Abstract

Introduction

Antibodies are a key component of the human adaptive immune system, and the elicitation of antibodies has been correlated with vaccine efficacy in many diseases (1). Passively infused antibodies have been used in anti-toxin, anti-viral, and anti-inflammatory treatments; and monoclonal recombinant antibodies are also currently being pursued for prevention of HIV-1 infection in large Phase IIb clinical trials (NTC02716675 and NCT02568215). Antibodies can exert their protective functions via a multitude of mechanisms. Some functions, such as neutralization, mainly depend on interaction of the Fv domain (Figure 1A) with antigen and are therefore predominantly Fc domain independent. Other functions, including antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), require interactions between the antibody Fc domain with other proteins or immune effector cells via recognition by Fc receptors (9–11) (Figures 1A–C). These Fc receptor-dependent antibody functions provide a direct link between the innate and adaptive immune systems, harnessing the potent anti-pathogen functions of the innate immune system, and overcoming its inherent limited pattern recognition capacity by utilizing the diversity and specificity of the adaptive immune response. Fc receptor-dependent antibody functions are important components of the immune response that provide mechanisms for clearance of infected host cells, immune complexes, or opsonized pathogens. Fc receptor-dependent antibody functions are also involved in activation of downstream adaptive immune responses by facilitating antigen presentation or by stimulating the secretion of inflammatory mediators (12, 13). This review is focused on the antibody Fc receptor-dependent effector function ADCP in immune responses against viruses and targets three areas of interest: (1) discussion of the biophysical factors that influence ADCP including the properties of the receptors, antibodies, and effector cells; (2) survey and interpretation of evidence supporting a potential role for ADCP in either inhibiting or enhancing viral infection; and (3) description of current approaches used to measure ADCP with consideration for the translation of studies performed in animal models.

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Figure 1

(A) Surface representation of human IgG1 indicating: the Fv regions, which are the portions of the Fab arms required for antigen binding; the Fc region, which interacts with immunoproteins and specific receptors for effector functions; and the hinge region, which provides flexibility for the Fab and Fc regions. The IgG heavy chain 1 is depicted in blue, heavy chain 2 in teal, light chain 1 in orange, and light chain 2 in gray. Glycans are represented by yellow sticks. Rendering was made in PyMOL software using protein data bank ID 1HZH. (B) Expanded view of human IgG1 Fc region after 180° rotation, indicating residues involved in binding to Fcγ receptors (2–5), and the glycosylation site at asparagine 297 (N297). (C) Binding affinities (6–8) of Fc receptors commonly implicated in ADCP and comparison of receptor composition and signaling domains. Absence of values indicates no detectable binding.

Antibody and Fc Receptor Interactions Involved in ADCP

Immune complexes formed between antigen and antibody are capable of engaging a diversity of Fc receptors on innate immune cells. The type I IgG Fc receptors are activatory FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, FcγRIIIb, and inhibitory FcγRIIb (Figure 1C). Other IgG Fc receptors include the non-classical (type II) IgG Fc receptors CD209 and CD23, neonatal FcR (FcRn) which is involved in IgG transport and recycling, and the cytosolic Fc receptor TRIM21. IgA antibodies are specifically engaged by FcαRI (Figure 1C), and the specific receptor for IgE is FcεRI—which is involved in rapid allergic responses. Immune complexes may also interact with other receptors that have been described to bind to immunoglobulins but have been relatively uncharacterized, including FCA/MR (14), FCMR (15), IgD-R (16), CD71 (17), secretory component receptors (18), asialoglycoprotein receptors (19), and M cell receptors (20).

When engaged, most Fc receptors are capable of cytoplasmic signaling. For instance, as shown in Figure 1C, FcγRIIa and FcγRIIc signal via their immunoreceptor tyrosine-based activation motif (ITAM) domains, whereas FcγRI and FcγRIIIa lack ITAM domains but associate with an FcRγ signaling chain (γ₂) and signal via its ITAM domain. The inhibitory FcγRIIb signals via an immunoreceptor tyrosine-based inhibition motif (ITIM) domain. The IgA receptor, FcαRI, also associates with the FcRγ signaling chain (Figure 1C), but this seems to be dispensable for signaling, and signaling is dependent on dephosphorylation of the intracellular domain of FcαRI (21). Downstream signaling pathways are complex and dependent on the Fc receptor, cell type, and stimulation mechanism, but generally act via increasing intracellular calcium cation concentration, activation of PKC, or activation of ras (22).

