Adenovirus type 5-expressing Gn induces better protective immunity than Gc against SFTSV infection in mice

Abstract

Severe fever with thrombocytopenia syndrome (SFTS) is caused by the SFTS virus (SFTSV) with high morbidity and mortality. The major immunodominant region of SFTSV surface glycoprotein (G) remains unclear. In this study, we constructed adenovirus type 5 (Ad5) vectored vaccine candidates expressing different regions of SFTSV G (Gn, Gc and Gn-Gc) and evaluated their immunogenicity and protective efficacy in mice. In wild-type mice, compared with Ad5-Gc or Ad5-Gn-Gc, Ad5-Gn recruited/activated more dendritic cells and B cells in lymph nodes or peripheral blood, causing Th1-/Th2-mediated responses in splenocytes and triggered a greater level of SFTSV-neutralizing antibodies. In IFNAR Ab-treated mice, immunization of Ad5-Gn exhibited better protection against SFTSV challenge than Ad5-Gc or Ad5-Gn-Gc. Furthermore, passive immunization revealed complete protective immunity of Gn-specific serum rather than Gc. Collectively, our data demonstrated that Gn is the immunodominant fragment of SFTSV G and could be a potential candidate for SFTSV vaccine development.

Introduction

Severe fever with thrombocytopenia syndrome (SFTS) occurs widely among many kinds of animals and is mainly distributed in East Asia, including China¹, South Korea², Japan³, Vietnam⁴ and other countries. SFTS virus (SFTSV), the causative agent of SFTS, belongs to the Bandavirus genus of the Phenuiviridae family. Hemaphysalis longicornis and Rhipicephalus microplus were identified as the predominant tick vectors. SFTSV transmission vectors have spread to Oceania and North America⁵, indicating a continuous increase in the endemic area. The main clinical features of SFTS are fever, thrombocytopenia, leukocytopenia and multiple organ failure, with a mortality rate of 5.1–31%⁶. Tick bite has been recognized as the main infection route, while animal-to-human or human-to-human transmission via blood or body fluid contact has also been reported⁷. The World Health Organization (WHO) has included SFTSV as a priority pathogen requiring urgent attention⁸. However, there are currently no licensed treatments or vaccines against SFTSV.

SFTSV is a lipid bilayer envelope-covered RNA virus whose genome has three single-stranded negative-sense RNA fragments: small (S; 1744 bp), medium (M; 3378 bp) and large (L; 6368 bp)⁹. The L fragment encodes RNA-dependent RNA polymerase (RdRp). The S fragment encodes nucleoprotein (NP) and nonstructural proteins (NSPs), which are important virulence factors¹⁰. The M fragment encodes glycoprotein precursors (GPCs), which can be processed into two fragments: glycoprotein N (Gn) and glycoprotein C (Gc). Gn and Gc are two major antigenic components on the surface of SFTSV¹¹, similar to many kinds of Bunyavirus, including Rift Valley fever virus¹², Crimean-Congo Hemorrhagic Fever Virus¹³, Schmallenberg virus¹⁴, and Hantavirus¹⁵. GPCs are responsible for host cell receptor attachment and membrane fusion^16,17 and are the predominant targets of related vaccine development. However, most vaccine studies on Bunyavirus have focused on full-length glycoprotein or only one of its two subunits, and few reports have compared the differences in antigenicity between Gn and Gc. Further studies need to be performed to identify more accurate major immunodominant regions.

In this study, we constructed recombinant adenovirus type 5 vector vaccines expressing SFTSV Gn, Gc and Gn-Gc and analyzed their biological characteristics, pathogenicity, and immunogenicity, providing a theoretical basis for the development of SFTSV vaccines. The purpose of this study was to elucidate the specific role of Gn or Gc in the immune response to SFTSV infection, to elucidate the possible pathogenic mechanism of Bunyavirus infection and to explore potential therapeutic targets for SFTSV infection.

