Modulating Antibody Functionality in Infectious Disease and Vaccination

TRMOME 1175 No. of Pages 14

Review

Modulating Antibody Functionality in Infectious Disease and Vaccination Bronwyn M. Gunn1 and Galit Alter1,* Induction of pathogen-specific binding antibodies has long been considered a signature of protective immunity following vaccination and infection. The humoral immune response is a complex network of antibodies that target different specificities and drive different functions, collectively acting to limit and clear infection either directly, via pathogen neutralization, or indirectly, via pathogen clearance by the innate immune system. Emerging data suggest that not all antibody responses are equal, and qualitative features of antibodies may be key to defining protective immune profiles. Here, we review the most recent advances in our understanding of protective functional antibody responses in natural infection, vaccination, and monoclonal antibody therapeutics. Moreover, we highlight opportunities to augment or modulate antibody-mediated protection through enhancement of antibody functionality. Constantly Functional: The Other End of the Antibody Humoral immune responses evolve following infection and serve as readout for vaccine responsiveness in all clinically approved vaccines to date. However, the mechanisms by which vaccineinduced antibodies act to provide protection is varied, where direct pathogen neutralization accounts for protective immunity in only a small fraction of clinically approved vaccines [1]. Thus, while vaccine design efforts often aim to induce ‘neutralizing’ antibodies (see Glossary), increasing evidence suggests that the development of non-neutralizing antibody responses may contribute to vaccine-mediated protection from infection/disease. Beyond their role in neutralization, mediated by the antibody antigen-binding arms (Fabs), antibodies also drive a remarkably wide array of additional antipathogen and immune-regulatory functions via their constant regions (Fc). The Fc domain of the antibody directs the effector functions of antibodies via Fc-binding proteins, including complement proteins, lectin-like proteins, and Fc-receptors found on all innate immune cells. Effector functions include: first, phagocytosis of antibody-coated pathogens and/or infected cells by monocytes, macrophages, neutrophils, and dendritic cells; and second, direct killing of infected cells by cytotoxic natural killer (NK) cells and complement-mediated lysis. Given that Fc-receptors are differentially expressed on innate immune cells (Figure 1 and Box 1), antibody binding to specific Fcreceptors enables the elicitation of distinct effector functions. In addition, antibodies initiate the complement cascade through both classical and lectin pathways, contributing to the direct destruction of target cells and their enhanced phagocytic clearance via complement receptors found on macrophage/monocytes, neutrophils, and dendritic cells. Thus, the production of qualitatively unique antibody profiles that selectively bind particular classes of Fc-binding receptor may enable the selective induction of particular antibody effector functions and protective mechanisms of action.

Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

Trends An antibody Fc domain interacts with innate immune cells, mediating a range of functions, including effector mechanisms, to limit and clear infection. Fc-mediated antibody functionality is increasingly being recognized as a critical aspect of humoral immunity against infectious diseases. Antibody functionality is modulated during disease and vaccination through both subclass selection and glycosylation of the antibody Fc domain. Broadly neutralizing monoclonal antibodies against HIV-1 and influenza viruses require Fc-mediated functionality to confer immune protection. Comprehensive profiling of the humoral response beyond titer and neutralization can identify underlying protective signatures of antibodymediated protection. Different vaccines, adjuvants, regimens, and vectors induce distinct antibody functional profiles, and future vaccines may be designed to direct antibody functionality.

1 Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA

*Correspondence: [email protected] (G. Alter).

http://dx.doi.org/10.1016/j.molmed.2016.09.002 © 2016 Published by Elsevier Ltd.

1

TRMOME 1175 No. of Pages 14

Cellular expression

Anbody affinity

Glossary FcγRI

FcγRIIA

FcγRIIB

FcεRI

FcαRI

IgG1

++++

++

+

+

+/–

–

–

IgG2

–

+/–

+/–

+/–

+/–

–

–

IgG3

++++

+

+

++

+

–

–

IgG4

+++

+/–

+

+

–

–

–

IgA

–

–

–

–

–

–

++++

IgE

–

–

–

–

–

++++

–

Monocytes

+

+++

+

+

–

–

++

DCs

+

+++

+

–

–

–

NK cells

–

–

+/–*

+++

–

–

–

Neutrophils

+ (ind)

+

+/–

–

+++

–

+++

Eosinophils

–

++

–

–

–

+++

+

Basophils

–

++

++

–

+/–

+++

–

Mast cells

+ (ind)

++

+/–

–

–

+++

–

–

–

+++

–

–

–

–

B cells

FcγRIIIA

FcγRIIIB

++++: high affinity/expression; +/–: low affinity/expression; ind: inducible expression; –: no affinity/expression; *: in FcγRIIC polymorphic individuals.

Figure 1. Antibody Affinity for FcRs and FcR Expression on Immune Cells. Antibody isotypes and subclasses have different affinities for various FcRs. The FcgR family of receptors binds IgG, FceRI binds IgE, and Fc/R binds IgA. Within IgG, IgG1 and IgG3 bind to all FcgRs with higher affinity compared with IgG2 and IgG4, and IgG2 does not bind FcgRI. Cellular expression of the different FcRs also varies on innate immune cells. Monocytes and macrophage express the high-affinity FcgRI, high levels of the FcgRIIA, low levels of FcgRIIB, and low levels of FcgRIIIA. Dendritic cells (DCs) express FcgRI, FcgRIIA, and FcgRIIB, as well as the type II Fc receptor DC-SIGN. NK cells predominantly express activating FcgRIIIA, although polymorphisms in the FCGR2C gene allow for FcgRIIC expression on NK cells a subset of individuals. [2_TD$IF]Neutrophils express high levels of the GPI-linked FcgRIIIB, low levels of FcgRIIA, and can induce expression of the high-affinity FcgRI. Eosinophils, basophils, and mast cells predominantly express the FceR to bind IgE, but also express FcgRIIA and FcgRIIB, and mast cells can induce FcgRI. B cells express only one FcgR, the inhibitory FcgRIIb, which provides negative feedback to the B cell, and plays a key role in immune tolerance.

Given that every circulating antibody has both a Fab and an Fc domain, every antibody must harbor an intrinsic ability to interact with the innate immune system. Moreover, since antibody functionality can be tuned up or down, an antibody must be considered as a whole, rather than just its Fab or Fc domains. This concept has been most recently demonstrated by studies Box 1. The Human FcRs Humans possess five major types of Fc-receptor, differentiated by the antibody isotype they bind: FcgR, Fc/R, FceR, FcdR, and FcmR binding IgG, IgA, IgE, IgD, and IgM, respectively. In addition, C-type lectin receptors, such as DC-SIGN and CD23, represent another class of Fc-receptors, termed type II Fc-receptors, shown to bind IgG and to mediate antiinflammatory as well as immune-regulatory functions in both mouse and human systems [76,89–91]. Human FcgRs can be further divided into inhibitory (FcgRIIB/CD32b) and activating receptors (FcgRI/CD64, FcgRIIA/CD32a, FcgRIIC/ CD32c, FcgRIIIA/CD16a, and FcgRIIIB/CD16b), and different innate immune cells express varying combinations and levels of FcgRs ([5_TD$IF]see Figure 1[6_TD$IF] in main text). With the exception of the high-affinity FcgRI, FcgRs are generally low in affinity for IgG, relying more heavily on avidity for activation. However, allelic polymorphisms found within the activating FcgRs, FcgRIIA (H131/R131) and FcgRIIIA (V158/F158), have been shown to confer higher and lower binding affinity for IgG, respectively [92–94]. Thus, individuals with the higher affinity polymorphisms generally exhibit higher levels of antibody function, which may play a role in protection or pathology, depending on the disease setting [95].

