Superantigen Recognition and Interactions: Functions, Mechanisms and Applications

Abstract

Superantigens are unconventional antigens which recognise immune receptors outside their usual recognition sites e.g. complementary determining regions (CDRs), to elicit a response within the target cell. T-cell superantigens crosslink T-cell receptors and MHC Class II molecules on antigen-presenting cells, leading to lymphocyte recruitment, induction of cytokine storms and T-cell anergy or apoptosis among many other effects. B-cell superantigens, on the other hand, bind immunoglobulins on B-cells, affecting opsonisation, IgG-mediated phagocytosis, and driving apoptosis. Here, through a review of the structural basis for recognition of immune receptors by superantigens, we show that their binding interfaces share specific physicochemical characteristics when compared with other protein-protein interaction complexes. Given that antibody-binding superantigens have been exploited extensively in industrial antibody purification, these observations could facilitate further protein engineering to optimize the use of superantigens in this and other areas of biotechnology.

Full text

Superantigen Recognition
and Interactions: Functions,
Mechanisms and Applications
Anthony M. Deacy
1
, Samuel Ken-En Gan
2,3
*and Jeremy P. Derrick
1
*
1
School of Biological Sciences, Faculty of Biology, Medicine, and Health, University of Manchester, Manchester,
United Kingdom,
2
Antibody & Product Development Lab, Experimental Drug Development Centre –Bioinformatics
Institute (EDDC-BII), Agency for Science Technology and Research (ASTAR), Singapore, Singapore,
3
James Cook
University,Singapore,Singapore
Superantigens are unconventional antigens which recognise immune receptors outside
their usual recognition sites e.g. complementary determining regions (CDRs), to elicit a
response within the target cell. T-cell superantigens crosslink T-cell receptors and MHC
Class II molecules on antigen-presenting cells, leading to lymphocyte recruitment,
induction of cytokine storms and T-cell anergy or apoptosis among many other effects.
B-cell superantigens, on the other hand, bind immunoglobulins on B-cells, affecting
opsonisation, IgG-mediated phagocytosis, and driving apoptosis. Here, through a review
of the structural basis for recognition of immune receptors by superantigens, we show
that their binding interfaces share specific physicochemical characteristics when
compared with other protein-protein interaction complexes. Given that antibody-
binding superantigens have been exploited extensively in industrial antibody purification,
these observations could facilitate further protein engineering to optimize the use of
superantigens in this and other areas of biotechnology.
Keywords: superantigen, T-cell, B-cell, cytokine storm, interface, antibody purification
INTRODUCTION
Superantigens are unconventional antigens in the sense that they elicit a response by binding outside
the complementary determining regions (CDRs) of their target immune receptor macromolecules
(antibodies or T-cell receptors). At their initial description in 1989, superantigens were originally
defined as proteins that hyper-stimulate T-cells via the crosslinking of T-cell receptors (TCRs) and
MHC Class II molecules (1,2). This definition required extension following the discovery of B-cell
superantigens. B-cell superantigens can hyper-stimulate a large population of B-cells without
necessarily having the ability to crosslink TCRs with MHC Class II receptors; they therefore have a
different mechanism and specificity compared to T-cell superantigens (3). B-cell superantigens are
commonly known to (i) stimulate a high proportion of B-cells, and (ii) bind outside of the CDRs (4).
An extended definition of the term ‘superantigen’was suggested to incorporate both functions, as a
molecule which has antigen-receptor mediated interactions with over 5% of the lymphocyte pool
(5). This functional definition is therefore based on the hyper-activity of the target receptor upon
exposure, and we will use the term in this context here.
Frontiers in Immunology | www.frontiersin.org September 2021 | Volume 12 | Article 7318451
Edited by:
Ester Boix,
Universitat Autònoma de Barcelona,
Spain
Reviewed by:
Marco Pio La Manna,
University of Palermo, Italy
Chozha V. Rathinam,
University of Maryland, United States
*Correspondence:
Jeremy P. Derrick
jeremy.derrick@manchester.ac.uk
Samuel Ken-En Gan
samgan@apdskeg.com
Specialty section:
This article was submitted to
Microbial Immunology,
a section of the journal
Frontiers in Immunology
Received: 28 June 2021
Accepted: 30 August 2021
Published: 20 September 2021
Citation:
Deacy AM, Gan SK-E
and Derrick JP (2021)
Superantigen Recognition and
Interactions: Functions,
Mechanisms and Applications.
Front. Immunol. 12:731845.
doi: 10.3389/fimmu.2021.731845
REVIEW
published: 20 September 2021
doi: 10.3389/fimmu.2021.731845

Here we review the current understanding of superantigens,
how they directly interact with immune receptors of T and B-
cells, what common features may be identified in recognition
interfaces and how such insights could be adapted to facilitate
further protein engineering of these versatile macromolecules for
therapeutic, diagnostic, and biotechnological applications.