As a result of the ubiquitous presence of antibody in both the systemic and mucosal microenvironments, regulatory systems are required to prevent constitutive Fc receptor signaling. This is achieved via several mechanisms that are either intrinsic to Fc receptor signal pathways, impacted by external soluble signals detected by the phagocyte, or established at the genetic level (Figure 2). A key intrinsic regulator is the inability of free antibody to activate Fc receptor signaling. The low-affinity Fc receptors, including FcγRIIa, FcγRIIb, FcγRIIc, and FcγRIIIa require multiple coordinated interactions for sufficient binding avidity, and thus can only be triggered by multivalent antibody-antigen immune complexes (22). Even for the high-affinity FcγRI, which is able to bind monomeric IgG, binding does not trigger signaling through its associated γ-chain; instead, signaling requires receptor clustering and cross-linking (23). Moreover, activatory Fc receptors can also produce inhibitory signals when engaged at a low level–the mechanisms for this phenomenon are not well-understood but may involve ITAM monophosphorylation which activates the inhibitory SHIP-1 (24) rather than the diphosphorylation required for activatory Syk engagement (25). Similarly, immunostimulatory and immunoinhibitory Fc receptors are often co-expressed on the same cell, thus the outcome of antibody-mediated signaling is often dependent on the balance of activating or inhibiting signals. The abundance of these receptors on the cell-surface and their ability to interact with immune complexes is influenced by soluble signaling molecules, which allows the local inflammatory milieu to also contribute to the regulation of Fc receptor-dependent responses of phagocytes (26–28). Finally, Fc receptor signaling is regulated at the genetic level. Single nucleotide polymorphisms in human Fc receptors affect interactions with antibody Fc, resulting in Fc receptor variants with lower or higher relative affinities for immune complexes (2, 6, 11, 12, 29). Several Fc receptor polymorphisms have been associated with the occurrence or progress of disease resulting from infection with viruses including dengue virus (30, 31), influenza virus (32), human coronavirus (33), Epstein-Barr virus (EBV) (34), Kaposi's Sarcoma virus (KSV) (35), and HIV-1 (36–38).

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Figure 2

Factors impacting ADCP and potential outcomes of the ADCP response to virus infection.

The specific Fc receptor engagements involved in ADCP of different viruses or virus-infected cells at sites of infection and throughout antiviral immune responses have not been completely defined, and are not expected to follow a generalizable rule. As will be described in the following sections, the receptors involved will differ depending on the characteristics of the antibody forming the complex —such as isotype, subclass, and glycosylation— as well as on the particular type of phagocyte encountering the immune complex.

Antibody Characteristics That Impact ADCP

In addition to the Fc receptor regulatory mechanisms described above, the ability of interactions between immune complexes and Fc receptors to result in Fc receptor signal transduction is further regulated by specific characteristics of the antibody Fc region. Antibody isotype serves as the principal level of regulation, as most Fc receptors are specific for only one isotype of antibody. Within isotype, there is additional regulation at the level of antibody subclass (Figure 1C). For the human IgG isotype, IgG3 has the highest affinity for most of the type I FcγRs, followed by IgG1, then IgG4, then IgG2 (6). In contrast, subclass is not a predominant source of regulation for ADCP by IgA, as human FcαR has been demonstrated to have similar affinity for IgA1, and IgA2 (7).

Additional regulation of Fc–Fc receptor interactions required for ADCP occurs via diversity in glycosylation of the antibody Fc (39). The IgG Fc region contains an N-linked glycan at asparagine 297 (Figure 1B) that impacts the conformation of the antibody and affinity for Fc receptors (39). As described in a comprehensive recent review by Jennewein and Alter, for IgG antibodies there are 36 possible glycoforms and 4 different subclasses, yielding a total of 144 possible unique Fc regions (40). Importantly, antibody glycovariation can be modulated by inflammatory responses, allowing the immune system to adapt and adjust antibody Fc glycoforms to modulate biologic activities in response to infection (40). Changes in antibody glycosylation have been described during the course of HIV-1 infection (41, 42), and in response to influenza vaccination (43, 44), but for many viral diseases the role of glycovariation in antiviral antibody responses has yet to be defined.

Fc Receptor Signaling for ADCP

Leukocytes involved in ADCP must express at least one type of Fc receptor. This includes monocytes, macrophages, neutrophils, and eosinophils; canonically referred to as professional phagocytes. In ADCP the phagocyte is engaged by antibody, either directly via Fc receptor or indirectly via antibody-fixed complement, to engulf one or more opsonized particles or molecules, which typically including pathogens, infected cells, and their derivatives (45, 46). The internalization in most cases leads to the destruction of the internalized target by phagolysosomal degradation, though it is important to note that several human pathogens have evolved to co-opt this process and survive within phagocytes (47–50). Phagocytosis leads to different immune outcomes depending on the cell type—for instance, antibody-mediated phagocytosis by macrophages leads to enhanced pathogen destruction and antigen presentation, whereas antibody-mediated phagocytosis by plasmacytoid dendritic cells leads to enhanced secretion of interferon alpha (51–53).

