SARS Mosaic S(N)

What are mosaic RBD nanoparticles and how do they differ from homotypic nanoparticles?

Mosaic RBD nanoparticles are vaccine candidates that display multiple different receptor binding domains from various sarbecoviruses on a single nanoparticle structure. For example, the mosaic-8b nanoparticle displays the SARS-CoV-2 Beta variant RBD plus seven other sarbecovirus RBDs attached to a 60-mer nanoparticle scaffold . In contrast, homotypic nanoparticles display multiple copies of a single type of RBD, such as 60 copies of only the SARS-CoV-2 RBD .

The key difference lies in antigen arrangement and diversity: mosaic nanoparticles present a variety of related but antigenically different viral antigens, while homotypic nanoparticles present multiple copies of the same antigen. The mosaic arrangement is specifically designed to increase the chances of stimulating production of antibodies against conserved regions, as adjacent antigens on the particle are unlikely to be identical .

What is the theoretical basis for using mosaic nanoparticles over traditional vaccine approaches?

The theoretical basis for the mosaic nanoparticle approach addresses the challenge of immunodominance in coronavirus vaccination. When exposed to a single coronavirus antigen (as in natural infection or traditional vaccines), the immune system tends to focus on immunodominant epitopes that may be variable across different coronaviruses, limiting cross-protection.

Mosaic nanoparticles counter this limitation by:

• Displaying multiple different RBDs simultaneously, which decreases the probability of adjacent RBDs being identical

• Favoring B cell interactions that can cross-link between adjacent different RBDs

• Using avidity effects to preferentially recognize conserved, but potentially sterically occluded, epitopes (such as class 3, class 4, and class 1/4 RBD epitopes)

• Presenting conserved T cell epitope peptides across different RBDs, potentially facilitating increased protection against mismatched sarbecoviruses

This approach effectively redirects the immune response toward conserved regions rather than variable immunodominant epitopes, providing broader protection against both known and emerging coronaviruses .

How are mosaic RBD nanoparticles manufactured?

The manufacturing process for mosaic RBD nanoparticles utilizes SpyTag/SpyCatcher technology to covalently attach RBDs to a protein nanoparticle scaffold. The specific methodology includes:

• Engineering RBDs from different sarbecoviruses with C-terminal SpyTag003 sequences

• Mixing these RBD-SpyTag003 proteins with a 60-mer nanoparticle scaffold (SpyCatcher003-mi3)

• Allowing SpyTag003 and SpyCatcher003 domains to form spontaneous covalent bonds, attaching the RBDs to the nanoparticle

• For mosaic nanoparticles, using a mixture of different RBD-SpyTag003 proteins, resulting in random attachment of different RBDs to the nanoparticle

• Purifying the conjugated nanoparticles using size-exclusion chromatography

• Performing quality control via SDS-PAGE to confirm conjugation efficiency, dynamic light scattering, and negative-stain electron microscopy to verify particle size and morphology

The resulting particles are monodisperse with a defined diameter and exhibit near 100% conjugation efficiency of RBDs to the nanoparticle scaffold .

What animal models have been used to evaluate mosaic RBD nanoparticles?

Researchers have utilized both mouse and non-human primate (NHP) models to evaluate the efficacy of mosaic RBD nanoparticles:

• Mouse models:

  • K18-hACE2 transgenic mice, which express human ACE2 and are susceptible to SARS-CoV-2 infection

  • BALB/c mice for immunogenicity studies and antibody mapping

  • These models were used for both immunization studies and viral challenge experiments with SARS-CoV-2 Beta and SARS-CoV-1

• Non-human primate models:

  • Rhesus macaques were used for immunization and subsequent challenge with either SARS-CoV-2 Delta variant or SARS-CoV-1

  • These animals were evaluated for antibody responses and viral titers following challenge

Both models demonstrated that mosaic-8b RBD nanoparticles provided protection against both matched and mismatched viral challenges, whereas homotypic SARS-CoV-2 RBD nanoparticles only fully protected against matched challenges .

How do immune responses to mosaic RBD nanoparticles compare to those elicited by homotypic nanoparticles?