T-CELL SUPERANTIGENS
T-cell superantigens are typically microbial proteins. They were
first identified from observation of the hyper-stimulation of T-
cells by Staphylococcal Enterotoxin B (SEB). This phenomenon
was caused by the crosslinking of T-cell receptors (TCRs) Vb
with MHC class II a
1
on antigen presenting cells (APC) by SEB
(1,2). By crosslinking MHC Class II to TCR, small amounts of
superantigens can stimulate extensive T-cell proliferation. In a
normal adaptive immune response, only around 0.0001% of T-
cells are activated. In contrast, superantigen exposure can
activate up to 30% of the T-cell pool, leading to severe
pathologies following infection (6,7).
Enterotoxins produced by Staphylococcus aureus and
Streptococcus pyogenes form a common family of T-cell
superantigens. These enterotoxins are small (20-28 kDa), two
domain proteins which are diverse in sequence (15-90%) (8).
Despite this variation, enterotoxins and enterotoxin-like proteins
from both Staphylococcus aureus and Streptococcus pyogenes are
structurally similar (Figure 1), possessing a conserved Greek key
motif at the N-terminus known as an oligonucleotide (OB)-fold
(9). The C-terminus consists of a conserved b-domain capped by
an a-helix (9) with the two b-folds separated by a cluster of a-
helices. Due to their structural similarity, it has been suggested by
others that the enterotoxins from Staphylococcus aureus and
Streptococcus pyogenes shared a common ancestor (8).
Enterotoxins are thermostable, can withstand extreme pH
and are resistant to degradation by proteolytic enzymes such as
pepsin and trypsin (10,11). Some can retain activity after the
cooking and digestive process to cause food poisoning (12):
FIGURE 1 | Comparison of selected Staphylococcal aureus and Streptococcus pyogenes enterotoxin and enterotoxin-like structures. Staphylococcal aureus
enterotoxins include SEA (PDB code: 1ESF), SEB (PDB code: 1SE4) and TSST-1 (PDB code: 2QIL). The Staphylococcal aureus enterotoxin-like protein shown is
SSL4 (PDB code: 4DXF). Two Streptococcal pyogenes enterotoxins are displayed: SpeA1 (PDB code: 1UUP) and SpeC (PDB code: 1KTK). The structures are
shown as a ribbon plot with a-helices, b-strands and loops coloured in red, yellow, and green, respectively.
Deacy et al. Superantigen Recognition and Interactions
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nearly 25% of food poisoning cases in the USA are attributed
to Staphylococcal enterotoxins (13). In addition, T-cell
superantigens also contribute to the development of systemic
inflammatory response syndrome (SIRS) known as sepsis (14),
toxic shock syndrome (15,16), scarlet fever (17) and atopic
dermatitis (18).
CELLULAR RESPONSES TO T-CELL
SUPERANTIGENS
Observations of the cellular responses of T-cells to superantigens
are inconsistent, depending on the type and maturity of the T-
cell populations studied. T-cell superantigens can cause
immature CD4
+
and CD8
+
T-cells to become depleted. Mature
CD4
+
and CD8
+
T-cells on the other hand, proliferate and
produce a cytokine storm (19–25) driving mature T-cells into
a state of anergy (26). TCR activation upregulates Fyn signalling,
preventing the protein tyrosine kinase ZAP-70 from associating
with TCRs via CD3, thus inhibiting TCR signalling (27). The
depletion of immature T-cells and anergy of mature T-cells
would potentially allow a pathogen to evade the innate
immune response, increasing pathogen survivability.
TCR binding to the MHC class II receptors on APCs results in
a variety of responses that is dependent on the APC type; the
principal pathways and components are summarized in
Figure 2.Duringinfection,neutrophilsarerecruitedalong
with other effector cells through the release of cytokines (e.g.
IFN-g, IL-17, IL-12) and CXC chemokines produced primarily
by CD4
+
T-cells (28,50–52). Counterintuitively, the recruitment
of leukocytes increases the survivability of Staphylococcus aureus,
due to the hyper-stimulation of T-cells, eventually leading to T-
cell anergy and cell death. S. aureus is known to survive within
neutrophils and macrophages in abscesses (29,30).
Alongside TCR/MHC Class II activation, signalling pathways
are co-stimulated by crosslinking CD28 on the T-cell with CD80/
B7-2 on APCs (53–56). T-cell superantigens can also crosslink the
a-subunit of laminin, LAMA2, with G-protein coupled receptor
(GPCR), resulting in T-cell stimulation (57–59)(Figure 2).
MHC CLASS II BINDING
T-cell superantigens first bind to MHC Class II receptors and
accumulate on the surface of the APC before binding to the TCR
(9). There are two possible binding sites on MHC Class II: a Zn-
dependent high affinity site (K
d
=10
-7
-10
-8
M) located on MHC
Class II bchain, and a low affinity site (K
d
= ~10
-5
M) located on
MHC Class II achain (60). Most superantigens bind via the Zn-
dependent binding site, forming a complex which is stable for
more than 40 hours (61). The high affinity interface between SEH
and MHC Class II achain is shown in Figure 3A, showing a
hydrophobic pocket surrounded by polar residues. In addition to
H-bonds and salt bridges, a Zn ion contributes to the high
binding affinity by stabilizing the complex through crosslinking
H81 on the MHC Class II b-strand and H206, N208 on the b-
strand 12 on SEH (62). This stabilization allows for the
formation of 4 extra H-bonds due to the proximity of the
chains where the removal of the Zn ion results in a decrease in
binding for SEA, SED, SEE and SEH (63).
