SARS MERS RBD

What is the sequence homology between SARS-CoV, MERS-CoV, and SARS-CoV-2, and how does it affect cross-reactivity?

Phylogenetic analysis reveals significant variations in sequence homology between these coronaviruses, which directly impacts cross-reactivity potential. SARS-CoV-2 shares approximately 78% sequence homology with SARS-CoV and about 50% with MERS-CoV . This pronounced similarity, particularly between SARS-CoV and SARS-CoV-2, creates conditions favorable for cross-reactive antibodies targeting cognate antigenic epitopes.

A growing body of evidence suggests that this homology translates to meaningful cross-reactivity. Studies have demonstrated that SARS-CoV RBD-specific antibodies can cross-react with SARS-CoV-2 RBD protein, and SARS-CoV RBD-induced antisera can cross-neutralize SARS-CoV-2 . This cross-reactivity is primarily observed between SARS-CoV-2, SARS-CoV, and to a lesser extent, MERS-CoV.

Methodologically, researchers assess cross-reactivity using techniques such as ELISA, pseudovirus neutralization assays, and bead-based multiplex assays that can simultaneously detect antibodies against multiple coronavirus antigens.

How do the binding affinities of SARS-CoV and SARS-CoV-2 RBDs to human ACE2 compare?

SARS-CoV-2 RBD exhibits significantly higher binding affinity to the human angiotensin-converting enzyme 2 (ACE2) receptor compared to SARS-CoV RBD . This enhanced binding affinity likely contributes to the higher transmissibility observed with SARS-CoV-2.

Experimental data has shown that SARS-CoV-2 RBD can block the binding and attachment of both SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells, thus inhibiting their infection of host cells . This competitive binding demonstrates the overlapping nature of the receptor-binding sites despite the differences in binding affinity.

The methodological approaches to measure these binding affinities typically include surface plasmon resonance (SPR), biolayer interferometry, or enzyme-linked immunosorbent assays (ELISA), allowing researchers to determine dissociation constants (Kd values) that quantify the strength of these interactions.

What are the primary cellular receptors for SARS-CoV, MERS-CoV, and SARS-CoV-2?

The three pathogenic human coronaviruses utilize different cellular receptors for host cell entry:

• SARS-CoV and SARS-CoV-2: Both use angiotensin-converting enzyme 2 (ACE2) as their primary cellular receptor . The RBD in their spike proteins directly interacts with ACE2, which is expressed in various human tissues, including respiratory epithelium, cardiovascular tissues, and the gastrointestinal tract.

• MERS-CoV: Utilizes dipeptidyl peptidase 4 (DPP4, also known as CD26) as its primary receptor . DPP4 is expressed on epithelial cells in the respiratory tract, kidneys, small intestine, liver, and on activated leukocytes.

These receptor differences partly explain the varied tissue tropism, transmission patterns, and clinical manifestations observed across these coronaviruses. Understanding these receptor interactions is crucial for developing therapeutic interventions targeting the virus-host cell interface.

How do clinical manifestations differ between SARS, MERS, and COVID-19 infections?

While these coronaviruses cause similar respiratory illnesses, they present distinct clinical profiles:

• Case Fatality Rates: MERS-CoV shows the highest fatality rate at approximately 37%, followed by SARS-CoV at around 10%, and SARS-CoV-2 at approximately 3.7% .

• Asymptomatic Infections: All three coronaviruses can cause asymptomatic infections, but the proportion varies. SARS-CoV had relatively few asymptomatic cases, while MERS-CoV and SARS-CoV-2 have larger numbers of asymptomatic cases . Research has shown that asymptomatic COVID-19 patients can carry high viral loads and have long virus shedding times, indicating transmission potential .

• Disease Progression Factors: The factors related to progression to severe disease differ. For SARS, advanced age and chronic hepatitis B virus infection were important independent risk factors for progression to acute respiratory distress syndrome (ARDS) . For COVID-19, advanced age, neutropenia, organ and coagulation dysfunctions are associated with progression to ARDS and death .

• Laboratory Findings: Common laboratory abnormalities across all three diseases include lymphocytopenia, elevated lactate dehydrogenase, and elevated liver transaminases. Thrombocytopenia and elevated creatine kinase are also observed .

What methods are most effective for evaluating cross-neutralization between different coronavirus RBDs?

Several methodological approaches are essential for comprehensive evaluation of cross-neutralization:

• Pseudovirus Neutralization Tests (pVNT): These assays use pseudotyped viruses expressing spike proteins from different coronaviruses to quantify neutralizing activity without requiring high biosafety level facilities. Studies have demonstrated that SARS-CoV RBD-induced antisera can cross-neutralize SARS-CoV-2 pseudoviruses .

• Bead-Based Multiplex Assays: This high-throughput approach allows simultaneous detection of antibodies against multiple coronavirus antigens. Research has utilized these assays to profile cross-reactive S1 antibody responses across all human coronaviruses, revealing significant cross-reactivity between betacoronaviruses, particularly SARS-CoV-2, SARS-CoV, and MERS-CoV .

• Binding Blockade Assays: These assess whether antibodies can prevent the interaction between viral RBDs and their cellular receptors. Studies have shown that some antibodies can block both SARS-CoV-2 RBD and SARS-CoV RBD attachment to ACE2-expressing cells .

• Competitive Binding Experiments: These determine whether antibodies targeting one coronavirus can compete with antibodies targeting another, suggesting recognition of overlapping epitopes.

How does prior MERS-CoV infection affect immune responses to subsequent SARS-CoV-2 vaccination?