In Fc receptor-mediated phagocytosis, ligated and aggregated Fc receptor become phosphorylated via Src family tyrosine kinases (54) on their ITAM domains (either their own or from an associated γ-subunit), forming a docking site for Syk (55) and triggering a signaling cascade involving PKC (56, 57), PI3K (58–60), and synthesis of PI(4,5)P2, (3,4,5)-PIP3, and DAG (61). These lead to actin cytoskeleton remodeling (62), allowing the advance of the phagocytic cell over the target. The strength of early signaling events is proportional to the number of engaged Fc receptors, whereas late signaling events required to complete phagocytosis require a concentration threshold of 3′PI to be satisfied (63).

Phagocytes Involved in Virus ADCP

The type of phagocyte involved in an ADCP responses to virus infection depends not only on the profile of Fc receptors expressed by the cell and characteristics of the antiviral antibodies as described above, but also on the phagocyte being present at, or recruited to, sites of infection. Professional phagocytes are differentially distributed in the circulation and tissues (64–67), and inflammatory signaling can promote both ingress and egress of immune effector cells (68). Therefore, cells present at the site of infection and involved in the antiviral response likely change over time. Transgenic mouse models and cell-type specific depletions can help to identify essential cell populations, but a clear view of the specific interactions involved in the tissue at the site of virus infection is often limited, especially in humans. Despite the difficulties inherent to accessing and studying immune cells in situ within human tissues, a remarkable study by Sips et al. defined the distribution and frequency of Fc receptor expressing immune cells in mucosal and lymphoid tissues (69). They identified differential distribution of professional phagocytes —with macrophages being the dominant phagocyte population in lymph nodes, and intestinal tissues and neutrophils representing the dominant phagocyte population in tissues from the lower female reproductive tract. Using a novel tissue phagocytosis assay, they compared the HIV-1-specific ADCP activity of neutrophils and macrophages from the colon and cervix. They found that although abundant in the colon, colon-resident macrophages were deficient in ADCP compared to colon- and cervix-resident neutrophils as well as cervix-resident macrophages. This seminal study likely only partially defines the diversity of professional phagocytes, both for phenotype and functionality, within tissues that can be encountered by antibody-virus immune complexes during virus infection, and that inevitably impacts outcome of these encounters.

Balance of Activatory and Inhibitory Signals Determine How Phagocytes Respond to Immune Complexes

Importantly, most phagocytes are capable of other Fc-dependent effector functions in addition to ADCP. Thus, the outcome of each interaction between phagocytes and immune complexes is determined by a combination of signals (Figure 2). Many phagocytes express more than one type of Fc receptor, often expressing both activatory and inhibitory Fc receptors. The balance of these divergent signal pathways is critical to simulating and regulating each potential effector response. Signals mediated by other types of receptors also contribute to determining the type of effector response a phagocyte will mount. Among them, information from pattern recognition receptors (PRRs) that can detect molecular patters associated with different types of pathogens are integrated in the response to Fc receptor stimuli. For example, Toll-like receptor 3, 7, 8, and 9 have the ability to detect viral nucleic acids and activate immune cells (70), and are therefore able to potentiate effective antiviral responses including ADCP. In contrast, alternative inhibitory signals, such as CD47 SIPα can negatively regulate phagocytosis. Intriguingly, some viruses express homologues of CD47, which may act to prevent activation of professional phagocytes as a strategy for immune evasion (71, 72).

Apart from direct antiviral activity through uptake and elimination of virus or infected cells, antibody-dependent phagocytosis is also important in the development and regulation of immune responses themselves (Figure 2) (12). Pathogen-associated molecular patterns (PAMPs) derived from virus antigens can be released upon phagocytosis and digestion of antibody–virus immune complexes. The released virus PAMPs can prime an inflammatory response upon sensing by PRRs, which may then stimulate additional immune cells and activate subsequent immune responses (12, 13). For instance, in adenovirus infection of the respiratory tract, alveolar macrophages are responsible for internalizing adenovirus and initiating early pro-inflammatory signaling (73). A similar response, termed antibody-induced inflammation, has been demonstrated to have an important role in protection against influenza infection (74). There are also direct roles for ADCP in modulation of adaptive immunity. ADCP of immune complexes by dendritic cells via FcγRIIa promotes MHC class I and II antigen presentation and induces cellular and humoral immune responses, while uptake through FcγRIIb prevents dendritic cell maturation and does not promote immune activation (75). Antibody production and affinity is also regulated by FcγRIIb-mediated induction of B-cell apoptosis, which helps to eliminate B cells with low affinity B cell receptors (75). Based on the diverse roles of ADCP in immune responses it is unsurprising that ADCP has been shown to be an important component of immune responses to infection by many different viruses.