Results

Generation and characterization of Ad5-Gn, Ad5-Gc, and Ad5-Gn-Gc

A schematic representation of how glycoprotein precursors are cleaved into Gn and Gc is presented in Fig. 1a. Gn, Gc and Gn-Gc were cloned and inserted into the Ad5 vector (Fig. 1b) to generate recombinant Ad5-Gn, Ad5-Gc, and Ad5-Gn-Gc, respectively, which were subsequently transfected into Vero cells to examine the expression of Gn and Gc. The insertion results were confirmed by PCR and sequencing (data not shown). Western blotting (Fig. 1c) revealed Gn in Ad5-Gn- and Ad5-Gn-Gc-infected HEK293 cells and Gc in Ad5-Gc- and Ad5-Gn-Gc-infected HEK293 cells. For the primary antibodies of Gn and Gc were both murine origins, immunofluorescence staining of Gn and Gc in Ad5-Gn-Gc were performed separately and presented in two different columns (Fig. 1d). The expression levels of Gn in Ad5-Gn were greater than those in Ad5-Gn-Gc, while for Gc, no significant difference was found between Ad5-Gc and Ad5-Gn-Gc. Direct GFP observations revealed that the transduction efficacy of all the experimental groups, including the Ad5-GFP group, reached nearly 100% (Fig. 1d). An indirect immunofluorescence assay (Fig. 1d) revealed that Ad5-Gn-, Ad5-Gc- and Ad5-Gn-Gc-infected Vero cells exhibited positive expression of Gn or Gc.

Fig. 1. Construction and characterization of the recombinant human adenoviruses Ad5-Gn, Ad5-Gc and Ad5-Gn-Gc.

a Schematic representation of the SFTSV structure and how Gn or Gc are formed from their precursors. b Construction strategy for pAd5-Gn, pAd5-Gc and pAd5-Gn-Gc. Gn, Gc and Gn-Gc were subsequently cloned and inserted into the Sgf I and Mlu I sites of pAd5, with pAd5-GFP serving as a negative control. The expression of Gn or Gc was confirmed by western blotting (c) and immunofluorescence assays, including direct GFP observations (d). Green represents Gn, and red represents Gc in IFA. Scale bar, 50 μm. Gn and Gc staining in Ad5-Gn-Gc are shown in separate columns because the primary antibodies for both are of murine origin (d).

The Pathogenicity and Immunogenicity of Recombinant Adenoviruses Ad5-Gn, Ad5-Gc and Ad5-Gn-Gc in mice

As shown in Fig. 2a, wild-type BALB/c mice were immunized with Ad5-Gn, Ad5-Gc, Ad5-Gn-Gc, Ad5-GFP, or DMEM. During the observation period, the behaviors of the mice were normal, and no obvious symptoms were observed. No significant differences in the body weights of the mice among the groups were found (Fig. 2b), illustrating that Ad5-Gn, Ad5-Gc, and Ad5-Gn-Gc exhibited no obvious pathogenicity.

Fig. 2. Pathogenicity and immunogenicity of recombinant adenoviruses Ad5-Gn, Ad5-Gc and Ad5-Gn-Gc in wild-type BALB/c mice.

a Schematic representation of immunization protocols used for wild-type mice. b After immunization, the body weights and physical states of the mice (n = 8 in each group) were monitored daily for 21 d. c Serum SFTSV-neutralizing antibody titers were detected at 2, 4, and 8 weeks after Ad5-Gn, Ad5-Gc and Ad5-Gn-Gc immunization (n = 6 in each group at each time point). The data are presented as the means ± SEMs, and significant differences between groups are indicated as *P < 0.05 and **P < 0.01.

To investigate the immunogenicity of SFTSV candidate vaccines, mouse serum samples were collected at 2, 4, and 8 weeks after immunization to detect SFTSV-neutralizing antibody (VNA) levels in response to Ad5-Gn, Ad5-Gc, and Ad5-Gn-Gc (Fig. 2c). The titers of SFTSV VNA in Ad5-Gn-immunized mice were 1:53, 1:373, and 1:309 at 2, 4 and 8 weeks, respectively, while those in Ad5-Gc-immunized mice were 1:45, 1:203, and 1:139, respectively, and those in Ad5-Gn-Gc-immunized mice were 1:63, 1:177, and 1:150, respectively. The titers of VNA at 4 and 8 weeks were greater than those at 2 weeks, and the titer of VNA in the Ad5-Gn group was the highest among all groups at all three-time points. The above results demonstrated that Ad5-Gn had good immunogenicity and could induce strong humoral immunity in mice.