2

Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

Adjuvant: a substance that augments an immune response against an antigen and enhances vaccine efficacy. Affinity: the strength of interaction between a single antibody and a single antigen. Antibody-dependent cellular cytotoxicity (ADCC): directed killing of an opsonized target cell by an innate immune cell, such as an NK cell, macrophage, or neutrophil. Antibody glycan: a carbohydrate post-translational modification that is added to the antibody heavy chain at position N297 during antibody synthesis in B cells. Antibodies are exclusively glycosylated with a biantennary glycan structure. Arthus reaction: a type III hypersensitivity reaction that is characterized by vasculitis and primarily caused by activation of the complement cascade by immune complexes. Avidity: the strength of interaction between several antibodies and antigen(s). B cell: a lymphocyte that develops in the bone marrow and the only cell that secretes antibodies. B cell priming: the process that promotes B cell differentiation and maturation through stimulation of B cells by activated CD4+ T helper (TH) cells. Complement cascade: an extracellular innate immune program that can directly lyse pathogens and infected cells, augment phagocytosis, enhance antigen presentation, and modulate inflammation. Effector functions: mechanisms of pathogen clearance mediated by innate immune cells, including phagocytosis, complement-mediated killing, and direct killing through release of cytotoxic granules. Epitope spreading: expansion of the adaptive immune response from the immunodominant epitope of an antigen to subdominant epitopes, allowing for an increase in the diversity of epitopes recognized by both T cells and antibodies. Fab domain: the part of the antibody that binds specifically to antigens. There are two Fab arms per IgG antibody. Fragment crystallizable (Fc) domain: the ‘constant’ domain of the antibody that interacts with innate immune cells, B cells, and

TRMOME 1175 No. of Pages 14

showing that many broadly neutralizing monoclonal antibodies (bNAbs) require Fc-mediated effector functions to provide maximum protection from infection [2,3]. In addition, mounting evidence in the vaccine field suggests that antibody mechanisms beyond neutralization may contribute to protection, because Fc characteristics and antibody-dependent cellular cytotoxicity (ADCC) have been among the key correlates of protection against HIV acquisition in the moderately protective HIV vaccine trial, RV144, where neither neutralizing antibodies nor cytotoxic T cells were, in fact, induced [4]. Moreover, while the development of neutralizing antibodies is a key vaccine target against infectious diseases, mounting evidence across clinically approved vaccines highlights that additional antibody modes of action are required to control infection and explain differential clinical outcomes. As a consequence, a refined understanding of antibody characteristics in the context of different health and disease scenarios may provide important and novel insights into mechanisms of protective immunity. Here, we review recent advances in our understanding of how antibodies are modified during natural disease and vaccination, and how these antibody modifications track with pathogen control, and outline the critical studies that define how antibodies can be modified to enhance functionality in the context of monoclonal antibody (mAb) therapeutic strategies. Finally, we highlight the gaps in our understanding of how antibody functionality is naturally regulated, which, if understood, may offer additional opportunities to augment or modulate antibody functionality through vaccination and immunotherapeutics.

The Immune System Actively Tunes Antibody Effector Activity During an immune response, B cells are able to tune antibody function in two major ways: first, they may irreversibly genetically select different Fc-domains via a process called class switch recombination (CSR) to establish one of four isotypes (IgM, IgG, IgA, or IgE), each with unique Fc-effector functions; or second, B cells may differentially glycosylate antibodies aimed at modulating antibody affinity for individual Fc-receptors. IgG is the most abundant antibody isotype produced in mammals, and humans have four distinct IgG subclasses (IgG1, IgG2, IgG3, and IgG4), which vary in their affinity for Fcg-receptors and functionality (Figure 1 and Box 2). The critical importance of subclass in the induction of innate immune effector functions was first noted in studies evaluating murine mAbs for cancer immunotherapy where IgG2a (analogous to human IgG1/IgG3) was shown to induce ADCC in human NK cells in vitro [5]. Later work using recombinant antibodies with the same Fab domain specific for a melanoma antigen but different murine subclass Fc domains revealed that the ability of each subclass to clear melanoma lung metastases was linked to differential capacities of each subclass to interact with various murine FcgR, and with differential antibody functionality [6]. Furthermore, studies

Box 2. Fc-Mediated Functionality Is Modulated by Subclass and Fc Glycosylation Among the human IgG subclasses, IgG1 and IgG3 are considered the most functional, due to their enhanced affinities for Fc receptors [94]. IgG1 is the highest-produced IgG, binding all FcgRs and, although IgG3 is present in lower abundance compared with IgG1 and IgG2, IgG3 is thought to be the most functional subclass due to an extended hinge region in its structure, endowing it with increased flexibility and binding affinity to all FcgRs [94]. However, the half-life of IgG3 in circulation is 7 days, which is considerably shorter than the 21-day half-life of IgG1 [96], presumably because of the need to limit the presence of highly functional and potentially damaging antibodies. By contrast, IgG2 and IgG4 are less functional than IgG1 and IgG3 owing to their reduced affinity for most FcgRs (IgG2), or their overall lower abundance in human serum (IgG4) [94]. In addition to subclass, the antibody Fc glycan modulates functional activity through alterations in affinity to various FcgRs. For example, glycans that lack the core fucose (afucosylated glycans) drive enhanced ADCC via increased affinity for FcgRIIIA, [97]. Similarly, the addition of a bisecting GlcNAc also enhances FcgRIIIA binding and, thus, ADCC [59,98]. Conversely, sialylation (the presence of sialic acids at the tips of the biantennary glycan) has been linked to antiinflammatory activity via interactions with DC-SIGN and the secretion of TH2 cytokines in the setting of intravenous immunoglobulin [56,89,99], and reduction of complement activation [100]. While many of these associations have been uncovered using mAbs with the removal/addition of entire sugar classes, subtler antibody effector functions may be conferred via the generation of any of the individual glycan substructures.

complement. The Fc glycosylation site and distinct IgG subclasses are encoded within the Fc domain. Fc receptors: extracellular receptors on the surface of innate immune cells and B cells that bind to the Fc domain of antibodies and initiate effector functions. Activating Fc receptors contain an intracellular immunoreceptor tyrosine-based activation motif (ITAM), whereas inhibitory Fc receptors contain an intracellular immunoreceptor tyrosinebased inhibitory motif (ITIM). IgG subclass: refers to one of four types of IgG (IgG1, IgG2, IgG3, IgG4) that is defined by the protein sequence heavy chain of the antibody Fc domain. Each subclass differs in abundance in plasma (IgG1>IgG2>IgG3>IgG4) and affinity for Fc-receptors (IgG3>IgG1>IgG4>IgG2). Immune complex: a protein complex formed between antibodies and antigen. Isotype: refers to the type of antibody (IgA, IgM, IgD, IgE, IgG), and each isotype varies in structure and binds to distinct Fc receptors. Monoclonal antibodies (mAb): antibodies that are identical in the antigen-binding domain that are produced from a single B cell. Natural Killer (NK) cells: innate lymphocytes that can mediate cytotoxicity through both antibodydependent and -independent mechanisms. Neutralizing antibody: an antibody that binds to a pathogen and directly prevents infection through the Fab domain. Non-neutralizing antibody: an antibody that binds to a pathogen, but does not directly prevent infection, yet can limit dissemination and accelerate clearance through recruitment of innate immune effector functions via the antibody Fc domain. Phagocytosis: ingestion/engulfment of a pathogen or infected cell by another cell (e.g., monocyte, macrophage, neutrophil, dendritic cells). Polyfunctionality: the ability to recruit multiple innate immune effector functions. T helper (TH) cells: differentiated CD4+ T cells critical in the development of humoral immune responses and cytotoxic T cell responses.

Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

3

TRMOME 1175 No. of Pages 14

following infection with RNA and DNA viruses have illustrated that the immune system naturally selects for distinct antibody subclass profiles, marked by the preferential selection of IgG2a in mice [7] and IgG1/IgG3 in humans [8]. By contrast, humans select for a dominant IgG2 response following bacterial infection [9], highlighting the involvement of different T helper (TH) cell subsets and responses in antibody subclass selection and, therefore, in function. Transcriptional regulators of TH1/TH2/TH17 immunity have been shown to play a key role in antibody isotype selection in mice, further dissecting the mechanisms by which antibody function, via subclass selection, is coordinated immunologically [10]. During B cell priming, a B cell receives signals to promote isotype/subclass switch through CSR. This process, mediated by the activation-induced deaminase enzyme (AID) and influenced by transcription factors, allows for the removal of entire gene segments from the immunoglobulin locus between switch regions in an irreversible process, resulting in the production of antibodies with a specific antibody Fc-domain [11]. Importantly, excised regions may contain the DNA encoding different subclasses and, thus, once removed, the B cell can no longer revert to those excised subclasses. Therefore, subclass selection represents an irreversible decision in the priming of a B cell that influences its function. Beyond subclass selection, B cells may more subtly tune antibody functionality within a given subclass through alterations in antibody glycosylation. Each heavy chain of an IgG contains a single N-glycosylation site located in the conserved heavy chain CH2 domain that is glycosylated with a biantennary N-glycan structure. The specific glycan structures present on a given antibody can differ with respect to the presence/absence of a fucose moiety, a bisecting Nacetylglucosamine (GlcNAc), and the number of galactoses and sialic acid residues (Figure 2), yielding up to 36 possible structures on a given antibody [12]. The array of possible glycan structures suggests that different glycans likely contribute to the diversity in antibody functionality, modifying the flexibility and structure of the Fc domain [13], and allowing differential interactions with FcRs (Box 2). Importantly, because the polyclonal immune response encompasses many antibody isotypes/subclasses, hundreds of specificities, and heterogeneous glycosylation profiles, together these features ultimately shape the efficacy of the humoral immune response.

Antibody Functional Changes in Disease While there has been much interest in modulating antibody functionality for increased efficacy of mAb therapeutics against cancer and autoimmune diseases, the role of antibody functionality in the context of a polyclonal immune response in disease is only beginning to be explored. Much of our understanding of the role of differential antibody functionality has come from work in mouse models of autoimmunity and cancer, where both induction of disease by autoantibodies and therapeutic treatment by mAbs have elucidated the mechanisms of antibody functionality. Specifically, genetic ablation of the FcR g-subunit in mice (thus eliminating all FcgRs) has demonstrated a critical role for FcgR in autoimmune-associated pathologies, such as observed by the Arthus reaction [14] and in lupus nephritis [15]. Furthermore, genetic ablation of specific FcgRs in mice has highlighted a central role for IgG binding to inhibitory FcgRIIB in the efficacy of anti-inflammatory mAb therapeutics [16,17]. Moreover, IgG binding to FcgRIIB on B cells has been shown to be essential for mediating immune tolerance in the context of autoimmune diseases, such as lupus [18,19], and additional studies have underscored the balance of engaging activating versus inhibitory receptors in driving differential autoimmune disease outcomes [20–22]. These data have provided a critical framework to better profile the functionality of a polyclonal humoral response. However, beyond the role of specific antibody/FcR interactions, characterization of the pathologies associated with autoimmune diseases, such as rheumatoid arthritis (RA), have pointed to

4

Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

Vaccinal effect: induction of longlived adaptive immune responses against a specific antigen by passive transfer of antigen-specific monoclonal antibodies. Viral vectors: recombinant live replicating viruses that express an antigen.

TRMOME 1175 No. of Pages 14

G1B′

G2B

G1S1B

G1S1B′

G2S1B′

G2S1B

G2S2B

G0

G1

G1′

G2

G1S1

G1S1′

G2S1′

G2S1

G2S2

G0FB

G1FB

G1FB′

G2FB

G1S1FB

G1S1FB′

G2S1FB′

G2S1FB

G2S2FB

G0F

G1F

G1F′

G2F

G1S1F

G1S1F′

G2S1F′

G2S1F

G2S2F

G0

G1/G2

Sialylated Aninflammatory

Inflammatory Bisected structures

Fucosylated

G1B

Afucosylated

G0B

Key: GlcNAc

Mannose

Fucose

Galactose

Sialic acid

Figure 2. IgG Glycan Substructures. There are 36 possible biantennary glycan structures present on each heavy chain of IgG, and these can be broadly divided into agalactosylated (G0) structures, galactosylated nonsialylated (G1/G2), and sialylated structures. Agalactosylated IgG are more associated with inflammatory activity, whereas sialylated IgG are associated with anti-inflammatory activity. The presence or absence of fucose (fucosylated or afucosylated, respectively) or the presence or absence of a bisecting GlcNAC modulate interactions with FcgRIIIA that impact [3_TD$IF]induction [4_TD$IF]of FcgRIIIAdependent effector functions, with afucosylated and bisected IgG having the highest affinity for FcgRIIIA.

the critical role of antibody glycosylation perturbations in disease. For instance, elevated serum levels of agalactosylated IgGs has been associated with active RA relative to healthy individuals [23]. These observations provided the first clues that immune complexes might mediate inflammation within the joints, driven by aberrant agalactosylated IgGs and innate immune inflammatory responses. Accordingly, pretreatment of autoantibodies with galactosidases to generate agalactosylated antibodies was shown to enhance RA disease in a murine model of collagen-induced arthritis (CIA) [24], whereas the administration of sialylated/fully galactosylated antibodies could attenuate CIA [25]. In addition, agalactosylated IgG levels have been linked with RA disease severity in humans [26], indicating a role for agalactosylated antibodies in disease pathology, and ascribing a ‘biomarker’ quality to evaluate disease severity. Similarly, genome-wide association studies together with IgG glycan analysis suggest that elevated levels of agalactosylated IgG are a common feature of many autoimmune diseases and cancers [27,28], and analysis of IgG glycosylation is currently being investigated as a biomarker tool to assess immune activation, and prognosis for certain cancers, such as colorectal cancer [29,30]. A similar increase in the levels of bulk circulating agalactosylated IgG has been observed in chronically infected HIV-1+ individuals relative to uninfected individuals [31], and has been found

Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

5

TRMOME 1175 No. of Pages 14

to increase from acute to chronic infection, remaining elevated even with treatment of antiretroviral therapy [32]. Given that agalactosylation of IgG has been linked to immune activation [30], the accumulation of agalactosylated antibodies with HIV-1 infection is likely the result of chronic inflammation observed in HIV-1 infection [33], potentially driving nonspecific alterations in galactose content on antibodies. However, elevated levels of agalactosylated IgG might not necessarily be detrimental, because the highest levels of circulating agalactosylated antibodies have been observed in the serum of spontaneous controllers of HIV-1 infection (i.e., controllers) [32]. Importantly, the increased levels of agalactosylated IgG have been shown to correlate with enhanced NK cell-mediated inhibition of virus production from HIV-infected T cells [32], likely contributing to control of virus FcgRIIB receptor [34]. Furthermore, controllers have been reported to bear a subclass selection bias towards an IgG1/IgG3 response [35,36], resulting overall in a more functional HIV-1-specific antibody pool capable of recruiting multiple innate immune effector functions, also known as polyfunctionality. While controllers are not able to fully clear an HIV infection, the presence of a functional antibody response may act to control active viral replication, allowing for long-term viral control in the absence of antiretroviral therapy. Beyond HIV infection, limited studies have examined the role of human antibody functionality in natural infection. Several studies suggest a role for FcR interactions in antibody-mediated protection from West Nile virus and herpes simplex virus [37,38], yet engagement of FcgR is not necessarily protective in all viral infections. The most notable example is in dengue virus (DENV) infection, where antibody-dependent enhancement (ADE) of DENV into Fc-bearing monocytes is thought to be a major driver of hemorrhagic fever upon secondary infection with a different DENV serotype [39]. ADE occurs in the presence of low levels of cross-reactive DENVspecific antibodies that are insufficient to neutralize incoming virus [40,41], but draw the virus into target cells via FcgR binding and endocytosis. This process is particularly nefarious in the setting of heterologous DENV infection and, alarmingly, other flaviviruses, such as Zika virus [42], where antibodies from a previous infection may bind but not neutralize incoming virus, providing limited protective immunity and, instead, facilitating the delivery of the heterologous virus to target cells, driving ADE and severe disease. Furthermore, recent studies of chronic viral infection in the lymphocytic choriomeningitis virus (LCMV) mouse model demonstrate that chronic viral infection, associated with chronic inflammation, drives dampened Fc-mediated effector activity through the presence of persistent immune complexes [43,44]. These findings are particularly relevant for other chronic infectious diseases, such as tuberculosis, hepatitis B, cytomegalovirus infection, HIV, and hepatitis C virus, that have chronic and acute manifestations of disease, potentially rendering them refractory to mAb-mediated therapeutic treatment. Moreover, these data also raise the possibility that, similar to autoimmune disease, infection-induced immune complexes might drive inflammation, chronicity, and anergy, and may offer exciting promising interventional targets that, if disrupted, might relieve chronic inflammation and enable control/ clearance of the target pathogen. These opportunities as well as additional points of intervention are likely to emerge as the role of antibody-mediated effector functions is explored in the context of other diseases.

Antibody Functionality in Vaccination Mounting evidence from human Fc-receptor knock-in mice and nonhuman primates (NHPs) strongly argue for a critical role for antibody effector function in protection from several infectious diseases, including influenza and HIV. Specifically, several HIV-specific bNAbs require Fceffector function to provide protective immunity against HIV in mice [3] as well as in NHPs [45]. Along the same lines, elevated antibody titers, linked to polyfunctional antibody effector activity, have been associated with protective immunity following vaccination with an adenovirus-26 (Ad26)-based vaccine in NHPs [46]. Moreover, immune correlates analysis of 17 vaccineinduced humoral immune features that interrogated antibody binding, neutralization, and ADCC in the moderately protective HIV vaccine efficacy trial, RV144, indicated that the IgG response

6

Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

TRMOME 1175 No. of Pages 14

against a particular region of the HIV envelope, V1V2, with enhanced ADCC activity, inversely correlated with risk of infection [4]. Subsequent analyses demonstrated that the induction of polyfunctional vaccine-specific IgG3 responses inversely correlated with risk of infection [47,48], similar to the Ad26-induced antibody profile observed in protected NHPs [46]. Moreover, a new Systems Serology profiling approach used to interrogate antibody function and biophysical profiles broadly, identified unique antibody network profiles induced by four distinct HIV vaccines, including RV144 [49]. Specifically, RV144, which used a canarypox vector (ALVAC) prime followed by a recombinant gp120 and alum boost, was marked by a well-coordinated highly functional IgG1 and polyfunctional IgG3 response against the gp120 V1V2 region. Conversely, the other vaccines exhibited antibody signature profiles that exhibited less coordinated IgG1/IgG3 responses, highlighting unique antibody networks induced by distinct vaccines, and suggesting ways in which these antibodies might interact to generate immune complexes, driving differential antibody functionality. Adjuvants, such as alum and oil-in-water emulsions (e.g., MF59 and AS01B), and most recently, toll-like receptor (TLR) agonists, are used to drive more robust immunity [50]. Recent data suggest that adjuvants can not only improve the magnitude of the vaccine-induced humoral immune response, but also qualitatively alter the response by broadening it to cover more regions of the vaccine antigen through a process known as epitope spreading [51,52]. Yet, beyond their effect on Fab-mediated activity, adjuvants also qualitatively shape the effector function of antibodies through subclass selection biases in a TH1/TH2-dependent manner, and also via changes in antibody glycosylation. Specifically, ALVAC-SIV gp120 vaccination in the presence of two distinct adjuvants, either alum or MF59, demonstrated that the alum-adjuvanted vaccine was more protective against SIV challenge in macaques than was the MF59adjuvanted vaccine [53]. While MF59-adjuvanted animals mounted stronger antibody responses overall, alum-vaccinated animals generated higher levels of mucosal antibodies directed against the V2 region of gp120 [53], suggesting that qualitative, rather than quantitative, differences in antibody quality might be more critical for predicting vaccine immunity. Interestingly, alum and MF59-induced gp120-specific antibodies were also differentially glycosylated. The alum arm could induce higher levels of gp120-specific antibodies with fucosylated monogalactosylated (G1F) and agalactosylated (G0F) glycan structures [53], and increased levels of these structures have been linked to enhanced antibody effector function for HIV-specific human mAbs and in HIV infected-subjects [54,55]. By contrast, MF59-adjuvanted macaques exhibited an increase in the levels of SIV gp120-specific antibodies with sialylated di-galactosylated glycan structures, which are predicted to have low antibody effector function due to the anti-inflammatory effect of sialylation on antibody function [56]. Similarly, analysis of human influenza-specific antibodies induced via vaccination with an MF59-adjuvanted influenza vaccine demonstrated a transient increase in galactosylated and sialylated influenza-specific antibodies [57], suggesting that MF59 itself may selectively drive highly galactosylated and sialylated vaccine-specific antibodies. However, whether adjuvants can selectively skew antibody glycosylation and enable a customized induction of antibodies with specific functional responses remains to be determined. Vaccination using viral vectors has shown promise against several different infectious diseases for which traditional vaccination strategies have failed. Delivery of antigens by adenovirus, poxviruses, and vesicular stomatitis virus have been used in preclinical trials in NHP to drive efficacy against HIV/SIV, Ebola virus, malaria, and influenza [58]. Studies analyzing the impact of different vaccine vectors on antibody functionality are limited, yet are beginning to be explored in the context of HIV/SIV, Ebola virus infection, and malaria. Interestingly, Ad26 vaccination in humans has been found to induce IgG1/IgG3-biased responses that are more analogous to the poxvirus/protein-based vaccine strategy that was used in the RV144 HIV vaccine trial, when compared with protein-based or DNA/Ad5-based vaccine strategies [49]. This suggests that viral vectors can skew Fc-effector profiles of vaccine-induced antibodies. Moreover, another

Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

7

TRMOME 1175 No. of Pages 14

study showed that, compared with an HIV alum-adjuvanted protein vaccine (VAX003), Ad26vectored immunized (IPCAVD001) humans developed gp120-specific antibodies that were more sialylated and galactosylated, yet also exhibited higher levels of bisecting GlcNAc, which is associated with enhanced ADCC [55,59]. These data indicate that select viral vector-based vaccines are able to skew both antibody subclass and glycosylation profiles. Thus, future studies comparing vaccine vectors, inserts, and/or adjuvants may provide exciting insights into the potential opportunities to drive potent antipathogen immunity via functional optimization of the humoral immune response.

