According to immune network theory, the adaptive immune system is regulated by interactions between idiotypes. The base of the Y plays a role in modulating immune cell activity. This region is called the Fc Fragment, crystallizable region, and is composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins the Fc region ensures that each antibody generates an appropriate immune response for a given antigen.

The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. By doing so, it mediates different physiological effects including opsonization, cell lysis, and degranulation of mast cells, basophils and eosinophils.

A single-chain variable fragment scFv is a fusion protein of the variable regions of the heavy VH and light chains VL of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa.

This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. These molecules were created to facilitate phage display , where it is highly convenient to express the antigen-binding domain as a single peptide.

As an alternative, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. ScFvs have many uses, e. Unlike monoclonal antibodies, which are often produced in mammalian cell cultures, scFvs are more often produced in bacteria cell cultures such as E. The fragment antigen-binding Fab fragment is a region on an antibody that binds to antigens.

It is composed of one constant and one variable domain of each of the heavy and the light chain. The two variable domains bind the epitope on their specific antigens. View All pathways. Protocols References Download Center. Customers outside the US, please order from local distributors. Relevance The fragment crystallizable region Fc region is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system.

This property allows antibodies to activate the immune system. InIgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains; IgM and IgE Fc regions contain three heavy chain constant domains CH domains 2—4 in each polypeptide chain.

The Fc regions of IgGs bear a highly conserved N-glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity. The N-glycans attached to this site are predominantly core-fucosylated diantennary structures of the complex type. Fc-mediated antibody effector functions play an important role in shaping the immune response and their active regulation is crucial to prevent excessive immune activation. A number of determinants have been found to influence Fc-mediated effector functions on both the cellular and antibody side of the Fc-Fc receptor FcR interaction.

Important antibody characteristics are the isotype, subclass, glycosylation pattern, and antigen specificity, while important cellular determinants are the epitope position relative to the target cell membrane and FcR expression and polymorphisms on the effector cell, which together determine the capacity of the antibody to interact with specific FcRs.

Most antibodies are not specifically eliciting a single effector function, and therefore the combination of all these characteristics determines the outcome of the various Fc-FcR interactions and the interaction with the complement system. Antibodies consist of two functional domains: the variable antigen-binding fragment Fab and the constant fragment Fc that interacts with FcRs and C1q. Whereas, the majority of antibodies in serum are of the IgG subtype, IgA is the major isotype present in mucosal secretions.

Other important isotypes to briefly mention are IgM, which is a potent complement activator 76 , and IgE, which has been linked to various allergic diseases.

Production of different isotypes and subclasses is tightly regulated and dependent on differentiation of the B cell, which can be influenced by cytokines and interactions with pattern-recognition receptors. The response to protein antigens usually involves T cell help and induces class switching to IgG1 or IgG3, whereas polysaccharide antigens induce class switching to IgG2 in the absence of T cell help In contrast, receptor-blocking antibodies are often of the IgG2 or IgG4 subclass to avoid Fc-mediated cytotoxic side effects RV recipients produced highly functional IgG3 antibodies that provided Severe RSV-mediated disease is most prevalent in infants below 6 months of age.

These children mainly rely on maternally-derived IgG for protection against infectious diseases, but the correlation between serum IgG levels and protection against RSV disease is poor 7 , 9 — A recent study by Habibi et al.

In accordance with these results, lower levels of nasal IgA were found in naturally RSV-infected adults compared to healthy controls However, it is questionable whether IgA plays a role in protection or disease during primary infection. IgA antibodies to RSV are only found in secretions after 4 months of age, confirming they are synthesized as a result of primary infection RSV-specific IgA has been shown to induce antibody-mediated effector functions.

Although palivizumab-IgA demonstrated slightly higher lysis of RSV-infected HEp2 cells by neutrophils but not monocytes in vitro , there was a somewhat decreased efficacy in vivo compared to palivizumab-IgG No further research with RSV-IgA immune complexes has been published to date and therefore their role in protection or disease remains to be investigated. Another interesting isotype is IgE, as the results from multiple studies suggest the involvement of this isotype in the development of RSV-mediated bronchiolitis and wheezing — Since all infants produce IgE in response to RSV infection , it is thought that the height and duration of the IgE response are important for the induction of subsequent immunopathology , , In addition to studies on isotypes, extensive studies have been performed on the presence of IgG subclasses during RSV infection.

Wagner et al. RSV infection led to a poor IgG4 antibody response in all subjects. This difference was thought to be due to the extensive glycosylation of the G protein, resulting in IgG2 antibodies. A recent study confirms the findings of Wagner et al. Besides human studies, several mouse studies have been performed to investigate the subclass antibody response.

Although some homology between mouse and human IgG subclasses has been found, it is unclear whether they induce the same downstream immune responses. These results indicate that both the age of the host and the antigens determine the subclass response. Thus, caution is warranted in the translation between human and mouse antibody studies. Although extensive studies have been performed on the presence of specific subclasses, evidence on the role of these different subclasses during RSV infection is limited.

