An mRNA-Based Multiple Antigenic Gene Expression System Delivered by Engineered Salmonella for Severe Fever with Thrombocytopenia Syndrome and Assessment of Its Immunogenicity and Protection Using a Human DC-SIGN-Transduced Mouse Model

Abstract

Currently, there are no commercial vaccines or therapeutics against severe fever with thrombocytopenia syndrome (SFTS) virus. This study explored an engineered Salmonella as a vaccine carrier to deliver a eukaryotic self-mRNA replicating vector, pJHL204. This vector expresses multiple SFTS virus antigenic genes for the nucleocapsid protein (NP), glycoprotein precursor (Gn/Gc), and nonstructural protein (NS) to induce host immune responses. The engineered constructs were designed and validated through 3D structure modeling. Western blot and qRT-PCR analyses of transformed HEK293T cells confirmed the delivery and expression of the vaccine antigens. Significantly, mice immunized with these constructs demonstrated a cell-mediated and humoral response as balanced Th1/Th2 immunity. The JOL2424 and JOL2425 delivering NP and Gn/Gc generated strong immunoglobulin IgG and IgM antibodies and high neutralizing titers. To further examine the immunogenicity and protection, we utilized a human DC-SIGN receptor transduced mouse model for SFTS virus infection by an adeno-associated viral vector system. Among the SFTSV antigen constructs, the construct with full-length NP and Gn/Gc and the construct with NP and selected Gn/Gc epitopes induced robust cellular and humoral immune responses. These were followed by adequate protection based on viral titer reduction and reduced histopathological lesions in the spleen and liver. In conclusion, these data indicate that recombinant attenuated Salmonella JOL2424 and JOL2425 delivering NP and Gn/Gc antigens of SFTSV are promising vaccine candidates that induce strong humoral and cellular immune responses and protection against SFTSV. Moreover, the data proved that the hDC-SIGN transduced mice as a worthy tool for immunogenicity study for SFTSV.

Keywords: SFTS; Salmonella delivery; hDC-SIGN; vaccine; viral protein.

Figure 1

Characterization of vaccine antigens. (A–C) Predicted 3D structure of vaccine constructs. (a–c) ProSA web with z-score indicates overall model quality and recognizes errors in 3D model of vaccine antigens. (d–f) Ramachandran plot describes the validation of the vaccine construct by showing amino acids in favored and allowed regions.

Figure 2

Construction of the recombinant eukaryotic expression vector. (A) Graphical representation of the recombinant SFTSV vaccine constructs. (B) HEK293T was transfected with expression plasmids: pJHL204-empty, pJHL203-NP-GnGc-epitope, pJHL205-NP-GnGc-epitope, pJHL204-NP, and GnGc-epitope. Cells were fixed and incubated with anti-NP-GnGc-epitope antibodies and labeled with alexafluor-488-labeled anti-rabbit IgG (green); nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (C) Relative mRNA expression of transfected HEK293T cells was evaluated by qRT-PCR using NP-specific primer, and relative gene expression was analyzed by the 2^−ΔΔCT method. p-values indicate statistical significance (* p ≤ 0.05, ** p ≤ 0.001, and *** p ≤ 0.005). (D) HEK293T cells were transfected with indicated plasmids, and the cell lysate was subjected to Western blot analysis. Protein bands were detected at 48 kDa (arrow), 28 kDa, 50 kDa, and 30 kDa, corresponding to the expression of NP-GnGc-epitope, NP, Gn/Gc, and NS, respectively.

Figure 3

Constructs for the SFTSV vaccine elicit cellular immune reactions. Two-week post-booster immunization splenocytes were collected and stimulated with each antigen. (A) At 48 h post-stimulation, changes in Th1 and Th2 cytokine profiles were assessed by qRT-PCR. (B) Splenocyte proliferation indices in immunized mice were compared with the control group. (C) Representative scatter plot diagrams of CD4⁺ and CD8⁺ sub-T cell populations were gated from a CD3⁺ population. (D) Upon stimulation with SFTSV antigens, the percentages in CD4⁺ and CD8⁺ T cell subpopulations were evaluated by flow cytometry. * p ≤ 0.05, ** p ≤ 0.001, *** p ≤ 0.005, and **** p ≤ 0.0001 indicate significant differences compared to the vector control. Error bar: mean ± S.D.

Figure 4

SFTSV vaccine constructs induce humoral immune responses and potent neutralizing antibodies. (A,B) Antigen-specific IgG and IgM antibody responses were assessed in serum samples collected at 0, 1, 2, 4, 6, and 8 week(s) post-immunization. * p ≤ 0.05 and ** p ≤ 0.01 indicate significant differences in comparison to vector control. (C) Neutralizing antibody titers generated by vaccine constructs against SFTSV were determined by FRNT₅₀. Individual serum samples were diluted 2-fold. Data were analyzed by unpaired Student’s t-test, and * p ≤ 0.05 indicates significant difference in comparison to vector control.

Figure 5

The protective effects of the vaccines were assessed in an hDC-SIGN transduced murine model. AAV-hDC-SIGN-transduced mice were examined daily during the four-day time course following challenge with 1 × 10³ FAID50 of SFTSV (n = 4 per group). (A) Body weights and (B) temperature were measured every day, and (C) blood cell counts were conducted using automated blood cell counter and analyzed the platelet count. (D) Viral RNA copy number was determined by qRT-PCR from serum at 4 dpi, (E) spleen and liver collected at 2 and 4 dpi. One-way ANOVA and unpaired Student’s t-test were used to determine the level of statistical significance. Significant differences were compared between vaccine and vector control groups. * p ≤ 0.05, ** p ≤ 0.001, *** p ≤ 0.005, (F) Representative sections (n = 4 per group) of challenged spleen and liver tissue were analyzed by H&E staining. Images of the spleen (magnification: 5×) and liver (magnification: 10× and 40×) were taken under the microscope. Decreased white pulp area (#) and infiltration of mononuclear cells (arrow) were observed in spleen and liver samples, respectively. Scale bars, 20 μm (left panel) and 10 μm (right panel).