
==== Front
eBioMedicine
EBioMedicine
eBioMedicine
2352-3964
Elsevier

S2352-3964(24)00354-2
10.1016/j.ebiom.2024.105318
105318
Review
The role of respiratory syncytial virus G protein in immune cell infection and pathogenesis
Anderson Jeremy jeremy.anderson@mcri.edu.au
ab∗
Do Lien Anh Ha ab
van Kasteren Puck B. c
Licciardi Paul V. paul.licciardi@mcri.edu.au
ab∗∗
a Infection, Immunity and Global Health, Murdoch Children's Research Institute, Melbourne, Australia
b Department of Paediatrics, University of Melbourne, Melbourne, Australia
c Center for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, the Netherlands
∗ Corresponding author. Murdoch Children's Research Institute, Melbourne, Australia. jeremy.anderson@mcri.edu.au
∗∗ Corresponding author. Murdoch Children's Research Institute, Melbourne, Australia. paul.licciardi@mcri.edu.au
31 8 2024
9 2024
31 8 2024
107 10531811 6 2024
12 8 2024
17 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Severe respiratory syncytial virus (RSV) disease is a significant contributor to the global burden of disease in infants and children. The RSV attachment protein (G) has been shown to be critical in invading airway epithelial cells through its CX3C motif interacting with the host receptor CX3CR1. The ubiquitous expression of this receptor on immune cells may explain their susceptibility to RSV infection. The RSV G protein may enhance disease severity through reprogramming of normal cellular functionality leading to inhibition of antiviral responses. While existing preventives targeting the RSV fusion (F) protein are highly effective, there are no RSV therapeutics based on the G protein to limit RSV pathogenesis. Monoclonal antibodies targeting the RSV G protein administered as post-infection therapeutics in mice have been shown to improve the antiviral response, reduce viral load and limit disease severity. Further research is required to better understand how RSV infection of immune cells contributes to pathogenesis for the development of more targeted and efficacious therapeutics.

Keywords

RSV
Infection
Pathogenesis
G protein
Therapeutics
==== Body
pmc Search strategy and selection criteria

Articles selected in this study were searched for between March 1st, 2023, and June 30th, 2024, and largely obtained from PubMed and Google Scholar. Search terms such as “RSV” was used in combination with “infection”, “F protein”, “G protein”, “immune cells”, “NK cells”, “dendritic cells”, “monocytes”, “B cells”, “T cells”, “CX3CR1”, “therapeutics”, “vaccine”. Articles obtained were in English only. As much as possible, the most recent articles were selected but impactful articles that were >5 years old were still included. Other articles in the reference were selected based on relevance.

Introduction

Respiratory syncytial virus (RSV) is the most common respiratory viral pathogen causing disease in infants. Globally, 1.4 million hospitalisations and 13,300 in-hospital deaths and 45,700 overall deaths occur in infants <6 months of age each year, with the majority occurring in low- and middle-income countries (LMICs).1 The role of the RSV fusion protein (F) and attachment protein (G) in infection and disease have been studied extensively. Since the elucidation of the two distinct conformations of the RSV F protein (pre-fusion and post-fusion), the RSV the prefusion F (preF) protein has been the primary target for vaccine and monoclonal antibody development due to its highly conserved sequence among RSV strains and its essential role for RSV entry. These include the two recently approved products for the prevention of RSV disease in infants: the maternal bivalent RSV pre-fusion F vaccine Abrysvo™ (Pfizer), administered in the third trimester to protect infants in the first 6 months of life, and the long-acting monoclonal Beyfortus™, nirsevimab (Sanofi, Astra Zeneca), administered directly to infants early after birth.2,3 The two products have shown efficacy at reducing medically attended RSV lower respiratory tract infections in clinical trials and are instrumental in reducing severe RSV disease.4 However, the high cost of the long-acting monoclonal antibody and the signal of enhanced premature birth from the maternal vaccine need to be considered, especially in LMICs where access to healthcare for preterm birth may be limited. Vaccination with the RSVPreF3-Mat (GSK) resulted in significantly higher preterm birth rates compared to the placebo group (6.8% versus 4.9%), leading to its discontinuation. Increased preterm births associated with maternal vaccination, though not statistically significant, were also observed with Abrysvo™. This led some countries to adjust the vaccination timeline to 32–36 weeks of gestation to mitigate further risk.5,6

Due to its highly diverse sequence between RSV strains, the RSV G protein has been less extensively explored as a vaccine or monoclonal antibody candidate.7 However, the G protein plays a critical role in promoting disease pathogenesis and may be a suitable target for future preventative/therapeutic strategies. The RSV G protein is a type II transmembrane glycoprotein that has a central highly conserved region across RSV strains, flanked by two hypervariable mucin-like regions that are heavily glycosylated. This central conserved domain (CCD) contains a CX3C chemokine motif facilitating RSV attachment to cells bearing the CX3C chemokine receptor, CX3CR1, which is ubiquitously expressed on airway epithelial cells and immune cells.8, 9, 10, 11 The G protein can also be secreted from infected cells (soluble G protein) to act as a decoy for neutralising antibody responses and inhibit anti-viral responses to promote viral replication.12,13 Pre-clinical studies in mice have shown that monoclonal antibodies targeting the RSV G protein have shown promising results, enhancing antiviral responses, and reducing disease severity by inhibiting binding to CX3CR1.14 However, further research in this area is required as we have a limited understanding of the capacity of RSV to infect immune cells through CX3CR1 resulting in altered function and promoting pathogenesis.

