SARS Mosaic S(M)

Basic Research Questions

• What is a SARS Mosaic S(M) protein and how does it differ from conventional spike proteins?

  A SARS Mosaic S(M) protein is a recombinant construct containing multiple immunodominant regions from coronavirus spike proteins, designed to present diverse epitopes in a single molecule. Unlike conventional full-length spike proteins, the mosaic approach selectively incorporates critical antigenic regions from different coronaviruses or variants.

  For example, the E. coli-derived 38 kDa recombinant mosaic protein contains specifically selected middle sections of the Spike protein (amino acids 408-470 and 540-573), which are immunodominant regions . This strategic selection of epitopes enables broader immunoreactivity while minimizing specificity problems in diagnostic applications.

  The mosaic approach represents a fundamental shift from homotypic antigen presentation (single variant) to heterotypic presentation (multiple variants), thereby addressing the challenge of viral diversity and evolution.

• What are the key structural components of mosaic RBD nanoparticles?

  Mosaic RBD nanoparticles typically consist of:

  • A nanoparticle scaffold (such as proliferating cell nuclear antigen or SpyCatcher003-mi3) that provides the structural framework

  • Multiple receptor-binding domains (RBDs) from different coronaviruses attached to the scaffold

  • Covalent attachment systems (such as SpyTag/SpyCatcher technology) to secure RBDs to the scaffold

  A prototypical example is the 6RBD-np design, which displays six RBDs derived from different α- and β-coronaviruses linked to a heterotrimeric scaffold composed of proliferating cell nuclear antigen (PCNA) subunits (PCNA1, PCNA2, and PCNA3). This creates a stable ring-shaped disk with six protruding antigens, resembling "jewels in a crown" .

  Similarly, other designs like mosaic-8b present the SARS-CoV-2 Beta RBD plus seven other sarbecovirus RBDs attached to 60 sites on a nanoparticle, creating a multivalent antigen display system .

• How are mosaic nanoparticles validated for research applications?

  Validation of mosaic nanoparticles involves multiple analytical approaches:

  • Size-exclusion chromatography (SEC) to confirm particle homogeneity

  • SDS-PAGE to verify conjugation efficiency (typically aiming for near 100%)

  • Dynamic light scattering (DLS) to measure particle size distribution

  • Negative-stain electron microscopy (EM) to visualize structure and confirm monodispersity

  • Binding assays with human ACE2 and monoclonal antibodies to ensure epitopes retain proper conformation

  For example, mosaic-8 and homotypic SARS-2 Beta nanoparticles are typically purified by SEC and validated with SDS-PAGE to show conjugation efficiency. DLS and negative-stain EM confirm that conjugated nanoparticles are monodisperse with a defined diameter .

  These validation methods ensure that the complex nanoparticle constructs maintain structural integrity and appropriate antigen presentation for immunological studies.

• What immunological readouts are used to evaluate mosaic nanoparticle vaccines?

  Evaluation of mosaic nanoparticle vaccines typically employs multiple immunological readouts:

  • Antibody titers (total and neutralizing) against homologous and heterologous antigens

  • T cell responses (particularly IFN-γ production)

  • Cross-reactivity against diverse coronavirus strains not included in the mosaic design

  • Protection in challenge models (viral load, clinical symptoms, survival)

  For instance, prime-boost immunizations with 6RBD-np in mice induced significantly high antibody titers against RBD antigens derived from both α- and β-coronaviruses and increased interferon (IFN-γ) production, with full protection against SARS-CoV-2 wild type and Delta variant challenges .

  Similarly, mice immunized with RBD nanoparticles, but not soluble antigen, elicited cross-reactive binding and neutralization responses, with mosaic RBD nanoparticles demonstrating superior cross-reactive recognition of heterologous RBDs compared to homotypic SARS-CoV-2–RBD nanoparticles .

Advanced Research Questions

• How does epitope accessibility and presentation differ between mosaic and homotypic nanoparticle designs?