FIGURE 2 | Principal components involved in the superantigen activation of T-cells, B-cells, macrophages, and neutrophils. The interactions displayed are based on
material from references (28–49). The responses contribute to and escalate the hyper-activation of T-cells and subsequent cytokine storm.
Deacy et al. Superantigen Recognition and Interactions
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The low affinity binding site is exemplified by a structure
containing the enterotoxin SEB, which forms a complex with
MHC class II (61): the low affinity interface is shown in
Figure 3B. In addition to cross-interface bonds, there is a
hydrophobic patch on SEB comprised of F44, L45 and F47
which inserts into a hydrophobic pocket on MHC Class II
achain.
The enterotoxin SEA can also bind to the low and high affinity
sites to crosslink two MHC Class II molecules (61,64,65).
Staphylococcal Enterotoxin H (SEH) was shown to bind the Zn-
dependent high affinity site on MHC class II (62), as well as to
TCR Vainstead of Vb(66,67). A list of T-cell superantigens and
their site specificities has been previously summarized by Proft
and Fraser (9). T-cell superantigen selectivity for the aor b
chains of the MHC Class II complex is dependent on the
presence of the Zn atom at the C-terminal bdomain. Its
absence leads to the binding of the a-chain of MHC Class II
via a hydrophobic ridge on the N-terminal OB-domain (9).
T-CELL RECEPTOR BINDING
Superantigens bind to the TCR after adhesion to MHC Class II;
there are also two sites on the TCRs in all superantigen
complexes studied to date. Some T-cell superantigens bind to
the achain [SEH (68)], although most recognize the bchain.
Unlike complexes with MHC Class II, both TCR interfaces bind
T-cell superantigens at low affinity (K
d
=10
-4
–10
-6
M) (68,69)
and yet both are capable of mediating activation of a cytokine
storm (8,70–73). SEB binding to the TCR bchain is shown in
Figure 4A where the interface is located at the TCR binding cleft
between the N-terminal b-barrel and the second a-helix. It is
characterised by several cross-interface bonds, with N23 playing a
crucial role, and a nearby hydrophobic patch formed of V26, Y79
and Y80 on SEH packing against the CDR2 loop of TCR Vb(74).
SEH binding to the TCR achain forms an interface
comprising hydrophobic and hydrophilic patches, with a
notable hydrophilic patch surrounding a Na ion (Figure 4B).
Comparing this to the SEB-TCR bchain interface, there are 7
fewer H-bonds and 2 fewer salt bridges, although the binding
affinities are similar (K
d
=10
-4
–10
-6
M) (68). The lack of
contacts between SEH and the TCR achain may be bolstered by
the presence of the Na ion. N16 found on the second a-helix and
the hydrophobic patch (Y79 and Y80) on SEH are well conserved
among T-cell superantigens whether they bind to the TCR aor b
chains (67). The mutation N23A (equivalent to N16 in SEH) in
SEC2 caused the loss of mitogenic activity (75) and the same
mutation in SEB resulted in poorer proliferation of T-cells (76).
BINDING OF B-CELL SUPERANTIGENS
B-cell superantigens bind immunoglobulins outside the CDRs;
proteins which would fit this definition of a superantigen were
first described in the early 1990s (3). Binding to the Fab fragment
drives B-cells into apoptosis by hyper-activation of B-cell
A
B
FIGURE 3 | Binding of SEH and SEB superantigens to -MHC Class II. (A) Left panel: ribbon plot of SEH (blue) bound to the high affinity site on MHC Class II a
Chain (purple) (PDB code: 2XN9). Middle Panel: MHC Class II showing polar (red) and hydrophobic residues (blue). Right Panel: SEH. (B). Left panel: ribbon plot of
SEB (green) bound to the low affinity site on MHC Class II bChain (orange) (PDB code: 1SEB). Middle panel: MHC Class II bChain. Right panel: SEB.
Deacy et al. Superantigen Recognition and Interactions
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receptors (BCRs). Considering that 20 to 50% of B-cells have
BCRs on their surfaces (77), B-cell superantigens can elicit a
potent immune response. However, B-cell superantigens are
better known for their ability to bind Fc and their applications
as affinity resins for antibody purification.
Staphylococcal Protein A (SpA), Streptococcal Protein G (SpG)
and Peptostreptococcal Protein L (PpL) are B-cell superantigens
located on the bacterial cell wall (78,79). SpA was classified as a
superantigen in 1995 due to its observed effect on B-cells (4).
However, SpA was first isolated in 1940 and identified in 1964 due
to its Fc binding ability (78,80). It comprises a 42 kDa protein
arranged into five homologous domains (E-D-A-B-C), each
forming a three a-helix bundle fold (Figure 5A)(81,82). The
domains are linked by conserved, flexible linkers (82). Native SpA
also includes region X, a 12 x 8-residue repeat sequence which
binds peptidoglycan. All 5 A-E domains can bind both Fc and Fab
fragments (83). The binding affinity for specific immunoglobulins
depends on the isotype and species origin. In humans, SpA binds
strongly to IgG1, IgG2, IgG4 and weakly to IgA1, IgA2 and IgM.