Ad5-Gn Promotes the Activation of DCs and B and T Cells and Enhances the Production of Ig

To determine whether Ad5-Gn, Ad5-Gc, and Ad5-Gn-Gc induce an adaptive immune response, the recruitment and/or activation of dendritic cells (DCs), the activation of B and T cells, and the production of Ig were investigated. Surface marker antigen (CD11c) and antigen-presenting molecules (CD80, MHC I, MHC II, and CD86) from activated DCs in the inguinal lymph nodes of immunized mice were detected by flow cytometry (Fig. 3a–c). No difference was observed among the groups at 3 dpi. At 6 dpi, the percentages of CD11c⁺ CD80⁺ DCs significantly increased in the mice immunized with Ad5-Gn, Ad5-Gc, and Ad5-Gn-Gc compared to those in the Mock and Ad5-GFP groups. At 9 dpi, immunization with Ad5-Gc induced significant upregulation of CD80, MHC-I, MHC-II and CD86, and immunization with Ad5-Gn-Gc induced significant upregulation of CD80 and MHC-I. The experimental results proved that Ad5-Gn can promote the early immune response of mice and promote the activation of DCs in the lymph nodes of mice.

Fig. 3. Activation of DCs in lymph nodes (LNs) in immunized wild-type mice.

Six- to eight-week-old female BALB/c mice (n = 5 in each group at each time point) were immunized intramuscularly with 100 μl of Ad5-GFP, Ad5-Gn, Ad5-Gc, or Ad5-Gn-Gc (equivalent to 1 × 10⁸ PFU/ml) or DMEM. After immunization, the inguinal lymph nodes (LNs) were collected at 3, 6 and 9 d postimmunization (dpi). Single-cell suspensions were prepared and stained separately with antibodies against DCs (CD80⁺, MHC I⁺, MHC II⁺, CD86 and CD11c⁺) and then detected by flow cytometry. a The gating strategies for analyzing DCs. b Representative flow cytometric plot of activated DCs. c Statistical analysis of the percentages of activated DCs (CD11c⁺CD80⁺, CD11c⁺CD86⁺, CD11c+MHC-I⁺, and CD11c+MHC-II⁺). The data are presented as the means ± SEMs, and significant differences between groups are indicated as *P < 0.05, **P < 0.01 and ***P < 0.001.

Subsequently, to detect the effects of Ad5-Gn, Ad5-Gc, and Ad5-Gn-Gc on B-cell activation, peripheral blood and inguinal lymph nodes of the immunized mice were collected for flow cytometry analysis at 3, 6 and 9 dpi. The percentages of CD19⁺ CD40⁺ B cells in the lymph nodes of mice in the Ad5-Gn, Ad5-Gc and Ad5-Gn-Gc groups increased significantly compared to those in the Mock and Ad5-GFP groups at 3, 6 and 9 dpi (Fig. 4a–c). At 6 dpi, the percentages of CD19⁺ CD40⁺ B cells in the Ad5-Gn and Ad5-Gc groups were significantly greater than those in the Ad5-Gn-Gc group. The percentage of CD19⁺ CD40⁺ B cells in the peripheral blood of mice in the Ad5-Gn group was greater than that in the other four groups, and the proportion of double-positive B cells accounted for approximately 40% of the total cells at 3 and 6 dpi; this difference was statistically significant (P < 0.05) (Fig. 4d–f). These results indicated that the recombinant virus Ad5-Gn can quickly recruit/activate B cells in peripheral blood and lymph nodes, triggering humoral immunity in mice.

Fig. 4. Activations of B cells and SFTSV-specific IgG1/IgG2a subtype responses in immunized wild-type mice.

