1 , Jason Goldstein  2 , Jan Pohl  2 , Jay W Hooper  3 , R Lee Pitts  2 , Michael B Townsend  2 , Dennis Bagarozzi  2 , Inger K Damon  2 , Kevin L Karem  2
Affiliations

A highly specific monoclonal antibody against monkeypox virus detects the heparin binding domain of A27

Laura J Hughes et al. Virology. 2014 Sep.

Abstract

The eradication of smallpox and the cessation of global vaccination led to the increased prevalence of human infections in Central Africa. Serologic and protein-based diagnostic assay for MPXV detection is difficult due to cross-reactive antibodies that do not differentiate between diverse orthopoxvirus (OPXV) species. A previously characterized monoclonal antibody (mAb 69-126-3-7) against MPXV [1] was retested for cross-reactivity with various OPXVs. The 14.5 kDa band protein that reacted with mAb 69-126-3 was identified to be MPXV A29 protein (homolog of vaccinia virus Copenhagen A27). Amino acid sequence analysis of the MPXV A29 with other OPXV homologs identified four amino acid changes. Peptides corresponding to these regions were designed and evaluated for binding to mAb 69-126-3 by ELISA and BioLayer Interferometry (BLI). Further refinement and truncations mapped the specificity of this antibody to a single amino acid difference in a 30-mer peptide compared to other OPXV homologs. This particular residue is proposed to be essential for heparin binding by VACV A27 protein. Despite this substitution, MPXV A29 bound to heparin with similar affinity to that of VACV A27 protein, suggesting flexibility of this motif for heparin binding. Although binding of mAb 69-126-3-7 to MPXV A29 prevented interaction with heparin, it did not have any effect on the infectivity of MPXV. Characterization of 69-126-3-7 mAb antibody allows for the possibility of the generation of a serological based species-specific detection of OPXVs despite high proteomic homology.

Keywords: Antibody; Heparin binding; Monkeypox virus; Orthopoxvirus; Vaccinia virus.

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Figures

Fig. 1
Fig. 1
Diagram of VACV A27 functional domains. Functional regions: signal peptide (29), HBS (4, 15), coiled-coil (34), and anchoring domains (37) are modeled highlighting the heparin binding domain in red. Interaction with VACV A26 and A17 are both shown. Interaction with VACV A26 is shown by the formation of two disulfide bonds between VACV A26 C441 and C442 with VACV A27 C71 and C72 respectively (3). Binding between VACV A27 and VACV A17 occurs at two binding sites on A17. VACV A27 amino acids R90 and R101 bind to one site on A17 through VACV A17 amino acids Q29 and D37. The other binding site is between VACV A27 F80 and E87 and VACV A17 L20 and Q29 (37).
Fig. 2
Fig. 2
Binding is specific to MPXV A29. (A) Western blot: MPXV proteins A29, A35, B6, M1 and VACV A27 were screened for binding by mAb 69-126-3-7. Due to limited reagent, B6 was loaded at ¼ of the concentration as the other proteins in the coomassie gel shown below. The antibody reacted with MPXV A29 only. (B) BLI analysis: MPXV A29 and VACV A27 were tested for binding on the octet system and binding was both strong and specific to MPXV A29. (C) IFAT: Expression plasmids containing either VACV A27 or monkeypox A29 were transfected into COS cells and the cells were screened for expression using 69-126-3-7. Empty vector and unrelated antibodies were used as controls. Binding was specific to cells transfected with the MPXV A29 expression plasmid.
Fig. 3
Fig. 3
Sequence variability in VACV A27. (A) Alignment: As new orthopoxvirus sequences have been identified since the initial studies were done, an alignment incorporating all currently identified orthopoxvirus sequences of MPXV A29 orthologs was done and all viruses that demonstrated sequence variation were analyzed by Western blot to confirm specificity. (B) Western blot: Viruses representing all possible sequence variations within the orthologs of VACV A27 were screened for binding by mAb 69-126-3-7. Lanes were loaded as follows: MPXV-USA2003-044, VACV-Wyeth, VARV-BGD75-Banu, CPXV-FIN2000-MAN, CPXV-NOR1994-MAN, CPXV-UK2000-K2984, CMLV-M96, ECTV-Mos, and TATV-DAH68. Binding was specific to MPXV. The bottom portion of the figure represents virus loading. Each semi-pure virus was loaded at equal loading concentrations and screened with VIG at a 1:500 dilution. Differences in band intensity are likely due to differential reactivity by VIG and slight variation in cellular protein contaminants during the virus purification process.
Fig. 4
Fig. 4
ELISA and BLI of peptides. (A) ELISA: 5 biotinylated peptides were plated onto a streptavidin coated ELISA plate in replicates of 4 or coated to octet sensor and assayed for binding by the antibody 69-126-3-7. 2 additional peptides were used as controls. The antibody bound to the MPX peptide (MPXpep) and did not bind to the consensus peptide (CONpep) confirming the use of peptides in these assays. When testing the single amino acid substitution peptides (CONpep1, CONpep2, and CONpep3), the antibody bound only to CONpep1. (B) BLI: 5 biotinylated peptides and the 2 control peptides were bound to the streptavidin coated sensors and tested for binding by mAb 69-126-3-7. The mAb bound strongly and specifically to MPXpep and CONpep1.
Fig. 5
Fig. 5
ELISA and BLI of truncated peptides. To try and identify the minimal region required for antibody binding, 2 truncations were made (5 amino acids from the N-terminus and separately 6 amino acids from the C-terminus of the peptide) and screened for binding. In both ELISA (A) and BLI analysis (B), the C-terminal truncation was tolerated while the N-terminal truncation was not. To further refine the role of the N-terminal amino acids in antibody binding, a single amino acid was added back to the N-truncated peptide one at time. Interestingly binding was minimally restored with addition of 3 amino acids with an increase in binding signal with the restoration of 4 amino acids, but all 5 n-terminal amino acids were required for binding signals equivalent to MPXpep.
Fig. 6
Fig. 6
Protein binding to heparin and heparin inhibition dot blots, MPXV A29 and VACV A27, were run over a heparin column. (A) The flow through (FT), wash (W) and eluate or elution (E) were along with purified protein (P) were dotted onto nitrocellulose membrane. This was probed with polyclonal rabbit α-A27 antibody at a 1:10,000 dilution. Protein was visible in E and the loading control, but none in the FT or the W for both proteins, indicating that A29 binds heparin regardless of the amino acid difference in the heparin binding site. (B) Antibody 69-126-3-7 was incubated with 10 µg of MPXV at a ratio of antibody:protein of 1:2, 1:1, 2:1, and 5:1 and run over a heparin column. Flow through (FT), representing what did not bind to the column, and Eluate or Elution, representing what did bind, were concentrated and screened with a polyclonal α-A27 antibody. Inhibition of binding to heparin was seen as early as 1:2, but almost complete inhibition of binding was not observed until 5:1.
Fig. 7
Fig. 7
Heparin inhibition dot blot. Antibody 69-126-3-7 was incubated with 10 µg of MPXV at a ratio of antibody:protein of 1:2, 1:1, 2:1, and 5:1 and run over a heparin column. Flow through (FT), representing what did not bind to the column, and Eluate or Elution, representing what did bind, were concentrated and screened with a polyclonal α-A27 antibody. Inhibition of binding to heparin was seen as early as 1:2, but almost complete inhibition of binding was not observed until 5:1.
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