Evidence For the Importance of ADCP in Immune Responses Against Viruses

Phagocytosis has traditionally been known for its role in clearance of bacteria and fungi, as evidenced by the fact that persons with defects in phagocytosis are susceptible to common bacterial and fungal infections (76). It is important to note that these observations relate to total phagocytosis (antibody dependent and antibody independent) and thus do not allow for dissection of the specific contribution of ADCP to protection from bacterial and fungal infection. The role of ADCP in immune responses against viruses is similarly complex and difficult to dissect given its association with other antibody functions in the settings of infection, vaccination, and passive immunization.

Potential for ADCP to Enhance Viral Infection

ADCP is not always associated with beneficial or protective immune responses. In some cases, ADCP has been demonstrated to promote infection. Termed antibody-dependent enhancement (ADE), this has predominantly been described for viruses from the genus Flavivirus including dengue virus, yellow fever virus, Japanese encephalitis virus, and zika virus (135–140). Fc receptor-dependent ADE is a particular concern for infections by dengue virus (141–143). There are four serotypes of dengue virus, and antibodies generated in response to infection with one serotype have variable degrees of cross reactivity to the other three serotypes. Upon re-challenge with the original serotype, antibodies are protective. However, the pre-existing antibody response will augment infection and promote more severe disease upon secondary infection with any of the other serotypes (143). Moreover, ADE resulting from cross reactivity may not be limited to secondary infection with another strain of the same virus; ADE might also occur when the secondary infection is a different but closely related virus. For example, the results of recent studies have suggested that dengue virus-specific antibodies may be capable of enhancing zika virus infection (144), and reciprocally, that zika virus-specific antibodies may be capable of enhancing dengue virus infection (140). These observations have been met with skepticism (145), and additional work will be required to demonstrate that this type of ADE has clinical relevance to dengue and zika incidence and disease. ADE is thought to occur when non-neutralizing antibodies, or suboptimal concentrations of neutralizing antibodies, bind and opsonize virus, and are subsequently internalized by Fc receptor expressing cells via ADCP. Within the cells these immune complexes may perturb normal antiviral immune functions resulting in the virus not being destroyed. Instead, the virus exploits ADCP as a tool to expand access to host cells, resulting in higher infection burden and often stimulating an overt inflammatory response that contributes to disease pathogenesis (143). How do viruses such as dengue subvert the process of ADCP to promote increased infection? One potential mechanism that has been proposed to allow for dengue virus to escape Fc receptor-mediated antiviral signaling is through engaging the inhibitory receptor leukocyte immunoglobulin-like receptor-B1 (LILRB1) (146). Thus, for dengue and other viruses, the outcome of each interaction between antibody-virus immune complexes and phagocytes depends on the balance of activating signals, which are expected to promote ADCP and virus elimination, and inhibitory signals, which have the potential to enhance infection.

In the studies of HIV-1 infection, early papers reported that complement and antibodies at low titers could enhance HIV-1 infection in vitro (147–150). Since monocytes and macrophages can be infected with HIV-1, one theory is that antibody-mediated phagocytosis may have caused increased viral infection of these phagocytes. More specifically, enhancement may have been due solely to weakly or non-neutralizing antibodies that remain capable of engaging but not neutralizing HIV-1 virus (151). However, it is notable that no enhancement of infection in the vaccine arm was observed in the VAX003 and VAX004 clinical trials, both of which elicited high levels of non-neutralizing antibodies (152, 153).

As described in a comprehensive review of the topic by Taylor and collaborators (143), there is evidence of ADE for many other viruses including influenza, SARS-CoV, RSV, and ebola virus—for all of which there also exists evidence of ADCP as being part of beneficial antiviral antibody responses. These dichotomous observations emphasize the balancing act faced by the immune system as it attempts to respond to virus infection. Immune responses of sufficient specificity and potency are required to protect against infection and disease, but suboptimal responses can be exploited by pathogens resulting in higher levels of infection and more severe disease. Because of this, ADE remains a concern for the development of vaccines against viruses for which potent neutralizing antibody response cannot be elicited.