  Epitope accessibility and presentation between mosaic and homotypic designs differ in several critical ways:

  • Epitope competition dynamics: In homotypic nanoparticles, identical epitopes compete for B cell recognition, potentially reinforcing immunodominance patterns. Mosaic designs disrupt this by presenting diverse epitopes in proximity.

  • Conserved epitope exposure: Mosaic nanoparticles enhance exposure of conserved epitopes that might be immunologically subdominant in homotypic presentations. RBD mapping experiments show that mosaic-8b primarily elicits antibodies against conserved RBD regions, whereas homotypic SARS-2 RBD-mi3 nanoparticles predominantly generate responses against immunodominant class 1 and class 2 RBD epitopes .

  • Spatial arrangement effects: By including eight different RBD antigens arranged randomly on a 60-mer nanoparticle, mosaic designs maximize the chances of stimulating production of cross-reactive antibodies against conserved regions because adjacent antigens are unlikely to be the same .

  This fundamental difference in epitope presentation explains why mosaic-8b nanoparticles protected against both matched (SARS-CoV-2) and mismatched (SARS-CoV) challenges, while homotypic SARS-2 RBD nanoparticles only fully protected against matched challenges .

• What strategies can be employed to modulate epitope immunodominance in mosaic nanoparticle designs?

  Several sophisticated strategies can be employed to modulate epitope immunodominance:

  1. Strategic glycosylation: N-linked glycosylation site sequons can be introduced at specific positions (e.g., RBD position 484) to selectively occlude immunodominant epitopes like class 1 and 2 RBD epitopes .

  2. Epitope masking: Specific immunodominant regions can be modified or deleted to redirect immune responses toward more conserved epitopes.

  3. Varying epitope density: Adjusting the ratio of different RBDs on the nanoparticle can fine-tune the immune response toward desired epitopes.

  4. Random versus directed display: Random arrangement of antigens on the nanoparticle surface (as in mosaic-8 designs with 60-mer particles) increases the probability that adjacent antigens will be different, enhancing the likelihood of stimulating cross-reactive antibody production .

  These approaches represent sophisticated immunological engineering to subvert natural immunodominance patterns that typically limit the breadth of antibody responses elicited by natural infection or conventional vaccination strategies.

• How do cross-reactive neutralizing antibody profiles differ between natural infection, homotypic vaccination, and mosaic nanoparticle vaccination?

  The profiles of cross-reactive neutralizing antibodies differ significantly based on immunization strategy:

  Immunization Type	SARS-CoV-2 Neutralization	Heterologous Sarbecovirus Neutralization	Cross-Clade Protection
  Natural Infection	Strong	Weak or absent	Limited
  Homotypic RBD Nanoparticles	Strong	Present after boosting	Partial
  Mosaic RBD Nanoparticles	Strong	Strong	Comprehensive

  Research has shown that soluble SARS-2 S immunization and natural infection with SARS-CoV-2 resulted in weak or no heterologous responses in plasmas . In contrast, homotypic SARS-2 nanoparticle immunization produces IgG responses that bind zoonotic RBDs and neutralize heterologous coronaviruses, but only after boosting .

  Mosaic nanoparticles demonstrated the most advantageous profile, eliciting neutralizing antibodies against zoonotic sarbecoviruses while maintaining robust responses against SARS-CoV-2, providing protection against both matched and mismatched viral challenges .

• What are the critical considerations for RBD selection when designing a pan-sarbecovirus mosaic vaccine?

  Designing an effective pan-sarbecovirus mosaic vaccine requires strategic RBD selection based on:

  1. Phylogenetic diversity: Selection should cover multiple clades within the sarbecovirus subgenus to ensure breadth of protection. For example, the mosaic-8b design incorporated RBDs from clade 1a, 1b, and 2 sarbecoviruses .

  2. Receptor usage patterns: Including RBDs with diverse receptor binding profiles (ACE2-dependent and independent) ensures broader protection against viruses with different cell entry mechanisms.