One study describes a direct comparison between the functionality of palivizumab-IgG1 and -IgG2 The neutralizing potential of both subclasses was comparable. This finding underscores the protective potential of IgG1-mediated effector functions during RSV infection. Glycosylation of the antibody Fc domain is another important regulator of Fc-mediated effector functions. Each IgG molecule contains a highly conserved asparagine at position N that functions as a glycosylation site that can harbor a variety of glycans, consisting of varying combinations of mannose, bisecting N-acetylglycosamine GlcNAc , fucose, galactose, and sialic acid Figure 8.

Figure 8. Antibody glycosylation. Each IgG molecule contains a glycosylation site that can harbor a variety of glycans, consisting of varying combinations of mannose, bisecting N-acetylglycosamine, fucose, galactose, and sialic acid. Antibody effector activity is substantially impaired in absence of this glycan.

Afucosylation has the most straightforward influence on antibody effector functions. Interestingly, afucosylated mAbs have shown to be more protective against various infectious agents , and more efficacious in cancer therapy , However, increased levels of afucosylation are also associated with severe disease during secondary dengue infection Another biologically important modification to the Fc glycan is sialylation.

Besides having anti-inflammatory properties, sialylated Fc glycans have also been shown to induce the production of high-affinity broadly neutralizing antibodies against influenza virus Besides its effect on Fc receptor interactions, Fc glycosylation also affects complement C1q binding to immune complexes.

A recent study shows that elevated galactosylation and sialylation increase C1q-binding, downstream complement deposition, and complement dependent cytotoxicity In contrast, agalactosylated IgG has also been suggested to elicit enhanced complement activation, considering its role in several autoimmune diseases These findings suggest that activation of complement potentially contributes to pathogen clearance, but can also contribute to inflammation in autoimmune disease, highlighting the dual role of complement.

Fc glycosylation is subject to active regulatory mechanisms that control the composition of the glycan structure. Major changes in glycosylation occur during pregnancy , , upon vaccination , , and during certain viral infections Therefore, insight in the glycosylation pattern during RSV infection and disease may provide valuable clues on the cause of severe RSV disease.

To our knowledge, only one group has investigated the effect of glycosylation in the response toward RSV infection. Hiatt et al. The in vivo protective capacity of the G0 glycovariant was improved compared to the original, as evidenced by decreased pulmonary viral titers. In conclusion, this study suggests that the influence of Fc-glycosylation may be important in the protective capacity of RSV-specific antibodies but this needs to be studied in more detail for other mAbs and virus- and vaccine-induced antibodies.

Next to antibody structure and glycosylation, the location of the antibody-bound epitope with respect to the membrane of the infected cell has been shown to be pivotal in determining Fc-mediated effector functions. Since the use of mAbs, it has been noticed that different mAbs binding the same target protein can elicit different effector mechanisms Antibodies binding to epitopes closer to the membrane membrane proximal epitopes mediate ADCC and CDC activity more efficiently, whereas antibodies that target membrane distal epitopes are often highly neutralizing and efficient ADCP-inducers 13 , — More specifically, recent research suggests that ADCP is most efficiently triggered when antibodies bind within 10 nm from the cell surface , indicating that the optimal ADCP-inducing epitope is located neither too close, nor too far away from the cell membrane.

For CDC activity, stabilization of complement components on the cell surface is essential. This would require a short distance from epitope to cell membrane. During ADCC, the formation of an immune synapse is essential.

This small synapse can only be formed when the NK cells engage antibodies bound in close proximity to the cell membrane, explaining the need for membrane proximal epitopes Antibodies against the SH and N protein have also been described , and although these antibodies are not involved in neutralization, they may have other important Fc-mediated functions Capella et al.

Pre-F-specific antibodies are better neutralizers than post-F-specific antibodies However, not all pre-fusion F antibodies have similar neutralizing activity The most potent neutralizing antibodies bind to distal epitopes, suggesting that the neutralizing potential of anti-RSV F antibodies not only relies on the conformation of F on which the epitope is present e.

As described above, the proximity to the membrane determines the efficiency of Fc-mediated effector functions 13 , , This suggests that potently-neutralizing antibodies, binding to distal epitopes, may also be efficient inducers of ADCP. Antibodies against this receptor-binding domain efficiently neutralize RSV infection and decrease pathogenesis by binding soluble G protein, an immune evasion protein secreted by RSV-infected cells 56 , 57 , Taken together, not only the antigen but also the epitope determines the efficacy of antibodies.

Interestingly, evidence suggests that targeted epitopes may differ between infants and adults , but the effect of these changes on the efficacy of the antibody response is unknown.

Further research may uncover the relation between antigenic site and effector functions against RSV infection, and thereby reveal preferred antibody-binding sites for protection against RSV disease. Additionally, co-engagement and signaling through other receptors such as TLRs may influence the activation threshold Altogether, this points out the importance of receptor expression patterns on effector cells. Although many SNPs have been identified, only few have been shown to impact receptor function Only the RH allelic variant of this receptor is capable of interacting with IgG2, enabling efficient phagocytosis , The presence of a valine residue at position increases the affinity for IgG1 and IgG3, augmenting for example NK cell activity , Currently there are no market-approved vaccines or antivirals available against RSV.