Protective Broadly Neutralizing Antibodies Require Fc-mediated Effector Functions The ultimate goal of most vaccines is to induce neutralizing antibodies that can block infection. However, in many cases, antibodies may not protect via neutralization alone, but may recruit additional antibody effector functions to control and kill the pathogen against which they are directed. Along these lines, several recent studies have uncovered unexpected, yet remarkable, biological activities of neutralizing antibodies in the restriction of pathogens, neutralization of toxins, and in modulating immunity. The protective activity of some neutralizing antibodies requires Fc-mediated effector functions, such as complement activation and phagocytic clearance. For example, neutralizing mAbs against vaccinia virus (VACV) require complement in order to neutralize the enveloped form of the virion, conferring protection, as shown in mouse models of VACV infection [60,61]. In the context of antibody-mediated protection from Bacillus anthracis, neutralizing antibody titers against anthrax toxin correlate with vaccine-mediated protection in guinea pigs [62], yet these antibodies require activating FcgRs in vivo in order to provide protection in mice via FcR-mediated clearance of toxin subunits, preventing the formation of anthrax lethal toxin [63,64]. In the context of HIV infection, more than three dozen HIV-specific bNAbs have been described to date, many of which confer sterilizing protection from infection in NHPs [65–69]. Early studies have demonstrated that even neutralizing antibodies may depend on Fc-effector activity to confer protection from infection. In particular, an Fc-mutated form of the neutralizing antibody b12 (unable to bind Fc-receptors) only conferred partial protection from infection in a macaque SIV challenge model [45]. However, subsequent studies aimed at augmenting b12-mediated protection through enhanced FcgRIIIA binding via antibody afucosylation did not result in increased protection from simian-HIV infection in macaques [70], suggesting that other FcgRs might be important for protection. Moreover, analysis of Fc-effector function dependence of HIVspecific bNAbs, including one of the most potent neutralizing mAbs described to date, 3BNC117, in a humanized mouse model have highlighted the importance of antibody binding to activating FcgR in protection from HIV infection [3]. Specifically, bNAbs with point mutations in the Fc-domain that enhance binding to activating FcgR conferred not only higher levels of protection against HIV infection compared with bNAbs with the wild-type Fc domain, but the Fcenhanced bNAbs were also able to control plasma viral loads in humanized mice more effectively following HIV infection compared with bNAbs that lacked the capacity to bind FcgRs. Similarly, protection against lethal influenza infection by influenza-specific bNAbs in mice was dependent on antibody engagement of activating FcgRs, and protection was augmented when neutralizing antibodies were Fc-optimized to enhance binding to FcgRIIA and FcgRIIIA [71]. While FcgR activity has been found to contribute to the protective efficacy of HIV-specific bNAbs targeting distinct regions of the HIV envelope [3], the dependence on FcgR activity for protective efficacy against influenza infection was shown to be dependent on the antigenic domain within influenza hemagglutinin (HA) targeted by the antibody. Specifically, the protective efficacy of antibodies targeting the variable head domain of HA was not reported to be dependent on the engagement of FcgRs, as opposed to antibodies targeting the conserved stalk domain of HA,

8

Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

TRMOME 1175 No. of Pages 14

which were shown to be dependent on FcgRs [2,71]. Such discrepant antibody bioactivities might reflect the viral infection stage at which these antibodies act to target the virus. FcgRindependent antibodies that recognize the head domain might prevent entry into a host cell, whereas FcgR-dependent mAbs targeting the stalk domain might trigger innate immune destruction within endosomes or on the surface of the infected cell. However, the exact mechanism(s) for the mode of action for antibody-mediated protection in the context of HIV and influenza infection remain to be defined. Nevertheless, these data clearly illustrate the critical nature of antibody Fc-effector activity in immunotherapies against viral infection. Beyond the relatively short-term activity conferred by passive transfer of bNAbs, recent studies demonstrate that there are also long-term protective activities of bNAbs partly mediated through FcgR interactions within the immune system. Specifically, single administration of 3BNC117 has been shown to reduce viral loads in HIV-infected individuals [72], while being able to increase neutralizing antibody potency against autologous viruses and increased neutralization breadth against heterologous viruses when compared with control-treated individuals, 6 months after initial treatment [73]. A potential mechanism that may underlie this finding is an antibody ‘vaccinal effect’, which was first observed in cancer immunotherapeutic treatments using the mAb rituximab, where targeting a tumor using rituximab induced cellular immunity that contributed to a long-lived control of the tumor [74]. Along these lines, recent work dissecting the mechanisms of the vaccinal effect in a CD20-expressing murine lymphoma model has indicated that immune complexes formed by the anti-CD20 antibody together with tumor antigens can promote cellular immunity through FcgRIIA-dependent activation of dendritic cells, driving development of T cells that can provide long-term protection against subsequent tumor challenge [75]. Moreover, immune complexes composed of sialylated influenza HA-specific human antibodies have been shown to stimulate higher affinity and broadly protective anti-HA antibodies in a CD23-dependent manner in mice compared with asialylated immune complexes [76], suggesting that particular glycosylated antibody profiles may act as critical adjuvants to trigger B cell immunity following vaccination. Together, these studies highlight the diversity of functions that the antibody Fc-domain mediates, impacting both T and B cells, providing further insight into how these functions could be leveraged to design putative therapeutic and/or vaccine strategies against infectious diseases. Together, these findings have prompted the analyses of Fc-mediated function against other viruses, such as Ebola virus [77]. Treatment of Ebola-infected NHPs with the highly protective mAb cocktail, ZMapp, which contains two neutralizing antibodies in addition to a non-neutralizing antibody, c13C6, has resulted in a remarkable reduction of viral loads, reversal of disease symptoms, and survival [78]. Importantly, removal of c13C6 from the ZMapp cocktail was shown to decrease protection from infection, suggesting that non-neutralizing functions conferred by c13C6, such as complement activation [79] or other innate immune effector functions, may be necessary for antibody-mediated protection against Ebola virus [80]. In addition, protective non-neutralizing mAbs have also been recently identified in the context of influenza virus, where several broadly cross-reactive non-neutralizing mAbs were isolated from vaccinated humans. Importantly, these non-neutralizing mAbs conferred protection from a lethal influenza infection in a mouse model in a FcgR-dependent manner, because protection was lost when Fc domain was mutated to abrogate binding to FcgR [81]. These results further support the concept that FcgR-recruiting non-neutralizing antibodies might be useful in therapeutic strategies against a variety of viral infections.

Harnessing Antibody-mediated Innate Immune Effector Functionality The mechanistic links between induction of specific antibody-mediated innate immune effector functions and biophysical features of antibodies (e.g., subclass and glycosylation) are becoming clearer, yet the mechanisms that govern the production of antibodies with a given functional

Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

9

TRMOME 1175 No. of Pages 14

capacity remain unclear. However, there are several steps in the development of an antibody response where vaccines have the opportunity to shape the functional humoral response, including promoting humoral immunity through induction of a TH response, and shaping antibody functionality through CSR and/or antibody glycosylation ([8_TD$IF]see Figure I[9_TD$IF] in Box 3). The natural immune signals that promote CSR to a particular isotype/subclass have not been completely defined. Cytokines such as IL-4, TGFb, and IFNg activate transcriptional programs that regulate CSR and class switch to different isotypes and IgG subclasses, yet additional stimuli, including TLR and BCR engagement, are required for CSR [11] ([8_TD$IF]see Figure I[9_TD$IF] in Box 3). Less is known about the immune signals that tune antibody glycosylation. Differential expression of glycosyltransferases and glycosidases within the Golgi apparatus in B cells can alter the composition of the antibody glycan ([8_TD$IF]see Figure I[9_TD$IF] in Box 3). This information has been used extensively in the field of mAb therapeutics, where advances in cellular and plant bioengineering have enabled the rapid

Box 3. Natural Modulation of Antibody Functionality Functionality of IgG can be modulated by subclass selection and glycosylation, but the immunologic signals that modify these IgG features remain undefined, although it will be important to define these in order to direct antibody functionality by vaccination. Different Th subsets (Th1/Th2, Th17, Tfh, etc.) together with certain cytokines, such as IL-4, TGF-b, and IFNg, are known to promote class switch to a given IgG subclass (Figure I). Thus, activation of a particular T helper cell subset through vaccination may determine the dominant IgG subclass induced following vaccination. In addition, TLR and BCR stimulation on B cells also drives class switch in the absence of T cell help, offering additional mechanisms by which vaccination might direct antibody functionality [101]. Modulation of IgG glycosylation likely occurs within antibody-secreting cells, and several glycosidases and glycotransferases control the glycosylation of human IgG in a manner that can impact antibody functionality. The fucosyltransferase FUT8 regulates the addition of fucose, the addition of galactose on each arm of the antibody glycan is regulated by B4GALT1, and addition of terminal sialic acid residues to the galactose is mediated by the sialyltransferase, ST6GAL1. Differential levels of these enzymes in the Golgi apparatus during antibody production is thought to determine the glycan structure of a given antibody, yet, how these enzyme might be regulated during an immune response is unclear. In vitro stimulation of differentiated human B cells with the TLR ligand CpG, IFNg, or IL-21 have been shown to result in increased galactosylation and sialylation of produced IgG1, whereas stimulation with other cytokines, such as TNF/ and IL-4, has been found to bear minimal impact on glycosylation [102]. Whether these stimuli impact antibody glycosylation in vivo has yet to be defined, but could offer an opportunity for vaccine development to further tune antibody functionality.