Viral Epitopes Recognized in ADCP

One of the best examples of how epitope specificity can impact ADCP was described for influenza-specific antibodies. Two distinct epitope regions of the influenza hemagglutinin protein are recognized by influenza-specific antibodies: the immunodominant and antigenically variable head domain; and the more antigenically conserved stalk region (Figure 3). DiLillo and colleagues demonstrated a requirement for antibody–Fcγ receptor interactions for protection by the stalk-specific antibodies, but not for antibodies recognizing the head domain (114). They further demonstrated that the stalk-specific antibodies were capable of activating NK cells for ADCC, and that the protective efficacy of stalk-specific antibodies could be improved with mutations that selectively enhance engagement with FcγRIIa and FcγRIIIa (114). Mullarkey and colleagues followed-up on these insights and identified a similar epitope-dependent dichotomy of anti-hemagglutinin antibody responses in antiviral assays performed with neutrophils as the source of innate effector cells. Using recombinantly-produced chimeric antibodies with mouse Fv and human Fc regions, they demonstrated that stalk-specific, but not head-specific, antibodies induced ADCP, and reactive oxygen production by neutrophils. Similar observations were made for antibodies made both as IgG1, and as IgA, indicating that the epitope dichotomy for interaction with Fc receptors is conserved for Fcγ and Fcα receptors (115). Their use of recombinantly produced antibodies with identical Fc regions confirms that at least for influenza virus, antibody epitope specificity can play an essential role in establishing downstream antiviral effector responses. A subsequent study by DiLillo and collaborators expanded upon their initial observations, and demonstrated that the requirement for Fc receptor involvement for protection in vivo was not restricted only to stalk-specific antibodies, but instead was a common feature of antibodies capable or recognizing a breadth of influenza strains. Using mouse models, Fc receptor dependent protection was shown for both broadly neutralizing stalk and head specific antibodies, as well as broadly-binding non-neutralizing head-specific antibodies (116). Thus, there exists a yet to be explained relationship between breadth and Fc receptor-dependence in protective antibody response to influenza. Sera from human donors has the ability to mediate anti-influenza ADCP (119, 120), it will therefore be important for future research to further map the specificities of natural human influenza ADCP antibodies, and to explain the mechanisms underlying the differential functions of the strain-specific head antibodies from multi-strain specific head and stalk antibodies.

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Figure 3

Surface representation of structures of the viral fusion proteins of influenza virus, ebola virus, and HIV-1 that are targets of antiviral antibodies. Perspective is from the side, such that the proteins are standing atop the virus envelope. These envelope proteins share a general structural architecture as trimers of dimeric proteins comprised of a receptor binding domain (head, represented by shades of green) and a viral membrane-bound domain (stem/stalk, represented by shades of orange). Renderings were made in PyMOL using protein data bank ID 3GBN (influenza), 5JQ3 (ebola), and 5FYL (HIV-1).

A similar epitope dichotomy has been described for ebola virus. The ebola virus envelope glycoprotein is similar to the envelope fusion glycoproteins of influenza and HIV-1 in that they share a common architecture of trimers of dimeric proteins. The dimers are comprised of a distal receptor-binding head region, and a membrane-bound stem/stalk region (Figure 3). The stem/stalk regions are less variable than the head regions, however the head regions are generally regarded as immunodominant. Ebola stalk-specific specific antibodies were demonstrated to be capable of mediating multiple Fc receptor-dependent effector functions including NK cell activation and ADCP by monocytes or neutrophils, while glycan cap-specific mAbs lacked this functionality (154). Thus, it will be interesting to study parallels and differences between broad-binding influenza and ebolavirus stalk-specific ADCP antibodies as the biomolecular interactions underlying the relationships between epitope and function are elucidated.

Diverse HIV-1 epitopes can be targeted for virion phagocytosis, including broadly neutralizing epitopes such as the gp41 MPER (stem region), and gp120 head-region targets such as the CD4 binding site, V2 glycan, and trimer apex. Non-broadly neutralizing epitopes such as the gp41 principal immunodominant domain (stem region), V1/V2 loop and V3 loop (gp120 head region) (104, 155–157) are also targets of ADCP antibodies. The CD4-induced epitopes found on HIV-infected cells are not engaged alone for phagocytosis, but can be engaged synergistically, at least with V2 epitopes, for virus engagement (98). The ability of these epitopes to be engaged for phagocytosis varies by strain, indicating the substantial heterogeneity between viruses. Furthermore, antibodies targeting the same epitope can vary in their phagocytosis potency, emphasizing the fact that the rules of antibody-epitope engagement that allow for ADCP are not completely understood. These may include antibody Fc availability which can be affected by antigen binding valency and angle of approach (158). It is also likely that there are non-neutralizing antigenic targets on the HIV-1 virion surface that are involved in ADCP or other Fc-dependent functions but have not been discovered. Our understanding of the HIV-1 envelope protein (Env) surface is strongly biased toward neutralizing epitopes, since the search for anti-HIV-1 antibodies typically involves the selection and characterization antibodies positive for neutralization. However, these typically make up only a portion of the antibodies that are elicited in response to HIV-1 infection or vaccination, but the non-neutralizing specificities capable of binding and engaging Env are typically not further studied. In addition, monomeric gp140 or gp120 proteins, and even the current generation of stabilized trimeric proteins that are typically used as hooks for antibody selection likely do not fully recapitulate the diversity of natural Env forms on the virion envelope, which may include both functional trimers and non-functional forms of envelope (159). If so, large-scale unbiased antibody screening may identify additional crucial antibody specificities for Fc-mediated effector functions against virions that may be targeted by vaccines or passive immunotherapy. It is important to note that most virion ADCP methods do not differentiate between infectious and non-infectious virion particles–thus, an open question remains as to whether non-neutralizing antibodies that appear to mediate virus ADCP do indeed cause uptake of infectious viruses, or whether they merely engage non-infectious epitopes on defective particles.

