key: cord-323685-gjocoa60
authors: Tsai, Shang-Jui; Guo, Chenxu; Atai, Nadia A.; Gould, Stephen J.
title: Exosome-Mediated mRNA Delivery For SARS-CoV-2 Vaccination
date: 2020-11-06
journal: bioRxiv
DOI: 10.1101/2020.11.06.371419
sha: 
doc_id: 323685
cord_uid: gjocoa60

Background In less than a year from its zoonotic entry into the human population, SARS-CoV-2 has infected more than 45 million people, caused 1.2 million deaths, and induced widespread societal disruption. Leading SARS-CoV-2 vaccine candidates immunize with the viral spike protein delivered on viral vectors, encoded by injected mRNAs, or as purified protein. Here we describe a different approach to SARS-CoV-2 vaccine development that uses exosomes to deliver mRNAs that encode antigens from multiple SARS-CoV-2 structural proteins. Approach Exosomes were purified and loaded with mRNAs designed to express (i) an artificial fusion protein, LSNME, that contains portions of the viral spike, nucleocapsid, membrane, and envelope proteins, and (ii) a functional form of spike. The resulting combinatorial vaccine, LSNME/SW1, was injected into thirteen weeks-old, male C57BL/6J mice, followed by interrogation of humoral and cellular immune responses to the SARS-CoV-2 nucleocapsid and spike proteins, as well as hematological and histological analysis to interrogate animals for possible adverse effects. Results Immunized mice developed CD4+, and CD8+ T-cell reactivities that respond to both the SARS-CoV-2 nucelocapsid protein and the SARS-CoV-2 spike protein. These responses were apparent nearly two months after the conclusion of vaccination, as expected for a durable response to vaccination. In addition, the spike-reactive CD4+ T-cells response was associated with elevated expression of interferon gamma, indicative of a Th1 response, and a lesser induction of interleukin 4, a Th2-associated cytokine. Vaccinated mice showed no sign of altered growth, injection-site hypersensitivity, change in white blood cell profiles, or alterations in organ morphology. Consistent with these results, we also detected moderate but sustained anti-nucleocapsid and anti-spike antibodies in the plasma of vaccinated animals. Conclusion Taken together, these results validate the use of exosomes for delivering functional mRNAs into target cells in vitro and in vivo, and more specifically, establish that the LSNME/SW1 vaccine induced broad immunity to multiple SARS-CoV-2 proteins.

COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Coronaviridae Study Group of the International Committee on Taxonomy of, 2020 ; Zhou et al., 2020b) . COVID-19 typically presents with symptoms common to many respiratory infections such as fever and cough (Gandhi et al., 2020) but can also progress to acute respiratory distress, disseminated disease, and death (Force et al., 2012; Guo et al., 2020; Richardson et al., 2020; Zhou et al., 2020a) . Humans have long been host to several mildly pathogenic beta-coronaviruses (OC43, HKU1, etc. (Corman et al., 2018) ) but SARS-CoV-2 entered the human population in late 2019 as the result of a zoonotic leap. SARS-CoV-2 is closely related to a pair of prior bat-to-human zoonoses that were responsible for the outbreaks of severe acute respiratory syndrome (SARS-CoV) in 2002 (Graham and Baric, 2010) and middle east respiratory syndrome (MERS-CoV) in 2012 (Memish et al., 2013) . While SARS-CoV-2 infection is associated with lower mortality than SARS-CoV or MERS-CoV, SARS-CoV-2 displays a higher rate of transmission and has become a major cause of morbidity and mortality worldwide (https://www.cdc.gov/coronavirus/2019-ncov/hcp/clinical-guidance-managementpatients.html)(coronavirus.jhu.edu) (Korber et al., 2020) .