This review will focus on how the RSV G protein promotes infection of immune cells and the consequence this has on pathogenesis. Although this information is tailored towards the infant population, this is likely to be relevant to older populations who are also at risk of severe RSV disease. Unravelling how infection of immune cells by RSV alters their function will be critical in understanding susceptibility to severe disease and will inform the development of future preventatives/therapeutics.

Virology

RSV is an enveloped, negative sense single-stranded RNA virus (ssRNA) that belongs to the Pneumoviridae family.7 RSV strains are classified into two distinct subgroups (RSV-A and RSV-B) based on epitope differences in the G protein.15 RSV encodes eleven proteins (Fig. 1 and Table 1) including three membrane-spanning envelope proteins: fusion (F), attachment (G) and the small hydrophobic (SH) protein, a surface transmembrane protein24 involved in delaying apoptosis through inhibition of the tumour necrosis factor alpha (TNF-α) signaling pathway.25 The matrix (M) protein stabilises the viral particle by forming a scaffold on the inside of the viral envelope. Furthermore, two nonstructural proteins (NS1 and NS2) are involved in promoting immune evasion through various mechanisms, including down-regulation of antiviral immunity.23 The RSV nucleocapsid (N) protein, phosphoprotein (P) and large polymerase (L) are essential for viral replication.7 Finally, M2.1 and M2.2 are expressed from overlapping open reading frames and have a role in regulating viral replication and transcription.21Fig. 1 Organisation of the RSV genome and G protein. The RSV genome consists of 10 genes with overlapping M2 and L genes encoding 11 proteins. These are the non-structural proteins 1 and 2 (NS), nucleocapsid protein (N), phosphoprotein (P), matrix protein (M2.1, M2.2), small hydrophobic protein (SH), G protein, fusion protein (F), M2.1 and M2.2 proteins and the large polymerase subunit L (L). For the RSV G protein, the cytosolic tail (CT), transmembrane domain (TMD), mucin-like regions I and II (MLR), central conserved domain (CCD), CX3C motif and heparin binding domain (HBD) are depicted.

Table 1 RSV proteins and their functions.

Protein	Function	Reference	
Fusion protein	Cellular entry through fusion with the host cell membrane	Walsh et al. 198316	
G protein	Cellular attachment through binding CX3CR1 which inhibits the anti-viral response, increases viral load and pulmonary inflammation	Levine et al. 198717
Chirkova et al. 201318	
Small hydrophobic protein	Delay apoptosis and promote viral replication	Fuentes et al. 200719	
Matrix protein	Virion formation	Mitra et al. 201220	
M2.1 and M2.2	Replication and transcription	Bermingham et al. 199921	
Nucleocapsid protein	Replication	Grosfeld et al. 199522	
Phosphoprotein	Replication	Grosfeld et al. 199522	
Large polymerase subunit L	Replication	Grosfeld et al. 199522	
Non-structural proteins	Inhibit type I IFN responses to reduce antiviral activity	Sedyn et al. 201923	

CX3CR1 and RSV infection

CX3CR1 is a G protein-coupled 7-transmembrane receptor that also binds the ligand fractalkine (CX3CL1), an important chemokine that promotes leukocyte migration. The RSV G protein contains a CX3C motif similar to CX3CL1, which competes with CX3CL1 for binding to its receptor to mediate viral infection (Fig. 1).26 In paediatric patients infected with RSV, CX3CR1 is present in both the upper and lower airways where CX3CR1+ cells are preferentially infected. This has been confirmed as blocking CX3CR1 resulted in a reduced viral load in vitro and in animal models. In cotton rats, mutating CX3CR1 and blocking CX3CR1 with an antibody prevents RSV replication in the lungs.27 The effects of the RSV G protein on viral load may be due in part to the inhibition of type I and type II IFNs by the CX3C motif following infection of airway epithelial cells.18 It is important to recognise that the interaction between the CX3C motif on the RSV G protein and CX3CR1 might not be the only mechanism driving infection of airway epithelial cells, as blocking CX3CR1 still leads to infection.8 Additionally, infection in cell lines still occurs in RSV mutants lacking the G protein, whilst the F protein remains essential to viral infection.28 The RSV G protein can also use heparan sulfate as a receptor to enter epithelial and immune cells as competitively binding cell surface heparan sulfate proteoglycans has been shown to reduce RSV infectivity of Hep-2 and A549 cell lines.24,29 These findings should however be interpreted with caution, as in contrast to cell lines, heparan sulfate binding appears to be less important in infection of more physiologically relevant primary differentiated epithelial cultures at air-liquid interface.30 This is because ciliated epithelial cells, which are the major cell type infected by RSV, poorly express heparan sulfate proteoglycans.31 As heparan sulfate binds many cytokines and chemokines to promote immune cell migration and activation, RSV binding may similarly limit the antiviral immune response, however studies have not investigated this in detail.32

The interaction between CX3CR1 and its ligand CX3CL1 communicates with many signaling pathways (JAK-STAT, toll-like receptor (TLR), MAPK and NF-κB) which are important in leukocyte chemotaxis and initiating inflammatory immune responses that are protective against RSV.33 Studies have shown that mutations in the CX3CR1 gene that reduce its affinity for CX3CL1 are associated with severe RSV disease.34 This has been supported by studies using CX3CR1-deficient mice, showing an increased pulmonary neutrophilic inflammation, and a bias towards IL-17+ γδ T cells which promotes severe RSV disease.35 During RSV infection, competitive binding of the G protein to CX3CR1 limits the interaction between CX3CR1 and CX3CL1 leading to reduced type I and type III IFN responses.36 Although CX3CR1 expression has mostly been linked to RSV infection of airway epithelial cells, it is also ubiquitously expressed on many immune cells such as dendritic cells (DCs), monocytes, natural killer (NK) cells, T cells and B cells.37

