HR2 Antibody

What is the HR2 domain and why is it a significant target for neutralizing antibodies?

The HR2 domain is a highly conserved region within the S2 subunit of the SARS-CoV-2 spike protein. It plays a critical role in viral membrane fusion by interacting with the HR1 domain to form a six-helix bundle (6-HB) structure. This process is essential for the virus to enter host cells. The HR2 domain shows remarkable conservation not only among SARS-CoV-2 variants but also across different coronaviruses, with 100% amino acid sequence identity between SARS-CoV-2, SARS-CoV, WIV1, and HKU3 . This conservation makes HR2 an attractive target for developing broad-spectrum antibodies that could potentially neutralize multiple variants and even related coronaviruses .

Unlike the more variable receptor-binding domain (RBD) in the S1 subunit, which is the target of most current antibody therapeutics, the HR2 domain represents a "coldspot" with infrequent amino acid changes (defined as >17 consecutive amino acids with a frequency of substitutions of <0.1%) . This stability across variants makes HR2-targeting antibodies potentially more resilient against viral escape mutations.

How do HR2-specific antibodies neutralize SARS-CoV-2 infection?

HR2-specific antibodies employ a distinct neutralization mechanism compared to RBD-targeting antibodies. While RBD antibodies primarily block receptor binding, HR2 antibodies interfere with the post-attachment phase of viral entry by inhibiting membrane fusion . Specifically, they:

• Bind to the HR2 domain in its pre-fusion state

• Prevent the interaction between HR1 and HR2 domains

• Block the formation of the six-helix bundle (6-HB) structure

• Inhibit the conformational rearrangement from "fusion intermediate" to "postfusion" structure

HR2 antibodies do not interfere with ACE2 binding to the spike protein but can effectively inhibit cell-cell fusion processes . This post-attachment neutralization mechanism makes them complementary to RBD-targeting antibodies in therapeutic applications. Flow cytometry and competitive ELISA experiments have confirmed that HR2 antibodies do not block ACE2 binding but can prevent the membrane fusion process necessary for viral entry .

What is the origin of naturally occurring HR2 antibodies in humans?

HR2-specific antibodies have been detected in COVID-19 convalescent patients but are relatively rare compared to antibodies targeting other regions of the spike protein. High levels of IgG antibodies binding to HR2 coldspot peptides have been found in convalescent individuals, while such antibodies were low to undetectable in samples from uninfected controls, pre-pandemic samples, and most vaccinated individuals (except some who received inactivated virus-based vaccines) .

The average number of V gene somatic nucleotide mutations for naturally occurring HR2 antibodies is relatively high (range: 8 to 92), suggesting that these antibodies undergo significant affinity maturation . This extensive mutation profile indicates these antibodies likely develop through prolonged antigen exposure and selection processes, which could explain their relative rarity in the antibody response to SARS-CoV-2 infection.

What methods are most effective for isolating and characterizing HR2-specific monoclonal antibodies?

Researchers have successfully isolated HR2-specific monoclonal antibodies using several complementary approaches:

• Flow cytometry-based B cell sorting: Isolating B cells specific for HR2 peptides from convalescent individuals with high antibody levels in plasma has proven effective. This approach involves:

  • Labeling HR2 peptides with fluorescent markers

  • Identifying and sorting single B cells that bind the labeled peptides

  • Sequencing paired heavy and light chain variable regions

  • Cloning and expressing recombinant antibodies

• Hybridoma technology: Creating stable antibody-producing cell lines by fusing B cells from immunized animals with myeloma cells. One study generated 18 hybridomas secreting S protein-specific monoclonal antibodies, with binding sites mapped to four linear epitopes, including two within the HR2 region .

• Competitive binding assays: Competitive ELISA has been used to detect serum antibodies that block the binding of HR1 to HR2. This method involves:

  • Coating plates with HR2 protein

  • Adding diluted serum samples

  • Measuring inhibition of HR1-HRP binding

Characterization typically involves epitope mapping through peptide scanning, binding kinetics measurement via surface plasmon resonance (SPR), and neutralization assays using both pseudotyped and authentic viruses .