Infection of a cell by SARS-CoV-2 results in the translation of its viral genomic RNA (gRNA) into large polyproteins, open reading frame 1 (orf1a) and orf1ab, which are processed to release 16 nonstructural proteins (nsp1-16) (V'Kovski et al., 2020) . These early proteins prime the host cell for virus replication and mediate the synthesis of subgenomic viral RNAs. These encode 12 additional proteins, including the SARS-CoV-2 structural proteins Nucleocapsid (N), Spike (S), Membrane (M), and Envelope (E). The coronavirus integral membrane proteins S, M, and E are co-translationally translocated into the endoplasmic reticulum (ER) and trafficked by the secretory pathway to Golgi and Golgi-related compartments (Ruch and Machamer, 2012; Ujike and Taguchi, 2015) , and perhaps other compartments of the cell as well (Ghosh et al., 2020) . During their intracellular trafficking, the S, M, and E proteins work together to recruit N protein-gRNA complexes into nascent virions and to drive the budding of infectious vesicles from the host cell membrane. The resulting SARS-CoV-2 virions are small, membrane-bound vesicles of ~100 nm diameter, with large, spike-like trimers of S that protrude from the vesicle surface .

The S protein interacts with a variety of cell surface proteins including its canonical receptor, angiotensin-converting enzyme II (ACE2) (Hoffmann et al., 2020; Matheson and Lehner, 2020; Zhou et al., 2020b) , and neuropilin-1 (Cantuti-Castelvetri et al., 2020; Daly et al., 2020) . SARS-CoV-2 receptors and other infection mediators (e.g. TMPRSS2 (Hoffmann et al., 2020) ) are expressed within the respiratory tract, consistent with its respiratory mode of transmission (Mason, 2020) . However, the surface proteins that facilitate SARS-CoV-2 binding and entry are also expressed in many other cell types, allowing SARS-CoV-2 to spread within the body and impact multiple organ systems, including the brain, heart, gastrointestinal tract, circulatory system, and immune system (Cantuti-Castelvetri et al., 2020; Daly et al., 2020; Li et al., 2020; Nicin et al., 2020; Singh et al., 2020; Ziegler et al., 2020) .

Studies show that COVID-19 patients generate potent cellular and humoral immune responses to the virus (Poland et al., 2020; St John and Rathore, 2020; Zost et al., 2020) .

Moreover, animal studies provide clear evidence that SARS-CoV-2 infection elicits immune responses that reverse the course of disease, clear the virus, and confer resistance to reinfection (Bosco-Lauth et al., 2020; Chandrashekar et al., 2020; Deng et al., 2020; Shan et al., 2020) . Taken together, these observations augur well for control of SARS-CoV-2 transmission and disease through vaccination. Although to date there are no approved vaccines for any human coronavirus, disease-preventing vaccines have been progressively developed for multiple animal coronaviruses (Tizard, 2020) . Most of these coronavirus vaccines are based on attenuated viruses, which elicit immune responses to all viral proteins, or inactivated virus particle vaccines, which induce immunity to the structural proteins of the virus (i.e. S, N, M, and E). Of the SARS-CoV-2 vaccines selected for rapid development, all are based on immunization with just a single viral protein, the large, spike-like S protein (Slaoui and Hepburn, 2020) (Samrat et al., 2020) .

Although S-based SARS-CoV-2 vaccines all target the same protein, they vary significantly in antigen structure and mode of antigen delivery. Forms of S in vaccine trials range from S protein fragments (Walsh et al., 2020) to full-length forms of S (Bos et al., 2020; Graham et al., 2020; Hassan et al., 2020; Jackson et al., 2020; Keech et al., 2020; Walsh et al., 2020; Zhu et al., 2020) though none deliver the kinds of full-length, functional form of S encoded by SARS-CoV-2. As for the modes of S antigen delivery, most enlist host cells to express the S antigen component of their vaccine, from either injected 8 mRNAs (Jackson et al., 2020; Walsh et al., 2020) or infectious viral vectors (Bos et al., 2020; Graham et al., 2020; Hassan et al., 2020; Zhu et al., 2020) while some involve direct injection of purified, recombinant S protein (Keech et al., 2020) . Here we describe an alternative approach to SARS-CoV-2 vaccination that combines the features of exosome-based mRNA delivery with the expression of viral antigens in forms designed for antigen presentation by major histocompatibility (MHC) Class I and Class II pathways (Imai et al., 2019) . Exosomes are small extracellular vesicles (sEVs) of ~30-150 nm in diameter that are made by all cells, abundant in all biofluids, and mediate intercellular transmission of signals and macromolecules, including genetic information such as RNAs (Pegtel and Gould, 2019) . Here we describe the results of a trial immunization study in which exosomes were used to deliver multiple mRNAs designed to express fragments of the S, N, M, and E proteins targeted to MHC Class I and Class II antigen processing compartment, as well as a full-length, functional form of S.