Mutations R435H and F436Y on hIgG3 have been identified as the
reason SpA cannot bind human IgG3 (84). Interestingly,
mutations in CDR2 from the therapeutic antibodies Herceptin
and Pertuzumab were shown to contribute to binding SpA (85).
SpG was first identified in 1984 by Björck and Kronvall (86)and
subsequently described as a B-cell superantigen. The sequence of SpG
differs depending on the Streptococcus strain of origin (Figure 5B).
SpG from group C Streptococcus sp. contains 2 immunoglobulin
binding domains (B1-B2) whereas group G has 3 (C1-C2-C3) (87–
89). Between each immunoglobulin binding domain are ‘spacers’,
known as D domains. All SpG immunoglobulin-binding domains
can bind both the Fc and Fab fragments (83). SpG has provided an
alternative to SpA in antibody manufacturing, due it its ability to bind
some antibody isotypes not recognised by SpA. It can strongly bind to
all four human IgG subclasses (IgG1, IgG2, IgG3 and IgG4).
PpL was shown to induce apoptosis in B-cells by binding to the
V
L
region outside of the CDRs of BCRs, fulfilling the definition of
a B-cell superantigen (90). It was first isolated in 1985 and
characterised as an immunoglobulin-binding protein capable of
binding to the variable light chain in 1988 (79,91). Of the two
most common strains of Peptostreptococcus magnus, strain 312
produces a 79 kDa, 5 domain (B1-B2-B3-B4-B5) protein whereas
strain 3316 expressesa 106 kDa 4 domain (C1-C2-C3-C4) protein
(Figure 5C)(92). PpL recognizes the light chain exclusively and
cannot bind to the Fc region. This makes it highly suitable for
affinity-purification of non-IgG antibodies (93,94).
All three B-cell superantigens (SpA, SpG and PpL) share
several common features; they form small, stable, multidomain
structures with a ‘beads on a string’type structure. Kim et al.
compared antibody levels of IgG and V
H
3+ IgM in mice when
infected with SpA mutants with one to 6 domains. The results
showed that the optimal number of immunoglobulin binding
domains to induce the largest B-cell response was 5 (95). This
observation suggests that B-cell superantigens are driven by the
need for multivalency of binding and the consequent improved
cross-linking of BCRs. These results were corroborated by a
similar study with PpL (92). Although SpG and PpL share no
significant sequence homology (15%), their immunoglobulin
binding domains have similar folds, forming a b-sheet packed
A
B
FIGURE 4 | Binding of SEB and SEH superantigens to -TCRs. (A) Left panel: ribbon plot of SEB (blue) bound to TCR (green) (PDB code: 4C56). Middle panel: TCR
showing polar (red) and hydrophobic residues (blue). Right panel: SEB. (B) Left panel: ribbon plot of SEH (black) bound to TCR (purple) (PDB code: 2XN9). Middle
panel: TCR. Right panel: SEH.
Deacy et al. Superantigen Recognition and Interactions
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against a single a-helix. A gene transfer event between
Streptococcus aureus and Peptostreptococcus magnus has been
proposed to explain a possible common evolutionary origin of
SpG and PpL (96). All three B-cell superantigens also utilise
regions W and M for crossing the cell membrane, featuring the
common Gram-positive protein anchoring motif LPXTG (97,
98). SpG and PpL also contain albumin binding domains (99,
100), which are absent in SpA.
CELLULAR RESPONSES TO B-CELL
SUPERANTIGENS
B-cell superantigens cross-link BCRs to activate BCR dependent
signalling (101,102). This initial signal transduction leads to the
downregulation of BCRs, and an upregulation of several cluster
of differentiation (CD) receptors (102), resulting in B-cell
capping (summarized schematically in Figure 6). MHC Class
A
B
C
FIGURE 5 | Schematic diagrams of SpA, SpG and PpL domain structures. (A) Left panel: Individual SpA domains including S (sorting peptide), Domains E-D-A-B-C, Region
X and Region M. Right panel: SpA Domain C (PDB code: 4WWI) Each SpA immunoglobulin binding domains consists of 3 a-helices (red). (B) Left panel: Individual SpG
domains including S (sorting peptide), Region E, Albumin Binding Domains A1-A2-A3, immunoglobulin binding domains B1-B2/C1-C2-C3 and Region W. Right panel: SpG
Domain B1 (PDB code: 3GB1) Each SpG immunoglobulin binding domain consists of 1 a-helix (red) and 4 anti-parallel b-strands (yellow). (C) Left panel: Individual PpL
domains including S (sorting peptide), Immunoglobulin Binding Domains B1-B2-B3-B4-B5/C1-C2-C3-C4, Albumin Binding Domains D1 to D4, Region W and M. Right panel:
PpL Domain B1 (PDB code: 1HEZ). Each PpL immunoglobulin binding domain consists of 1 a-helix (red) and 4 anti-parallel b-strands (yellow).