How can researchers design recombinant proteins to elicit potent HR2-targeting antibodies?

Recent advances in vaccine design have focused on recombinant proteins that can expose the HR2 domain in an immunogenic conformation. Notable approaches include:

• HR212 design: A recombinant protein consisting of HR2–linker1–HR1–linker2–HR2 that mimics the conformation of three HR2s in the "fusion intermediate." This protein:

  • Forms a trimer with three HR2s exposed

  • Shows high affinity to HR1 (K_D = 1.2 × 10^-10 M)

  • Demonstrates notable α-helix features

• Structural stabilization strategies: To improve immunogenicity, researchers have proposed:

  • Covalent stabilization at N-terminals of the three free HR2s through interchain disulfide bonds

  • Conjugation with immunopotentiators

  • Optimization of linker sequences

• Adjuvant selection: Studies have shown that Freund's adjuvant enhances the immunogenicity of HR2-containing recombinant proteins, with Complete Freund's Adjuvant (CFA) used in prime immunization and Incomplete Freund's Adjuvant (IFA) in boosts .

Immunization protocols typically involve three doses at 2-3 week intervals, with 3-week intervals generating approximately 2.7-fold higher antibody titers compared to 2-week intervals in some studies .

What are the comparative neutralization potencies of HR2 antibodies against different coronavirus variants?

HR2 antibodies demonstrate broad neutralization capabilities against SARS-CoV-2 variants due to the conserved nature of their target. Specific data includes:

Interestingly, some HR2 antibodies show broader reactivity than expected, with certain FP antibodies recognizing coronaviruses across all four genera (alpha to delta), while some HR2 antibodies cross-react with alpha-, beta-, and gammacoronaviruses . This extensive cross-reactivity underscores the potential of HR2-targeting approaches for developing pan-coronavirus countermeasures.

What are the optimal assay systems for evaluating HR2 antibody neutralization mechanisms?

Several complementary assay systems have proven valuable for characterizing HR2 antibody functions:

• Pseudovirus neutralization assays: These provide a safe, high-throughput system for initial screening and comparative analysis. Key aspects include:

  • Using lentivirus-based pseudoviruses expressing SARS-CoV-2 spike variants

  • Testing neutralization against multiple variants simultaneously

  • Determining IC50/NT50 values for quantitative comparison

• Authentic virus neutralization: For confirming pseudovirus findings with replication-competent viruses:

  • Cell protection assays measuring cytopathic effect (CPE) inhibition

  • Plaque reduction neutralization tests

  • Testing in relevant cell types like human pulmonary alveolar epithelial cells (HPAEpiCs)

• Membrane fusion inhibition assays: Specifically designed to assess the fusion-blocking mechanism:

  • Cell-cell fusion assays with spike-expressing effector cells and receptor-expressing target cells

  • Quantification by reporter gene expression or microscopic observation

• Competitive binding assays: To elucidate molecular mechanisms:

  • Measuring inhibition of HR1-HR2 interaction

  • ELISA-based competition studies

  • Surface plasmon resonance (SPR) for binding kinetics

Researchers should employ multiple assay systems as each provides complementary information about neutralization mechanisms and potency.

What animal models are most appropriate for evaluating HR2 antibody protection in vivo?

Several animal models have been validated for evaluating the protective efficacy of HR2 antibodies and vaccines:

• hACE2 transgenic mice:

  • Suitable for initial proof-of-concept studies

  • Can be used for both pre- and post-exposure prophylaxis evaluation

  • ACE2-humanized by inhalation of modified adeno-associated virus (AAV-hACE2)

• Syrian golden hamsters:

  • Gold standard small animal model for SARS-CoV-2 pathogenesis

  • Develops pronounced weight loss and lung pathology

  • Used to demonstrate that transfer of rabbit anti-HR212 sera or immunization with HR212 offered efficient protection against SARS-CoV-2 ancestral strain and Omicron BA.2 variant infections

• Rhesus macaques:

  • Most suitable model for evaluating SARS-CoV-2 infection and COVID-19 vaccines

  • More closely approximates human immune responses

  • Used to validate HR121 vaccine efficacy, though HR212 showed relatively weak activity in this model

Recommended experimental endpoints include:

• Viral load determination in respiratory tissues

• Histopathological examination

• Weight loss and clinical scoring

• Antibody response measurements (binding and neutralizing titers)

• T-cell responses assessment through IFNγ and TNFα production

How can epitope mapping be effectively performed for HR2-targeting antibodies?