TH cells/TFH cells

TLR smuli BCR smuli other?

TLR smuli Cytokines? Other?

IL-4, TGF-β, IFN γ, others?

Acvated B cell

Cμ

IgG3

Cγ3

? Anbody-secreng cell

?

Cγ1

Cγ2

Cγ4

IgG2 FUT8

IgG1

IgG4

Figure I. Natural Modulation of Antibody Functionality.

10

Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

MGAT3

B4GALT1

ST6GAL1

TRMOME 1175 No. of Pages 14

Box 4. The Clinician's Corner

Outstanding Questions

 Beyond direct pathogen neutralization, antibodies can provide protection from infectious disease through recruitment

What are the functional profiles of antibodies induced by different vaccines, vaccine vectors, and adjuvants against the one same pathogen? Is there a particular profile associated with protective efficacy?

of innate immune effector functions that can clear pathogens and infected cells. This aspect of antibody functionality is modulated primarily through antibody isotype/subclass selection and antibody glycosylation.  Antibody functionality is altered in specific disease settings, such as in autoimmunity and HIV infection. Analysis of differential antibody glycosylation is currently being developed as a putative ‘biomarker’ for certain autoimmune diseases.  Different vaccines, adjuvants, and vaccine vectors induce distinct antibody profiles that likely contribute to vaccine efficacy. Thus, comprehensive profiling of the humoral response is needed in order to define mechanisms of vaccineinduced protection.  Modification of bNAbs antibodies to increase recruitment of innate immune effector functions might provide enhanced protection against HIV-1 and influenza infections.

production of Fc-optimized glycoengineered mAbs for a diverse range of diseases [82–85]. However, defining the natural immune signals and regulators of antibody isotype/subclass and glycosylation may hold the key to the rational development of functionally customizable antipathogen and anticancer vaccines of the future. Finally, the ultimate generation of customized Fcoptimized therapeutics or vaccines will likely benefit from a deeper understanding of the specific cell type(s) and FcRs that are present at relevant sites of an infection/malignancy. For example, HIV infects at mucosal sites, such as the female genital tract and the rectal mucosa, yet the expression of FcRs on mucosal innate immune cells is strikingly different from that observed on blood innate immune cells [86]. Furthermore, increasing the presence of pathogen-specific antibodies at the sites of interest, such as at mucosal sites for HIV preventative strategies, either through increased binding to the neonatal Fc-receptor, FcRn [87], or mucosal proteins [88], may additionally improve the bioactivity of antibodies to prevent transmission.

Concluding Remarks The growing interest in understanding how antibody functionality beyond neutralization impacts control of infectious diseases over the past few years has demonstrated the remarkable diversity in antibody bioactivity, and has highlighted their critical role in protection from infectious diseases. Importantly, because functional optimization of currently available bNAbs against HIV, influenza, and Ebola virus increases protective efficacy, these exciting data provide a rationale and framework for the rapid development of effective therapeutic antibodies against infectious diseases, including emerging viruses, such as Zika virus, which may be useful in limiting outbreaks during epidemics. Moreover, comprehensive, profiling approaches of humoral immunity that are able to incorporate measures of Fab and Fc bioactivities simultaneously, and are linked to humanized animal models, may offer a unique opportunity to define the mechanisms of action of antibodies against devastating infectious diseases. However, several important questions remain regarding the impact of different vaccine strategies, adjuvants, and diseases on antibody functionality (see Outstanding Questions; Boxes 3 and 4), particularly with regard to induction of specific functions. Moreover, many of the natural immune signals that regulate the development of functional antibodies remain undefined, and should be characterized in order to maximize antibody functionality in next generation vaccines. Finally, as our appreciation and understanding of the remarkably broad bioactive capacity of antibodies grows in parallel with the tools to interrogate their function and protective efficacy, new and unexpected mechanisms of antibody-mediated protection are likely to be described, offering additional exciting opportunities to leverage the humoral immune response to treat an array of diseases.

What are the immune signals that alter antibody glycosylation, and what are the signals that promote proinflammatory or anti-inflammatory glycan structures? Are these signals induced in specific disease settings or induced by particular vaccines? Is antibody functionality differentially modulated in acute versus chronic viral and bacterial infections? Does antibody-mediated recruitment of specific innate immune effector functions contribute to natural control of infection? Can highly functional neutralizing and non-neutralizing monoclonal antibodies be used as therapeutics to treat and clear infections? What are the specific innate immune effector function(s) that contribute to enhanced efficacy of broadly neutralizing monoclonal antibodies against HIV-1, influenza, and Ebola? Are there additional antibody Fc-mediated effector functions that modulate immunity? Is there a role for Fc-mediated effector functions that are induced by other antibody isotypes (IgA, IgM, IgE) in infectious diseases?

Acknowledgments We would like to thank the members of the Alter laboratory for discussion and support, with specific acknowledgement of Saheli Sadanand and Todd Suscovich for their critical insight and discussion. We would also like to acknowledge funding support from the Bill and Melinda Gates Foundation (OPP1032817 and OPP1114729 awarded to G.A.) and the National Institutes of Allergy and Infectious Diseases (R37 A1080389 and R01 AI102660 awarded to G.A., and F32 AI114406 awarded to B.G.).

Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

11

TRMOME 1175 No. of Pages 14

References 1.

Pulendran, B. and Ahmed, R. (2011) Immunological mechanisms of vaccination. Nat. Immunol. 12, 509–517

2.

DiLillo, D.J. et al. (2016) Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection. J. Clin. Invest. 126, 605–610

3.

Bournazos, S. et al. (2014) Broadly neutralizing anti-HIV-1 antibodies require Fc effector functions for in vivo activity. Cell 158, 1243–1253

4.

Haynes, B.F. et al. (2012) Immune-correlates analysis of an HIV-1 vaccine efficacy trial. N. Engl. J. Med. 366, 1275–1286

5.

Kipps, T.J. et al. (1985) Importance of immunoglobulin isotype in human antibody-dependent, cell-mediated cytotoxicity directed by murine monoclonal antibodies. J. Exp. Med. 161, 1–17

6.

Nimmerjahn, F. and Ravetch, J.V. (2005) Divergent immunoglobulin g subclass activity through selective Fc receptor binding. Science 310, 1510–1512

7.

Coutelier, J.P. et al. (1987) IgG2a restriction of murine antibodies elicited by viral infections. J. Exp. Med. 165, 64–69

8.

Beck, O.E. (1981) Distribution of virus antibody activity among human IgG subclasses. Clin. Exp. Immunol. 43, 626–632

9.