Virus-infected cells and virion particles are distinct biological targets of antibody-mediated internalization. The epitope specificities involved for each process may differ, given different epitope exposure on viruses and infected cells. For instance, in the context of HIV-1, the gp41 principal immunodominant domain targeted by antibodies such as 7B2 and F240 is frequently found on virions, but not on infected cells, whereas the converse is true for the conserved region 1 conformational domain targeted by antibodies such as A32 and C11 (104, 160, 161). The majority of studies describing the ADCP response to HIV-1 have been focused on gp120/gp140 proteins or whole virions, thus there is much less known about ADCP of HIV-1 infected cells and the epitopes that may be involved in this process. Additional research in this area is needed for HIV-1 and other viruses.

Comparatively less has been described regarding the fine epitope specificity of ADCP antibodies in the immune responses to other virus infections or vaccination. As a general rule, for ADCP of virions the epitope must be present on the surface of the virions, while for ADCP of infected cells any epitope expressed on the surface of a virus-infected cell may be a potential target. For most viruses limited antibody specificities have been described to mediate ADCP: CMV gB (124) protein, RSV G protein (126), ebola virus glycoproteins (127), HPV L2 protein (128), and WNV NS1 protein (132). In most of these studies, the ADCP activity was identified by testing available antibodies, or antibodies selected for alternative functionality such as neutralization. Therefore, many potential specificities remain unexplored and it is likely that antibodies that target other epitope regions are capable of ADCP activity but have not yet been identified. Overall, there is a dearth of knowledge regarding specific epitopes that can be targeted by ADCP antibodies against most viruses, and more insight into this area may inform new strategies for rational design of vaccines intended to elicit ADCP responses.

Approaches to Measure Virus ADCP

Virus ADCP is typically measured via a cell-based assay, where the amount of viral target internalized by a phagocyte is quantified. Sample monoclonal or polyclonal antibodies are first pre-mixed with the viral target to form immune complexes. Phagocytes are then introduced for antibody-dependent phagocytosis to occur. Such phagocytes include primary monocytes, macrophages, neutrophils, or corresponding cell lines (104, 162, 163). Phagocytes can be spinoculated (164) with the immune complexes to increase the signal-to-noise ratio, though this is not necessary to achieve detectable signal in the case of antibody-dependent phagocytosis of HIV-1 virions (165). Non-internalized virus particles are then washed off, and the amount of internalized viral target is quantified.

In theory, any method capable of quantifying target particles (e.g., nucleic-acid based RT-PCR, protein-based ELISA, or fluorescence) can be used as the assay readout, as long as internalized virus particles can be separated from extracellular or cell-attached particles. In practice, fluorescence-based approaches are the most popular due to the capability for high-throughput readout via flow cytometry (162). In fluorescence-based approaches, the target particle could be a live fluorescently-labeled virion (104, 120, 166) or a virus-derived protein conjugated to a fluorescent bead (120, 162). Where virions are labeled, this can be done with direct labeling [including viral membrane labeling which would exclude detection of membrane-fused viruses (167)], or internal labeling via co-transfection of genes encoding fluorescent non-structural proteins, resulting in their random incorporation into packaged virus particles (168). Fluorescence labels can also be engineered to report internalization, either by the addition of a non-cell-permeable fluorescence quencher during the wash step (169), or by using a pH-sensitive dye.

It is as yet unclear how results compare between different types of viral targets. Notably, phagocytosis signaling pathways and particle uptake dynamics are impacted by the physical properties of the target, including ligand spacing (170, 171), size (170, 172), shape (173), stiffness (174), and antigen height (175). Since virus particles differ from protein-coated beads in a number of these attributes, it remains unclear how these differences affect Fc-Fc receptor signaling, or how they influence the range of virus epitope-Fab conformations that are capable of mediating phagocytosis. In the case of HIV-1, one group has argued that HIV-1 virus particles cannot undergo ADCP due to insufficient surface ligand density (176). This was based on their observation of undetectable internalization of HIV-1 viruses by HIV-specific antibodies as compared with an anti-phosphatidylserine antibody. However, this result is at odds with our work demonstrating epitope-specific internalization of HIV-1 virus particles using a variety of HIV-specific monoclonal antibodies of different Env specificities (100), as well as polyclonal antibodies from sera and breast milk (165). Since the experimental procedures used by both groups were similar, the reasons for this discrepancy are unclear, and may involve the method of virus preparation.