The immune responses to mosaic RBD nanoparticles differ from those elicited by homotypic nanoparticles in several key ways:

• Antibody breadth:

  • Mosaic-8b immunization elicits antibodies with superior cross-reactive recognition of heterologous RBDs compared to homotypic SARS-CoV-2 RBD nanoparticles

  • Antisera from mosaic-8b-immunized animals neutralize both matched and mismatched pseudotyped coronaviruses, while homotypic immunization primarily neutralizes matched viruses

• Epitope targeting:

  • Antibody-escape mapping shows that mosaic-8b immunization elicits antibodies primarily targeting conserved RBD epitopes

  • Homotypic SARS-CoV-2 RBD immunization elicits antibodies primarily targeting variable immunodominant class 1 and class 2 RBD epitopes

  • This difference in epitope targeting explains the broader cross-protection provided by mosaic nanoparticles

• Protection from challenge:

  • Mosaic-8b and homotypic SARS-2 Beta RBD-mi3 nanoparticles are equally protective against SARS-CoV-2 challenge

  • Only mosaic-8b provides protection against SARS-CoV-1 challenge, with homotypic SARS-CoV-2 immunized animals experiencing similar weight loss to control animals

What immunization protocols have demonstrated efficacy for mosaic RBD nanoparticles?

Research has demonstrated efficacy with the following immunization protocols:

• Mouse protocol:

  • Prime: Mosaic-8b RBD-mi3 or homotypic SARS-CoV-2 Beta RBD-mi3 (5 μg) with AddaVax adjuvant administered intraperitoneally

  • Boost: Same formulation at week 3

  • Challenge: SARS-CoV-2 Beta or SARS-CoV-1 at week 7

• Non-human primate protocol:

  • Prime: Mosaic-8b RBD-mi3 (30 μg) with aluminum hydroxide (alum) adjuvant

  • First boost: Same formulation at day 28

  • Second boost: Mosaic-8b RBD-mi3 with EmulsiPan (an MF59-like squalene-based oil-in-water emulsion adjuvant) at day 92

  • Challenge: SARS-CoV-2 Delta or SARS-CoV-1 four weeks after the second boost

These protocols demonstrated that mosaic-8b immunization protected against both matched SARS-CoV-2 variants and mismatched SARS-CoV-1, while homotypic immunization only provided full protection against the matched SARS-CoV-2 challenge .

What are the critical factors in selecting RBDs for inclusion in a mosaic nanoparticle?

Critical factors in selecting RBDs for inclusion in a mosaic nanoparticle include:

• Phylogenetic diversity:

  • Including RBDs from different clades of sarbecoviruses to maximize diversity

  • The mosaic-8b included RBDs from clade 1a, 1b, and 2 sarbecoviruses, as well as SARS-CoV-2

• Receptor usage:

  • Including RBDs that use human ACE2 as well as those that use different receptors

  • This helps elicit antibodies against conserved epitopes that might be common across diverse receptor-binding mechanisms

• Antigenic distinctiveness:

  • Selecting RBDs that represent different antigenic surfaces to maximize exposure to diverse epitopes

  • For SARS-CoV-2, using variant RBDs (such as Beta) that represent the most antigenically distinct variants

• Potential zoonotic threat:

  • Prioritizing RBDs from viruses with zoonotic potential that could potentially cause future outbreaks in humans

  • SARS-like viruses circulating in bats represent a reservoir of potential future pandemic viruses

The specific RBDs included in mosaic-8b were from SARS-CoV-2 Beta variant and seven animal sarbecoviruses from different clades, providing broad coverage of the sarbecovirus phylogenetic tree .

How does epitope accessibility in mosaic nanoparticles influence immune responses?

Epitope accessibility in mosaic nanoparticles plays a crucial role in shaping immune responses:

What methodological challenges exist in evaluating cross-reactive neutralization against diverse sarbecoviruses?

Evaluating cross-reactive neutralization against diverse sarbecoviruses presents several methodological challenges:

• Receptor usage:

  • Many sarbecoviruses do not use human ACE2 for entry, making traditional pseudovirus neutralization assays challenging

  • Researchers must evaluate neutralization against viruses that use human ACE2 (like SARS-CoV-1) or create chimeric spikes (RBDs from other viruses with remaining spike protein from ACE2-using viruses)

  • For some viruses, engineered mutations are required to enable human ACE2 usage, such as a mutant form of BtKY72 (K493Y/T498W)

• Biosafety concerns:

  • Working with diverse live sarbecoviruses requires high biosafety level facilities

