SARS MERS, Sf9 Active

How do coronavirus transmission dynamics differ between SARS-CoV and MERS-CoV?

MERS-CoV primarily spreads through airborne and droplet transmission, particularly through coughing . The virus shows limited sustained human-to-human transmission, with most human-to-human spread occurring in healthcare settings through close contact with infected individuals . Community-acquired MERS-CoV infections typically result from direct or indirect contact with infected dromedary camels .

In contrast, SARS-CoV is hypothesized to have originated in bats and then infected humans either indirectly through intermediate hosts like palm civets or raccoon dogs, or potentially directly without an intermediate host . Both viruses are primarily transmitted via respiratory droplets, though their efficiency of transmission and reservoir hosts differ significantly .

What animal models are available for studying SARS and MERS coronaviruses?

Several animal models have been developed for studying coronavirus pathogenesis:

• Mouse models:

  • Transgenic mice expressing human ACE2 (hACE2) for SARS-CoV studies

  • Mouse-adapted (MA) strains of SARS-CoV (MA15, MA20, v163) that replicate to high titers in mouse lungs

  • Transgenic mice expressing human DPP4 (hDPP4) for MERS-CoV studies

  • Ad5-hDPP4 transduced mice for MERS-CoV research

• Immune-deficient mouse models:

  • STAT1−/− mice in the 129S background supported prolonged SARS-CoV viral replication

  • Rag1−/−, CD1−/−, and Beige knockout mice have been used to determine the role of immune effectors in SARS disease

Mouse models are particularly valuable due to their low cost, small size, availability, and potential for genetic manipulation. The mouse-adapted strains of SARS-CoV can produce disease in mice that resembles severe human cases, with high viral titers in the lungs, pathological changes, viral dissemination to extrapulmonary sites, and mortality .

What are the optimal conditions for expressing SARS-CoV-2 spike proteins in insect cells?

The expression of SARS-CoV-2 spike proteins in insect cells requires optimization of several parameters. Researchers have developed robust protocols using two primary insect cell lines: Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tni) .

When expressing SARS-CoV-2 spike proteins, researchers should consider the following constructs:

• S-RBD-eGFP: Spike protein receptor-binding domain with SD1 domain coupled to a fluorescent tag

• S-Ecto-eGFP: Spike ectodomain coupled to a fluorescent tag

• S-Ecto-HexaPro(+F): Spike ectodomain with six proline mutations and a foldon domain

• S-Ecto-HexaPro(-F): Spike ectodomain with six proline mutations without the foldon domain

These expression systems allow for the production of functional glycosylated spike protein of high purity. The choice between Sf9 and Tni cells should be based on the specific construct and desired yield, as different cell lines may provide varying expression levels for different protein constructs .

How can researchers effectively isolate and characterize neutralizing antibodies against coronavirus spike proteins?

The isolation and characterization of neutralizing antibodies require a systematic approach:

• Isolation: Start by collecting B cells from infected individuals. Single B cell isolation from peripheral blood mononuclear cells (PBMCs) of convalescent patients has proven effective. In one study, 206 RBD-specific monoclonal antibodies were isolated from single B cells of 8 SARS-CoV-2 infected individuals .

• Screening: Test antibodies for binding to viral proteins, particularly the RBD. Competition assays with ACE2 (for SARS-CoV and SARS-CoV-2) or DPP4 (for MERS-CoV) are valuable for identifying potentially neutralizing antibodies .

• Neutralization assays: Evaluate antibody neutralization potency using pseudovirus or live virus neutralization assays. Importantly, neutralization activity correlates strongly with competition with ACE2 for binding to RBD in SARS-CoV-2 .

• Structural analysis: Perform crystal structure analysis of RBD-bound antibodies to understand the mechanisms of neutralization. Studies have revealed that steric hindrance inhibiting viral engagement with ACE2 is a key mechanism for blocking viral entry .

• Cross-reactivity testing: Examine antibody cross-reactivity with related coronaviruses. Research has shown that anti-SARS-CoV-2 antibodies generally do not cross-react with the RBDs of SARS-CoV or MERS-CoV, suggesting viral-species specificity .

What molecular adaptations enable cross-species transmission of MERS-CoV?