To develop a system for exosome-mediated mRNA delivery we first established a protocol for exosome purification from cultured human cells. Towards this end, 293F cells were grown in suspension in chemically-defined media, free of animal products and antibiotic supplements. Cells and cell debris were removed by centrifugation and filtration to generate a clarified tissue culture supernatant (CTCS), followed by purification of the CTCS by filtration and chromatography. This process yielded a population of small EVs that have the expected ultrastructure and size distribution profile of human exosomes and contain the exosomal marker proteins CD9 and CD63 (Fig. 1) . This process concentrated exosomes ~500-fold, to ~2 x 10 12 exosomes/mL, with an average recovery of 35%.

To determine whether the treatment of cells with exosome-mRNA formulations could induce cells to express these mRNAs, we synthesized an mRNA containing a codonoptimized open reading frame (ORF) for Antares2, a reporter protein comprised of the luciferase teLuc fused to two copies of the fluorescent protein CyOFP1 (CyOFP1-teLuc-CyOFP1) (Yeh et al., 2017) . Antares2 expression can be measured via its luciferase activity (diphenylterazine-induced, bioluminescence resonance energy transfer (BRET)mediated bioluminescence) or via its CyOFP1-mediated fluorescence (excitation range of 475nm-535 nm; emission range of 565nm-610nm). This mRNA was loaded into exosomes and then incubated with cultures of HEK293 cells overnight. The cells were then processed for Antares2 luciferase activity and fluorescence microscopy (Fig. 2) .

Treated cells displayed high levels of Antares2 luciferase activity that was dependent on the specific order of component addition during the exosome formulation process. When interrogated by fluorescence microscopy, the cells displayed the expected expression of Antares2-mediated fluorescence.

To test whether exosome-mRNA formulations can elicit immune responses to proteins of SARS-CoV-2, we first synthesized a pair of mRNAs designed to express SARS-CoV-2 antigens. The first of these mRNAs encoded a membrane protein (LSNME) comprised of the receptor binding domain (RBD) of S, the entire N protein, and soluble portions of the M and E proteins, all expressed within the extracellular domain of the human Lamp1 protein. This protein is predicted to be degraded into peptides for antigen presentation by the MHC Class I system, and if expressed in antigen-presenting cells (APCs), to be degraded into peptides for antigen presentation by MHC Class II molecules (Gupta et al., 2006 ) (Imai et al., 2019) . Expression of such a protein in a non-APC cell type such as HEK293 is expected to result in its accumulation in the ER, and consistent with this hypothesis, staining HEK293 cells transfected with this mRNA expressed with a COVID-19 patient plasma identified an ER-localized protein (Fig. 3A, B) . The second of these in vitro synthesized mRNAs was designed to express the full-length, functional form of S from the original Wuhan-1 isolate of SARS-CoV-2 (S W1 ) (Zhou et al., 2020b) . Transfection of this mRNA into HEK293 cells led to expression of a distinct protein that was also recognized by antibodies present in a COVID-19 patient plasma (Fig. 3C, D) . Taken together, these results demonstrate that these mRNAs encode that are reactive with COVID-19 patient plasmas and have expected subcellular distributions.