Precise epitope mapping is essential for understanding the molecular basis of neutralization by HR2 antibodies. Effective approaches include:

• Peptide scanning:

  • Using overlapping peptide libraries covering the HR2 region

  • 15-amino acid overlapping peptide pools that span the entire HR2 sequence have been effective

  • ELISA binding assays to identify reactive peptides

• Mutagenesis studies:

  • Alanine scanning mutagenesis of the HR2 region

  • Testing binding and neutralization with mutated proteins

  • Identifying critical contact residues

• X-ray crystallography and cryo-EM:

  • Determining high-resolution structures of antibody-HR2 complexes

  • Identifying precise molecular contacts

  • Critical for rational design of improved immunogens

• Competition assays:

  • Competitive binding with known HR2-targeting antibodies

  • Defining epitope clusters based on competition patterns

One study mapped the binding sites of HR2-specific monoclonal antibodies to four linear epitopes, with two located within the HR2 region and two immediately upstream of the HR2 domain . This mapping revealed novel neutralizing epitopes that are important targets for antibody development.

How should researchers interpret the discrepancy between binding affinity and neutralization potency of HR2 antibodies?

Researchers have observed that binding affinity does not always correlate directly with neutralization potency for HR2 antibodies. Several factors explain this phenomenon:

• Epitope accessibility in different conformations:

  • The "fusion intermediate" conformation exists for only several minutes during membrane fusion, limiting the window for antibody action

  • Antibodies may bind strongly to HR2 peptides but encounter accessibility challenges on intact virions

• Kinetic considerations:

  • Off-rate constants (kd) may be more predictive of neutralization than equilibrium dissociation constants (KD)

  • The humanized mAb hMab5.17 exhibited a strikingly slower off-rate constant (10^-6/s) in binding with the S2 protein, indicating strong antigen-binding ability that correlated with neutralization

• Mechanistic factors:

  • Antibodies must compete kinetically with the rapid conformational changes during fusion

  • Binding to HR2 must occur during the short-lived pre-hairpin intermediate state

When analyzing HR2 antibody data, researchers should consider both binding and neutralization assays in parallel. For example, when evaluating the anti-HR212 sera, the antibody titers to HR12 were 10-fold higher than those in anti-HR212 sera, but the neutralizing antibody titers were 5.21-fold lower than those in HR212 sera . This suggests that high binding titers to certain conformations (like the 6-HB structure) may not translate to effective neutralization.

What immune correlates best predict protection for HR2-targeting vaccine approaches?

Multiple immune parameters contribute to protection conferred by HR2-targeting vaccines and should be evaluated comprehensively:

• Neutralizing antibody titers:

  • NT50 values against pseudoviruses correlate with protection

  • Breadth of neutralization against diverse variants is particularly important for HR2 approaches

  • Titers of 2.9 × 10^3 against the ancestral strain achieved with HR212 immunization provided effective protection in animal models

• Binding inhibition activity:

  • Ability to block HR1-HR2 interaction correlates with fusion inhibition

  • Competition ELISA measuring inhibition of HR1-HRP binding to HR212-coated plates provides a functional readout

• T-cell responses:

  • HR2 peptide pools have been shown to evoke strong CD8+ T cell responses in mice

  • IFNγ and TNFα double-positive cells increase following HR2 peptide stimulation

  • CD8+ T cell responses may complement antibody-mediated protection

• Antibody-dependent cellular cytotoxicity (ADCC):

  • Antibodies targeting the S2 subunit do not show significant ADCC evasion against variants

  • ADCC induced by NK cells can facilitate SARS-CoV-2 control

  • This mechanism may provide additional protection beyond direct neutralization

When evaluating HR2-targeting vaccines, researchers should assess all these parameters rather than focusing solely on neutralizing antibody titers, as multiple immune mechanisms likely contribute to protection.