Molecular adaptations in both the virus and host contribute to cross-species transmission of MERS-CoV:

• Receptor binding domain adaptations: The RBD of MERS-CoV can interact with CD26 (DPP4) receptors from different species, albeit with varying affinities. Bat CD26s (bCD26s) from different species exhibit substantial diversity, especially in regions responsible for binding to the MERS-CoV RBD .

• Host receptor polymorphisms: Despite their diversity, bat CD26 receptors can still interact with MERS-RBD at varied affinities and support viral entry. These polymorphisms likely create evolutionary pressure that drives adaptation of CD26-binding viruses, leading to diversified coronavirus strains .

• Intermediate host adaptation: Dromedary camels serve as the primary reservoir host for MERS-CoV, with multiple independent transmissions occurring from camels to humans. Additionally, alpacas have been identified as potentially susceptible to natural MERS-CoV infection, suggesting they may be another animal reservoir .

This receptor-virus co-evolution provides strong evidence supporting bats as the reservoir of MERS-CoV and similar viruses, highlighting the importance of surveying these coronaviruses among bat populations .

How should researchers design vaccination studies against coronaviruses using animal models?

When designing vaccination studies against coronaviruses using animal models, researchers should consider:

• Model selection: Choose appropriate animal models that reflect human disease. Mouse-adapted SARS-CoV strains (MA15, MA20, v163) in older mice produce clinical disease reminiscent of acute respiratory distress syndrome (ARDS) in humans .

• Antigen selection: The spike protein, particularly its RBD, is a primary target for vaccine development. Animal studies with RBD-based vaccines against SARS-CoV and MERS-CoV have demonstrated strong polyclonal antibody responses that inhibit viral entry .

• Immune response assessment: Evaluate both humoral and cellular responses. The spike protein plays key roles in inducing neutralizing antibody and T-cell responses, as well as protective immunity during coronavirus infection .

• Challenge studies design: For SARS-CoV, use transgenic mice expressing human ACE2 or mouse-adapted viral strains. For MERS-CoV, use transgenic mice expressing human DPP4 that are fully permissive to infection .

• Age considerations: Include older animals in studies as coronavirus diseases often show age-dependent severity. Infection of older mice with the MA15 virus produces clinical disease particularly reminiscent of ARDS in humans .

• Duration of immunity: Design long-term studies to assess the durability of protective immune responses, as this is crucial for effective vaccine development.

What are the critical factors to consider when evaluating cross-reactivity between different coronavirus antibodies?

When evaluating cross-reactivity between coronavirus antibodies, researchers should consider:

• Target protein domains: Examine reactivity against specific viral protein domains separately. Studies have shown that while anti-SARS-CoV-2 antibodies may not cross-react with the RBDs of SARS-CoV or MERS-CoV, there can be substantial plasma cross-reactivity to their trimeric spike proteins .

• Detection methods: Use multiple complementary methods such as ELISA, Western blotting, and functional assays. Cross-reactivity of antibodies has been demonstrated between SARS-CoV–positive human plasma and SARS-CoV-2 recombinant nucleocapsid protein with specific 45 kD band observation and confirmed via ELISAs .

• Neutralization vs. binding: Distinguish between binding antibodies and neutralizing antibodies. An antibody may bind to multiple coronavirus proteins but neutralize only specific strains.

• Epitope mapping: Identify specific epitopes recognized by cross-reactive antibodies to understand structural similarities between viruses.

• Pre-existing immunity: Consider how previous coronavirus infections might influence antibody responses to new viruses. Patients with cross-reactive antibodies may have been previously infected with related coronaviruses .

• Species variation: Examine antibody cross-reactivity across different host species to understand zoonotic potential and viral evolution.

How can researchers reconcile conflicting data on coronavirus receptor binding affinities?

When faced with conflicting data on coronavirus receptor binding affinities, researchers should:

• Standardize experimental conditions: Ensure comparable conditions across studies, including protein constructs, expression systems, and measurement techniques.

• Consider evolutionary context: Analyze different viral strains chronologically. Research has shown that "2019-nCoV likely uses human ACE-2 less efficiently than human SARS-CoV (year 2002) but more efficiently than human SARS-CoV (year 2003)" .