The only available treatment is the administration of a prophylactic F protein-specific mAb Palivizumab to reduce hospitalization in high-risk infants However, the use of Palivizumab is restricted and its cost-effectiveness is often discussed Improved mAbs with higher efficacy rates are thus highly needed and many research efforts are ongoing to develop these mAbs. A recent clinical trial with a pre-F-specific mAb Suptavamab failed to demonstrate efficacy in pre-term infants although the mAb was superior to Palivizumab in neutralization tests in vitro and in reducing viral load in the cotton rat model press release Regeneron, August 14, The failure of this highly neutralizing mAb indicates that protection against RSV-mediated disease, which is known to be immunopathological in nature, depends on more than just neutralization of the virus.

The development of vaccines is of great importance, especially for developing countries where RSV-related mortality is high and mAb therapy is inaccessible due to high costs. The majority of vaccine candidates currently in clinical trials are designed to induce systemic IgG, mostly against the RSV F protein. The results of the pre-F-specific Suptuvamab and the recent failures of two F-specific vaccine candidates tested in elderly, imply that a broader and more polyfunctional immune response may be needed to confer protection against RSV-mediated disease , press release Novavax, September 15, To this date, no accurate correlate of protection has been defined for RSV infection as virus-specific antibody levels or neutralization titers do not seem of use in this respect.

The lack of a well-defined correlate of protection complicates the development of new vaccines, as efficacy now has to be demonstrated in expensive large-scale clinical trials. Mounting evidence suggests that antibody effector functions beyond neutralization can contribute to both protection and disease , , , A balanced activation of different Fc-mediated effector functions is key to prevent excessive inflammation and tissue damage Figure 9. It will be of importance to implement assays that identify Fc-mediated effector functions of mAbs and vaccine-induced antibodies.

Systems serology captures a wide array of antibody characteristics and effector functions. It has proven effective in identifying antibody features that contribute to protection for various viral pathogens 19 , , Such an approach will provide detailed information on the characteristics that are required for a protective RSV antibody response. Figure 9.

The balance between Fc-mediated protection and enhanced disease. Antibody effector functions, regulated by differences in antibody characteristics, are suspected to play a role in disease outcome upon RSV infection. Immune activation by Fc-mediated effector functions is likely needed for efficient viral clearance. However, excessive activation may lead to inflammation and tissue damage. A balanced and contained immune response is most likely the key to protection upon infection.

Recent developments now allow targeted modifications to mAbs that can lead to enhancement or inhibition of specific Fc-mediated antibody effector functions through glyco-engineering or the induction of specific antibody subclasses or isotypes In the future, this might also be possible for vaccines.

One can conclude from the studies presented above that Fc-FcR interactions are an integral component of the immune response against RSV and should be considered in the rational design of next generation RSV-specific mAbs and vaccines.

Only limited data is available on the effect of specific Fc-mediated antibody effector functions during RSV infection, but it is clear that these can be both beneficial and detrimental for protection against RSV infection and disease outcome.

In the future, Fc-mediated effector functions might be harnessed to optimize the efficacy of RSV-specific mAbs and vaccine-induced antibodies.

However, our current knowledge on the precise role of individual effector functions in RSV disease is too limited to rationally design such antibodies and vaccines. Therefore, until the individual contributions of Fc-mediated effector functions to protection and disease are unraveled, aiming to induce highly neutralizing antibodies seems the safest approach.

These antibodies will need to halt the infection at the site of entry and thereby prevent excessive antibody-mediated immune activation. It remains to be seen whether complete neutralization can be achieved via the induction of serum IgG alone, or whether the induction of mucosal IgA is necessary for reliable neutralization activity. The many clinical trials that are currently ongoing with maternal and neonatal vaccine candidates will show whether these approaches indeed result in protection during the first, most vulnerable, months of life.

Neutralizing antibody titers do not adequately correlate with protection against RSV disease. Interestingly, antibodies have additional Fc-mediated effector functions besides neutralization, but this area of research is currently underappreciated in the RSV field. With this review, we aim to encourage a paradigm shift from neutralization-based studies toward functional studies examining the precise role of Fc-mediated antibody effector functions in vaccine efficacy and RSV disease.

We have evaluated the current literature on the effect of RSV-specific antibodies on NK cells, phagocytes, the complement system, cytokine production, and B- and T-cell skewing.

However, most studies into vaccine and mAb efficacy still only report antibody neutralization titers and disregard any Fc-mediated effector functions. The importance of these antibody effector functions has already been shown for multiple clinically important viral pathogens and is only starting to be explored for RSV.

In our view, a better understanding of the broad range of effector mechanisms that are induced by RSV-specific antibodies will greatly contribute to the much-needed development and testing of next generation mAbs and vaccines against this virus. EvE researched the literature and designed the figures. EvE and PvK drafted the first version of the manuscript. WL and GF critically reviewed the manuscript. All authors approved the final manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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