A single exosome-mRNA formulation containing both the LSNME and S W1 mRNAs (hereafter referred to as the LSNME/S W1 vaccine) was injected (intramuscular) into 13 weeks-old male C57BL/6J mice (Fig. 4) . The vaccine was dosed at 4 ug or 0.25 ug equivalents of each mRNA and injections were performed on day 1 (primary immunization), day 21 (1st boost), and day 42 (2nd boost). Blood (0.1 mL) was collected on days 14, 35, 56, 70 and 84. On day 84 the animals were sacrificed to obtain tissue samples for histological analysis and splenocytes for blood cell studies. Using ELISA kits adapted for the detection of mouse antibodies, we observed that vaccinated animals displayed a dose-dependent antibody response to both the SARS-CoV-2 N protein and S protein. These antibody reactions were not particularly robust but they were longlasting, persisting to 7 weeks after the final boost with little evidence of decline. It should be noted that the modest antibody production was expected in the case of the N protein, as the LSNME mRNA is designed to stimulate cellular immune responses rather than the production of anti-N antibodies.

Vaccinated and control animals were also interrogated for the presence of antigenreactive CD4+ and CD8+ T-cells. This was carried out by collecting splenocytes at the completion of the trial (day 84) using a CFSE proliferation assay in the presence or absence of recombinant N and S proteins. These experiments revealed that vaccination had induced a significant increase in the percentages of CD4 + T-cells and CD8 + T-cells that proliferated in response to addition of either recombinant N protein or recombinant S protein to the culture media ( Fig. 5A-D) . These vaccine-specific, antigen-induced proliferative responses demonstrate that the LSNME/S W1 vaccine achieved its primary goal, which was to prime the cellular arm of the immune system to generate N-reactive CD4 + and CD8 + T-cells, and also S-reactive CD4 + and CD8 + T-cells. In additional experiments, we stained antigen-induced T-cells cells for the expression of interferon gamma (IFNg) and interleukin 4 (IL4). These experiments revealed that the S-reactive CD4 + T-cell population displayed elevated expression of the Th1-associated cytokine IFNg, and to a lesser extent, the Th2-associated cytokine IL4 (Fig 6) . In contrast, Nreactive T-cells failed to display an N-induced expression of either IFNg or IL4.

Control and vaccinated animals were examined regularly for overall appearance, general behavior, and injection site inflammation (redness, swelling). No vaccine-related differences were observed in any of these variables, and animals from all groups displayed similar age-related increases in body mass (supplemental figure 1).

Vaccination also had no discernable effect on blood cell counts (supplemental figure 2) .

Histological analyses were performed on all animals at the conclusion of the study by an independent histology service, which reported that vaccinated animals showed no difference in overall appearance of any of the tissues that were examined. Representative 13 images are presented for brain, lung, heart, liver, spleen, kidney, and side of injection skeletal muscle in an animal from each of the trial groups (Fig. 7) .

Exosomes represent a novel drug delivery vehicle capable of protecting labile cargoes from degradation and delivering them into the cytoplasm of target cells (Kamerkar et al., 2017; Li et al., 2017; O'Brien et al., 2020) . This is particularly relevant for the development of RNA-based vaccines and therapeutics, as unprotected RNA-based drugs are subject to rapid turnover, poor targeting, and in some cases unwanted side effects arising from naked nucleic acid injection. Encapsulating RNAs in liposomes and other types of lipid nanoparticles (LNPs) is one approach to solving these problems (Witzigmann et al., 2020; Yu et al., 2020) , but LNPs are known to pose risks of their own (Peer, 2012) , and in some cases LNP-RNA drugs have been associated with severe adverse effects (Hong et al., 2020) . In contrast, exosomes are continually released by all cells, are abundant components of human blood and all other biofluids (Coumans et al., 2017; Pegtel and Gould, 2019) , and are therefore well-tolerated drug-delivery vehicles in human (Kamerkar et al., 2017; Li et al., 2017; O'Brien et al., 2020) . In addition, exosomes play critical roles in the intercellular delivery of signals and macromolecules, including the functional delivery of mRNAs and other RNAs, making RNA-loaded exosomes an attractive candidate for clinical applications of RNA therapeutics (O'Brien et al., 2020; Ratajczak et al., 2006; Skog et al., 2008) .