• Evaluate mutation effects: Identify key mutations that affect binding affinities. For example, a single N501T mutation (corresponding to S487T in SARS-CoV) may significantly enhance binding affinity between SARS-CoV-2 RBD and human ACE-2 .

• Use multiple measurement techniques: Employ complementary approaches such as surface plasmon resonance, bio-layer interferometry, and cell-based assays to validate findings.

• Consider host factors: Receptor polymorphisms across species or individuals can affect binding affinities and explain apparent discrepancies.

• Contextualize with infectivity data: Correlate binding affinity measurements with actual infectivity or transmissibility data, as ACE-2-binding affinity has been shown to be one of the most important determinants of SARS-CoV infectivity .

What are the most reliable methods for diagnosing coronavirus infections in research settings?

In research settings, reliable diagnostic methods for coronavirus infections include:

• Molecular detection:

  • PCR-based tests using respiratory samples to detect active viral infection

  • This approach is considered the gold standard for detecting active coronavirus infection

• Serological testing:

  • Blood samples to detect antibodies to coronaviruses

  • Indicates recent or past infection

  • Particularly useful for surveillance studies

• Viral isolation:

  • Culture of virus from patient samples

  • Provides viable virus for further characterization

  • Important for identifying new variants

• Genomic sequencing:

  • Full or partial genome sequencing to identify specific viral strains

  • Critical for tracking viral evolution and emergence of new variants

  • Helps identify mutations that may affect transmission, pathogenicity, or immune evasion

• Antigen detection:

  • Detection of viral proteins in clinical samples

  • Offers rapid results but may be less sensitive than PCR

Researchers should select diagnostic methods based on specific research questions, considering factors such as sensitivity, specificity, turnaround time, and the ability to differentiate between active and past infections .

What are the promising research avenues for developing broadly neutralizing antibodies against multiple coronaviruses?

Several promising research avenues for developing broadly neutralizing antibodies include:

• Conserved epitope targeting: Identify and target highly conserved epitopes across different coronaviruses, particularly in the S2 domain which tends to be more conserved than the RBD.

• Structure-based immunogen design: Use structural information about spike proteins to design immunogens that elicit antibodies targeting conserved vulnerability sites across coronaviruses.

• Sequential immunization strategies: Employ vaccination regimens that use antigens from different coronaviruses in sequence to guide antibody maturation toward conserved epitopes.

• Germline-targeting approaches: Design immunogens that activate B cell receptors with the potential to develop into broadly neutralizing antibodies.

• Combination antibody therapy: Develop cocktails of monoclonal antibodies targeting different epitopes to increase breadth of coverage and reduce the likelihood of escape mutations.

Despite substantial plasma cross-reactivity to trimeric spike proteins of different coronaviruses, current evidence suggests that anti-SARS-CoV-2 antibodies are largely viral-species-specific inhibitors, highlighting the challenge and importance of developing broadly neutralizing antibodies .

How can Sf9 insect cell expression systems be optimized for high-yield production of coronavirus antigens?

To optimize Sf9 insect cell expression systems for high-yield production of coronavirus antigens:

• Construct optimization:

  • Include appropriate signal peptides for secretion

  • Consider adding stabilizing mutations (e.g., proline substitutions in the spike protein)

  • Incorporate purification tags that minimally impact protein function

  • Test various constructs (RBD-only, full ectodomain, etc.) to determine optimal expression

• Vector selection:

  • Choose between baculovirus expression vector system (BEVS) and stable cell line approaches

  • Optimize promoter strength and regulatory elements

• Culture conditions:

  • Compare expression in different insect cell lines (Sf9 vs. Tni)

  • Optimize cell density, infection multiplicity, and harvest timing

  • Adjust temperature, pH, and oxygen levels during expression

• Purification strategy:

  • Develop multi-step purification protocols with minimal yield loss

  • Consider on-column folding or refolding strategies if needed

  • Implement quality control measures to ensure protein functionality

• Glycosylation considerations:

  • Although insect cells produce simpler glycosylation patterns than mammalian cells, they can produce functional glycosylated spike proteins

  • Consider glycoengineered insect cell lines for more mammalian-like glycosylation if needed

The robust protocol developed for SARS-CoV-2 spike expression in insect cells demonstrates the potential for quick and inexpensive production of functional glycosylated spike protein of high purity .