Siber, G.R. et al. (1980) Correlation between serum IgG-2 concentrations and the antibody response to bacterial polysaccharide antigens. N. Engl. J. Med. 303, 178–182

10. Wang, N.S. et al. (2012) Divergent transcriptional programming of class-specific B cell memory by T-bet and RORalpha. Nat. Immunol. 13, 604–611 11. Xu, Z. et al. (2012) Immunoglobulin class-switch DNA recombination: induction, targeting and beyond. Nat. Rev. Immunol. 12, 517–531 12. Pucic, M. et al. (2011) High throughput isolation and glycosylation analysis of IgG-variability and heritability of the IgG glycome in three isolated human populations. Mol. Cell Proteomics 10, M111010090 13. Krapp, S. et al. (2003) Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity. J. Mol. Biol. 325, 979–989 14. Sylvestre, D.L. and Ravetch, J.V. (1994) Fc receptors initiate the Arthus reaction: redefining the inflammatory cascade. Science 265, 1095–1098 15. Clynes, R. et al. (1998) Uncoupling of immune complex formation and kidney damage in autoimmune glomerulonephritis. Science 279, 1052–1054 16. Samuelsson, A. et al. (2001) Anti-inflammatory activity of IVIG mediated through the inhibitory Fc receptor. Science 291, 484– 486

26. Ercan, A. et al. (2010) Aberrant IgG galactosylation precedes disease onset, correlates with disease activity, and is prevalent in autoantibodies in rheumatoid arthritis. Arthritis Rheum. 62, 2239–2248 27. Lauc, G. et al. (2013) Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers. PLoS Genet. 9, e1003225 28. Wuhrer, M. et al. (2015) Skewed Fc glycosylation profiles of antiproteinase 3 immunoglobulin G1 autoantibodies from granulomatosis with polyangiitis patients show low levels of bisection, galactosylation, and sialylation. J. Proteome Res. 14, 1657–1665 29. Theodoratou, E. et al. (2016) Glycosylation of plasma IgG in colorectal cancer prognosis. Sci. Rep. 6, 28098 30. de Jong, S.E. et al. (2016) IgG1 Fc N-glycan galactosylation as a biomarker for immune activation. Sci. Rep. 6, 28207 31. Moore, J.S. et al. (2005) Increased levels of galactose-deficient IgG in sera of HIV-1-infected individuals. AIDS 19, 381–389 32. Ackerman, M.E. et al. (2013) Natural variation in Fc glycosylation of HIV-specific antibodies impacts antiviral activity. J. Clin. Invest. 123, 2183–2192 33. Deeks, S.G. et al. (2013) Systemic effects of inflammation on health during chronic HIV infection. Immunity 39, 633–645 34. Ackerman, M.E. et al. (2013) Enhanced phagocytic activity of HIV-specific antibodies correlates with natural production of immunoglobulins with skewed affinity for FcgammaR2a and FcgammaR2b. J. Virol. 87, 5468–5476 35. Lai, J.I. et al. (2014) Divergent antibody subclass and specificity profiles but not protective HLA-B alleles are associated with variable antibody effector function among HIV-1 controllers. J. Virol. 88, 2799–2809 36. Ackerman, M.E. et al. (2016) Polyfunctional HIV-specific antibody responses are associated with spontaneous HIV control. PLoS Pathog. 12, e1005315 37. Vogt, M.R. et al. (2011) Poorly neutralizing cross-reactive antibodies against the fusion loop of West Nile virus envelope protein protect in vivo via Fcgamma receptor and complement-dependent effector mechanisms. J. Virol. 85, 11567–11580 38. Balachandran, N. et al. (1982) Protection against lethal challenge of BALB/c mice by passive transfer of monoclonal antibodies to five glycoproteins of herpes simplex virus type 2. Infect. Immun. 37, 1132–1137 39. Guzman, M.G. et al. (2013) Secondary infection as a risk factor for dengue hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibody-dependent enhancement of infection. Arch. Virol. 158, 1445–1459

17. Clynes, R.A. et al. (2000) Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat. Med. 6, 443–446

40. de Alwis, R. et al. (2014) Dengue viruses are enhanced by distinct populations of serotype cross–reactive antibodies in human immune sera. PLoS Pathog. 10, e1004386

18. Fukuyama, H. et al. (2005) The inhibitory Fcgamma receptor modulates autoimmunity by limiting the accumulation of immunoglobulin G+ anti-DNA plasma cells. Nat. Immunol. 6, 99–106

41. Halstead, S.B. (2003) Neutralization and antibody-dependent enhancement of dengue viruses. Adv. Virus Res. 60, 421–467

19. McGaha, T.L. et al. (2005) Restoration of tolerance in lupus by targeted inhibitory receptor expression. Science 307, 590–593

42. Dejnirattisai, W. et al. (2016) Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus. Nat. Immunol. 17, 1102–1108

20. Kaneko, Y. et al. (2006) Pathology and protection in nephrotoxic nephritis is determined by selective engagement of specific Fc receptors. J. Exp. Med. 203, 789–797 21. Kalergis, A.M. and Ravetch, J.V. (2002) Inducing tumor immunity through the selective engagement of activating Fcgamma receptors on dendritic cells. J. Exp. Med. 195, 1653–1659 22. Baudino, L. et al. (2008) Differential contribution of three activating IgG Fc receptors (FcgammaRI, FcgammaRIII, and FcgammaRIV) to IgG2a- and IgG2b-induced autoimmune hemolytic anemia in mice. J. Immunol. 180, 1948–1953 23. Parekh, R.B. et al. (1985) Association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum IgG. Nature 316, 452–457 24. Rademacher, T.W. et al. (1994) Agalactosyl glycoforms of IgG autoantibodies are pathogenic. Proc. Natl. Acad. Sci. U.S.A. 91, 6123–6127 25. Ohmi, Y. et al. (2016) Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis. Nat. Commun. 7, 11205

12

Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

43. Wieland, A. et al. (2015) Antibody effector functions mediated by Fcgamma-receptors are compromised during persistent viral infection. Immunity 42, 367–378 44. Yamada, D.H. et al. (2015) Suppression of Fcgamma-receptormediated antibody effector function during persistent viral infection. Immunity 42, 379–390 45. Hessell, A.J. et al. (2007) Fc receptor but not complement binding is important in antibody protection against HIV. Nature 449, 101–104 46. Barouch, D.H. et al. (2015) Protective efficacy of adenovirus/ protein vaccines against SIV challenges in rhesus monkeys. Science 349, 320–324 47. Chung, A.W. et al. (2014) Polyfunctional Fc-effector profiles mediated by IgG subclass selection distinguish RV144 and VAX003 vaccines. Sci. Transl. Med. 6, 228ra238 48. Yates, N.L. et al. (2014) Vaccine-induced Env V1-V2 IgG3 correlates with lower HIV-1 infection risk and declines soon after vaccination. Sci. Transl. Med. 6, 228ra239

TRMOME 1175 No. of Pages 14

49. Chung, A.W. et al. (2015) Dissecting polyclonal vaccine-induced humoral immunity against HIV using systems serology. Cell 163, 988–998

72. Caskey, M. et al. (2015) Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117. Nature 522, 487–491

50. Bergmann-Leitner, E.S. and Leitner, W.W. (2014) Adjuvants in the driver's seat: how magnitude, type, fine specificity and longevity of immune responses are driven by distinct classes of immune potentiators. Vaccines (Basel) 2, 252–296

73. Schoofs, T. et al. (2016) HIV-1 therapy with monoclonal antibody 3BNC117 elicits host immune responses against HIV-1. Science 352, 997–1001

