Nanograms of SARS-CoV-2 spike protein delivered by exosomes induce potent neutralization of both delta and omicron variants.

Exosomes are emerging as potent and safe delivery carriers for use in vaccinology and therapeutics. A better vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to provide improved, broader, longer lasting neutralization of SARS-CoV-2, a more robust T cell response, enable widespread global usage, and further enhance the safety profile of vaccines given the likelihood of repeated booster vaccinations. Here, we use Capricor's StealthXTM platform to engineer exosomes to express native SARS-CoV-2 spike Delta variant (STX-S) protein on the surface for the delivery of a protein-based vaccine for immunization against SARS-CoV-2 infection. The STX-S vaccine induced a strong immunization with the production of a potent humoral immune response as demonstrated by high levels of neutralizing antibody not only against the delta SARS-CoV-2 virus but also two Omicron variants (BA.1 and BA.5), providing broader protection than current mRNA vaccines. Additionally, both CD4+ and CD8+ T cell responses were increased significantly after treatment. Quantification of spike protein by ELISA showed that only nanograms of protein were needed to induce a potent immune response. This is a significantly lower dose than traditional recombinant protein vaccines with no adjuvant required, which makes the StealthXTM exosome platform ideal for the development of multivalent vaccines with a better safety profile. Importantly, our exosome platform allows novel proteins, or variants in the case of SARS-CoV-2, to be engineered onto the surface of exosomes in a matter of weeks, comparable with mRNA vaccine technology, but without the cold storage requirements necessary for mRNA vaccines. The ability to utilize exosomes for cellular delivery of proteins, as demonstrated by STX-S, has enormous potential to revolutionize vaccinology by rapidly facilitating antigen presentation at an extremely low dose resulting in a potent, broad antibody response.

Exosome-Based Multivalent Vaccine: Achieving Potent Immunization, Broadened Reactivity, and Strong T-Cell Responses with Nanograms of Proteins.

Currently approved vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have focused solely on the spike protein to provide immunity. The first vaccines were developed rapidly using spike mRNA delivered by lipid nanoparticles but required ultralow-temperature storage and have had limited immunity against variations in spike. Subsequently, protein-based vaccines were developed, which offer broader immunity but require significant time for development and the use of an adjuvant to boost the immune response. Here, exosomes were used to deliver a bivalent protein-based vaccine in which two independent viral proteins were used. Exosomes were engineered to express either SARS-CoV-2 delta spike (Stealth X-Spike [STX-S]) or the more conserved nucleocapsid (Stealth X-Nucleocapsid [STX-N]) protein on the surface. When administered as a single product (STX-S or STX-N) or in combination (STX-S+N), both STX-S and STX-N induced strong immunization with the production of potent humoral and cellular immune responses. Interestingly, these results were obtained with the administration of only nanograms of protein and without an adjuvant. In two independent animal models (mouse and rabbit), the administration of nanograms of the STX-S+N vaccine resulted in increased antibody production, potent neutralizing antibodies with cross-reactivity to other variants of spike, and strong T-cell responses. Importantly, no competition of immune responses was observed, allowing the delivery of nucleocapsid with spike to offer improved SARS-CoV-2 immunity. These data show that the StealthX exosome platform has the enormous potential to revolutionize vaccinology by combining the advantages of mRNA and recombinant protein vaccines into a superior, rapidly generated, low-dose vaccine resulting in potent, broader immunity. IMPORTANCE The pandemic emergency has brought to light the need for a new generation of rapidly developed vaccines that induce longer-lasting, potent, and broader immune responses. While the mRNA vaccines played a critical role during the emergency in reducing SARS-CoV-2 hospitalization rates and deaths, more efficient approaches are needed. A multivalent, protein-based vaccine delivered by exosomes could meet this urgent need due to the high speed of development, manufacturability, and the ability to produce a strong antibody response, with neutralizing antibodies and a strong T-cell response able to broadly combat viral infection with a minimum number of injections.

Single Cell Mechanotype and Associated Molecular Changes in Urothelial Cell Transformation and Progression.

Cancer cell mechanotype changes are newly recognized cancer phenotypic events, whereas metastatic cancer cells show decreased cell stiffness and increased deformability relative to normal cells. To further examine how cell mechanotype changes in early stages of cancer transformation and progression, an in vitro multi-step human urothelial cell carcinogenic model was used to measure cellular Young's modulus, deformability, and transit time using single-cell atomic force microscopy, microfluidic-based deformability cytometry, and quantitative deformability cytometry, respectively. Measurable cell mechanotype changes of stiffness, deformability, and cell transit time occur early in the transformation process. As cells progress from normal, to preinvasive, to invasive cells, Young's modulus of stiffness decreases and deformability increases gradually. These changes were confirmed in three-dimensional cultured microtumor masses and urine exfoliated cells directly from patients. Using gene screening and proteomics approaches, we found that the main molecular pathway implicated in cell mechanotype changes appears to be epithelial to mesenchymal transition.

Impact of isolation methods on the biophysical heterogeneity of single extracellular vesicles.

Extracellular vesicles (EVs) have raised high expectations as a novel class of diagnostics and therapeutics. However, variabilities in EV isolation methods and the unresolved structural complexity of these biological-nanoparticles (sub-100 nm) necessitate rigorous biophysical characterization of single EVs. Here, using atomic force microscopy (AFM) in conjunction with direct stochastic optical reconstruction microscopy (dSTORM), micro-fluidic resistive pore sizing (MRPS), and multi-angle light scattering (MALS) techniques, we compared the size, structure and unique surface properties of breast cancer cell-derived small EVs (sEV) obtained using four different isolation methods. AFM and dSTORM particle size distributions showed coherent unimodal and bimodal particle size populations isolated via centrifugation and immune-affinity methods respectively. More importantly, AFM imaging revealed striking differences in sEV nanoscale morphology, surface nano-roughness, and relative abundance of non-vesicles among different isolation methods. Precipitation-based isolation method exhibited the highest particle counts, yet nanoscale imaging revealed the additional presence of aggregates and polymeric residues. Together, our findings demonstrate the significance of orthogonal label-free surface characteristics of single sEVs, not discernable via conventional particle sizing and counts alone. Quantifying key nanoscale structural characteristics of sEVs, collectively termed 'EV-nano-metrics' enhances the understanding of the complexity and heterogeneity of sEV isolates, with broad implications for EV-analyte based research and clinical use.