Neutralization of Human Respiratory Syncytial Virus Infectivity by Antibodies and Low-Molecular-Weight Compounds Targeted against the Fusion Glycoprotein

Abstract

Human respiratory syncytial virus (HRSV) fusion (F) protein is an essential component of the virus envelope that mediates fusion of the viral and cell membranes, and, therefore, it is an attractive target for drug and vaccine development. Our aim was to analyze the neutralizing mechanism of anti-F antibodies in comparison with other low-molecular-weight compounds targeted against the F molecule. It was found that neutralization by anti-F antibodies is related to epitope specificity. Thus, neutralizing and nonneutralizing antibodies could bind equally well to virions and remained bound after ultracentrifugation of the virus, but only the former inhibited virus infectivity. Neutralization by antibodies correlated with inhibition of cell-cell fusion in a syncytium formation assay, but not with inhibition of virus binding to cells. In contrast, a peptide (residues 478 to 516 of F protein [F478-516]) derived from the F protein heptad repeat B (HRB) or the organic compound BMS-433771 did not interfere with virus infectivity if incubated with virus before ultracentrifugation or during adsorption of virus to cells at 4°C. These inhibitors must be present during virus entry to effect HRSV neutralization. These results are best interpreted by asserting that neutralizing antibodies bind to the F protein in virions interfering with its activation for fusion. Binding of nonneutralizing antibodies is not enough to block this step. In contrast, the peptide F478-516 or BMS-433771 must bind to F protein intermediates generated during virus-cell membrane fusion, blocking further development of this process.Human respiratory syncytial virus (HRSV), a member of the Pneumovirus genus of the Paramyxoviridae family, is the main cause of severe lower respiratory tract infections in very young children (36), and it is a pathogen of considerable importance in the elderly (24, 26) and in immunocompromised adults (22). Currently, there is no effective vaccine against the virus although it is known that passive administration of neutralizing antibodies to individuals at high risk is an effective immunoprophylaxis (37,38).The HRSV genome is a single-stranded negative-sense RNA molecule of approximately 15 kb that encodes 11 proteins (16, 53). Two of these proteins are the main surface glycoproteins of the virion. These are (i) the attachment (G) protein, which mediates virus binding to cells (44), and (ii) the fusion (F) protein, which promotes both fusion of the viral and cell membranes at the initial stages of the infectious cycle and fusion of the membrane of infected cells with those of adjacent cells to form characteristic syncytia (72). These two glycoproteins are the only targets of neutralizing antibodies either induced in animal models (19,63,65,70) or present in human sera (62).The G protein is a highly variable type II glycoprotein that shares neither sequence identity nor structural features with the attachment protein of other paramyxoviruses (75). It is synthesized as a precursor of about 300 amino acids (depending on the strai...

Structure of a Major Antigenic Site on the Respiratory Syncytial Virus Fusion Glycoprotein in Complex with Neutralizing Antibody 101F

“…We have demonstrated that 101F does not block virus attachment over a wide range of concentrations (Fig. 5B), which agrees with recently published data showing a similar result at a single antibody concentration (29). We have also shown that 101F is capable of preventing infection once the virus has attached to the cell (Fig.…”

Structure of Respiratory Syncytial Virus Fusion Glycoprotein in the Postfusion Conformation Reveals Preservation of Neutralizing Epitopes

“…Though we demonstrate binding to the postfusion state, these antibodies must neutralize prior to membrane fusion and formation of the six-helix bundle, and therefore, we infer that they can bind intermediate states that at least partially resemble the postfusion conformation. Recently, Magro et al showed that 101F and an antibody specific for antigenic site II bound virus before incubation with target cells, remained bound during ultracentrifugation, and neutralized the virus (26). This result is consistent with 101F and motavizumab being able to bind the prefusion conformation, as well.…”

“…Thus, 101F and motavizumab can likely bind the fusion glycoprotein in the prefusion, postfusion, and intermediate states, which results from their epitopes residing in domains that are not expected to undergo large structural rearrangements during the fusion process (44). This is different from the modes of action of other RSVneutralizing compounds, such as antigenic site I-directed antibodies, which appear not to bind the prefusion state (26), and some small molecules and heptad repeat B-derived peptides that target various intermediate states of the F glycoprotein (24,35) (Fig. 7).…”

Neutralizing epitopes of RSV and palivizumab resistance in Japan

“…Palivizumab is licensed and widely used throughout the world to suppress severe RSV infection in high -risk children 24) . Site IV is recognized by mAb 19 25) or 101 F 26) with moderate NT activity. A recently isolated human mAb, 54G10, shows NT activity for human metapneumovirus (hMPV), which belongs to genus paramyxovirus as RSV 27) .…”