51. Khurana, S. et al. (2010) Vaccines with MF59 adjuvant expand the antibody repertoire to target protective sites of pandemic avian H5N1 influenza virus. Sci Transl Med 2, 15ra15 52. Shukla, N.M. et al. (2012) Potent adjuvanticity of a pure TLR7agonistic imidazoquinoline dendrimer. PLoS ONE 7, e43612 53. Vaccari, M. et al. (2016) Adjuvant-dependent innate and adaptive immune signatures of risk of SIV acquisition. Nat. Med. 22, 762–770 54. Chung, A.W. et al. (2014) Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function. AIDS 28, 2523–2530 55. Mahan, A.E. et al. (2016) Antigen-specific antibody glycosylation is regulated via vaccination. PLoS Pathog. 12, e1005456 56. Kaneko, Y. et al. (2006) Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science 313, 670–673 57. Selman, M.H. et al. (2012) Changes in antigen-specific IgG1 Fc N-glycosylation upon influenza and tetanus vaccination. Mol Cell Proteomics 11, M111014563 58. Draper, S.J. and Heeney, J.L. (2010) Viruses as vaccine vectors for infectious diseases and cancer. Nat. Rev. Microbiol. 8, 62–73 59. Davies, J. et al. (2001) Expression of GnTIII in a recombinant antiCD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FC gamma RIII. Biotechnol. Bioeng. 74, 288–294 60. Benhnia, M.R. et al. (2009) Vaccinia virus extracellular enveloped virion neutralization in vitro and protection in vivo depend on complement. J. Virol. 83, 1201–1215 61. Benhnia, M.R. et al. (2013) Unusual features of vaccinia virus extracellular virion form neutralization resistance revealed in human antibody responses to the smallpox vaccine. J. Virol. 87, 1569–1585

74. Cartron, G. et al. (2004) From the bench to the bedside: ways to improve rituximab efficacy. Blood 104, 2635–2642 75. DiLillo, D.J. and Ravetch, J.V. (2015) Differential Fc-receptor engagement drives an anti-tumor vaccinal effect. Cell 161, 1035–1045 76. Wang, T.T. et al. (2015) Anti-HA glycoforms drive B cell affinity selection and determine influenza vaccine efficacy. Cell 162, 160–169 77. Corti, D. et al. (2016) Protective monotherapy against lethal Ebola virus infection by a potently neutralizing antibody. Science 351, 1339–1342 78. Qiu, X. et al. (2014) Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature 514, 47–53 79. Wilson, J.A. et al. (2000) Epitopes involved in antibody-mediated protection from Ebola virus. Science 287, 1664–1666 80. Qiu, X. et al. (2016) Two-mAb cocktail protects macaques against the Makona variant of Ebola virus. Sci Transl Med 8, 329ra333 81. Henry Dunand, C.J. et al. (2016) Both neutralizing and non-neutralizing human H7N9 influenza vaccine-induced monoclonal antibodies confer protection. Cell Host Microbe 19, 800–813 82. Strasser, R. et al. (2008) Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. Plant Biotechnol. J. 6, 392–402 83. Zeitlin, L. et al. (2011) Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant. Proc. Natl. Acad. Sci. U.S.A. 108, 20690–20694 84. Hiatt, A. et al. (2014) Glycan variants of a respiratory syncytial virus antibody with enhanced effector function and in vivo efficacy. Proc. Natl. Acad. Sci. U.S.A. 111, 5992–5997

62. Reuveny, S. et al. (2001) Search for correlates of protective immunity conferred by anthrax vaccine. Infect. Immun. 69, 2888–2893

85. Umana, P. et al. (1999) Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat. Biotechnol. 17, 176–180

63. Abboud, N. et al. (2010) A requirement for FcgammaR in antibody-mediated bacterial toxin neutralization. J. Exp. Med. 207, 2395–2405

86. Cheeseman, H.M. et al. (2016) Expression profile of human Fc receptors in mucosal tissue: implications for antibody-dependent cellular effector functions targeting HIV-1 transmission. PLoS ONE 11, e0154656

64. Bournazos, S. et al. (2014) Human IgG Fc domain engineering enhances antitoxin neutralizing antibody activity. J. Clin. Invest. 124, 725–729 65. Moldt, B. et al. (2012) Highly potent HIV-specific antibody neutralization in vitro translates into effective protection against mucosal SHIV challenge in vivo. Proc. Natl. Acad. Sci. U.S.A. 109, 18921–18925 66. Pegu, A. et al. (2014) Neutralizing antibodies to HIV-1 envelope protect more effectively in vivo than those to the CD4 receptor. Sci Transl Med 6, 243ra288 67. Mascola, J.R. et al. (2000) Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat. Med. 6, 207–210

87. Ko, S.Y. et al. (2014) Enhanced neonatal Fc receptor function improves protection against primate SHIV infection. Nature 514, 642–645 88. Gunn, B.M. et al. (2016) Enhanced binding of antibodies generated during chronic HIV infection to mucus component MUC16. Mucosal Immunol. Published online March 9, 2016. http://dx.doi. org/10.1038/mi.2016.8 89. Anthony, R.M. et al. (2008) Recapitulation of IVIG antiinflammatory activity with a recombinant IgG Fc. Science 320, 373–376 90. Schwab, I. et al. (2012) IVIg-mediated amelioration of ITP in mice is dependent on sialic acid and SIGNR1. Eur. J. Immunol. 42, 826–830

68. Barouch, D.H. et al. (2013) Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys. Nature 503, 224–228

91. Sondermann, P. et al. (2013) General mechanism for modulating immunoglobulin effector function. Proc. Natl. Acad. Sci. U.S.A. 110, 9868–9872

69. Hessell, A.J. et al. (2009) Effective, low-titer antibody protection against low-dose repeated mucosal SHIV challenge in macaques. Nat. Med. 15, 951–954

92. Clark, M.R. et al. (1989) Molecular basis for a polymorphism involving Fc receptor II on human monocytes. J. Immunol. 143, 1731–1734

70. Moldt, B. et al. (2012) A nonfucosylated variant of the anti-HIV-1 monoclonal antibody b12 has enhanced FcgammaRIIIa-mediated antiviral activity in vitro but does not improve protection against mucosal SHIV challenge in macaques. J. Virol. 86, 6189–6196

93. Ravetch, J.V. and Perussia, B. (1989) Alternative membrane forms of Fc gamma RIII(CD16) on human natural killer cells and neutrophils. Cell type-specific expression of two genes that differ in single nucleotide substitutions. J. Exp. Med. 170, 481–497

71. DiLillo, D.J. et al. (2014) Broadly neutralizing hemagglutinin stalkspecific antibodies require FcgammaR interactions for protection against influenza virus in vivo. Nat. Med. 20, 143–151

94. Bruhns, P. et al. (2009) Specificity and affinity of human Fcgamma receptors and their polymorphic variants for human IgG subclasses. Blood 113, 3716–3725

Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

13

TRMOME 1175 No. of Pages 14

95. Bournazos, S. et al. (2009) Functional and clinical consequences of Fc receptor polymorphic and copy number variants. Clin. Exp. Immunol. 157, 244–254

99. Anthony, R.M. et al. (2011) Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway. Nature 475, 110–113

96. Morell, A. et al. (1970) Metabolic properties of IgG subclasses in man. J. Clin. Invest. 49, 673–680

100. Quast, I. et al. (2015) Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity. J. Clin. Invest. 125, 4160–4170

97. Ferrara, C. et al. (2011) Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose. Proc. Natl. Acad. Sci. U.S. A. 108, 12669–12674 98. Shields, R.L. et al. (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J. Biol. Chem. 277, 26733– 26740

14

Trends in Molecular Medicine, Month Year, Vol. xx, No. yy

101. Pone, E.J. et al. (2012) BCR-signalling synergizes with TLRsignalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-kappaB pathway. Nat. Commun. 3, 767 102. Wang, J. et al. (2011) Fc-glycosylation of IgG1 is modulated by Bcell stimuli. Mol. Cell Proteomics 10, M110004655