. In the present report, we established that formulations of purified exosomes, in vitrosynthesized mRNAs, and polycationic lipids can mediate mRNA transport into human cells, and functional expression of mRNA-encoded protein products. This was established first for Antares2, a bioluminescent and fluorescent protein that served as a reporter protein for interrogating the effect of exosome-mRNA formulation variables that affect exosome-mediated mRNA delivery. It was then extended to the functional delivery of mRNAs encoding membrane proteins, including the multi-antigen carrier protein LSNME and S W1 , a functional spike protein. Taken together, these results indicate that mRNAs delivered via exosome-mRNA formulations can support cargo protein synthesis, regardless of whether the protein is predicted to be synthesized on free cytosolic ribosomes (e.g. Antares2) or on membrane-bound ribosomes that mediate cotranslational translocation of the protein into the endoplasmic reticulum (e.g. LSNME and S W1 ).

We also explored the ability of an exosome-RNA formulation to drive functional mRNA expression in vivo by injecting an exosome complex containing the LSNME and S W1 mRNAs (LSNME/S W1 ) into mice and monitoring immune responses to the SARS-CoV-2 N and S proteins. This vaccine was administered at relatively low doses of 4 µg mRNA equivalents and 0.25 µg mRNA equivalents in the absence of adjuvant. Injections were spaced at three-week intervals, and blood samples were collected over the course of 12 weeks. The animals were then sacrificed and tissues were harvested for analysis of cellular immune responses and organ histology. Consistent with the goal of vaccineinduced development of balanced T-cell responses, vaccinated animals displayed antigen-induced proliferation of CD4 + and CD8 + T-cell responses to both the N and S proteins. These antigen-responsive CD4+ and CD8+ populations were present nearly two months after the final boost injection, indicating that LSNME/S W1 vaccination had elicited a sustained cellular immune response to both of these SARS-CoV-2 structural proteins.

Furthermore, when these cell populations were interrogated for antigen-induced expression of the cytokines IFNg and IL4, we detected elevated expression of IFNg in CD4+ T-cells exposed to exogenous S protein, as well as a more modest S-induced expression of IL4. These results raise the possibility that the LSNME/S W1 induces the kind of Th1-skewed cellular response desired for vaccine-induced immunity. These results are consistent with the design of the LSNME open reading frame, which is engineered to drive antigen processing by the MHC Class I and Class II pathways (Gupta et al., 2006; Imai et al., 2019; Wu et al., 1995) . Vaccinated animals also developed durable antibody responses to the N and the S proteins. While the titers of these antibody responses were modest, they were sustained at relatively constant levels over the 7 weeks following the final boost injection. The relative strength of these immune responses is likely a consequence of the low mRNA dose of the LSNME/S W1 vaccine, and is likely to be amplified significantly by the >20-fold increase in dose projected in large animal models and human trials.

In conclusion, the results presented in this study validate the use of exosome-mRNA formulations for functional delivery of mRNAs both in cultured cells and in live animals.

The successful use of exosomes to deliver Antares2 mRNA opens the door to follow-on studies aimed at optimizing exosome-RNA formulation conditions, as well as for characterizing the time-dependence of Antares2 expression, biodistribution of exosomemediated RNA expression, injection site effects, and exosome-mediated tissue. As for the future development of the LSNME/S W1 vaccine, we anticipate that follow-on studies in larger animal models at doses comparable to other mRNA vaccines will demonstrate a desirable combination of safety, balanced immune responses, and when challenged, protection against SARS-CoV-2 infection and/or disease.