CX3CR1 and immune cells

While few studies have investigated the relationship between immune cell expression of CX3CR1 and RSV disease, many studies have elucidated the function of CX3CR1 on immune cells. In alveolar macrophages, CX3CR1 is required to facilitate clearance of eosinophilia.38 A murine study showed that interactions between CX3CR1 on monocytes and CX3CL1 is critical for cell survival and the absence of this interaction leads to increased cell death.9 CX3CR1 expression on human NK cells relates to fully mature NK cell phenotypes that are more cytotoxic.39 The expression of CX3CR1 has also been related to the differentiation of cytotoxic and antiviral CD8+ T cell responses.11 In B cells, the expression of CX3CR1 has been linked to the suppression of CD4+ T cell activation.10 In the context of Japanese encephalitis virus, CX3CR1 expression on mouse DCs is important in generating effective antiviral NK cell and antigen-specific T cell responses to reduce disease severity.40

The important role that CX3CR1 has on immune cell function is of interest to RSV research as its natural interaction with CX3CL1 can be inhibited by the RSV G protein. Therefore, infection of immune cells by RSV is likely to impair antiviral activity and promote disease pathogenesis.

Infection of immune cells by RSV

The expression of CX3CR1 on immune cells suggests that RSV has the potential to infect all immune cells to promote pathogenesis (Fig. 2 and Table 2). However, comprehensive studies evaluating how RSV reprograms immune cells both functionally and mechanistically are limited.Fig. 2 Infection of immune cells by RSV. This likely occurs through the interaction between the CX3C motif of the RSV G protein and CX3CR1 which is ubiquitously expressed on immune cells. RSV infection of immune cells reduces the antiviral response which promotes severe disease through increased pulmonary inflammation and increased viral load.

Table 2 Immune cells infected by RSV/A in human studies.

Immune cell	In vitro or In vivo	Abortive or productive	Reference	
Alveolar macrophage	In vitro	Productive	Panuska et al. 199041	
Monocytes	In vitro	Not studied	Midulla et al. 198942	
Dendritic cells	In vitro	Not studied	Johnson et al. 201143	
NK cells	In vitro	Abortive	Van Erp et al. 201944	
T cells	In vitro and in vivo	Productive	Raiden et al. 201745	
B cells	In vitro and in vivo	Not studied	Zhivaki et al. 201746	

Alveolar macrophages and monocytes

Alveolar macrophages are critical in phagocytosis, antigen presentation and limiting viral replication through type I IFN production during early RSV infection. These cells also promote the recruitment of inflammatory monocytes into the lung to prevent severe lung inflammation.47 One of the earliest studies to investigate RSV infection of immune cells showed that RSV can productively infect alveolar macrophages. For 25 days post-infection of alveolar macrophages, infectious RSV was released into supernatants.41 However, whether productive infection of alveolar macrophages influenced their antiviral capacity was not studied.

Similar to alveolar macrophages, monocytes engage in phagocytosis, antigen presentation and the release of pro-inflammatory cytokines early during RSV infection. When comparing the capacity of RSV to infect cord blood or peripheral blood monocytes, it was found that cord blood-derived monocytes were more susceptible to infection. While cellular function was not assessed in infected monocytes, it was found that secreted IFN-γ and TNF, in response to LPS stimulation, were able to reduce monocyte infection by RSV.42 However, it has been shown that monocytes exposed to the CX3C motif in the G protein produce less TNF-α, promoting viral infection.18

Dendritic cells

DCs are often classified into conventional dendritic cells (cDCs) or plasmacytoid dendritic cells (pDCs). Following RSV exposure, cDCs undergo maturation through the upregulation of CD80 and CD86 co-stimulatory molecules. This promotes antigen presentation which activates virus-specific T cell responses.48 The infection rate is relatively low in cDCs, with ∼15% of cells being RSV+ following infection with RSV-GFP in vitro.43,49 However, when cDCs are infected by RSV they upregulate IL-10 which leads to the inhibition of CD4+ T cell proliferation, subsequently reducing antiviral responses.50 One study comparing the capacity of RSV to infect adult peripheral blood and cord blood derived cDCs showed a similar level of infection and similar gene expression profile. Notably, a relatively weak type I IFN response was seen in RSV-infected cDCs, from both cord blood and adult blood, possibly due to the NS1/2 proteins which inhibit this response.49 While not required for infection of cDCs, the RSV G protein binds to DC-SIGN and L-SIGN which inhibits the activation and maturation of cDCs resulting in reduced IFN-α production.51

During RSV infection, pDCs are major producers of IFN-α which limits viral replication and controls infection during the early response phase. Infection of pDCs by RSV occurs in ∼5% of pDCs in vitro.43 Infected pDCs show increased expression of CD86 and CD40, however, no data concerning cytokine differences between infected and non-infected pDCs was explored.43 While not directly related to infection of pDCs, pDCs stimulated with a CX3C mutant virus (CX4C) produced higher amounts of IFN-α, suggesting that the CX3C interaction with pDCs inhibits IFN-α production.18 Similar to cDCs, the activation of pDCs can be inhibited through binding of RSV to DC-SIGN and L-SIGN reducing the production of IFN-α.51

Some studies have also focused on the infection of monocyte-derived dendritic cells (moDCs) by RSV. These cells can be classified as immunogenic or tolerogenic. Immunogenic moDCs promote phagocytosis, antigen presentation and secrete many pro-inflammatory cytokines (e.g. IL-6, IL-23, TNF-α), while tolerogenic moDCs produce IL-10 to promote Treg expansion.52 It has been shown that moDCs can be infected by RSV.53 Infection of moDCs by RSV leads to a reduced capacity to stimulate CD4+ T cell proliferation and cells induced by RSV-infected moDCs exhibited an impaired Th1 and Th2 response.54

