SARS Spike (14-1195)

What is the SARS-CoV-2 spike protein and what is its primary function?

The SARS-CoV-2 spike protein is a surface glycoprotein found on the viral envelope responsible for host cell entry . It binds to the angiotensin-converting enzyme 2 (ACE2) receptor on host cells, facilitating viral attachment and subsequent infection . The spike protein not only mediates virus-cell binding but can also promote cell-to-cell fusion, a phenomenon observed in the lungs of COVID-19 patients . This protein represents one of the most important therapeutic targets for COVID-19 intervention strategies, including vaccines and antiviral drugs .

The spike protein's structural characteristics enable it to undergo conformational changes during the infection process, transitioning from a prefusion to a postfusion state after receptor binding . These conformational changes are essential for membrane fusion and viral genome delivery into the host cell cytoplasm.

How stable is the SARS-CoV-2 spike protein, and what implications does this have for research?

Research conducted at the University of Kentucky College of Medicine has demonstrated that the majority of SARS-CoV-2 spike protein degrades within 24 hours . This finding has significant implications for understanding both the infection process and vaccination mechanisms. For mRNA vaccines that instruct cells to produce the spike protein, this degradation timeline provides crucial insight into how long the newly generated protein remains present in the body .

The protein's relatively short half-life necessitates specific experimental considerations when working with it in laboratory settings. Researchers should account for this degradation when designing experiments involving spike protein detection, purification, or functional assays. This characteristic also highlights the importance of proper sample handling and storage conditions to maintain protein integrity during experimental procedures.

What are the key differences between spike proteins of various SARS-CoV-2 variants?

The spike proteins of different SARS-CoV-2 variants, particularly the Omicron lineages, exhibit distinct mutation profiles that affect their properties and interactions with host cells . The Omicron sublineages (BA.1, BA.2, BA.4, and BA.5) display unique epidemic characteristics and immune escape capabilities .

BA.2 has demonstrated approximately 30% higher transmissibility than BA.1, though it does not appear to cause more severe disease . In South Africa, BA.1 was dominant from November 2021 to January 2022, followed by BA.2 dominance in February and March 2022 . Subsequently, BA.4 and BA.5 rapidly replaced BA.2, accounting for more than 50% of sequenced cases by April 2022 .

These sequential waves of variant predominance reflect the evolutionary advantage conferred by specific spike protein mutations, which affect receptor binding affinity, fusion efficiency, and immune evasion capabilities. Understanding these differences is crucial for developing targeted therapeutic approaches and updating vaccine formulations.

How can researchers enhance the stability and expression of SARS-CoV-2 spike protein for experimental applications?

Structure-based design approaches have significantly improved the yield and stability of SARS-CoV-2 spike ectodomain in its prefusion conformation . Multiple strategies have proven effective:

High-resolution cryo-EM structural analysis confirms that these modifications, particularly the proline substitutions, adopt the designed conformations without disrupting the S2 subunit structure, thus preserving antigenicity . These approaches facilitate the production of prefusion spike proteins for diagnostic kits, subunit vaccines, and structural studies.

When implementing these strategies, researchers should consider:

• The specific experimental application and required protein yield

• Whether native glycosylation patterns need to be maintained

• The impact of modifications on epitope presentation

• Expression system compatibility with the designed construct

What evidence exists for persistent SARS-CoV-2 spike antigen in post-acute COVID-19 patients, and how might this serve as a biomarker?

A significant study analyzing plasma samples from post-acute sequelae of COVID-19 (PASC) and COVID-19 patients (n = 63) detected SARS-CoV-2 spike antigen in a majority of PASC patients up to 12 months post-diagnosis . This finding suggests the presence of an active persistent SARS-CoV-2 viral reservoir in these individuals .

Temporal antigen profiles for many patients showed spike protein presence at multiple time points over several months, highlighting the potential utility of full spike protein as a biomarker for PASC . This persistence may explain some of the prolonged symptoms experienced by long COVID patients, potentially through continued immune stimulation or direct tissue effects.

For researchers investigating PASC biomarkers, these findings suggest:

• The need for highly sensitive detection methods capable of measuring low levels of circulating spike protein

• The importance of longitudinal sampling to establish temporal profiles

• The potential value of correlating spike protein levels with specific symptom clusters

• Opportunities to investigate whether targeting this persistent viral reservoir could provide therapeutic benefit

How do the prefusion spike protein conformational changes differ between SARS-CoV-1 and SARS-CoV-2, and what implications does this have for viral transmissibility?

Despite striking similarities between SARS-CoV-1 and SARS-CoV-2, their differential transmissibility has not been fully explained at the molecular level . Extensive microsecond-level all-atom molecular dynamics simulations of prefusion spike proteins from both viruses reveal differences in their dynamic behavior .

