SARS MERS Spike S2 Antibody

What is the S2 subunit of coronavirus spike proteins and why is it a target for broadly protective antibodies?

The S2 subunit is the most evolutionarily conserved region of the coronavirus spike protein, compared to the more variable S1 subunit that encompasses the receptor-binding domain (RBD) and N-terminal domain (NTD). S2 can elicit broadly neutralizing and protective antibodies because it contains highly conserved sequences across different coronavirus species .

Unlike the S1 subunit which accumulates most sequence modifications in emerging variants of concern, S2 is less prone to mutations, making it an attractive target for developing pan-coronavirus vaccines and therapeutics . Functionally, the S2 subunit mediates fusion between viral and host membranes after cleavage at the S1/S2 furin site, shedding of S1, further proteolytic cleavage at the S2' site, and refolding from a prefusion to postfusion conformation .

How do S2-targeting antibodies differ from RBD-targeting antibodies in terms of neutralization mechanisms?

While RBD-targeting antibodies primarily neutralize viruses by blocking receptor binding, S2-targeting antibodies operate through different mechanisms:

• S2 antibodies can target various conserved regions such as the fusion peptide, the S2 stem proximal to the viral membrane, and the S2 hinge region

• They can neutralize by preventing conformational changes required for fusion

• Mutations in the hinge epitope that ablate antibody binding have been shown to compromise pseudovirus infectivity

• S2 antibodies like RAY53 can mediate antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) against SARS-CoV-2 spike

This provides additional protection mechanisms beyond direct neutralization, potentially contributing to broader protection across coronavirus variants.

What molecular features enable cross-reactivity of S2 antibodies between SARS-CoV-2 and MERS-CoV?

The cross-reactivity of S2 antibodies between SARS-CoV-2 and MERS-CoV is primarily enabled by targeting highly conserved epitopes within the S2 subunit:

• Antibody IgG22 binds with high affinity to both MERS-CoV and SARS-CoV-2 S proteins by targeting a conserved coiled-coil region in the S2 subunit

• Cryo-EM and crystal structures revealed that IgG22 binds a region in the S2 stalk that undergoes substantial changes during the pre-to-postfusion conformational transition

• RAY53 binds a conformational S2 domain epitope (residues 980-1006) present only in the prefusion core of β-coronaviruses

• This epitope is part of the flexible hinge region that converts from a bent hairpin to an extended alpha helix during the pre-to-post-fusion spike conformational change

What evidence exists for natural cross-reactive immunity between seasonal coronaviruses and SARS-CoV-2/MERS-CoV?

Several lines of evidence support natural cross-reactive immunity:

• SARS-CoV-2-infected children and young adolescents with recent endemic HCoV infections develop higher titers of S2 antibodies than SARS-CoV-2-infected adults without recognized antecedent endemic HCoV infection

• In individuals not previously infected with SARS-CoV-2, a majority of the anti-SARS-CoV-2 IgG repertoire targets the S2 subunit, underscoring the potential for cross-reactivity directed at conserved epitopes on human coronaviruses

• Blood samples from Sierra Leoneans exposed to seasonal coronavirus contained antibodies cross-reactive to both SARS-CoV-2 and MERS-CoV

• While only 8% of the general population elicited antibodies against MERS-CoV S protein, most sera from COVID-19 convalescent individuals (>60%) showed binding to MERS-CoV S protein, indicating that SARS-CoV-2 infection boosted antibody response with cross-reactivity against MERS-CoV

What are the most effective immunization strategies for eliciting broadly protective S2 antibodies?

Based on recent research, effective immunization strategies include:

• Stabilized S2-only constructs: MERS-CoV stabilized stem (SS) constructs, MERS SS.V1 and MERS SS.V2, both lacking the S1 subunit, were highly immunogenic in mice, inducing broadly cross-reactive antibodies to MERS-CoV, HCoV-HKU1, SARS-CoV, and SARS-CoV-2 S

• Engineered S2 stability: Simulation-driven design of stabilized S2-only antigens retaining a closed prefusion conformation through tryptophan substitutions (V991W and T998W) in the central helices produced antigens with increased protein expression, superior thermostability, and preserved immunogenicity against sarbecoviruses

• Prime-boost regimens: Balb/c mice immunized with stabilized MERS-CoV S2 protein and boosted 4 weeks later developed robust serum antibody titers against the immunogen detectable at >1:10,000 dilution

These strategies focus on ensuring that S2 is presented in its native prefusion conformation, which is important for eliciting antibodies that recognize functionally important, conserved epitopes.

How can single B cell screening technologies be optimized for isolating S2-targeting cross-reactive antibodies?

Several successful approaches have been described:

• Microfluidic single B cell screening: Two monoclonal antibodies (mAbs), IgG22 and IgG72, were isolated using microfluidic single B cell screening technology from MERS SS.V1-immunized mice

• Memory B cell sorting from convalescent individuals: Researchers sorted single memory B cells from COVID-19 convalescents and constructed 38 mAbs, finding that 11 showed binding activity with MERS-CoV S2

• Phage display libraries: An immune antibody library with ~3.1 × 10^8 individual clones expressed as scFv-c-myc tag-pIII fusion proteins generated and displayed on M13 bacteriophage yielded >80 clones binding both MERS S2 and SARS-2 spike after 3-4 panning rounds

For optimization, researchers should:

• Use stabilized S2 antigens that maintain the prefusion conformation

• Implement counter-selection strategies to remove antibodies targeting non-conserved regions

• Employ multiplexed binding assays to identify antibodies with broad cross-reactivity

• Combine binding assays with functional assays to select antibodies with protective potential

What are the three main classes of S2-binding antibodies and their respective epitopes?

