The Receptor-Binding Site of the Measles Virus Hemagglutinin Protein Itself Constitutes a Conserved Neutralizing Epitope

Abstract

e Here, we provide direct evidence that the receptor-binding site of measles virus (MV) hemagglutinin protein itself forms an effective conserved neutralizing epitope (CNE). Several receptor-interacting residues constitute the CNE. Thus, viral escape from neutralization has to be associated with loss of receptor-binding activity. Since interactions with both the signaling lymphocyte activation molecule (SLAM) and nectin4 are critical for MV pathogenesis, its escape, which results from loss of receptor-binding activity, should not occur in nature. Measles virus (MV) is an enveloped virus that belongs to the genus Morbillivirus of the family Paramyxoviridae and possesses two types of surface glycoprotein spikes, the hemagglutinin (H) and fusion (F) proteins. MV infection is initiated by binding of the H protein to cellular receptors on the target host cells. The binding triggers membrane fusion between the virus envelope and the host cell plasma membrane mediated by the F protein. The signaling lymphocyte activation molecule (SLAM) on a subset of immune cells and nectin4 at adherens junctions are the principal receptors for MV (1-4). The H and F proteins are both neutralizing targets, but greater amounts of antibodies (Abs) are elicited against the H protein than the F protein, and the H protein-specific antibodies mainly contribute to neutralization of MV infection in vivo (5-8). All the available data suggest that measles eradication is biologically feasible (9, 10), and one of the major factors that would ensure measles eradication is the single-serotype nature of MV. Our previous paper suggested that the receptor-binding site (RBS) on the H protein, which is exposed outside the heavy N-glycan shields, may constitute a major neutralizing epitope that contributes to the single-serotype nature of MV (11). However, our recent paper (12) showing the detailed locations of five epitopes (I, II, iv, v, and vi) on the H protein provided insufficient evidence that the RBS acts as a neutralizing epitope. Here, we provide direct and concrete evidence that the RBS itself forms an effective conserved neutralizing epitope (CNE) which provides a strong functional constraint against change.In the present study, we characterized a new mouse monoclonal antibody (MAb), 2F4. MAb 2F4 was produced using a cell line expressing the H protein of the wild-type IC-B strain as an antigen. For competitive binding enzyme-linked immunosorbent assays (cELISAs), MV antigens precoated on cELISA plates (Denka Seiken) were incubated with previously reported MAbs (E81, E128, E185, E39, and E103) (12, 13), 2F4, or phosphate-buffered saline for 1 h and then incubated with MAb 2F4 labeled with biotin using a biotin labeling kit (NH2; Dojindo Laboratories). After three washes, the MV antigens bound by the MAbs were incubated with a streptavidin-horseradish peroxidase (HRP) conjugate (Thermo Scientific) for 1 h before addition of the TMB substrate (3,3=5,5=-tetramethylbenzidine; Denka Seiken). The plates were incubated for 25 min, and the colorimetric...

Biophysical characterization and single‐chain Fv construction of a neutralizing antibody to measles virus

“…Several neutralizing antibodies and the detailed locations of neutralizing epitopes ( I , II , iv , v , vi and vii ) on the H protein were reported for measles neutralization . Among the epitope regions, the epitope vii is thought to be recognized by 2F4 based on the neutralizing experiment using mutant strains and the current study.…”

Antibody neutralization of retargeted measles viruses

“…MV H glycoproteins are the major target for neutralizing antibodies. The receptor-binding surface is a major target for neutralizing monoclonal antibodies and is a conserved neutralizing antigenic site [48, 51, 68]. Since, retargeted MV derivatives use a separate domain displayed on H to bind selected receptors and encode mutations ablating native MV receptor binding, it is hypothesized that retargeted MV derivatives may be less susceptible to human serum neutralization.…”

“…However, we cannot explain why MV-H AALS -Wue1 but not MV-H-Wue1 is neutralized and why the degree of escape by the other viruses varies between experiments. Due to the fact that the receptor-binding surface of the H protein is a hot spot for neutralizing antibodies [47–51] we were surprised that the H AALS mutations protected this antigenic site from mAbs but provided no advantage in escaping neutralization by human sera. We tested pooled human sera available commercially and sera from five individuals diagnosed with malignant neoplasms, as this would be the patient population for oncolytic virotherapy using retargeted MVs.…”

“…Measles oncolytic virotherapy is limited by preexisting immunity due to widespread global vaccination against measles [45]. The hemaggluntinin attachment protein is the major target for neutralizing antibodies [46] that tend to cluster at the receptor binding surface targeting a conserved neutralizing antigenic region [47–51]. Retargeted MV derivatives have two modifications that could potentially destroy or shield epitopes within the receptor-binding surface.…”

Enhanced immunosurveillance for animal morbilliviruses using vesicular stomatitis virus (VSV) pseudotypes

“…During the adaptation of the virus for growth in cell culture, the biological properties of the virus may alter dramatically to suit the new ex vivo environment and the availability of potential viral receptors [11], [12], [13], [14], [15], [16], [17], [18], [19]. As the receptor binding domains of the morbilliviral haemagglutinins are targets for neutralising antibodies [20], alterations in receptor binding that facilitate infection in vitro may alter the conformation of the viral haemagglutinin, modulating sensitivity to neutralising antibodies. If biologically-relevant neutralising antibody responses are to be quantified accurately, assays that utilize primary field strains of virus and their cognate receptors are required.…”