Deacy et al. Superantigen Recognition and Interactions
Frontiers in Immunology | www.frontiersin.org September 2021 | Volume 12 | Article 7318456

II is also upregulated (102). The upregulation of these receptors
leads to the activation of pro-apoptotic signals, such as Caspase
3, causing mitochondrial permeabilization and apoptosis (5,101,
102). Recently it has been shown that SpA B-cell superantigen
activity is dependent on the presence of the LPXTG anchoring
motif as well as the ‘LysM domain’between region X and the
LPXTG motif (103). These observations imply SpA must be
bound to peptidoglycan to cause B-cell stimulation.
The precise functional role of microbial B-cell superantigens
binding to Fc is obscure, although it has been shown recently that
soluble IgG is a requirement for the successful activation of BCRs by
SpA (104). The efficiency of BCR activation was dependent on the
strength of Fc binding to each IgG subclass (104). The binding of
SpA-IgG complex to BCR is predicted to increase the functional
valency of the complex (104). SpA-IgG is thought to form a ‘lattice’
structure around the B-cells by crosslinking BCR Fab with IgG Fc
and other BCR Fab regions promoting a sustained stimulation.
Other potential functions of the B-cell superantigen-
immunoglobulin interaction are the blocking of immunoglobulin
effector functions, opsonization and immunoglobulin-mediated
phagocytosis, antibody-dependent cell mediated cytotoxicity
(ADCC) and complement-dependent cytotoxicity (CDC) (105–
107). Expression of B-cell superantigens ultimately leads to B-cell
depletion and evasion of the immune system: in this sense, they can
be considered as virulence factors (108–111).
SpA, SpG and PpL bind to BCRs at different sites on the Fab
fragment, although the activation results in similar cellular
responses. SpG binds to the C
H
1domain(112), implying
isotype dependent binding, whereas SpA binds to the V
H
3
family only. A comparison of the conservation of key residues
betweenthesevenV
H
families shows that, although many
residues are conserved, there are several which are key and
which, when mutated, result in the loss of binding for SpA
(113), including in the V
H
-CDR2 (85). PpL domains only bind
the klight chain V
L
region and therefore lacks the ability to bind
lchains. The binding affinity of PpL differs between the families
of klight chain, specifically to FW1: it can bind to human VkI,
III and IV, but not II (114,115).
Several T-cell superantigens have the ability to bind BCRs,
although generally in a weak and non-specific manner (4), and
without a B-cell response. Exceptions have been noted, for
example, SEA increased the survival of V
H
3 B-cells (116). SED
has also been shown to increase survival of V
H
4 B-cells (117).
However, the in vivo response is yet to be determined.
Recent research suggests that B-cell superantigens also
enhance immune defences (118). Two superantigens have been
identified from the commensal bacteria Lachnospiraceae sp:
Immunoglobulin Binding Proteins A (IbpA) and B (IbpB). Both
were observed to activate BCRs by binding V
H
3 leading to the
increased secretion of IgA, although this was only shown in vitro.
B-CELL SUPERANTIGEN-FAB
COMPLEX INTERFACES
The crystal structure of the SpA and IgM Fab complex is
illustrated in Figure 7A, showing that the interface occurs at the
V
H
domain (involving residues from b-strands B to E) of the Fab
fragment and a-helices 2 and 3 of SpA (113). The interface is
dominated by polar residues with three negatively charged
residues from SpA and two positively charged residues from Fab
forming electrostatic interactions (113). All SpA domains can bind
FIGURE 6 | Proposed mechanisms for the activation of B-cell receptors by SpA. Activation leads to B-cell capping and B-cell apoptosis.
Deacy et al. Superantigen Recognition and Interactions
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to the Fab fragment (119), and each domain varies in its affinity
towards V
H
3. The interacting residues form a predominantly
hydrophilic interface forming several cross-interface bonds.
SpG domain C2 co-crystalized with IgG Fab (Figure 7B): the
interface forms an antiparallel alignment between the last b-
strand of the C
H
1 domain and the second b-strand of SpG (120).
The antiparallel complex also results in interactions occurring
between first b-strand of C
H
1 and the C-terminal end of the a-
helix of SpG (121). The interface is formed by mostly hydrophilic
residues flanking a small hydrophobic patch.
The first of two binding sites of PpL to IgM Fab is shown in
(Figure 7C). The majority of the interface occurs at framework
region 1 (FR1) of the V
L
region, with several contacts occurring
outside of the V
L
region: K107 between the V
L
and C
L
regions,
E143 from the C
L
region and R24 on the b-strand of CDR-L1 of
IgM Fab. The interface includes residues from the a-helix and
second b-strand of PpL domain B1 (122). The interface has a high
affinity (K
d
:110nM)(123), forming a predominantly hydrophilic
interaction characterised by 9 H-bonds, although several residues
have been proposed as hotspots from in silico alanine scanning of
the Fab and PpL (115,123,124). Interestingly, recent evidence
showed distal FWR3 effects on the PpL binding site at the FW1
(125) adding to the considerations for the light chain pairing with
the heavy chain (126,127).
