Immunogenic Properties of MVs Containing Structural Hantaviral Proteins: An Original Study

Abstract

Hemorrhagic fever with renal syndrome (HFRS) is an emerging infectious disease that remains a global public health threat. The highest incidence rate is among zoonotic disease cases in Russia. Most cases of HFRS are reported in the Volga region of Russia, which commonly identifies the Puumala virus (PUUV) as a pathogen. HFRS management is especially challenging due to the lack of specific treatments and vaccines. This study aims to develop new approaches for HFRS prevention. Our goal is to test the efficacy of microvesicles (MVs) as PUUV nucleocapsid (N) and glycoproteins (Gn/Gc) delivery vehicles. Our findings show that MVs could deliver the PUUV N and Gn/Gc proteins in vitro. We have also demonstrated that MVs loaded with PUUV proteins could elicit a specific humoral and cellular immune response in vivo. These data suggest that an MV-based vaccine could control HFRS.

Keywords: delivery system; hemorrhagic fever with renal syndrome; microvesicles; orthohantavirus; vaccine.

Figure 1

Immunophenotyping analysis of adipose-tissue-derived mMSCs by flow cytometry. Adipose-tissue-derived mMSCs were incubated in anti-mouse-CD29-PE, anti-mouse-Sca-1-AmCyan-A, anti-mouse CD90-BV421, anti-mouse-CD49-PE, and anti-mouse CD73-Alexa Fluor 647 antibodies. Cells were analyzed using flow cytometry on a FACS Aria III (Becton, Dickinson and Company, Becton Drive Franklin Lakes, Franklin Lakes, NJ, USA). A minimum of 300,000 events were collected for each sample. Results represent the percentage of cells expressing the surface markers.

Figure 2

The structure and size distribution of MVs. (A)—TEM analysis was used to analyze the structure of mMSC-derived MVs (scale bar 1 µm). The diameter of the MVs (black lines) in each experimental group was calculated individually (five images per group) using ZEN 2 Blue Edition software. One example figure was demonstrated for each group: I—control MVs; II—MVs-Katushka2S; III—MVs-PUUV N; IV—MVs PUUV Gn/Gc; and V—MVs-PUUV N and Gn/Gc. (B)—The size distribution of MVs: control (blue); Katushka2S (green); PUUV N (orange); Gn/Gc (red); and a combination of N and Gn/Gc proteins (pink). MVs from non-transduced cells were used as the control. Data are presented as the percentage of MVs in each size range $\pm$ SD.

Figure 3

Western blot analysis of N and Gn/Gc protein load in MV cargo. Total proteins (10 µg) from MVs carrying PUUV N, Gn/Gc as well as a combination of PUUV N and Gn/Gc proteins were analyzed by Western blot. MVs from non-transduced mMSCs were used as the control. Proteins were probed with primary rabbit anti-N protein or mouse anti-Gc protein antibodies detecting PUUV N or Gc proteins, respectively. Antibody–antibody complexes were visualized using Clarity ECL substrate solution. (A)—PUUV Gc (56 kDa) in MVs. Lane A1—control; lane A2—MVs PUUV Gn/Gc; lane A3—PUUV N and Gn/Gc. (B)—PUUV N protein (50 kDa) in MVs. Lane B1—control; lane B2—MVs PUUV N; lane B3—PUUV N and Gn/Gc.

Figure 4

Cytokine and chemokine levels in mMSCs supernatant. mMSCs were transduced with LV-PUUV-S, LV-PUUV-M, as well as combined LV-PUUV-S and PUUV-M lentiviruses. Supernatants from non-transduced as well as cells transduced with LV-Katushka2S were used as the control. Data are represented as the median ± SD. * p < 0.05. A p value < 0.05 was considered statistically significant.

Figure 5

Cytokines and chemokine analysis of MVs cargo. Cytokine and chemokine levels were analyzed in MVs carrying PUUV N, PUUV Gn/Gc and combined PUUV N and Gn/Gc proteins, using Multiplex analysis. MVs generated from non-transduced as well as transduced with LV-Katushka2S mMSCs were used as control. MVs (50 µL in each well) with total protein (10 µg) were loaded into the well. (A)—Interleukin levels; (B)—cytokine and chemokine levels. Data is represented as median ± SD. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001. A p-value < 0.05 was considered statistically significant.