293F cells (Gibco, Cat.# 51-0029) were tested for pathogens and found to be free of viral 

Exosome and cell lysates were separated by SDS-PAGE using pre-cast, 4-15% gradient gels (Bio-Rad 4561086) and transferred to PVDF membranes (ThermoFisher, #88518).

Membranes were probed using antibodies directed against CD9, CD63 (System Biosciences EXOAB-CD9A-1 and EXOAB-CD63A-1, respectively), and actin (Sigma A2066), with HRP-conjugated goat anti-rabbit secondary antibody used for detection (Cell Signaling, #7074). Target proteins were visualized by chemiluminescence, and images were captured using a ChemiDoc imager (Bio-Rad).

Exosomes were fixed by addition of formaldehyde to a final concentration of 4%. Carboncoated grids were placed on top of a drop of the exosome suspension. Next, grids were 20 placed directly on top of a drop of 2% uranyl acetate. The resulting samples were examined with a Tecnai-12 G2 Spirit Biotwin transmission electron microscope (John Hopkins University, USA).

mRNAs were purified using RNeasy columns (Qiagen) and reuspended in DNase-free, RNase-free water using nuclease-free tips and tubes. RNAs were then combined with different combinations and amounts of polycationic lipids and exosomes, as well as in different orders of addition. RNA loading of exosomes for vaccine formulation involved pre-mixing of mRNAs with polycationic lipids followed by addition of exosomes.

HEK293 cells were incubated with exosome-mRNA formulations overnight under standard culture conditions. Antares2 luciferase activity was measured by Live cell bioluminescence was collected after incubating with substrate diphenylterazine (MCE, HY-111382) at final concentration of 50 µM for 3 minutes. Readings were collected using a SpectraMax i3x (Molecular Devices). Fluorescence micrographs of Antares2 expression in transfected HEK293 cells were captured as PNG files using an EVOS M7000 microscope equipped with an Olympus UPlanSAPo 40x/0.95 objective.

Immunization RNA-loaded exosome formulations were generated and then stored for 24 hours at 4°C prior to injection of mice. Injection doses were at either 4 µg equivalents of each mRNA, or 0.25 µg equivalents of each mRNA. Immunizations were initiated on thirteen weeksold, male C57BL/6J mice (Jackson Laboratory) housed under pathogen-free conditions at the Cedars-Sinai Medical Center animal facility. All animal experimentation was performed following institutional guidelines for animal care and were approved by the Cedars-Sinai Medical Center IACUC (#8602). All injections were at a volume of 50 µls.

Blood (~0.1 mL) was collected periodically from the orbital vein. At day 84, mice were deeply anesthetized using isoflurane, euthanized by cervical dislocation, and processed using standard surgical procedures to obtain spleen, lung, brain, heart, liver, kidney, muscle, and other tissues. Spleens were processed for splenocyte analysis, and all tissues were processed for histological analysis by fixation in 10% neutral buffered formalin. Histological analysis was performed by the service arm of the HIC/Comparative Pathology Program of the University of Washington.

Mouse IgG antibody production against SARS-CoV-2 antigens was measured by enzyme-linked immunosorbent assays (ELISA). For antigens S1 (RBD) and N, precoated ELISA plates from RayBiotech were utilized (IEQ-CoV S RBD-IgG; IEQ-CoVN-IgG), and the experiments were performed according to the manufacturer's instructions, with modification. Briefly, mouse plasmas at dilutions of 1:50 were added to antigen precoated wells in duplicates and incubated at room temperature (RT) for 2 hours on a shaker (200 rpm). The plates were washed 4 times with wash buffer followed by blocking for 2 hours at RT with 1% BSA in PBS. Mouse antibodies bound to the antigens coated on the ELISA plates were detected using HRP-conjugated goat anti-mouse secondary antibodies (Jackson Immuno Research Inc.) Plates were washed 4 times with washing buffer, and developed using TMB substrate (RayBiotech). Microplate Reader was used to measure the absorbance at 650 nm (SpectraMaxID3, Molecular Devices, with SoftMax Pro7 software).