A cohort study investigating patients with prior MERS-CoV infection who subsequently received COVID-19 mRNA vaccination revealed several important findings:

• Enhanced Cross-Reactive Antibodies: Vaccination significantly boosted cross-reactive immune responses toward other human coronaviruses, with the strongest cross-recognition between SARS-CoV-2, SARS-CoV, and MERS-CoV .

• Boosted Neutralizing Antibodies: A significant increase in neutralizing antibodies against MERS-CoV was observed following COVID-19 vaccination, suggesting that vaccination can enhance preexisting immunity against MERS-CoV .

• Antibody Isotype Patterns: The study found high antibody responses against SARS-CoV-2 spike protein and receptor-binding domain (RBD), including total immunoglobulins, IgA, and IgG, without a boost in IgM levels after vaccination .

• Antibody-Dependent Cellular Cytotoxicity (ADCC): Interestingly, no significant increase in ADCC activity was observed following vaccination in these patients .

These findings suggest that COVID-19 vaccination might enhance preexisting immunity against MERS-CoV and SARS-CoV, potentially through the stimulation of memory B cells that recognize conserved epitopes between these coronaviruses.

What experimental approaches can identify cross-reactive epitopes between SARS-CoV, MERS-CoV, and SARS-CoV-2?

Identifying cross-reactive epitopes requires sophisticated experimental approaches:

• Structural Biology Techniques:

  • X-ray crystallography and cryo-electron microscopy to resolve atomic-level structures of antibody-antigen complexes

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map epitope footprints

• Epitope Mapping Methods:

  • Alanine scanning mutagenesis to identify critical binding residues

  • Peptide arrays to identify linear epitopes

  • Competitive binding assays to determine overlapping epitopes

• Domain Swapping and Chimeric Proteins:

  • Creating chimeric spike proteins with domains from different coronaviruses to localize cross-reactive regions

  • Testing truncated protein constructs to narrow down epitope locations

• Computational Approaches:

  • Sequence alignment and conservation analysis

  • Structural modeling of potential cross-reactive epitopes

  • Epitope prediction algorithms

Research has shown that cross-reactive epitopes between SARS-CoV and SARS-CoV-2 are often found in conserved regions of the spike protein, particularly in the S2 subunit rather than the more variable RBD region . The cross-reactivity between MERS-CoV and other betacoronaviruses is typically weaker but still detectable, especially in patients exposed to multiple coronavirus antigens through infection and vaccination .

What are the optimal strategies for isolating broadly neutralizing antibodies from patients exposed to multiple coronaviruses?

Isolating broadly neutralizing antibodies requires a systematic approach:

• Optimal Patient Selection: Targeting individuals with documented exposure to multiple coronaviruses (e.g., prior MERS-CoV infection followed by SARS-CoV-2 vaccination) who show evidence of broad neutralizing activity in their sera .

• Antigen-Specific B Cell Isolation:

  • Using fluorescently labeled RBD antigens from multiple coronaviruses for flow cytometry-based sorting

  • Targeting B cells that bind to RBDs from multiple coronaviruses simultaneously

• Single B Cell Antibody Cloning:

  • Single-cell RNA sequencing to obtain paired heavy and light chain sequences

  • Cloning recovered sequences into expression vectors

  • Recombinant antibody production in mammalian cell lines

• Comprehensive Screening Pipeline:

  • Initial screening for binding to multiple coronavirus RBDs

  • Secondary screening for neutralization against pseudotyped viruses

  • Confirmation of broadly neutralizing activity using live virus neutralization assays

  • Epitope mapping to characterize the molecular basis of cross-reactivity

This approach has successfully identified broadly neutralizing antibodies targeting conserved epitopes in the spike protein, particularly in patients exposed to both MERS-CoV (through infection) and SARS-CoV-2 (through vaccination) . These antibodies typically target conserved epitopes outside the receptor-binding motif, where sequence conservation is higher.

What are the implications of cross-reactive immunity for pan-coronavirus vaccine development?

Cross-reactive immunity observed in patients exposed to multiple coronaviruses has significant implications for developing pan-coronavirus vaccines:

• Target Epitope Selection: The identification of cross-reactive epitopes between SARS-CoV, MERS-CoV, and SARS-CoV-2 provides rational targets for vaccine design. Research has shown that vaccination can enhance preexisting immunity against MERS-CoV and SARS-CoV, suggesting that vaccines targeting conserved epitopes could provide broader protection .

• Prime-Boost Strategies: The enhanced cross-reactivity observed in patients with MERS-CoV infection followed by COVID-19 vaccination suggests that heterologous prime-boost approaches may be particularly effective for inducing broadly protective immunity . This could involve priming with antigens from one coronavirus followed by boosting with antigens from another.

• Immunogen Design Approaches:

  • Consensus sequence immunogens that represent conserved features across multiple coronaviruses

  • Multivalent vaccines incorporating RBDs from different coronaviruses

  • Structure-based design of immunogens that present conserved epitopes while minimizing exposure of variable regions

• Evaluation Metrics: Vaccine candidates should be evaluated not only for protection against the target coronavirus but also for the breadth of cross-neutralization against diverse coronaviruses, including currently circulating strains and potential zoonotic threats.

The isolation of broadly reactive antibodies from patients exposed to multiple coronaviruses could help identify cross-reactive epitopes and guide the structure-based design of pan-coronavirus vaccines and therapeutic molecules . This approach holds promise for developing preventive strategies against future coronavirus outbreaks.