SARS-CoV-2 has proven more easily transmissible between humans compared to SARS-CoV-1 . The spike protein plays a crucial role in the infection process and has been the primary target for drugs and vaccines . Molecular dynamics studies suggest that conformational changes in the prefusion spike protein during ACE2 binding may occur at different rates between the two viruses, potentially contributing to their differential transmissibility .

These findings highlight the importance of protein dynamics, not just static structures, in understanding viral infectivity. Researchers investigating viral entry mechanisms should consider:

• The temporal aspects of receptor binding and conformational changes

• How mutations might alter the energy landscape of these conformational transitions

• The potential for targeting specific intermediate conformations with therapeutics

• How these dynamic properties might predict the behavior of emerging variants

What is the role of TMPRSS2 in SARS-CoV-2 spike protein activation, and how does it affect viral entry across different species?

Transmembrane Serine Protease 2 (TMPRSS2) plays a significant role in SARS-CoV-2 spike protein activation and viral entry . Experimental evidence shows that co-expression of TMPRSS2 with ACE2 receptors can enhance SARS-CoV-2 pseudoparticle entry, particularly for non-cognate receptors that show limited entry capability when expressed alone .

A similar trend was observed in cell-cell fusion assays, where co-expression of human ACE2 and TMPRSS2 in target cells led to larger syncytia compared to ACE2 alone . TMPRSS2 co-expression significantly enhanced the use of non-cognate receptors (fruit bat, ferret, turkey, and chicken) .

For researchers investigating cross-species transmission potential:

• TMPRSS2 expression levels in target tissues should be considered alongside ACE2 compatibility

• In vitro systems should account for both receptor binding and proteolytic activation

• The potential for TMPRSS2 to "rescue" otherwise inefficient ACE2-spike interactions suggests a more complex host range determinant than receptor binding alone

• Species-specific variations in TMPRSS2 activity may contribute to differences in susceptibility

What is known about T-cell mediated immune responses against Omicron lineage infections, and how do they compare to responses against previous variants?

T-cell mediated immune responses play a crucial role in the body's defense against SARS-CoV-2 infection. Early induction of antigen-specific CD4+ T-cells following vaccination is associated with generation of antibody and CD8+ T-cell responses against SARS-CoV-2 . Patients with mild COVID-19 show increased numbers of CD8+ T-cells in bronchoalveolar lavage fluid, which effectively eliminate the virus and strengthen reactivity to SARS-CoV-2 antigen .

Recent studies have investigated T-cell reactivity against different SARS-CoV-2 variants:

Following booster vaccination, T-cell response increases by 20.1-fold against wild-type and 20.4-fold against Omicron . Memory mediated by T cells in individuals with prior infection (wild-type or previous variants of concern) and vaccination has preserved reactivity to the Omicron variant, especially when enhanced by booster vaccination [82-87].

Interestingly, the percentage of participants with prior infection and/or vaccination who had a >50% reduction in T-cell response to Omicron spike was higher than for Delta (21.2% vs. 9.7%), potentially due to escape of HLA-I restricted epitopes . This suggests that protection against variants of concern may rely on cross-reactive SARS-CoV-2-specific T-cell mediated immunity.

For researchers studying T-cell responses:

• The differential impact of spike mutations on antibody vs. T-cell recognition should be assessed

• HLA diversity should be considered when evaluating population-level immune protection

• The durability of T-cell memory compared to antibody responses may inform vaccination strategies

• Special attention should be given to immunocompromised patients who may have impaired T-cell responses

What techniques are most effective for detecting persistent spike protein in patient samples?

Detection of persistent SARS-CoV-2 spike protein in patient samples requires highly sensitive methods. The study that identified spike antigen in PASC patients up to 12 months post-diagnosis utilized ultra-sensitive digital immunoassay platforms . When designing studies to detect persistent spike protein, researchers should consider:

• Sample selection: Plasma samples are commonly used, but other biofluids or tissues may harbor spike protein. Multiple specimen types may provide a more comprehensive assessment.

• Assay sensitivity: Ultra-sensitive digital immunoassays, such as Simoa technology, offer superior detection limits compared to conventional ELISA methods .

• Epitope targeting: Using antibodies that target conserved regions of the spike protein ensures detection even if mutations are present.

• Temporal sampling: Collecting samples at multiple time points increases the likelihood of detection, as spike protein levels may fluctuate over time.

• Controls: Proper negative and positive controls are essential for assay validation, including samples from pre-pandemic individuals and acute COVID-19 patients.