NK cells

NK cells contribute to the cytotoxic lysis of RSV-infected cells through granzyme B and perforin secretion and further limit viral replication through IFN-γ production.44 It has been demonstrated that RSV can infect and replicate within NK cells, however this does not lead to the release of infectious viral particles.44 While RSV infection of NK cells did induce IFN-γ production, it was found that RSV infection of NK cells did not enhance cytotoxicity, suggesting a selective inhibitory effect of RSV infection. Interestingly, the capacity of RSV to infect NK cells was similar between cord blood and adult peripheral blood-derived NK cells, although considerable variation between donors was observed.44 While increased IFN-γ may be beneficial, it is also important to recognise that elevated IFN-γ has been associated with RSV bronchiolitis in infants.55

T cells

T cells play a diverse role in the pathogenesis of RSV from viral clearance to helping the generation of antibody responses. Efficient clearance of RSV is driven by an effective Th1 response, while skewing towards a Th2 type response has been suggested to promote disease severity and longer-term adverse lung health outcomes.56 Th17 cells have been thought to contribute to disease severity by acting synergistically with Th2, while Tregs are thought to limit pulmonary inflammation and limit disease progression.57 The contribution of CD8+ T cells is also important in viral clearance as they directly lyse virus-infected cells through perforin and granzyme B and produce IFN-γ and TNF-α.57,58

Murine infection studies have shown that infection with mutant RSV lacking the G protein or CX3C motif is associated with increased CX3CR1+ T cell migration to the lung, increasing cytotoxic and IFN-γ expressing T cells compared to infection with the wildtype.59 Similarly, in peripheral blood mononuclear cells (PBMCs), the CX3C motif inhibits the production of IFN-γ from memory CD4+ T cells.18 It has been shown that T cells are susceptible to infection by RSV and this infection can lead to the infection of human epithelial cells in vitro.45 The amount of infected T cells by RSV has been shown to differ in cord blood and peripheral blood from adults and children, with cord blood being more susceptible. Additionally, RSV+ T cells produced less IL-2 and IFN-γ and exhibited decreased CD25 expression compared to RSV and UV-irradiated RSV conditions.45 In peripheral blood from RSV-infected infants with severe bronchiolitis, RSV+ CD4+ T cells were found, and this was associated with disease severity.45

B cells

B cells are primarily responsible for the production of antibodies against RSV infection. Neutralising immunoglobulin G (IgG) is correlated with protection against RSV and are the basis of efficacious vaccines and monoclonal antibodies.60 It has been demonstrated that RSV also infected neonatal regulatory B cells (nBreg) in infants.46 nBregs exhibit immunosuppressive activity by producing IL-10 to reduce Th1 activation. This was demonstrated following RSV stimulation of nBregs which led to a high production of IL-10. RSV can infect up to 50% of nBregs in vitro, and RSV+ nBregs, but not UV-inactivated Bregs, upregulate IL-10 production. This infection is thought to occur through CX3CR1 on nBregs, which is upregulated only after RSV exposure. The importance of the G protein in promoting infection of nBregs was demonstrated by the finding that G protein mutants exhibited a poor capacity to infect these cells. This is critical as infection of nBregs was associated with severe RSV bronchiolitis in infants under 3 months of age.46 Further studies in mice have shown that nBregs colonise the lungs during RSV infection and secrete IL-10 which inhibits type I IFN production by alveolar macrophages.61

Targeting the G protein

The G protein has been known to induce Th2-biased immune responses. Potent Th2 responses, such as those induced by the formalin-inactivated RSV vaccine, led to an 80% hospitalisation rate following infection in vaccinated infants.62 Mice primed with vaccinia virus expressing secreted RSV G protein produced high levels of IL-5 and IL-13 mRNA and induced pulmonary eosinophilia which enhanced disease severity.63,64 Additionally, mice infected with recombinant vaccinia virus expressing the G protein produced higher levels of IL-4 compared to mice infected with the same viral vector expressing the F protein.65 Considering the detrimental effect of a Th2 bias in RSV disease severity, and the capacity of the RSV G protein to suppress protective inflammatory responses, targeting the G protein may be beneficial strategy in reducing the burden of disease. However, a proper understanding of the mechanisms underlying the role of the RSV G protein in pathogenesis is pivotal for the rational design of safe and effective interventions.