The second binding site is formed from 15 residues at b-
strands A, B, C and D of the V
L
region of IgM Fab, and the a-helix
and third b-strand of PpL (Figure 7D). Although the second
binding site is slightly larger and composedof more cross-interface
contacts, it has a lower binding affinity (3.4 µM) (115). The first
and second binding sites of PpL share only one common residue
from PpL (R52) but 10 out of 15 residues from IgM Fab.
B-CELL SUPERANTIGEN-FC BINDING
The crystal structure of a single domain from SpA was determined
in complex with IgG Fc: it showed that the protein-protein interface
occurs between a-helix 1 and 2 of SpA domain B and C
H
2andC
H
3
of the Fc (113)(Figure 8A). The residues forming the interface are
generally hydrophilic (128). SpA residues Q9, Q10, D36 and D37,
are conserved in the five immunoglobulin binding domains of SpA
and are required for Fc binding (129). Mutating residue H435 in
IgG eliminates SpA binding, as this residue is situated on the C-
terminus of the C
H
3 region and protrudes into the C
H
2-C
H
3cleft
forming surface contacts with SpA (84).
The crystal structure of SpG C2 in complex with IgG Fc
showed that it binds at the same site as SpA, with SpG binding to
IgG Fc at the C
H
2-C
H
3 interface (Figure 8B). SpG fits within the
C
H
2-C
H
3 cleft and binds through residues on the a-helix and
third b-strand. As they recognise essentially the same site, SpA
and SpG bind competitively to IgG Fc (130,131). The strong
binding affinity of SpG for IgG Fc is contributed by a
hydrophobic pocket surrounded by hydrophilic residues.
Comparing the binding sites of SpG for Fab and Fc, Fab
binding uses b-strands 1 and 2 as well as the a-helix, whereas
Fc binding uses b-strand 3 as well as a more prominent
contribution of a-helix residues.
PHYSICOCHEMICAL CHARACTERISTICS
OF T AND B CELL SUPERANTIGEN
INTERFACES
The structures of the complexes of T and B-cell superantigens
with immune macromolecules were examined to compare the
nature of the interfaces with all other structures of protein-
protein complexes. A list was compiled from the Protein Data
Bank, extracting specific data on hydrophobicity, number of
hydrogen bonds, salt bridges, interface area, binding affinity,
and charges at the interface. These values were then condensed
onto a two-dimensional plot using t-distributed stochastic
neighbor embedding, such that each point represents a
complex (Figure 9).
Complexes were categorized according to function: most were
well dispersed by functional category, although peptide
complexes tended to predominate in the upper half of the plot
(Figure 8A). The superantigen complexes are grouped in
the central and right side of the plot, indicating that their
binding interfaces share some physicochemical characteristics
(Figure 9B). An explanation for this phenomenon is that T-cell
and B-cell superantigen interactions are transient-type complexes,
as defined by Noreen and Thornton (132). Such complexes tend to
be small and less hydrophobic than obligate, homo oligomeric
complexes. Both T- and B-cell superantigen interfaces form
interface areas less than 1000 Å
2
and range from slightly to
very hydrophilic.
The T-cell superantigens are located on the centre and right-
hand side of the plot and are more scattered than the B-cell
superantigen interfaces (Figure 9B). The interface areas of T-cell
and B-cell superantigens have similar ranges: 436 –944 Å
2
and
517 –714 Å
2
respectively. Fractional hydrophobicity of the T-cell
superantigen interfaces range from 18 –49%, similar to those for
B-cell superantigens (9 –40%).
Although B-cell superantigens recognise different binding
sites within the Fab molecule (V
H
,V
L
and C
H
1) they share
very similar interface physicochemical properties, which align
closely to those seen in peptide complexes.
The fact that superantigens are promiscuous and capable of
recognition of different binding partners indicates there is scope for
improving binding affinity and extending specificity for specific
targets. The observation that B-cell superantigens, and more
specifically superantigen-Fab complexes, are physicochemically
similar may allow for development of engineering strategies
which makes use of this facility. Nonetheless, just as we expanded
the definition of superantigens in this review to include B-cell
activation based on new findings, we are also aware of novel
superantigen-like behaviours by non-proteins e.g., nickel (133)
that may in time be included as superantigens in the future.
SUPERANTIGEN APPLICATIONS
Superantigens have been employed in multiple applications, both
clinically and industrially. Though many improvements have
been made, there is room to engineer and expand their scope
Deacy et al. Superantigen Recognition and Interactions
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and applications. Understanding the biochemistry of the
superantigen-antibody interfaces provides an information
resource for the development of novel biotechnological and
pharmaceutical applications.
INDUSTRIAL
Since the approval of the first therapeutic monoclonal antibody in
1986 (Muromonab-CD3), the use of antibody-based drugs has
A
B
D
C
FIGURE 7 | Binding of SpA, SpG and PpL to antibody Fab fragments: (A) Left panel: ribbon plot of SpA (Red) bound to V
H
3 domain of IgM Fab (Green) (PDB code:
1DEE). Middle panel: IgM Fab showing polar (red) and hydrophobic residues (blue). Right panel: SpA. (B) Left panel: ribbon plot of SpG (Orange) bound to C
H
1
domain of IgG Fab (Cyan) (PDB code: 1QKZ). Middle panel: IgG Fab. Right panel: SpG. (C) Left panel: ribbon plot of PpL Domain B
1
(Purple) interface 1 bound to
IgM Fab (Green) at the V
L
domain (PDB code: 1HEZ). Middle panel: IgM Fab. Left panel: PpL. (D) Left panel: ribbon plot of PpL Domain B
1
(Purple) interface 2
bound to IgM Fab (Blue) at the V
L
domain (PDB code: 1HEZ). Middle panel: IgM Fab. Right panel: PpL.