Six- to eight-week-old female BALB/c mice (n = 5 in each group at each time point) were immunized intramuscularly with 100 μl of Ad5-GFP, Ad5-Gn, Ad5-Gc, or Ad5-Gn-Gc (equivalent to 1 × 10⁸ PFU/ml) or DMEM. After immunization, the LNs and peripheral blood of the immunized mice were collected at 3, 6 and 9 dpi. Single-cell suspensions were prepared and stained with antibodies against B cells (CD40⁺ and CD19⁺) and then detected by flow cytometry. The gating strategies for B cells from LNs (a) or peripheral blood (d). A representative flow cytometric plot for analyzing activated B cells (b, e). Statistical analysis of activated B-cell (CD19⁺CD40⁺) percentages in LNs (c) or peripheral blood (f). The relative ratios of SFTSV-specific IgG1, IgG2a, and IgG2a/IgG1 (g) in Ad5-Gn-, Ad5-Gc- and Ad5-Gn-Gc-immunized mice are shown. The data are presented as the means ± SEMs, and significant differences between groups are indicated as *P < 0.05, **P < 0.01 and ***P < 0.001.

Th1-dependent IFN-γ induces the production of IgG2a, whereas the Th2 cytokine IL-4 stimulates the expression of IgG1 in mice. Consequently, immunoglobulins (Igs) of these specific isotypes are indicators of the underlying Th-cell responses¹⁸. At 2, 4 and 8 weeks after immunization, we detected the relative IgG1 and IgG2a antibody levels in the serum of immunized mice by ELISA. The results are presented in Fig. 4g. The average IgG1 relative antibody levels in the serum of the Ad5-Gn group were 1:600, 1:1333 and 1:2133; those in the Ad5-Gc group were 1:467, 1:800, and 1:1733; those in the Ad5-Gn-Gc group were 1:533, 1:933, and 1:2000; and the average IgG2a relative antibody levels in the serum of the Ad5-Gn group were 1:667, 1:1467, and 1:3733; those in the Ad5-Gc group were 1:600, 1:1067, and 1:2667; and those in the Ad5-Gn-Gc group were 1:667, 1:1600, and 1:3467. These results further indicated that Ad5-Gn can induce Th1 and Th2 cellular immune responses and is more inclined toward Th1-biased immune responses, which play a critical role via interferon-γ (IFN-γ) production, in mediating intracellular killing against a variety of infectious pathogens.

To further investigate the T-cell immune response induced by Ad5-Gn, Ad5-Gc and Ad5-Gn-Gc immunization, we harvested the spleens of the mice at 28 dpi and assessed the numbers of T cells secreting IFN-γ (Fig. 5a, b) or IL-4 (Fig. 5c, d) against intact SFTSV particles or Gn proteins by enzyme-linked immunospot (ELISpot) assays. Representative plots of IFN-γ-secreting T cells and IL-4-secreting T cells are presented below the graphs in Fig. 5. The IFN-γ and IL-4 ELISpot results demonstrated that mice immunized with Ad5-Gn and Ad5-Gn-Gc produced significantly more IFN-γ SFCs (Fig. 5a, P < 0.05) in the presence of intact SFTSV particles according to the enzyme-linked immune spot (ELISpot) assay. The numbers of IFN-γ SFCs against Gn proteins and IL-4 SFCs against intact SFTSV particles or Gn proteins were not significantly different among the groups (Fig. 5b–d, P > 0.05).

Fig. 5. Detection of the production of antigen-specific T cells in the spleens of immunized mice by ELISpot.

Six- to eight-week-old female BALB/c mice (n = 6 in each group at each time point) were immunized intramuscularly with 100 μl of Ad5-GFP, Ad5-Gn, Ad5-Gc, or Ad5-Gn-Gc (equivalent to 1 × 10⁸ PFU/ml) or DMEM. Splenocytes were collected at 28 d postimmunization. The inactivated purified SFTSV virion-specific IFN-γ⁺ spot-forming cells (SFCs) (a) and IL-4⁺ SFCs (c) were detected by ELISpot kits. SFTSV Gn protein-specific IFN-γ⁺ SFCs (b) and IL-4⁺ SFCs (d) were also detected by ELISpot kits. Representative images of IFN-γ⁺ or IL-4⁺ SFCs are presented below each graph. The data are presented as the means ± SEMs, and significant differences between groups are indicated as *P < 0.05, **P < 0.01 and ***P < 0.001.