After terminal blood collection mice were euthanized, and part of fresh spleens were harvested. Single cell splenocyte preparation was obtained by machinal passage through a 40 µm nylon cell strainer (BD Falcon, #352340). Erythrocytes were depleted using Ammonium-Chloride-Potassium (ACK) lysis buffer (Gibco, #A10492-01), and splenocytes were washed using R10 media by centrifuging at 300x g for 5 minutes at RT. 

Splenocytes (2 x 10 5 cells/mouse) were resuspended in 100 µL of 10% FBS in 1x PBS and incubated with fluorochrome-conjugated antibodies for surface staining of CD3 (Invitrogen, #17-0032-82) CD4 (Biolegend, #100433), CD8 (Biolegend, #100708), B220 (BD, #552771) CD11c (Invitrogen, #17-0114-81), F4/80 (Invitrogen, #MF48004) Ly6G (Invitrogen, #11-9668-80) and Ly6C (BD, #560592)) for 30 minutes at 4 °C in the dark.

Following incubation, samples were washed twice with 200 µLs 10% FBS in 1x PBS and centrifuged at 300 x g for 5 minutes at RT to remove unbound antibodies. Next the cells were fixed with 100 µLs ICS fixation buffer (Invitrogen, #00-8222-49). Samples were analyzed on a FACS Canto II (BD Biosciences) with 2,000 -10,000 recorded lymphocytes . The data analysis was performed using FlowJo 10 software (FlowJo, LLC) and presented as a percentage change in the immune cell population compared to the vehicle-treated group.

Splenocytes were resuspended at 10 6 cells/mL in 10% FBS in 1xPBS and stained with carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen, #C34554) by rapidly mixing equal volume of cell suspension with 10 µM CFSE in 10% FBS in 1x PBS for 5 minutes at 37°C. The labeled cells were washed three times with R10 complete medium. The cells were incubated for 96 hours in the presence of 10 µg/mL SARS-CoV-2 antigens N or S1 The stained cells were analyzed on a BD FACS Canto II with 5,000 -10,000 recorded lymphocytes. The data analysis was performed using FlowJo 10 software. individual six control mice, (orange bars and black squares) six mice immunized with 0.25 µg equivalents of each mRNA, and (rust bars and black triangles) six mice immunized with 4 µg equivalents of each mRNA. Height of bars represents the mean, error bars represent +/-one standard error of the mean, and the statistical significance of differences between different groups is reflected in Student's t-test values of * for <0.05 and ** for <0.005. Figure 6 . LSNME/S W1 vaccination leads to S-induced expression of IFNg and IL4 by CD4 + T-cells. Splenocytes were interrogated by flow cytometry following incubation in the absence or presence of (A, B) purified, recombinant N protein or (C, D) purified, recombinant S protein, and labeling with antibodies specific for CD4 or CD8, and for IFNg or IL4. Differences in labeling for IFNg or IL4 in CD4 + CD8 + cell populations were plotted for (grey bars and black circles) individual six control mice, (orange bars and black squares) six mice immunized with 0.25 µg equivalents of each mRNA, and (rust bars and black triangles) six mice immunized with 4 µg equivalents of each mRNA. Height of bars represents the mean, error bars represent +/-one standard error of the mean, and the statistical significance of differences between different groups is reflected in Student's ttest values of * for <0.05. Figure 7 . Absence of tissue pathology upon LSNME/S W1 vaccination. Representative micrographs from histological analysis (hematoxylin and eosin stain) of lung, brain, heart, liver, kidney, spleen, and muscle (side of injection) of animals from (upper row) control mice, (middle row) mice immunized with the lower dose of the LSNME/S W1 vaccine, and (lower row) mice immunized with the higher dose of the LSNME/S W1 vaccine. CD3 + cells were further differentiated by staining for (E) CD4 and (F) CD8. No statistically significant differences were detected in these subpopulations of white blood cells.