  • Researchers often use pseudotyped viruses as safer alternatives, but these may not fully recapitulate all aspects of authentic virus neutralization

• Standardization across diverse viruses:

  • Different viruses may have different inherent sensitivities to neutralization

  • Establishing appropriate controls and normalization methods is challenging when comparing neutralization across diverse viruses

• Correlating in vitro neutralization with in vivo protection:

  • In vitro neutralization titers may not always predict in vivo protection, especially across diverse viruses with different pathogenesis mechanisms

  • Challenge studies with diverse viruses are needed to confirm protection, which has practical and ethical limitations

How can antibody escape mapping be used to evaluate mosaic nanoparticle effectiveness?

Antibody escape mapping provides a powerful approach to evaluate mosaic nanoparticle effectiveness by revealing which epitopes are targeted by the immune response:

• Methodology:

  • Deep mutational scanning creates libraries of RBD variants with single amino acid mutations

  • These libraries are screened to identify mutations that escape binding by antisera from immunized animals

  • The escape profiles reveal which epitopes are targeted by antibodies elicited by different immunization strategies

• Interpretation for mosaic versus homotypic nanoparticles:

  • Antisera from mice immunized with homotypic SARS-CoV-2 RBD nanoparticles show escape mutations primarily in variable, immunodominant class 1 and class 2 epitopes

  • In contrast, antisera from mosaic-8b-immunized mice show escape mutations distributed across more conserved regions of the RBD

  • This pattern confirms that mosaic nanoparticles successfully redirect the immune response toward conserved epitopes

• Correlation with protection:

  • The antibody escape mapping results correlate with the observed protection patterns

  • Targeting conserved epitopes through mosaic immunization leads to broader protection against both matched and mismatched challenges

  • The focus on variable epitopes after homotypic immunization corresponds with protection limited to matched challenges

What metrics and assays best assess the potential breadth of protection offered by mosaic RBD nanoparticles?

Multiple complementary metrics and assays are needed to comprehensively assess the breadth of protection offered by mosaic RBD nanoparticles:

• Binding assays:

  • ELISA against diverse RBDs, including matched (those represented on the nanoparticle) and mismatched (not represented) RBDs

  • Binding breadth against SARS-CoV-2 variant RBDs has been shown to be predictive of neutralization breadth across variants

• Neutralization assays:

  • Pseudovirus neutralization assays using diverse sarbecovirus spikes

  • Authentic virus neutralization for select representative viruses

  • Both matched and mismatched viruses should be included to assess breadth

• Challenge studies:

  • Viral load measurements in tissues and swabs following challenge

  • Weight loss and survival outcomes

  • Challenges with both matched and mismatched viruses are critical to demonstrate breadth

• Antibody epitope mapping:

  • Deep mutational scanning to identify escape mutations

  • Competition assays with monoclonal antibodies of known epitope specificity

  • These approaches reveal which epitopes are targeted and how conserved they are across sarbecoviruses

The combination of these assays has demonstrated superior breadth for mosaic-8b compared to homotypic immunization, with protection from both SARS-CoV-2 and SARS-CoV-1 challenges, and antibody mapping showing targeting of conserved epitopes .

How can researchers distinguish between sterilizing immunity and disease-modifying protection in mosaic nanoparticle studies?

Distinguishing between sterilizing immunity and disease-modifying protection requires careful experimental design and comprehensive analysis:

• Viral load measurements:

  • Sterilizing immunity is characterized by complete or near-complete prevention of viral replication

  • In NHP studies, mosaic-8b-immunized animals challenged with SARS-1 had no detectable viral titers in bronchoalveolar lavage (BAL) or nasal swabs, suggesting potential sterilizing immunity

  • Both genomic and subgenomic RNA should be measured, as the latter indicates active viral replication

  • Infectious virus isolation provides stronger evidence of sterilizing immunity than RNA detection alone

• Timing of viral load assessment:

  • Early time points (e.g., 2-4 days post-challenge) are critical for detecting transient viral replication

  • Multiple time points help distinguish between delayed viral clearance and true sterilizing immunity

• Sampling multiple sites:

  • Comprehensive sampling of both upper respiratory tract (nasal swabs) and lower respiratory tract (BAL, lung tissue)

  • Some vaccines may provide sterilizing immunity in the lungs but allow transient replication in the upper airway

• Clinical parameters:

  • Weight loss, survival, and other clinical parameters indicate disease severity

  • Disease-modifying protection may still allow some viral replication but prevent severe outcomes

  • In mouse studies, both mosaic-8b and homotypic immunizations prevented weight loss after SARS-CoV-2 challenge, but only mosaic-8b prevented weight loss after SARS-CoV-1 challenge

How might mosaic nanoparticle technology be adapted to address emerging coronavirus variants?