Vaccines

The high genetic variance of the G protein has made it a difficult protein to target for vaccine development. Soluble G protein from infected cells acts as a decoy for neutralising antibody responses further reducing the efficacy of G protein vaccination strategies.66 Another consideration is that targeting the G protein does not entirely prevent infection of epithelial cells. However, it has been shown that infants hospitalised with RSV disease develop a robust G specific humoral response.67 Targeting the CCD of the RSV G protein with a recombinant protein vaccine (BBG2Na) in healthy adults led to the generation of a moderate neutralising antibody response, however, the vaccine did not progress due to safety concerns.68 The phase-I human trial with the BARS13 vaccine (recombinant G protein vaccine) led to an increase in anti-CCD neutralising antibodies 60 days post vaccination compared to the placebo and a suppression of over-reactive CD4+ T cells thought to induce vaccine enhanced disease. Additionally, as no serious adverse events were recorded this vaccine may be further explored in future clinical trials.69 A recent study using a bivalent vaccine targeting the central conserved domain of the G protein and a stabilised pre-fusion-F nanoparticle in macaques and mice have shown that this can induce potent neutralising IgG titres, leading to a significantly reduced viral load.70 However, viral load did not differ between the bivalent vaccine and monovalent pre-F vaccine. Another study in mice vaccinated with an RSV A2 recombinant unglycosylated G protein ectodomain vaccine, found a reduction in pulmonary viral load without negatively impacting the inflammatory response.71 Similarly, in a cotton rat model, bivalent immunisation against the RSV G and pre-fusion F protein with a virus-like particle vaccine led to higher levels of IgG neutralising antibodies and a reduced viral titre compared to monovalent F protein vaccination.72 In mice, mutating the CX3C motif to CX4C which impairs RSV G protein binding to CX3CR1, resulted in a reduction in pulmonary inflammation, and an increase in IFN-α, IFN-β and IFN-γ production which are critical for the antiviral response.73 This was suggested as an effective method to improve the development of live attenuated RSV vaccines. TLR2 and TLR6 have been shown to recognise the RSV G protein to initiate an innate immune response. Mice vaccinated with a fusion peptide containing the CX3C motif and a CD8 epitope of the RSV matrix protein 2 with the addition of a TLR2 agonist had a reduced Th2 biased immune response shown through the reduction of IL-4, IL-5, IL-13, and eosinophils. Additionally, they had an increased secretion of RSV G-specific IgG2a and lower pulmonary viral loads.74

Monoclonal antibodies

Alternative strategies have also aimed to target the G protein as a post-infection treatment (Fig. 3). Strategies targeting the G protein are effective due to their inhibition of CX3C-CX3CR1 while maintaining interaction with CX3CL1, which is required for protective inflammatory responses.75 Monoclonal antibody studies in mice targeting the G protein have shown that this can successfully reduce the infection and replication of RSV and limit disease severity by reducing inflammation.14 Two high-affinity broadly neutralising antibodies to the RSV G CCD, 3D3 and 2D10, have been shown to reduce RSV G protein induced chemotaxis by blocking interactions between the RSV CX3C motif and CX3CR1. Importantly, this interaction had no effect on chemotaxis induced by CX3CL1.76 Another study in mice using the 3D3 and 2D10 anti-RSV G protein human monoclonal antibodies showed a reduction in pulmonary viral titre. Blocking of the CX3C-CX3CR1 interaction by 3D3 led to an increase in type I IFNs, promoting antiviral activity.36 Prophylactic treatment with anti-G protein monoclonal antibodies in mice have been shown to switch the T cell response from a Th2 bias to a Th1 bias. This was demonstrated by a shift from IgG1 and IgG2b antibody production to IgG2a which favours a Th1 response. Additionally, a decrease in IL-4+ T cells and an increase in IFN-γ+ T cells was identified in these mice.77Fig. 3 Blocking RSV infection of immune cells by anti-G protein monoclonal antibodies. Blocking the interaction between CX3C and CX3CR1 allows CX3CL1 to bind more efficiently and promote antiviral inflammatory responses. This results in increased IFN-α and IFN-γ production, reduced Th2 cytokines (IL-4, IL-5 and IL-13) which reduces pulmonary inflammation, viral load and disease severity.

The demonstrated effectiveness of monoclonal antibodies against the RSV G protein by reducing viral load and promoting protective inflammatory responses in small animal models suggests further research into its efficacy in humans as a post-infection treatment is warranted. Additionally, by targeting a highly conserved domain, it is unlikely that escape mutants would result following introduction of monoclonal antibodies targeting the G protein, especially in the context of limited therapeutic use.78

Conclusion

RSV remains a significant contributor to the global burden of disease experienced by infants and children. Understanding its pathogenesis on a mechanistic level is critical to developing therapeutics to reduce the global burden of disease. The CX3C motif on the G protein of RSV plays a significant role in pathogenesis, allowing RSV to infect and reprogram airway epithelial cells and immune cells. Reprogramming of immune cells to reduce their antiviral function promotes disease severity and further exploration of this topic is needed. The high cost of the newly developed RSV long-acting monoclonal will likely lead to poor access in LMICs where severe disease is most prevalent. Alternative strategies such as novel therapeutics targeting the G protein may be highly valuable especially in a post-infection situation.

Outstanding questions

To what extent does infection of immune cells by RSV impact anti-viral immunity and how does this translate into clinical outcomes?

Will monoclonal antibodies and vaccines targeting the G protein be effective therapeutics in human studies?

If vaccines or monoclonal antibodies targeting the G protein become available, will mutations in the CCD of the G protein arise?

Will a combination of therapeutics targeting the F and G protein be more beneficial than either alone?

Contributors

JA and PVL conceived the idea for this manuscript. JA prepared the initial draft with contributions from PBVK, LAHD and PVL. All authors provided important intellectual input and critically revised the manuscript. All authors approve the final version of this manuscript to be submitted.

Declaration of interests

All authors declare they have no competing interests.