Moving forward, it will be important to determine how the nature of the immune complex affects phagocytosis. Current assays do not routinely measure the size of the immune complex engulfed, but only the number of intracellular or intravesicular virions, which may be either a single immune complex or an amalgamation of multiple co-phagocytosed viruses. In fact, for many viruses, it is not even clear whether antibody-dependent virion internalization is strictly a process of phagocytosis, or whether other processes including endocytosis and macropinocytosis are involved. It will also be important to determine the signaling outcomes of such phagocytosis in multiple cell types and inflammatory conditions, in order to determine the role of phagocytosis in the recruitment of other effector functions as well as the subsequent development of the local inflammatory response and longer term adaptive immune priming. For highly polymorphic viruses including HIV-1 and influenza, it will also be useful to develop a panel of representative viral strains (fluorescent if necessary) for use as a reproducible measure of the breadth of ADCP activity which can be compared across different settings and clinical trials. Indeed, just as HIV-1 antibody neutralization potency against tier 1 viruses did not necessarily predict neutralization breadth, ADCP breadth may provide surprising clues for the development of vaccines that elicit strong Fc effector function.

In contrast to ADCP of virus particles, methodology for ADCP of virus-infected cells remains underexplored, as most current ADCP assays have been developed to measure internalization of immune complexes formed with virions, or protein-coated beads. Thus, it remains difficult to quantify how antibody epitope specificity and Fc profile contribute toward their potency for ADCP of virus-infected cells. Methodologies to investigate ADCP of virus-infected cells both in vitro and in vivo, in particular differentiating such events from non-antibody-mediated phagocytosis of virus-infected cells, will also be important to determine whether infected-cell ADCP results in virus inhibition or spread.

In addition to ADCP, trogocytosis is another potential outcome of Fc-dependent interactions between antibodies and virus-infected cells. Two assays developed to measure HIV/SIV-specific ADCC responses have been shown to also detect antibody-mediated interactions between monocytes and virus-infected or protein-coated cells (177, 178). Both assays use fluorescent dyes to label infected or protein-coated target cells. Flow cytometric analysis of cell populations after incubation of these target cells with antiviral antibodies and innate effector cells identified CD14⁺ cells present in the effector cell population as having acquired the fluorescent dye used to label the target cells —indicating transfer of target cell membrane (trogocytosis) and/or ADCP of cells or cell fragments. More recently, an in vitro HIV-1 trogocytosis assay has been developed, using flow cytometry to specifically measure the transfer of membrane fragments from a gp120-coated CD4⁺ T cell line to the THP-1 monocytic cell line (179). The relationship between trogocytosis and phagocytosis remains unclear. It may be that these two processes are not mutually exclusive, and that trogocytosis occurs as a result of “aborted phagocytosis” (180). Alternatively, antibody-dependent trogocytosis may represent an alternative mechanism for elimination of target cells (181–183). Presently, the roles of trogocytosis in immune responses to virus infection are not known, and additional research will be needed to determine how this process impacts virus pathogenesis and to further explore potential integrations with ADCP.

Considerations For Translation of Studies Performed in Animal Models

There are significant barriers to the translation of Fc effector functions between animals and humans. The most notable is the difference between the Fc/Fc receptor systems of rhesus, mice, and humans. The differences between the mouse and human Fc/Fc receptor system have been reviewed previously (184). While rhesus and human Fc/Fc receptor systems might be expected to be more similar, there remain substantial differences. Fc differences are apparent when comparing IgA and IgG subclasses between rhesus macaques and humans (Figure 4). Rhesus macaques have only one IgA gene, whereas humans have two IgA genes, IgA1 and IgA2. While both rhesus macaques and humans have four IgG subclasses, rhesus IgGs are not structurally or functionally analogous to human IgG1-4. In fact, rhesus IgG subclasses are genetically more similar to each other than they are to their human homologues (Figure 4A), and the diversity in the germline immunoglobulin genes is higher in rhesus macaques than in humans (188, 189). Structural differences between rhesus and human antibodies are best exemplified by the IgG3 subclass. The human IgG3 C_H domain encodes repeats of a hinge region exon, resulting in a hinge that is approximately four times longer (in number of amino acids) than that of human IgG1 (Figure 4B) (190). There is no such exon duplication and hinge elongation in rhesus macaque IgG3 (188). The structure of the hinge dictates the flexibility of the antibody (185), and therefore impacts biological functions and has implications for translation of studies performed in humans and rhesus macaques. For example, vaccine-elicited HIV-specific IgG3 was identified as a correlate of reduced risk of infection in the RV144 clinical trial (87, 89), and in vitro studies have demonstrated higher ADCP activity for IgG3 HIV monoclonal antibodies compared to other IgG subclasses (104). Given the differences between human and rhesus IgG3 it is unlikely that preclinical studies performed in the rhesus model would have predicted that vaccine-elicited IgG3 could play a crucial role in reducing the risk of HIV infection as observed in RV144. Caveats such as this must be considered when using animal models.