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expanded significantly with technological developments such as scFv,
antibody-drug conjugates (ADCs) and bispecifics. In 2019, 70% of all
biopharmaceutical products sold were monoclonal antibodies in a
market worth over $150 billion. Antibody-based drugs continue to
increase their market share, with current estimates predicting global
revenue to increase to over $300 billion by 2025 (134). The expansion
of antibody-based drugs has therefore created a need for improved
manufacturing and purification processes.
The most prominent industrial application of B-cell
superantigens is their use as affinity resins for the purification of
antibodies, allowing highly efficient separation of antibodies for
clinical and research applications. Improvement of these affinity
resins has allowed pharmaceutical companies to develop cost-
effective antibody purification techniques, increasing the
feasibility of large-scale manufacturing of antibodies, resulting in
the expansion of the industry. There are, however, some limitations;
80% of the downstream processing cost occurs at the capture and
purification phase (135), and there is no single resin which can bind
all antibody isotypes from all species of interest. Furthermore,
antibodies are eluted from the affinity resins at low pH values,
frequently causing aggregation.
Some investigators have engineered superantigens to optimize
their application in antibody purification (136,137). For example
“Domain Z”was developed in 1987- a mutant of Domain B with
two mutations, A1V and G29A (138), which resulted in SpA
losing the ability to bind V
H
3-Fab while retaining Fc affinity (139);
this innovation allows for the selective purification of the Fc
fragment after pepsin digestion. One such Z domain affinity
resin is Cytiva’s mAb Select SuRe (140). When testing mutant
N23T, the stability of the SpA Z domain resin increased (141).
Recently a new SpA resin has been developed: AviPure. This resin
is formed of two B domains with two cysteine and histidine
residues at the C-terminus to the steric hindrance, increasing
binding capacity and increasing its resistance to pHs extremes,
while retaining high binding affinity (135). Affinity resins undergo
cleaning in place (CIP) procedures commonly using 0.5 M NaOH;
therefore, affinity resins with high alkaline stability are desirable.
To address the issue with CIP procedures, SpA was shown to have
higher alkaline stability with a single mutation at position 29, with
G29W being the most stable (142). Two further mutations N23T
and F30A to the SpA Z domain resulted in a higher alkaline
resistance when compared to wild type (143). SpA Z domain was
also engineered to include six glycine residues on the second loop,
which resulted in an increase in the elution pH (143). Wild type
SpA is less susceptible to extreme alkaline conditions, with a half-
life of 16 h (141) compared to SpG, which has a half-life of under
10 mins (144). Asn residues were identified as the most susceptible
to deamination: mutation of all three Asn residues of SpG (N8T,
N35A and N37A) improved alkaline stability by 8-fold (145). SpG
was further demonstrated to have higher alkaline stability when
three other mutations Y3F, T16I and T1I were introduced (145).
By increasing alkaline stability, the lifespan of affinity resins can be
increased, lowering the overall cost of antibody production. It has
also been recently demonstrated that adding an additional alkaline
A
B
FIGURE 8 | Binding of SpA and SpG superantigens to -IgG Fc. (A) Left panel: ribbon plot of SpA Domain B (Orange) bound to IgG Fc (Pink) at the C
H
2-C
H
3
interface (PDB code: 5U4Y). Middle panel: IgG Fc showing polar (red) and hydrophobic residues (blue). Right panel: SpA. (B) Left panel: ribbon plot of SpG Domain
C2 (Blue) bound to IgG Fc (Orange) at the C
H
2-C
H
3 interface (PDB code: 1FCC). Middle panel: IgG Fc. Right panel: SpG.
Deacy et al. Superantigen Recognition and Interactions
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wash step after the antibody capture step results in a decrease in
antibody aggregation, lower impurity levels and an increase in
antibody yield (146).
With the development of new formats such as single chain
variable fragments (scFvs), strong Fab binding is required.
Unfortunately, SpA and SpG have lower binding affinities for
the Fab fragment compared to Fc. PpL has the advantage over
other B-cell superantigens of binding strongly to the Vkof scFv,
(K
d
= 4.5nM) (147). The scFv structural arrangement consists of
the V
H
and V
L
domains connected by a short linker. scFv
molecules have the advantage of retaining the CDRs while
being significantly smaller than whole antibodies. SpA can also
bind scFv but at a lower binding affinity than PpL, whereas SpG
is unable to bind scFv. The most significant downside for the use
of PpL as an affinity resin is its inability to bind llight chains.
Therefore, in human antibody production, roughly 34% of the
antibodies will not bind to the resin, suggesting that engineering
PpL to bind llight chains could be valuable.