Acknowledgements

JA is supported by an Australian 10.13039/501100000925 NHMRC Emerging Leadership Investigator Grant. This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. Figures were created using BioRender.com.
==== Refs
References

1 Li Y. Wang X. Blau D.M. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in children younger than 5 years in 2019: a systematic analysis Lancet 399 10340 2022 2047 2064 35598608
2 Hammitt L.L. Dagan R. Yuan Y. Nirsevimab for prevention of RSV in healthy late-preterm and term infants N Engl J Med 386 9 2022 837 846 35235726
3 Harris E. FDA approves maternal RSV vaccine JAMA 330 11 2023 1029
4 Hodgson D. Wilkins N. van Leeuwen E. Protecting infants against RSV disease: an impact and cost-effectiveness comparison of long-acting monoclonal antibodies and maternal vaccination Lancet Reg Health Eur 38 2024 100829
5 Dieussaert I. Hyung Kim J. Luik S. RSV prefusion F protein-based maternal vaccine - preterm birth and other outcomes N Engl J Med 390 11 2024 1009 1021 38477988
6 Kampmann B. Madhi S.A. Munjal I. Bivalent prefusion F vaccine in pregnancy to prevent RSV illness in infants N Engl J Med 388 16 2023 1451 1464 37018474
7 Collins P.L. Fearns R. Graham B.S. Respiratory syncytial virus: virology, reverse genetics, and pathogenesis of disease Curr Top Microbiol Immunol 372 2013 3 38 24362682
8 Anderson C.S. Chu C.Y. Wang Q. CX3CR1 as a respiratory syncytial virus receptor in pediatric human lung Pediatr Res 87 5 2020 862 867 31726465
9 Landsman L. Bar-On L. Zernecke A. CX3CR1 is required for monocyte homeostasis and atherogenesis by promoting cell survival Blood 113 4 2009 963 972 18971423
10 Wu Z. CX3CR1(+) B cells show immune suppressor properties J Biol Chem 289 33 2014 22630 22635 24970890
11 Yamauchi T. Hoki T. Oba T. T-cell CX3CR1 expression as a dynamic blood-based biomarker of response to immune checkpoint inhibitors Nat Commun 12 1 2021 1402 33658501
12 Shingai M. Azuma M. Ebihara T. Soluble G protein of respiratory syncytial virus inhibits Toll-like receptor 3/4-mediated IFN-beta induction Int Immunol 20 9 2008 1169 1180 18611945
13 Arnold R. König B. Werchau H. König W. Respiratory syncytial virus deficient in soluble G protein induced an increased proinflammatory response in human lung epithelial cells Virology 330 2 2004 384 397 15567433
14 Boyoglu-Barnum S. Todd S.O. Chirkova T. An anti-G protein monoclonal antibody treats RSV disease more effectively than an anti-F monoclonal antibody in BALB/c mice Virology 483 2015 117 125 25965801
15 Yu J.M. Fu Y.H. Peng X.L. Zheng Y.P. He J.S. Genetic diversity and molecular evolution of human respiratory syncytial virus A and B Sci Rep 11 1 2021 12941
16 Walsh E.E. Hruska J. Monoclonal antibodies to respiratory syncytial virus proteins: identification of the fusion protein J Virol 47 1 1983 171 177 6345804
17 Levine S. Klaiber-Franco R. Paradiso P.R. Demonstration that glycoprotein G is the attachment protein of respiratory syncytial virus J Gen Virol 68 Pt 9 1987 2521 2524 3655746
18 Chirkova T. Boyoglu-Barnum S. Gaston K.A. Respiratory syncytial virus G protein CX3C motif impairs human airway epithelial and immune cell responses J Virol 87 24 2013 13466 13479 24089561
19 Fuentes S. Tran K.C. Luthra P. Teng M.N. He B. Function of the respiratory syncytial virus small hydrophobic protein J Virol 81 15 2007 8361 8366 17494063
20 Mitra R. Baviskar P. Duncan-Decocq R.R. Patel D. Oomens A.G. The human respiratory syncytial virus matrix protein is required for maturation of viral filaments J Virol 86 8 2012 4432 4443 22318136
21 Bermingham A. Collins P.L. The M2-2 protein of human respiratory syncytial virus is a regulatory factor involved in the balance between RNA replication and transcription Proc Natl Acad Sci U S A 96 20 1999 11259 11264 10500164
22 Grosfeld H. Hill M.G. Collins P.L. RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA J Virol 69 9 1995 5677 5686 7637014
23 Sedeyn K. Schepens B. Saelens X. Respiratory syncytial virus nonstructural proteins 1 and 2: exceptional disrupters of innate immune responses PLoS Pathog 15 10 2019 e1007984
24 McLellan J.S. Ray W.C. Peeples M.E. Structure and function of respiratory syncytial virus surface glycoproteins Curr Top Microbiol Immunol 372 2013 83 104 24362685
25 Ivancic-Jelecki J. Slovic A. Ljubin-Sternak S. Mlinarić Galinović G. Forcic D. Variability analysis and inter-genotype comparison of human respiratory syncytial virus small hydrophobic gene Virol J 15 1 2018 109 30021648
26 Tripp R.A. Jones L.P. Haynes L.M. Zheng H. Murphy P.M. Anderson L.J. CX3C chemokine mimicry by respiratory syncytial virus G glycoprotein Nat Immunol 2 8 2001 732 738 11477410
27 Green G. Johnson S.M. Costello H. CX3CR1 is a receptor for human respiratory syncytial virus in cotton rats J Virol 95 16 2021 e0001021
28 Techaarpornkul S. Barretto N. Peeples M.E. Functional analysis of recombinant respiratory syncytial virus deletion mutants lacking the small hydrophobic and/or attachment glycoprotein gene J Virol 75 15 2001 6825 6834 11435561
