Heterologous vaccination utilizing viral vector and protein platforms confers complete protection against SFTSV

Abstract

Severe fever with thrombocytopenia syndrome virus was first discovered in 2009 as the causative agent of severe fever with thrombocytopenia syndrome. Despite its potential threat to public health, no prophylactic vaccine is yet available. This study developed a heterologous prime-boost strategy comprising priming with recombinant replication-deficient human adenovirus type 5 (rAd5) expressing the surface glycoprotein, Gn, and boosting with Gn protein. This vaccination regimen induced balanced Th1/Th2 immune responses and resulted in potent humoral and T cell-mediated responses in mice. It elicited high neutralizing antibody titers in both mice and non-human primates. Transcriptome analysis revealed that rAd5 and Gn proteins induced adaptive and innate immune pathways, respectively. This study provides immunological and mechanistic insight into this heterologous regimen and paves the way for future strategies against emerging infectious diseases.

Figure 1

Generation and characterization of Gn protein and rAd5-Gn. (a) Schematic diagram of the SFTSV GnGc, Gn∆TM, and Gn∆STEM. (b) Predicted structure of Gn∆TM and Gn∆STEM by SWISS-MODEL. A black arrow indicates a different site for GnΔTM and GnΔSTEM. (c) SDS-PAGE of purified GnΔTM and GnΔSTEM. (d) ELISA binding curves of MAb4-5 to Gn∆TM, Gn∆STEM, and Gn coated at equimolar concentrations. The average ± SD from at least two independent experiments performed is shown. (e) Examining the incorporation of GnΔTM into the adenovirus type 5 by western blot analysis. Un.

Figure 2

Comparison of humoral immune responses induced by homologous or heterologous vaccination with Ad5-Gn and Gn protein. (a) The immunization procedure and serum collection. Six-week-old C57BL/6 mice (n = 5/group) are immunized intramuscularly with 1 × 10⁹ infectious unit (IU) of rAd5-Gn or alum and RNA-adjuvanted Gn protein at 0 and 2 weeks. Sera are collected at indicated time points. (b and c) Total IgG, IgG1, and IgG2c are measured by indirect ELISA. (d) SFTSV-specific neutralizing activity of plasma (n = 25) from immunized mice is analyzed using the standard 50% focus reduction neutralization test (FRNT₅₀). (e) The immunization procedure and serum collection. 16-month-old C57BL/6 mice (n = 5/group) are immunized intramuscularly with 1 × 10⁹ IU of rAd5-Gn or alum and RNA-adjuvanted Gn protein at 0 and 2 weeks. Sera are collected at indicated time points. (f and g) IgG1 and IgG2c were measured by indirect ELISA. (h) NHPs (n = 3/group) are intramuscularly immunized 1 × 10⁹ IU of rAd5-Gn or alum and RNA-adjuvanted Gn protein at 0 and 4 weeks. Sera are collected at indicated time points. (i) Total IgG was measured by indirect ELISA (j) SFTSV-specific neutralizing activity of plasma from immunized NHPs is analyzed using the FRNT₅₀. (b to c, f to g, i) Data are presented as mean values ± SD. Mean fold changes relative to PBS control group. P-values were calculated using one-way ANOVA with Bonferroni multiple comparison test.

Figure 3

Increased T cell-mediated immune responses by rAd5-Gn priming and Gn protein boosting. Six-week-old C57BL/6 mice (n = 5/group) are immunized intramuscularly with 1 × 10⁸ infectious units of rAd5-Gn or alum and RNA-adjuvanted Gn protein at 0 and 2 weeks. Mice are sacrificed one week after boosting. (a, d, g), and (j) Representative flow cytometry plots showing (a) follicular helper T cells, (d) effector T cells, central memory t cells, (g) interferon γ cytokine-producing T cells, (j) activated T cells. Frequencies of (b) CXCR5, (c) PD-1 expression in CD4⁺ T cell, and ICOS expression in CD4 + PD-1 + populations from immunized mice spleen. Frequencies of (e) effector memory T cells and (f) central memory T cells in CD4⁺ T cells were analyzed using flow cytometry. IFN-γ-producing (h) CD4⁺ and (i) CD8⁺ T cells in the spleen were analyzed using flow cytometry. (k) CD69 and (l) CD25 expression in CD4 + T cells in the spleen was analyzed using flow cytometry. Data are presented as mean values ± SD. P values are calculated using one-way ANOVA with Bonferroni multiple comparison test.

Figure 4

Protective efficacy of Ad5-Gn priming and Gn protein boosting. Six-week-old C57/BL6 wildtype mice (PBS/PBS (n = 5), Gn/Gn (n = 5), rAd5-Gn (n = 5), Gn/rAd5 (n = 4)) immunized with rAd5-Gn (1 × 10⁸ infectious unit) and/or Gn protein intramuscularly at 2-week intervals and challenged with SFTS virus at 5 weeks after the second immunization (Fig. S1c). The mice received the anti-interferon receptor antibody and IL-10 at 4 days and 1 day before the virus challenge. (a) Weight loss of mice. (b) Weights of the spleen of mice. (c) Quantitative real-time PCR was used to measure viral genomic RNA from the spleen of challenged mice. (d to k) Pathological changes in the liver and spleen of mice infected with SFTSV. Representative Hematoxylin and Eosin-stained tissue sections of the (d) liver and (e) spleen from SFTSV-infected mice. (f) The scores for inflammatory cell infiltration, (g) thrombus, and (h) necrosis in the liver. (i) Area of the cross-section of the spleen and (k) number of megakaryocytes evaluated using histological images obtained from a slide scanner. (j) Diffuse white pulp in the spleen was determined using a 5-point score system, as follows: 0 = no abnormality detected (NAD), 1 = minimal, 2 = mild, 3 = moderate, 4 = moderately severe, and 5 = severe. P-values are calculated using one-way ANOVA with Bonferroni multiple comparison and Tukey’s multiple comparison or Mann–Whitney U test.

Figure 5

RNA-seq analysis reveals that rAd5 and Gn each regulate distinct biological pathways. Differential expression analysis (a) between rAd5 + rAd5 (n = 5) and Gn + Gn (n = 5) and (b) between rAd5 + Gn (n = 4) and Gn + rAd5 (n = 5) vaccinated mice. The horizontal dashed line indicates the significant threshold (adjusted P-value < 0.01). Differentially expressed genes related to adaptive or innate immune response are marked in black. (c) Normalized expression values of adaptive and innate immune genes. Z-scores were computed for each gene for data visualization. (d and e) Immune cell composition estimation by CIBERSORTx and (f) immune KEGG pathway-level analysis by PLIER from RNA-seq data of splenic tissues of homologous and heterologous vaccinated mice.