CLINICAL USAGE
Diagnostics Potential
Superantigens are used to detect IgG in serum (148), making use
of their immunoglobulin binding specificity. On the contrary,
superantigens recognized by IgGs allow for the detection of
Staphylococcus aureus (149,150) in disease states.
Engineering of superantigens to be specific to regions of TCRs
or antibody V-region families or isotypes for the development of
diagnostic kits could be applied to the quantification of disease-
associated proteins e.g. IgE in allergy. The ability to specifically
bind antibodies can also allow its development in easy-to-use, non-
technical point-of-care testing home-use devices (151), as recently
applied during the COVID-19 pandemic. Such superantigen-based
diagnostics can be coupled with colorimetric, home-made devices
[e.g. mobile spectrophotometers (152,153)]. Given the increasing
association of antibody V
H
families with certain diseases e.g. [V
H
5
in nickel allergy (85)], superantigens that can differentiate antibody
V
H
families have clear potential in diagnostic kit development.
Therapeutic Potential
The role of superantigens in sepsis, a leading cause of death listed
by the WHO, makes them an important target for toxic-shock
syndrome (154). Several short peptide regions (~40 residues)
from SEA and SPEA have been identified as causes of vasodilation
(155), suggesting an application in the development of
antihypertension drugs.
Superantigens can also be used as a target for an anti-
Staphylococcus aureus vaccine. There have been several attempts
atproducinga vaccineagainstS. aureus,withoutsuccess,althoughit
A
B
FIGURE 9 | t-SNE plot of protein-protein interactions highlighting superantigen-antibody complexes. The position of each complex was determined using the following
parameters: “Buried Surface Area (A
2
)”,“Buried Surface Area Hydrophobicity (A
2
)”,“Number of Interface Residues”,“H-Bonds”,“Salt Bridges”,Category”,“Total Positive
Charge at Interface (A
2
)”,“Total Negative Charge at Interface (A
2
)”with a perplexity of 30. (A) Distribution of general protein-protein interactions categorized by function
(T-cell superantigen complex, B-cell superantigen complex, enzyme PPI, immune PPI, inhibitory PPI, peptide PPI, receptor PPI, signaling PPI, structural PPI, toxin-
antitoxin PPI and transport PPI). (B) The same plot as (A), but coloured for T-cell superantigen complexes (green) and B-cell superantigen complexes (red).
Deacy et al. Superantigen Recognition and Interactions
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has been shown that the use of anti-SpA antibodies leads to the
promotion of opsonophagocytic clearance of Staphylococcus aureus
(156,157).
Superantigens have also shown promise in the treatment of
cancer through a synergistic effect with antibodies in the
recruitment of T-cells (158). The ability of SEB to hyper-
stimulate and proliferate CAR T-cells led to a more effective
antitumour response when used in combination (159). PpL has
been shown to induce apoptosis in malignant k
+
Bcell
lymphomas in humans and mice (160), demonstrating the
potential use of superantigens as anti-cancer drugs, particularly
when sagaciously paired with a suitable V
k
light chain (94). A
range of potential T-cell superantigen-based anticancer drugs
have been recently reviewed (161), including SEB, demonstrating
the ability to inhibit metastasis and tumour growth (162). Several
Fab-superantigen fusion proteins show promising Phase I/II
clinical trial results. A major drawback with using
superantigens is their potential to elicit a toxic response. One
way to prevent this is to reduce the over-stimulation of T-cells.
SEA was split into two functionally inactive domains and
attached to a scFv. When used in combination, the two SEA
fragments reassemble, forming a functionally active superantigen
and resulting in the selective activation of T-cells (163). Another
way to avoid superantigen toxicity is to utilize superantigen-like
proteins which, as mentioned previously, are very similar in
structure and function, although they do not result in emesis.
They have been shown to inhibit tumour growth by 30% without
significant toxicity (164).
The importance of understanding superantigens goes beyond
bacterial sepsis to viruses, where for example, SARS-CoV2 spike
protein displays superantigen properties (165,166)causing
multisystem inflammatory syndrome in children through its
unspecific activation of T-cells (167).
SUMMARY
With the development of new clinical therapeutics, B-cell
superantigen engineering presents an opportunity to develop
novel applications, as well as improving current superantigen-
based technology, such as purification resins. Structural
information on B-cell superantigen interfaces has been useful in
providing a basis for the engineering of binding characteristics.
The application of protein engineering principles offers
considerable scope for directed modification of superantigen
binding properties and harnessing for applications in medicine
and the pharmaceutical industry.
AUTHOR CONTRIBUTIONS
AD wrote the manuscript, with critical revisions from SG and JD.
AD and JD prepared the figures. All authors contributed to the
article and approved the submitted version.
FUNDING
AD is a PhD student funded by The University of Manchester,
UK and the Agency for Science, Technology and Research
(A*STAR), Singapore. This review was partially funded by the
National Research Foundation (NRF) Singapore grant to
Experimental Drug Development Centre (EDDC), ASTAR.
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Deacy et al. Superantigen Recognition and Interactions
Frontiers in Immunology | www.frontiersin.org September 2021 | Volume 12 | Article 73184516