  3. Sequence conservation mapping: Analysis of sequence identity across RBDs (typically ranging from 68% to 95%) helps identify conserved regions that might elicit cross-protective responses, with particular attention to the receptor binding motif where sequence variability is highest .

  4. Immunological bridging: Including RBDs that share conserved epitopes but differ in immunodominant regions can help "bridge" immune responses from one variant to another.

  5. Spillover potential: Priority should be given to RBDs from viruses with demonstrated or predicted human spillover potential based on receptor usage and cell tropism studies .

  This systematic approach to RBD selection is essential for developing vaccines that protect not only against current viruses but also against future emerging coronaviruses with pandemic potential.

• How do T cell responses differ between homotypic and mosaic nanoparticle immunization strategies?

  The differential T cell responses between immunization strategies center on epitope diversity and conservation:

  Mosaic nanoparticle approaches offer potential advantages for T cell responses by presenting conserved T cell epitope peptides across multiple RBDs, which may facilitate increased protection from disease caused by mismatched sarbecoviruses . This is particularly important because:

  1. While antibody responses tend to target variable regions, T cell responses can recognize more conserved internal viral peptides

  2. The presentation of multiple RBD variants increases the probability of including peptides that bind to diverse MHC alleles in outbred populations

  3. Prime-boost immunizations with 6RBD-np in mice have been shown to increase interferon (IFN-γ) production, indicating robust T cell activation

  The breadth of T cell responses may be particularly crucial for protection against severe disease when neutralizing antibodies are evaded by emerging variants, making this an important consideration in mosaic vaccine design.

• What animal models are most informative for evaluating cross-protective efficacy of mosaic nanoparticle vaccines?

  Several animal models have proven valuable for evaluating mosaic nanoparticle vaccines, each with specific advantages:

  1. K18-hACE2 transgenic mice: These mice express human ACE2 and are susceptible to SARS-CoV-2 and SARS-CoV infections, making them ideal for cross-protection studies. Research has demonstrated that mosaic-8b, but not homotypic SARS-2 RBD-mi3 nanoparticles, protected K18-hACE2 mice against lethality with a mismatched SARS-1 challenge .

  2. hACE2-transgenic mice: Similar to K18-hACE2 mice but with different expression patterns, these animals have been used to show that immunization with 6RBD-np provided full protection against SARS-CoV-2 wild type and Delta challenges .

  3. Syrian hamsters: These animals develop respiratory disease upon SARS-CoV-2 infection and can be used to assess both virological and clinical endpoints.

  4. Nonhuman primates: Though more resource-intensive, these models provide the closest approximation to human immune responses and disease course.

  When selecting animal models, researchers should consider:

  • The receptor usage of target viruses

  • The ability to assess both virological and clinical endpoints

  • The availability of immunological reagents for the species

  • The relevance of the model to human disease pathogenesis

• What methodological approaches can address the challenge of antigenic imprinting when developing mosaic coronavirus vaccines?

  Antigenic imprinting (also known as original antigenic sin) presents a significant challenge for coronavirus vaccines, as prior exposure to one variant may bias responses toward familiar epitopes when exposed to new variants. Several methodological approaches can address this challenge:

  1. Epitope masking strategies: Introducing N-linked glycosylation at RBD position 484 can occlude class 1 and 2 RBD epitopes, forcing the immune system to recognize new epitopes .

  2. Heterologous prime-boost regimens: Using different mosaic compositions for prime and boost immunizations may help overcome imprinting by presenting new epitopes in each immunization.

  3. Adjuvant selection: Certain adjuvants can promote more diverse B cell responses and may help overcome imprinting effects.

  4. Nanoparticle presentation: The physical arrangement of antigens on nanoparticles subverts immunodominance compared to soluble antigens, as demonstrated by the superior cross-reactive responses to RBD nanoparticles compared to soluble antigens .

  These approaches allow researchers to strategically engineer immune responses that overcome pre-existing biases, which is particularly important for developing vaccines against rapidly evolving pathogens like SARS-CoV-2.