Nanograms of SARS-CoV-2 Spike Protein Delivered by Exosomes Induce Potent Neutralization of Both Delta and Omicron Variants
Mafalda Cacciottolo, Yujia Li, Justin B. Nice, Michael J. LeClaire, Ryan Twaddle, Ciana L. Mora, Stephanie Y. Adachi, Meredith Young, Jenna Angeles, Kristi Elliott, Minghao Sun
Abstract
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.
Introduction
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created an urgent need for vaccine development strategies to produce safe, effective, readily available and accessible vaccines that can be efficiently produced to combat the emergence of evolving SARS-CoV-2 variants [1] and any future outbreaks. Many vaccines against SARS-CoV-2 have been developed; all candidates try to achieve immunity to the virus, mainly directing the immune response to surface spike protein which binds to the host cell receptor angiotensin-converting enzyme 2 (ACE2), mediating viral cell entry and infection [2]. The spike (S) protein is a class I fusion glycoprotein, the major surface protein on the SARS-CoV-2 virus, which mediates binding to the ACE receptor on the cell surface, promoting its propagation and infection. It is the primary target for neutralizing antibodies. Spike protein is also the primary site of mutations identified in SARS-CoV-2 virus, potentially reducing the efficacy of the immune-response induced by certain vaccines. Since the beginning of the COVID-19 pandemic, five variants of concern (VOC) and eight variants of interest (VOI) have been reported by the World Health Organization, posing challenges to current vaccines, and creating the need for more effective vaccines.
Materials and methods
Cell lines
Human embryonic kidney 293 T cells (293T) were purchased from ATCC (CRL-3216). 293T cells were maintained in culture using Dulbecco’s Modified Eagle Medium (DMEM), high glucose, Glutamax™ containing 10% fetal bovine serum. 293T cells were incubated at 37°C /5% CO2. FreeStyle™ 293F cells (Gibco, 51–0029) were purchased from ThermoFisher. 293F cells were used as a parental cell line to generate spike SARS-CoV-2 delta spike expressing stable cell lines: Stealth X-Spike cells (STX-S). 293F and STX-S cells were maintained in a Multitron incubator (Infors HT) at 37°C, 80% humidified atmosphere with 8% CO2 on an orbital shaker platform rotating at 110 rpm.
Results
SARS-CoV-2 spike expression on the surface of STX-S cells and exosomes
To facilitate expression of naïve SARS-CoV-2 delta spike (STX-S) on the exosomes, STX-S cells were generated to express SARS-CoV-2 spike on their surface by lentiviral transduction of 293F cells. Expression of SARS-CoV-2 spike on the cell surface was evaluated by flow cytometry. As shown in Fig 2B, >97% of STX-S cells expressed spike on their surface. Non-engineered, parental 293F cells do not express spike, as expected (Fig 2A).
Discussions
Exosomes have gained great interest from the scientific community throughout the years for their high bioavailability, exceptional biocompatibility, and low immunogenicity, identifying them as promising drug delivery candidates [18]. Exosomes can cross biological barriers via their lipid membrane and endogenously expressed cargos and thus are able to effectively deliver engineered cargos as well [10]. As an endogenous product, highly produced by all cell types, exosomes are less prone to induce an adverse response thereby increasing safety. Together with their high stability at physiological pH and temperature, these unique exosome advantages make them an ideal delivery system for vaccinology.
Conclusions
In summary, the STX-S vaccine pre-clinical data presented here show that a potent B cell response was induced in immunized mice with less than 1 ng of spike protein displayed on exosomes, which was enough to elicit full neutralization of SARS-CoV-2 delta variant. Amazingly, neutralization of the Omicron variant was also observed using only 3 ng of delta spike protein, which has not been found using human serum immunized with two doses of the current mRNA vaccine, suggesting that our STX-S vaccine has a superior advantage as a broader vaccine candidate compared with current mRNA vaccines. The extremely low dose of spike protein (less than 1 ng dose compared with current recombinant protein vaccine dose at 5 ug or higher) without the use of an adjuvant or lipid nanoparticle, further enhances the safety profile of the STX-S vaccine for clinical development. Engineered exosome technology presented in this study has the ability to revolutionize the next generation of vaccines by providing a rapidly engineered, readily accessible vaccine which produces a potent, broader neutralization of the target virus, a robust T cell response, enhanced safety profile, and unlimited multi-valent vaccine cocktailing power by naturally delivering an extremely low dose of antigen via exosomes.
Citation: Cacciottolo M, Li Y, Nice JB, LeClaire MJ, Twaddle R, Mora CL, et al. (2023) Nanograms of SARS-CoV-2 spike protein delivered by exosomes induce potent neutralization of both delta and omicron variants. PLoS ONE 18(8): e0290046. https://doi.org/10.1371/journal.pone.0290046
Editor: Paulo Lee Ho, Instituto Butantan, BRAZIL
Received: April 1, 2023; Accepted: July 31, 2023; Published: August 22, 2023
Copyright: © 2023 Cacciottolo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: Capricor Therapeutics, Inc. is a NASDAQ listed company (Nasdaq: CAPR) and receives its funding primarily through the issuances of stock. The capital raised provides support in the form of salaries for all authors and pays for the acquisition of study materials and supplies, but the specific investors did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.
Competing interests: The authors have read the journal’s policy and have the following competing interests: All authors are employees of Capricor Therapeutics, Inc. This does not alter the adherence to PLOS ONE policies on sharing data and materials.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0290046#abstract0