According to current research, three main classes of S2-binding antibodies have been described:

These different epitope classes represent functionally important regions that are conserved across β-coronaviruses and may be targeted for broadly protective immunity. Fewer than 5% of the ~7000 anti-SARS-CoV-2 spike monoclonal antibody sequences in databases bind S2, demonstrating that this area remains underexplored .

How do conformational changes in the spike protein affect accessibility of S2 epitopes?

Spike conformational dynamics significantly impact S2 epitope accessibility:

• The S2 hinge epitope (residues 980-1006) is present only in the prefusion core of β-coronaviruses

• Changes that affect spike opening dynamics, including those found in Omicron BA.1, can occlude the S2 hinge epitope and may evade pre-existing serum antibodies targeting the S2 core

• Weighted ensemble simulations have characterized how the S2 trimer apex splays open along an asymmetric pathway, revealing that the S2 trimer transitions from a closed to open conformation through asymmetric protomer-protomer separation

• Antibody RAY53 binds the native hinge in MERS-CoV and SARS-CoV-2 spikes on the surface of mammalian cells , suggesting that this epitope is accessible in the native prefusion state

Understanding these conformational dynamics is crucial for designing immunogens that present S2 epitopes in their native, accessible conformations and for developing antibodies that can effectively target these regions on intact viruses.

What in vitro assays are most informative for assessing the protective potential of S2-targeting antibodies?

Several complementary assays provide valuable information:

A comprehensive assessment should combine multiple assays to evaluate both direct neutralization and Fc-mediated effector functions.

How can structural biology techniques contribute to understanding S2 antibody binding mechanisms?

Structural biology techniques have been instrumental:

• Cryo-electron microscopy (cryo-EM): Single-particle cryo-EM studies revealed that IgG22 binds a region in the S2 stalk of MERS-CoV and SARS-CoV-2 S proteins that undergoes substantial changes during the pre-to-postfusion conformational transition

• X-ray crystallography: High-resolution crystal structures of stalk peptide-antibody complexes provided a basis for understanding the binding of stalk-targeting antibodies to highly conserved sequences in human β-CoVs

• Molecular dynamics simulations: Weighted ensemble molecular dynamics simulations characterized how the S2 trimer apex splays open along an asymmetric pathway

• Free energy perturbation calculations: Computational methods confirmed that V991W and T998W substitutions in the central helices of S2 stabilize the trimer in the closed prefusion conformation

By combining these techniques, researchers can gain comprehensive insights into antibody binding mechanisms, conformational dynamics of S2, and rational design of stabilized immunogens.

What are the key challenges in designing stabilized S2-only immunogens for vaccine development?

Several significant challenges remain:

• Inherent instability of S2: S2's usage as an alternative vaccine strategy is hampered by its general instability; when isolated from S1, S2 can easily transition to its postfusion conformation

• Conformational dynamics: The S2 trimer apex shows flexibility and can splay open, transitioning from a closed to open conformation through asymmetric protomer-protomer separation

• Epitope accessibility: Changes affecting spike opening dynamics can occlude important epitopes like the S2 hinge region

• Balancing stability and immunogenicity: Modifications to improve stability must not compromise the immunogenicity of conserved epitopes

To address these challenges, researchers have employed computational simulations to understand S2 dynamics and design stabilizing mutations , created constructs with additional prefusion-stabilizing modifications , and utilized structural biology to guide rational design of immunogens that present conserved epitopes in their native conformations.

How do molecular dynamics simulations inform the design of stabilized S2 antigens?

Molecular dynamics simulations have been crucial for designing stabilized S2 antigens:

• Weighted ensemble (WE) simulations: Investigated the conformational plasticity of HexaPro-SS-Δstalk, revealing that the prefusion-stabilized S2 trimer transitions from a closed to open conformation through an asymmetric protomer-protomer separation

• Contact map analysis: Revealed key residue-residue interactions taking place in the opening pathway of the interfacial helices (CHs, UHs, and HR1)

• Prediction of stabilizing mutations: Identified two residues located in the CH apex, V991 and T998, as candidates for cavity-filling substitutions with tryptophan

• Validation of designs: Additional WE simulations showed that T998W and V991W+T998W provided kinetic stabilization by slowing down the S2 trimer opening

This simulation-driven approach provided mechanistic insights into S2 dynamics and guided the design of mutations that impart both kinetic and thermodynamic stabilization to S2 antigens, leading to improved immunogens.

How do antibodies isolated from MERS-CoV S2-immunized mice compare to those from COVID-19 convalescent individuals in terms of cross-reactivity?

The two sources yield antibodies with different characteristics:

What are the differential effects of S2 antibodies on various coronavirus strains and SARS-CoV-2 variants of concern?

S2 antibodies show varying effects across coronavirus strains and variants:

• Broad cross-reactivity: 9 mAbs from COVID-19 convalescents showed cross-reactivity with spike proteins from alphacoronaviruses (229E and NL63) and betacoronaviruses (SARS-CoV-1, SARS-CoV-2, OC43, and HKU1)

• Cross-protection: Antibody IgG22 protected mice against both MERS-CoV and SARS-CoV-2 challenge , demonstrating cross-protection across different betacoronavirus species

• Variant evasion potential: Changes in Omicron BA.1 that affect spike opening dynamics can occlude the S2 hinge epitope and may evade pre-existing serum antibodies targeting the S2 core

• Neutralization of variants: Sera from mice immunized with newly designed stabilized S2 antigens significantly neutralized recombinant vesicular stomatitis viruses bearing spikes from SARS-CoV-2 Wuhan-1 and Omicron BA.1

While S2 antibodies generally show broader cross-reactivity than S1 antibodies due to the higher conservation of S2, their effectiveness can still be influenced by variant-specific conformational changes that affect epitope accessibility or presentation.