Mosaic nanoparticle technology offers several adaptation strategies to address emerging coronavirus variants:

• Updating RBD composition:

  • The modular nature of the SpyTag/SpyCatcher system allows for rapid updating of the RBDs displayed on the nanoparticle

  • New variants of concern could be incorporated into updated mosaic nanoparticles alongside conserved sarbecovirus RBDs

  • This would maintain breadth while enhancing protection against specific emerging threats

• Increasing RBD diversity:

  • Current research used mosaic-8 (eight different RBDs), but the approach could be expanded to include more diverse RBDs

  • Including RBDs from a wider range of clades or from newly discovered sarbecoviruses could further enhance breadth

• Epitope-focused design:

  • Further research could optimize the exposure of specific conserved epitopes

  • Strategic glycan placement might shield immunodominant variable epitopes while maintaining exposure of key conserved epitopes

  • Research suggests that complete occlusion of class 1 and 2 epitopes may impede protection, so a balanced approach is needed

• Adjuvant optimization:

  • Different adjuvants might further enhance cross-reactive immune responses

  • NHP studies showed that EmulsiPan (an MF59-like adjuvant) effectively boosted responses that had contracted after the initial immunizations

  • Tailoring adjuvant selection to maximize responses against conserved epitopes could enhance breadth

What are the potential limitations of using RBD-only constructs for coronavirus vaccines?

Despite their promising results, RBD-only constructs have several potential limitations as coronavirus vaccines:

• Missing epitopes:

  • RBD-only constructs lack epitopes present in other regions of the spike protein

  • Neutralizing epitopes in the N-terminal domain (NTD) and S2 region are not included

  • Some broadly neutralizing antibodies target these non-RBD regions, which could enhance protection

• Conformational differences:

  • The RBD in isolation may adopt conformations different from those in the context of the full spike

  • Some epitopes might be presented differently, potentially affecting the quality of antibodies elicited

  • The RBD in the spike alternates between "up" and "down" conformations, which is not recapitulated in isolated RBD constructs

• T cell epitope limitations:

  • RBD-only constructs contain a subset of potential T cell epitopes

  • The full spike protein contains additional CD4+ and CD8+ T cell epitopes that might contribute to protection

  • T cell responses against conserved non-RBD regions could enhance cross-protection

• Durability of protection:

  • The long-term durability of protection from RBD-only constructs versus full spike vaccines remains to be fully established

  • Differences in epitope presentation and T cell responses might affect the generation and maintenance of immune memory

How might the mosaic nanoparticle approach be applied to other viral families with high diversity?

The mosaic nanoparticle approach demonstrates potential for application to other viral families with high diversity:

• Influenza viruses:

  • Mosaic nanoparticles displaying hemagglutinin (HA) domains from diverse influenza strains could address the challenge of antigenic drift and shift

  • This approach might enhance breadth compared to current seasonal influenza vaccines

  • Similar to coronaviruses, influenza has immunodominant variable epitopes and subdominant conserved epitopes that could be targeted through a mosaic approach

• HIV:

  • HIV's extreme diversity has challenged vaccine development for decades

  • Mosaic nanoparticles displaying diverse envelope protein domains might address this challenge

  • HIV vaccine research has already explored mosaic antigen approaches, which could be enhanced by nanoparticle display

• Methodological considerations for adaptation:

  • Identifying conserved neutralizing epitopes within each viral family

  • Determining the appropriate protein domains for display (equivalent to the RBD in coronaviruses)

  • Optimizing the spacing and arrangement of antigens on the nanoparticle

  • Selecting appropriate adjuvants to enhance responses to conserved epitopes

The mosaic nanoparticle platform, using technologies like SpyTag/SpyCatcher for modular assembly, offers a flexible approach that could be adapted to various viral families where diversity and immunodominance of variable epitopes present challenges to traditional vaccine approaches .