Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein

Abstract

Respiratory syncytial virus (RSV) infections remain a major cause of respiratory disease and hospitalizations among infants. Infection recurs frequently and establishes a weak and short-lived immunity. To date, RSV immunoprophylaxis and vaccine research is mainly focused on the RSV fusion (F) protein, but a vaccine remains elusive. The RSV F protein is a highly conserved surface glycoprotein and is the main target of neutralizing antibodies induced by natural infection. Here, we analyzed an internalization process of antigen-antibody complexes after binding of RSV-specific antibodies to RSV antigens expressed on the surface of infected cells. The RSV F protein and attachment (G) protein were found to be internalized in both infected and transfected cells after the addition of either RSV-specific polyclonal antibodies (PAbs) or RSV glycoprotein-specific monoclonal antibodies (MAbs), as determined by indirect immunofluorescence staining and flow-cytometric analysis. Internalization experiments with different cell lines, well-differentiated primary bronchial epithelial cells (WD-PBECs), and RSV isolates suggest that antibody internalization can be considered a general feature of RSV. More specifically for RSV F, the mechanism of internalization was shown to be clathrin dependent. All RSV F-targeted MAbs tested, regardless of their epitopes, induced internalization of RSV F. No differences could be observed between the different MAbs, indicating that RSV F internalization was epitope independent. Since this process can be either antiviral, by affecting virus assembly and production, or beneficial for the virus, by limiting the efficacy of antibodies and effector mechanism, further research is required to determine the extent to which this occurs in vivo and how this might impact RSV replication.IMPORTANCE Current research into the development of new immunoprophylaxis and vaccines is mainly focused on the RSV F protein since, among others, RSV F-specific antibodies are able to protect infants from severe disease, if administered prophylactically. However, antibody responses established after natural RSV infections are poorly protective against reinfection, and high levels of antibodies do not always correlate with protection. Therefore, RSV might be capable of interfering, at least partially, with antibody-induced neutralization. In this study, a process through which surface-expressed RSV F proteins are internalized after interaction with RSVspecific antibodies is described. One the one hand, this antigen-antibody complex internalization could result in an antiviral effect, since it may interfere with virus par-

Antibody-Induced Internalization of HIV-1 Env Proteins Limits Surface Expression of the Closed Conformation of Env

“…Primary CD4 ϩ T cells were infected with NL4.3 ADA green fluorescent protein (GFP) or CH58 T/F and dynamin was inhibited with the dynamin inhibitory peptide (DIP). This peptide blocks the binding of dynamin to amphiphysin and has been shown to reduce endocytic events (27,45,46). The addition of DIP significantly reduced bNAb-induced Env internalization in infected cells as measured by flow cytometry (Fig.…”

“…It has been previously suggested that Ab-mediated cross-linking facilitates internalization of RSV fusion proteins. In this study, RSV Env endocytosis was significantly reduced when Fab fragments were used instead of their full MAb counterpart (27). To verify whether this was the case for HIV-1 Env, we performed side-by-side comparisons on the ability of full-length Abs versus their Fab fragments to reduce Env levels at the cell surface upon incubation at 37°C, as measured by flow cytometry.…”

“…The ability of antigen-Ab complexes to remain on the surfaces of infected cells might be another factor modulating humoral responses against different viruses. It has been reported previously that the interaction of some Abs with Env glycoproteins expressed on the surfaces of respiratory syncytial virus (RSV) (27)and feline coronavirus (28)-infected cells accelerates their internalization and decreases their exposure to the immune system. We evaluated here the fate of HIV-1 Env expression at the surfaces of cells upon antibody binding and made the surprising observation that the binding of broadly neutralizing antibodies (bNAbs) but not that of nonneutralizing antibodies (nNAbs) induced Env-Ab complex internalization and reduced ADCC responses mediated by bNAbs.…”

A Revised View on the Role of Surface AMPAR Mobility in Tuning Synaptic Transmission: Limitations, Tools, and Alternative Views

“…More importantly, the encephalitis patients’ CSF reduced the amplitude and frequency of miniature EPSCs (mEPSCs), an effect that became evident 1 h after antibody application. Interestingly, this effect is not isolated to AMPARs, as antibodies against NMDARs ( Dalmau et al, 2011 ), potassium channels ( Sun and Li, 2013 ; Sun et al, 2016 ), FGFR1 ( Opaliński et al, 2017 ), ErbB3 ( Belleudi et al, 2012 ), Human Respiratory Syncytial Virus Fusion Protein ( Leemans et al, 2017 ), and acetylcholine receptors ( Lee et al, 2014 ) also trigger a reduction of their respective target proteins. These studies suggest that antibodies against surface proteins induce internalization after the proteins are crosslinked if the samples are not maintained in the cold.…”

Antibody‐mediated complement activation in pathology and protection

“…Other complement evasion mechanisms include the incorporation of host membrane complement regulatory proteins by budding enveloped virions, 88 molecular mimicry 89 or recruitment of such host regulatory factors, 87,90 and production of other complement‐disrupting factors such as complement‐cleaving enzymes, 91 and interference with C1q‐associated serine proteases 92 . Another mechanism of complement evasion, and evasion of antibody‐mediated effector function more generally, is antibody‐induced antigen internalization on virally infected host cells 93,94 . These pathogen evasion mechanisms, and others, have been extensively reviewed recently 95 .…”