Figure 6

PUUV N and Gn/Gc proteins in A549 cells after MVs treatment. Proteins (10 µg) were loaded in each well and separated using electrophoresis. Primary rabbit anti-N protein or mouse anti-Gc protein antibodies were used to detect PUUV N or Gn/Gc proteins, respectively. PUUV proteins were visualized using Clarity ECL Substrate solution. (A)—PUUV Gc (56 kDa) protein in A549 cells treated with MVs carrying PUUV Gn/Gc (lane A2) or PUUV N and Gn/Gc (lane A3); (B)—PUUV N (50 kDa) protein in A549 cells treated with MVs carrying PUUV N (lane B2) or PUUV N and Gn/Gc (lane B3). A549 cells treated with MVs from non-transduced MSCs, were used as control (lane A1-B1).

Figure 7

PUUV N and Gn/Gc protein detection in A549 cells treated with MVs. A549 cells (5 × 10⁴ cells/per well) were treated with MVs, containing PUUV N, Gn/Gc or their combination together with red fluorescent protein Katushka2S. Treated cells were incubated with primary rabbit anti-N protein or mouse anti-Gc protein antibodies followed by secondary donkey anti-rabbit IgG (H + L) Alexa Fluor 647 or donkey anti-mouse IgG (H + L) Alexa Fluor 555 to previous primary antibodies, respectively. (A)—A549 cells treated with MVs derived from non-transduced MSCs; (B)—The nuclei of A549 cells treated with MVs derived from non-transduced MSCs; (C)—Merged images (A,B); (D)—A549 cells treated with MVs-Katushka2S; (E)—The nuclei of A549 cells treated with MVs-Katushka2S; (F)—Merged images (D,E); (G)—A549 cells treated with MVs expressing PUUV N protein; (H)—The nuclei of A549 cells treated with MVs expressing PUUV N protein; (I)—Merged images (G,H); (J)—A549 cells treated with MVs expressing PUUV Gn/Gc protein; (K)—The nuclei of A549 cells treated with MVs expressing PUUV Gn/Gc protein; (L)—Merged images (J,K); (M)—A549 cells treated with MVs expressing PUUV N and Gn/Gc proteins; (N)—A549 cells treated with MVs expressing PUUV N and Gn/Gc proteins; (O)—The nuclei of A549 cells treated with MVs expressing PUUV N and Gn/Gc proteins; (P)—Merged images (M–O). Red fluorescence–PUUV N protein expression revealed by anti-rabbit AlexaFluor 647 antibodies. Yellow fluorescence–PUUV Gc protein expression revealed by anti-mouse AlexaFluor 546 antibodies. DAPI staining was used to demonstrate the nucleus. A549 cells treated with MVs derived from non-transduced MSCs, served as negative control. Scale bar 10 µm.

Figure 8

Serum anti-orthohantavirus IgG in mice treated with MVs carrying PUUV N, Gn/Gc as well as their combination. Serum samples were collected 14 and 28 after injection of MVs expressing PUUV N, Gn/Gc and combination of these proteins. Serum samples from the mice treated with 0.9% NaCl solution as well as from mice treated with MVs-Katushka2S were used as control. Anti-orthohantavirus proteins antibodies were detected using ELISA. (A)—anti-orthohantavirus antibody level at 14 days after MVs treatment; (B)—anti-orthohantavirus antibody level at 28 days after MVs treatment. Results are presented as OD₄₅₀ values. Data is represented as median ± SD. * p < 0.05, ** p < 0.01, *** p < 0.005. p value < 0.05 was considered statistically significant.

Figure 9

Analysis cytotoxic T lymphocytes at 14 and 28 days after MVs injection. ELISpot method was used for detection of INF-γ secretion by the activated T cells. Mice lymphocytes were isolated after treatment with MVs containing PUUV N, Gn/Gc, or their combination. Control lymphocytes were obtained from mice treated with 0.9% NaCl solution as well as from mice treated with MVs-Katushka2S. Lymphocytes (1 × 10⁵ cells) were placed into each well and treated with 1 μg of PUUV N25, N29, N43, M26, M44 or M82 peptides. Number of spots was counted to demonstrate the cytotoxic T cells activation. (A)—Cytotoxic T cell activation 14 days after MVs treatment; (B)—Cytotoxic T cell activation at 28 days after MVs treatment. Data is represented as median ± SD. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001. A p-value < 0.05 was considered statistically significant.

Figure 10

Serum cytokine analysis in mice treated with MVs at 14 and 28 days. Serum level of cytokines and chemokines was determined by using Multiplex method (BioRad). Serum was collected from mice treated with MVs containing PUUV N and Gn/Gc proteins, as well as their combination. Serum from mice treated with 0.9% NaCl solution as well as from mice treated with MVs-Katushka2S served as control. (A)—Serum cytokines and chemokines levels 14 days after MVs treatment; (B)—Serum cytokines and chemokines levels 28 days after MVs treatment. Data is represented as median ± SD. * p < 0.05, ** p < 0.01. p value < 0.05 was considered statistically significant.