29 Donalisio M. Rusnati M. Cagno V. Inhibition of human respiratory syncytial virus infectivity by a dendrimeric heparan sulfate-binding peptide Antimicrob Agents Chemother 56 10 2012 5278 5288 22850525
30 Kwilas S. Liesman R.M. Zhang L. Walsh E. Pickles R.J. Peeples M.E. Respiratory syncytial virus grown in vero cells contains a truncated attachment protein that alters its infectivity and dependence on glycosaminoglycans J Virol 83 20 2009 10710 10718 19656891
31 Cagno V. Tseligka E.D. Jones S.T. Tapparel C. Heparan sulfate proteoglycans and viral attachment: true receptors or adaptation bias? Viruses 11 7 2019 596 31266258
32 Simon Davis D.A. Parish C.R. Heparan sulfate: a ubiquitous glycosaminoglycan with multiple roles in immunity Front Immunol 4 2013 470 24391644
33 Zhuang Q. Ou J. Zhang S. Ming Y. Crosstalk between the CX3CL1/CX3CR1 axis and inflammatory signaling pathways in tissue injury Curr Protein Pept Sci 20 8 2019 844 854 30843484
34 Amanatidou V. Sourvinos G. Apostolakis S. Tsilimigaki A. Spandidos D.A. T280M variation of the CX3C receptor gene is associated with increased risk for severe respiratory syncytial virus bronchiolitis Pediatr Infect Dis J 25 5 2006 410 414 16645504
35 Das S. Raundhal M. Chen J. Respiratory syncytial virus infection of newborn CX3CR1-deficient mice induces a pathogenic pulmonary innate immune response JCI Insight 2 17 2017 e94605
36 Bergeron H.C. Kauvar L.M. Tripp R.A. Anti-G protein antibodies targeting the RSV G protein CX3C chemokine region improve the interferon response Ther Adv Infect Dis 10 2023 20499361231161157
37 Lee M. Lee Y. Song J. Lee J. Chang S.Y. Tissue-specific role of CX(3)CR1 expressing immune cells and their relationships with human disease Immune Netw 18 1 2018 e5
38 Lamkioued B. Gounni A.S. CX3CR1(+) alveolar macrophages: a new player in lung eosinophil homeostasis Am J Respir Crit Care Med 207 11 2023 1413 1415 37053469
39 Hamann I. Unterwalder N. Cardona A.E. Analyses of phenotypic and functional characteristics of CX3CR1-expressing natural killer cells Immunology 133 1 2011 62 73 21320123
40 Choi J.Y. Kim J.H. Hossain F.M.A. Indispensable role of CX(3)CR1(+) dendritic cells in regulation of virus-induced neuroinflammation through rapid development of antiviral immunity in peripheral lymphoid tissues Front Immunol 10 2019 1467 31316515
41 Panuska J.R. Cirino N.M. Midulla F. Despot J.E. McFadden E.R. Jr. Huang Y.T. Productive infection of isolated human alveolar macrophages by respiratory syncytial virus J Clin Invest 86 1 1990 113 119 2365811
42 Midulla F. Huang Y.T. Gilbert I.A. Cirino N.M. McFadden E.R. Jr. Panuska J.R. Respiratory syncytial virus infection of human cord and adult blood monocytes and alveolar macrophages Am Rev Respir Dis 140 3 1989 771 777 2476959
43 Johnson T.R. Johnson C.N. Corbett K.S. Edwards G.C. Graham B.S. Primary human mDC1, mDC2, and pDC dendritic cells are differentially infected and activated by respiratory syncytial virus PLoS One 6 1 2011 e16458
44 van Erp E.A. Feyaerts D. Duijst M. Respiratory syncytial virus infects primary neonatal and adult natural killer cells and affects their antiviral effector function J Infect Dis 219 5 2019 723 733 30252097
45 Raiden S. Sananez I. Remes-Lenicov F. Respiratory syncytial virus (RSV) infects CD4+ T cells: frequency of circulating CD4+ RSV+ T cells as a marker of disease severity in young children J Infect Dis 215 7 2017 1049 1058 28199704
46 Zhivaki D. Lemoine S. Lim A. Respiratory syncytial virus infects regulatory B cells in human neonates via chemokine receptor CX3CR1 and promotes lung disease severity Immunity 46 2 2017 301 314 28228284
47 Goritzka M. Makris S. Kausar F. Alveolar macrophage-derived type I interferons orchestrate innate immunity to RSV through recruitment of antiviral monocytes J Exp Med 212 5 2015 699 714 25897172
48 Jung H.E. Kim T.H. Lee H.K. Contribution of dendritic cells in protective immunity against respiratory syncytial virus infection Viruses 12 1 2020 102 31952261
49 Le Nouën C. Hillyer P. Levenson E. Lack of activation marker induction and chemokine receptor switch in human neonatal myeloid dendritic cells in response to human respiratory syncytial virus J Virol 93 22 2019 e01216 e01219
50 Gupta M.R. Kolli D. Garofalo R.P. Differential response of BDCA-1+ and BDCA-3+ myeloid dendritic cells to respiratory syncytial virus infection Respir Res 14 1 2013 71 23829893
51 Johnson T.R. McLellan J.S. Graham B.S. Respiratory syncytial virus glycoprotein G interacts with DC-SIGN and L-SIGN to activate ERK1 and ERK2 J Virol 86 3 2012 1339 1347 22090124
52 Zhao Y. Gao C. Liu L. Wang L. Song Z. The development and function of human monocyte-derived dendritic cells regulated by metabolic reprogramming J Leukoc Biol 114 3 2023 212 222 37232942
53 Jones A. Morton I. Hobson L. Evans G.S. Everard M.L. Differentiation and immune function of human dendritic cells following infection by respiratory syncytial virus Clin Exp Immunol 143 3 2006 513 522 16487251
54 de Graaff P.M. de Jong E.C. van Capel T.M. Respiratory syncytial virus infection of monocyte-derived dendritic cells decreases their capacity to activate CD4 T cells J Immunol 175 9 2005 5904 5911 16237083