• Correlation with viral RNA: Where possible, correlating spike protein detection with viral RNA testing provides stronger evidence of viral persistence.

What expression systems are optimal for producing stable, prefusion-locked SARS-CoV-2 spike proteins?

Production of stable, prefusion-locked SARS-CoV-2 spike proteins can be achieved through various expression systems, each with advantages and limitations:

• Mammalian cell expression: Systems such as HEK293 and CHO cells provide appropriate post-translational modifications, particularly glycosylation patterns that match native spike protein. The HexaPro construct with four proline substitutions has demonstrated a 10-fold increase in expression in mammalian systems .

• Insect cell expression: Baculovirus expression systems offer scalability and potentially higher yields, though with altered glycosylation patterns.

• Yeast expression: Can provide cost-effective production but may require additional optimization to achieve proper folding and post-translational modifications.

Key considerations for expression system selection include:

• Required scale of production

• Need for native-like glycosylation

• Downstream application requirements (structural studies, immunogens, etc.)

• Available purification strategies

For stabilization and prefusion locking, researchers have successfully employed:

Purification typically involves affinity chromatography using tags (His, Strep, etc.) followed by size exclusion chromatography to isolate properly folded trimeric spike proteins.

How can researchers effectively compare conformational changes between SARS-CoV-1 and SARS-CoV-2 spike proteins?

Comparing conformational changes between SARS-CoV-1 and SARS-CoV-2 spike proteins requires sophisticated biophysical and computational approaches:

• Molecular dynamics simulations: Microsecond-level all-atom molecular dynamics simulations have been valuable in revealing differences in dynamic behavior between these spike proteins . These simulations can capture transient conformational states and energy barriers between them.

• Cryo-electron microscopy (Cryo-EM): This technique can capture different conformational states and is particularly useful for large protein complexes like the spike trimer. Time-resolved cryo-EM can potentially capture intermediate states during conformational transitions.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This approach identifies regions of differential flexibility or solvent exposure between the two spike proteins, providing insights into dynamics.

• Single-molecule FRET: By strategically placing fluorophores on the spike protein, researchers can monitor distance changes during conformational transitions in real-time.

• Structural analysis: Comparing crystal or cryo-EM structures of the spike proteins in different conformational states can identify key differences in hinge regions or interdomain contacts.

When implementing these approaches, researchers should consider:

• The physiological relevance of experimental conditions (pH, temperature, membrane environment)

• The impact of stabilizing mutations on native dynamics

• Correlation between observed conformational changes and functional outcomes (receptor binding, fusion)

• Integration of multiple complementary techniques to build a comprehensive understanding

What are the most promising approaches for targeting persistent spike protein in PASC patients?

The discovery of persistent spike protein in PASC patients opens several potential therapeutic avenues that warrant investigation:

• Monoclonal antibody therapy: Development of antibodies specifically targeting the spike protein could help clear persistent antigen. These approaches might require extended treatment courses compared to acute COVID-19 applications.

• Antiviral strategies: Investigating whether antivirals like nirmatrelvir/ritonavir (Paxlovid) could eliminate persistent viral reservoirs in PASC patients, potentially addressing the source of spike protein production.

• Immunomodulatory approaches: If persistent spike protein drives ongoing inflammation, targeted immunomodulation might alleviate symptoms while viral clearance occurs.

• Circulating spike protein sequestrants: Engineering molecules that bind circulating spike protein and facilitate its clearance without triggering immune responses.

Research priorities should include:

• Identifying the cellular and tissue reservoirs of persistent virus or spike protein

• Determining whether persistent spike is actively produced or stable in circulation

• Establishing whether spike persistence correlates with specific PASC symptom clusters

• Developing animal models of spike persistence to test therapeutic approaches

How might future SARS-CoV-2 variants evolve to alter spike protein dynamics and what implications would this have?

Understanding how SARS-CoV-2 variants might continue to evolve is critical for pandemic preparedness. Future research should explore:

• Evolutionary constraints on spike protein: Identifying regions under strong selective pressure versus conserved functional domains could predict future mutation patterns.

• Impact of immune pressure: How population immunity shapes viral evolution, particularly in regions of the spike targeted by vaccines or common neutralizing antibodies.

• Dynamics-function relationships: Further characterizing how conformational dynamics affect transmissibility, immune evasion, and pathogenicity to anticipate functional consequences of emerging mutations.

• Cross-species transmission potential: Investigating how spike protein adaptations might facilitate transmission to new host species, potentially creating new reservoirs.

Computational approaches integrating evolutionary analysis, structural biology, and molecular dynamics could help predict emerging variants of concern before they achieve widespread circulation. Such predictive modeling would be valuable for proactive vaccine updates and therapeutic development.