What factors contribute to the rarity of HR2-targeting antibodies in natural infection?

Despite the conservation and functional importance of the HR2 domain, naturally occurring HR2-targeting antibodies are relatively rare. Several factors explain this phenomenon:

• Transient exposure during fusion:

  • The "fusion intermediate" conformation exists for only several minutes during membrane fusion

  • This brief exposure limits the opportunity for B cell recognition and affinity maturation

• Competition with immunodominant epitopes:

  • The RBD and other regions of the S1 subunit contain immunodominant epitopes that may divert the immune response

  • B cells targeting these regions may outcompete HR2-specific B cells during affinity maturation

• Structural constraints:

  • In the prefusion spike, HR2 may be partially occluded or in a conformation that differs from the fusion-intermediate state

  • The 6-HB postfusion structure induced very weak neutralizing antibodies against SARS-CoV-2

• Glycan shielding:

  • The HR2 domain of SARS-CoV-2 contains two glycosylation sites that may shield potential epitopes

  • Prokaryotically expressed HR212 lacks these glycans, which may affect immunogenicity compared to native HR2

Understanding these factors can inform strategies to overcome the limitations of natural immunity and design vaccines that specifically elicit HR2-targeting antibodies. For example, stabilizing the fusion-intermediate conformation through protein engineering and extending the immunization interval to 3 weeks (which produced approximately 2.7-fold higher antibody titers compared to 2-week intervals) may enhance HR2-specific responses .

How might HR2 antibodies be combined with other modalities for optimal therapeutic efficacy?

Combination approaches leveraging the unique attributes of HR2 antibodies alongside other therapeutic modalities hold significant promise:

• Antibody cocktails targeting distinct epitopes:

  • Pairing HR2 antibodies with RBD-targeting antibodies could provide complementary mechanisms of action

  • Such combinations could prevent escape through mutations in either domain

  • The distinct neutralization mechanisms (receptor blocking vs. fusion inhibition) would provide multiple barriers to infection

• HR2 antibodies with small molecule fusion inhibitors:

  • HR2-derived peptides like EK1 and EK1C can potently combine with HR1 trimer and block 6-HB generation

  • These peptides have shown antiviral effects against multiple SARS-CoV-2 variants

  • Combining such peptides with HR2 antibodies might produce synergistic effects

• Bispecific antibody approaches:

  • Engineering bispecific antibodies that simultaneously target HR2 and another conserved epitope

  • This approach could increase avidity and reduce the likelihood of escape

• Integration with cellular immunity enhancers:

  • HR2 peptide pools have been shown to evoke strong CD8+ T cell responses

  • Combining HR2 antibody therapy with vaccines or adjuvants that enhance T cell responses could improve clearance of infected cells

Future research should evaluate these combination approaches through in vitro synergy studies and in vivo efficacy testing against multiple variants to identify optimal therapeutic strategies.

What structural modifications could enhance the potency and breadth of HR2-targeting immunogens?

Several promising structural modifications could improve HR2-based immunogens:

• Covalent stabilization strategies:

  • Creating interchain disulfide bonds at the N-terminals of the three free HR2s in recombinant proteins

  • This approach could stabilize the desired conformation and improve presentation of neutralizing epitopes

• Improved linker design:

  • Optimizing the length and composition of linkers in constructs like HR212 (HR2–linker1–HR1–linker2–HR2)

  • This could improve folding and presentation of HR2 epitopes

• Enhanced display platforms:

  • Presenting HR2 domains on nanoparticles or virus-like particles for multivalent display

  • Such presentation could improve B cell receptor crosslinking and enhance immunogenicity

• Glycan engineering:

  • Strategic removal or modification of glycans that shield neutralizing epitopes

  • Alternatively, expressing HR212 in eukaryotic systems to include native glycans for authentic presentation

• Heterologous prime-boost strategies:

  • Priming with HR2-based immunogens and boosting with whole spike or different HR2 constructs

  • This approach could focus the immune response on shared conserved epitopes

Future studies should systematically evaluate these modifications through structural analysis, immunogenicity studies in small animals, and neutralization assays against a panel of coronaviruses to identify the most promising candidates for further development.