Ad26 vector-based COVID-19 vaccine encoding a prefusion-stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses

Experimental infection of domestic dogs and cats with SARS-CoV-2: Pathogenesis, transmission, and response to reexposure in cats

Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity

SARS-CoV-2 infection protects against rechallenge in rhesus macaques

Hosts and Sources of Endemic Human Coronaviruses

Coronaviridae Study Group of the International Committee on Taxonomy of

The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

Methodological Guidelines to Study Extracellular Vesicles

Neuropilin-1 is a host factor for SARS-CoV-2 infection

Primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques

Acute respiratory distress syndrome: the Berlin Definition

Mild or Moderate Covid-19

Recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission

Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19

Cardiovascular Implications of Fatal Outcomes of Patients With Coronavirus Disease 2019 (COVID-19)

SARS coronavirus nucleocapsid immunodominant T-cell epitope cluster is common to both exogenous recombinant and endogenous DNA-encoded immunogens

A Single-Dose Intranasal ChAd Vaccine Protects Upper and Lower Respiratory Tracts against SARS-CoV-2

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Phase 1 study of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumours

Distinct Subcellular Compartments of Dendritic Cells Used for Cross-Presentation

An mRNA Vaccine against SARS-CoV-2 -Preliminary Report

Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer

Phase 1-2 Trial of a SARS-CoV-2 Recombinant Spike Protein Nanoparticle Vaccine

Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus

Extracellular vesicles carry microRNA-195 to intrahepatic cholangiocarcinoma and improve survival in a rat model

Expression of the SARS-CoV-2 cell receptor gene ACE2 in a wide variety of human tissues

Thoughts on the alveolar phase of COVID-19

How does SARS-CoV-2 cause COVID-19?

Cell type-specific expression of the putative SARS-CoV-2 receptor ACE2 in human hearts

RNA delivery by extracellular vesicles in mammalian cells and its applications

Immunotoxicity derived from manipulating leukocytes with lipid-based nanoparticles

SARS-CoV-2 immunity: review and applications to phase 3 vaccine candidates

Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery

Presenting Characteristics, Comorbidities, and Outcomes Among 5700 Patients Hospitalized With COVID-19 in the New York City Area

The coronavirus E protein: assembly and beyond

Prospect of SARS-CoV-2 spike protein: Potential role in vaccine and therapeutic development

Infection with novel coronavirus (SARS-CoV-2) causes pneumonia in Rhesus macaques

A Single-Cell RNA Expression Map of Human Coronavirus Entry Factors

Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers

Developing Safe and Effective Covid Vaccines -Operation Warp Speed's Strategy and Approach

Early Insights into Immune Responses during COVID-19

Vaccination against coronaviruses in domestic animals

Incorporation of spike and membrane glycoproteins into coronavirus virions

Coronavirus biology and replication: implications for SARS-CoV-2

Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates

Clinical Characteristics of 138 Hospitalized Patients With

Novel Coronavirus-Infected Pneumonia in Wuhan, China

Lipid nanoparticle technology for therapeutic gene regulation in the liver

Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens

Molecular Architecture of the SARS-CoV-2 Virus

Red-shifted luciferase-luciferin pairs for enhanced bioluminescence imaging

RNA Drugs and RNA Targets for Small Molecules: Principles, Progress, and Challenges

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues

Potently neutralizing and protective human antibodies against SARS-CoV-2

Statistical analysis was performed using GraphPad Prism 8 software for Windows/Mac (GraphPad Software, La Jolla California USA). Results were reported as mean ± standard deviation or mean ± standard error, differences were analyzed using Student's t-test and one-way analysis of variance.

Sources of Support: This study was conducted with support from Capricor and from Johns Hopkins University.Disclosures: S.J.G is a paid consultant for Capricor, holds equity in Capricor, and is coinventor of intellectual property licensed by Capricor. S.J.T. is co-inventor of intellectual property licensed by Capricor. C.G. is co-inventor of intellectual property licensed by Capricor. N.A. is an employee of Capricor.