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Figure 4

(A) Phylogenic tree of human (Hu) and rhesus macaque (RM, red) IgG and IgA C_H genes. Scale bar represents the percentage sequence dissimilarity as a measure of phylogenetic distance. (B) The human IgG3 hinge region is elongated when compared to IgG1. The hinge region in the hybrid surface/schematic rendering of human IgG3 is drawn to scale, with an estimated hinge length of 80 angstroms, as estimated by Lu and colleagues using small angle x-ray scattering data, and consistent with prior estimates from electron microscopy data (185, 186). Disulfide bonds (n = 11) are represented by rungs in the ladder structure (187).

There are also differences in how human and rhesus macaque antibodies interact with Fc receptors. For instance, rhesus IgG2 and IgG4 retain strong binding to Fc receptors (191, 192), whereas human IgG2 and IgG4 have severely attenuated binding (6). Thus, there is more similarity in Fc receptor interactions and effector functions (191) across rhesus IgG compared to that observed for humans. This suggests that rhesus macaques do not use IgG subclasses to tune antibody responses to the same extent as humans, and highlights the need for caution when comparing subclass profiles in humoral responses across these species. In addition, rhesus macaque Fc receptors are more highly polymorphic than human Fc receptors (189, 192) and the functional implications of many of these polymorphisms have yet to be defined. Importantly, while rhesus and human Fc and Fc receptors can cross-react in binding, antibody binding to FcγRIIa is different between humans and rhesus macaques when comparing antibody Fc mutants, suggesting that antibodies may not behave similarly against human and rhesus macaque FcγRIIa (193), which is commonly engaged for ADCP (162). Despite these differences, our preliminary data indicate that there remains cross-reactivity and some level of functional homology for phagocytosis between humans and rhesus macaques, with similar rank order of phagocytosis activity across species when human IgG and IgA isotypes/subclasses are tested against human or rhesus monocytes and neutrophils (Pollara et al., unpublished observations).

Further studies are required on the interactions between Fc and Fc receptors in different species, their effects on phagocytosis, and the subsequent effects of phagocytosis to determine what findings from preclinical studies can be translated. Passive immunotherapy may be simpler to model since there appears to be functional homology for phagocytosis between human and rhesus systems when human Ig is used. Humanized mice will also be valuable for these types of studies. Specifically, the mouse model developed by Smith and collaborators in the laboratory of Dr. Jeffrey Ravetch is expected to provide the most utility (194). In their model, the mouse Fcγ receptor genes have been deleted and human Fcγ receptor transgenes have been inserted. This results in mice that express functional human Fcγ receptors on the same types of cells and at the same levels as found in humans. The primary limitations of this model are that only human Fcγ receptors are expressed. Thus, Fcα-receptors, FcεRI, Fc receptor neonatal, Type II Fc receptors, and other Fc-binding proteins remain as native mouse forms. Moreover, repeated infusion of human IgG can result the development of anti-human IgG antibodies in these mice (195).

Conclusions and Future Perspectives

ADCP is an Fc receptor-dependent function of antibodies that is likely common to immune responses elicited by virus infection and in response to vaccination. Importantly, there is substantial evidence that supports ADCP as contributing either to protection from infection, or reduction in disease severity for diverse types of viruses. In most cases, it is likely that ADCP works in tandem with additional Fc-independent and Fc-dependent antiviral activities as part of an effective polyfunctional humoral response. In fact, for many viruses that have been demonstrated to have antibody-dependent correlates of protection it is highly likely that ADCP is involved, but perhaps was not thoroughly explored. Additional investigation into the role of ADCP in protective viral responses, and the specific virus epitopes targeted by ADCP antibodies, may provide insight into strategies for rational vaccine design to elicit these types of antibody responses while avoiding deleterious ADE activity. Additionally, identifying the types of phagocytes and Fc receptors involved in ADCP at sites of virus infection within tissues, throughout the course of infection and virus clearance, remains an understudied aspect of host and virus interactions. Finally, as most ADCP assays used to measure this immune response in vitro quantify uptake of virus subunits, virions, and in some cases infected cells, there remains a gap in knowledge regarding the outcome of phagocytosis. Further research into this area could determine if protection by ADCP is dependent on clearance and elimination of virus and virus infected cells, or by potentiating subsequent immune responses via antigen presentation or immune signaling. As our understanding of ADCP grows, it is likely that approaches to successfully leverage this important immune response for improved antiviral immunity will be discovered.

Author Contributions

MT and JP conceived and wrote the manuscript. KW performed protein modeling for the figures and wrote the manuscript.

Acknowledgments

Footnotes

References