Preclinical Studies on Convalescent Human Immune Plasma-Derived Exosome: Omics and Antiviral Properties to SARS-CoV-2

Abstract

The scale of the COVID-19 pandemic forced urgent measures for the development of new therapeutics. One of these strategies is the use of convalescent plasma (CP) as a conventional source for passive immunity. Recently, there has been interest in CP-derived exosomes. In this report, we present a structural, biochemical, and biological characterization of our proprietary product, convalescent human immune plasma-derived exosome (ChipEXO), following the guidelines set forth by the Turkish Ministry of Health and the Turkish Red Crescent, the Good Manufacturing Practice, the International Society for Extracellular Vesicles, and the Gene Ontology Consortium. The data support the safety and efficacy of this product against SARS-CoV-2 infections in preclinical models.

Keywords: COVID-19; SARS-CoV-2; convalescence plasma; exosome; extracellular vehicles (EVs); viral treatment.

Graphical Abstract

Figure 1

Characterization of convalescent human immune plasma-derived exosomes (ChipEXOs). (A) Individual size distribution measurements. (B) Mean size distribution measurements. (C) SEM micrograph. (D) Dynamic light scattering image. (E) Bead-assisted flow cytometry measurements of key exosome markers (CD81 and TSG 101) and a negative control marker (CANX). (F) Geometric MFI values are provided above the peaks. MFI, mean fluorescent intensity.

Figure 2

miRNA analysis of ChipEXOs. (A) Heatmap demonstration of miRNA signals from two different sources of ChipEXOs and healthy-EXOs. (B) Enrichment and depletion of different miRNAs between the ChipEXO and healthy-EXO samples. (C) Venn diagram of GO pathways from three different databases (microT-CDS, TarBase, and TargetScan) of miRNAs. (D) GO pathway graph of miRNA found in all three databases.

Figure 3

Proteomics of ChipEXO; Gene Ontology (GO) analysis according to functional enrichment networks: (A) biological process (green), (B) molecular function (blue), and (C) cellular component (green).

Figure 4

(A) KEGG Ontology (KO) data of proteomic analysis of ChipEXO. (B) STRING relation scheme of the ChipEXO protein-enriched pathway.

Figure 5

Cytotoxicity of ChipEXOs from donor samples—sample 1 (A) and sample 2 (B)—on Vero E6 cells by real-time cytotoxicity assay on RTCA MP real-time cell analysis system. The data in the figure have been adjusted to the time point when the virus was added to the experiment. Histology: (C) control and (D) day 1 and (E) day 5—the exosome-administered animal showed no pathological changes in lung tissue of hematoxylin–eosin (H&E)-stained sections; ×4 magnification. Plethysmography: time activity for intratracheal instillation ChipEXOs. Exposure to normoxia (21.0% O₂) groups does not affect respiratory frequency (fR) (F). Tidal volume (Vt) (G) and minute ventilation (V) (H) during whole-body plethysmography measurement. Bonferroni after repeated measures two-way ANOVA; all data presented as mean ± SEM: N = 1 for the control group and N = 2 for the exosome group. (The x-axis shows time in minutes). The difference for the plethysmography data points between the treated and untreated control was not statistically significant.

Figure 6

Antiviral activity of ChipEXOs. Undiluted (A), 1/2 (B), 1/4 (C), and 1/8 (D) dilutions of ChipEXOs were mixed with 100 FFU of the SARS-CoV-2 and incubated at 37°C for 1 h. Infected non-treated (E) and mock-infected (F) controls were also included. (G) Bar graphical demonstration of green fluorescent levels of the virus antigen. (H) Comparison of TCID50 values of the virus-infected control and ChipEXO-treated cells. (I) Comparison of FFU values of virus-infected control and ChipEXO-treated cells. The antibody-labeled cells were detected and analyzed by immunofluorescence microscopy (Leica, DFC450C). Scale bars = 200 μm (*p < 0.05).

Figure 7

The antiviral efficacy of ChipEXO was evaluated using the xCELLigence RTCA MP real-time cell analysis equipment.The xCELLigence system’s cell index (CI) for Vero E6 cells in media (red line), or after viral inoculation (3.5 × 105 PFU/ml), or alone (green line) (A). CI in the presence of virus and ChipEXO from two different concentrations, respectively (1/2 and 1/4) (B). In the top right corner, a smaller second graph displays the same data with the standard deviation added. The bar graph and table depicted the antiviral activity rate of ChipEXO and the CITmed and CPE delay hours, respectively. Each curve was obtained from at least three separate duplicates of normalized cell index (NCI) values.