55 Thwaites R.S. Ito K. Chingono J.M.S. Nasosorption as a minimally invasive sampling procedure: mucosal viral load and inflammation in primary RSV bronchiolitis J Infect Dis 215 8 2017 1240 1244 28368490
56 Anderson J. Do L.A.H. Wurzel D. Severe respiratory syncytial virus disease in preterm infants: a case of innate immaturity Thorax 76 9 2021 942 950 33574121
57 Russell C.D. Unger S.A. Walton M. Schwarze J. The human immune response to respiratory syncytial virus infection Clin Microbiol Rev 30 2 2017 481 502 28179378
58 Jozwik A. Habibi M.S. Paras A. RSV-specific airway resident memory CD8+ T cells and differential disease severity after experimental human infection Nat Commun 6 2015 10224
59 Harcourt J. Alvarez R. Jones L.P. Henderson C. Anderson L.J. Tripp R.A. Respiratory syncytial virus G protein and G protein CX3C motif adversely affect CX3CR1+ T cell responses J Immunol 176 3 2006 1600 1608 16424189
60 Wilkins D. Yuan Y. Chang Y. Durability of neutralizing RSV antibodies following nirsevimab administration and elicitation of the natural immune response to RSV infection in infants Nat Med 29 5 2023 1172 1179 37095249
61 Laubreton D. Drajac C. Eléouët J.F. Regulatory B lymphocytes colonize the respiratory tract of neonatal mice and modulate immune responses of alveolar macrophages to RSV infection in IL-10-dependant manner Viruses 12 8 2020 822 32751234
62 Kim H.W. Canchola J.G. Brandt C.D. Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine Am J Epidemiol 89 4 1969 422 434 4305198
63 Johnson T.R. Graham B.S. Secreted respiratory syncytial virus G glycoprotein induces interleukin-5 (IL-5), IL-13, and eosinophilia by an IL-4-independent mechanism J Virol 73 10 1999 8485 8495 10482601
64 Johnson T.R. Johnson J.E. Roberts S.R. Wertz G.W. Parker R.A. Graham B.S. Priming with secreted glycoprotein G of respiratory syncytial virus (RSV) augments interleukin-5 production and tissue eosinophilia after RSV challenge J Virol 72 4 1998 2871 2880 9525607
65 Alwan W.H. Openshaw P.J. Distinct patterns of T- and B-cell immunity to respiratory syncytial virus induced by individual viral proteins Vaccine 11 4 1993 431 437 8470427
66 Graham B.S. Biological challenges and technological opportunities for respiratory syncytial virus vaccine development Immunol Rev 239 1 2011 149 166 21198670
67 Nziza N. Jung W. Mendu M. Longitudinal humoral analysis in RSV-infected infants identifies pre-existing RSV strain-specific G and evolving cross-reactive F antibodies Immunity 57 7 2024 1681 1695.e4 38876099
68 Power U.F. Nguyen T.N. Rietveld E. Safety and immunogenicity of a novel recombinant subunit respiratory syncytial virus vaccine (BBG2Na) in healthy young adults J Infect Dis 184 11 2001 1456 1460 11709789
69 Cheng X. Zhao G. Dong A. A first-in-human trial to evaluate the safety and immunogenicity of a G protein-based recombinant respiratory syncytial virus vaccine in healthy adults 18-45 Years of age Vaccines (Basel) 11 5 2023 999 37243103
70 Rainho-Tomko J.N. Pavot V. Kishko M. Immunogenicity and protective efficacy of RSV G central conserved domain vaccine with a prefusion nanoparticle NPJ Vaccines 7 1 2022 74 35773301
71 Fuentes S. Coyle E.M. Golding H. Khurana S. Nonglycosylated G-protein vaccine protects against homologous and heterologous respiratory syncytial virus (RSV) challenge, while glycosylated G enhances RSV lung pathology and cytokine levels J Virol 89 16 2015 8193 8205 26018164
72 McGinnes Cullen L. Luo B. Wen Z. Zhang L. Durr E. Morrison T.G. The respiratory syncytial virus (RSV) G protein enhances the immune responses to the RSV F protein in an enveloped virus-like particle vaccine candidate J Virol 97 1 2023 e0190022
73 Boyoglu-Barnum S. Todd S.O. Meng J. Mutating the CX3C motif in the G protein should make a live respiratory syncytial virus vaccine safer and more effective J Virol 91 10 2017 e02059 28275196
74 Powell T.J. Jacobs A. Tang J. Microparticle RSV vaccines presenting the G protein CX3C chemokine motif in the context of TLR signaling induce protective Th1 immune responses and prevent pulmonary eosinophilia post-challenge Vaccines (Basel) 10 12 2022 2078 36560488
75 Bergeron H.C. Murray J. Nuñez Castrejon A.M. DuBois R.M. Tripp R.A. Respiratory syncytial virus (RSV) G protein vaccines with central conserved domain mutations induce CX3C-CX3CR1 blocking antibodies Viruses 13 2 2021 352 33672319
76 Fedechkin S.O. George N.L. Wolff J.T. Kauvar L.M. DuBois R.M. Structures of respiratory syncytial virus G antigen bound to broadly neutralizing antibodies Sci Immunol 3 21 2018 eaar3534
77 Boyoglu-Barnum S. Chirkova T. Todd S.O. Prophylaxis with a respiratory syncytial virus (RSV) anti-G protein monoclonal antibody shifts the adaptive immune response to RSV rA2-line19F infection from Th2 to Th1 in BALB/c mice J Virol 88 18 2014 10569 10583 24990999
78 Tripp R.A. Power U.F. Openshaw P.J.M. Kauvar L.M. Respiratory syncytial virus: targeting the G protein provides a new approach for an old problem J Virol 92 3 2018 e01302 e01317 29118126