Ad5-Gn protected IFNAR Ab mice from SFTSV challenge

To examine the specific immune responses induced by Gn or Gc against lethal SFTSV infection, IFNAR Ab mice were used as an animal model for virus challenge. The adult immunocompetent mouse model can only partially replicate the characteristics of human SFTSV infections, as mice do not develop severe clinical manifestations or succumb to SFTSV infection, which limits the use of this model for evaluating the safety and efficacy of candidate vaccines and therapeutics. Among existing immunocompromised mouse models, the IFNAR Ab-treated mouse model can develop lethal symptoms and exhibit severe clinical manifestations similar to those observed in humans with moderate infection durations, even with a low dosage of SFTSV¹⁹. The physical states of the IFNAR mAb-treated mice were monitored for 21 d after the SFTSV challenge (Fig. 6a). As shown in Fig. 6b, c, d, mice in the Mock, Ad5-GFP and Ad5-Gc groups experienced symptom aggravation until death within 8 d. Most of the mice in the Ad5-Gn-Gc group died within 10 d, with only one surviving at 21 d. The mice in the Ad5-Gn group exhibited mild or moderate symptoms but recovered quickly, and all survived for 21 d.

Fig. 6. Ad5-Gn protected IFNAR Ab mice from the SFTSV challenge.

(a) Schematic representation of SFTSV challenge protocols in IFNAR Ab mice. Wild-type mice (n = 8 in each group) were immunized with 100 μl of 10⁸ PFU/ml Ad5-GFP, Ad5-Gn, Ad5-Gc or Ad5-Gn-Gc in DMEM. At 27 dpi, the MAR1-5A3 monoclonal antibody was intraperitoneally injected. At 28 dpi, the IFNAR Ab mice were challenged with 1 × 10^5.25TCID₅₀ of SFTSV. The body weights (b), survival rates (c) and clinical symptom scores (d) of the IFNAR Ab-treated mice were monitored daily for 21 d after the SFTSV challenge.

The spleen, liver, brain, and lung tissues from five groups of IFNAR Ab-treated mice challenged with SFTSV were collected for qRT-PCR to detect the viral loads of SFTSV (Fig. 7a). Almost no SFTSV RNA (detection value ≤ 0.1 TCID₅₀/mg was judged as negative) was detected in the Ad5-Gn-immunized mice throughout the challenge. Tissues from the Mock-, Ad5-GFP-, Ad5-Gc-, and dead Ad5-Gn-Gc-immunized mice all exhibited detectable viral loads (one surviving mouse immunized with Ad5-Gn-Gc had no detectable viral load). The viral loads in the liver and lung in the Mock and Ad5-GFP groups were significantly greater than those in the Ad5-Gn group. Differences among tissues from the Mock, Ad5-GFP, Ad5-Gc and Ad5-Gn-Gc groups were not statistically significant.

Fig. 7. qRT-PCR detection and histopathological examination of IFNAR Ab-treated mice challenged with SFTSV.

a The spleen, liver, brain, and lung tissues from five groups of IFNAR Ab-treated mice challenged with SFTSV were collected for qRT-PCR to detect the viral loads of SFTSV. b Liver and spleen tissues from five groups of IFNAR Ab-treated mice infected with SFTSV were collected for histopathological examination.

Livers and spleens from five groups of IFNAR Ab-treated mice infected with SFTSV were collected for histopathological examination (Fig. 7b). No obvious abnormalities were found in the livers of Ad5-Gn mice, but pathological changes, including hepatic lobular central vein and hepatic sinusoid dilation, congestion, hemorrhage, hepatic cellular atrophy, multifocal necrosis of the liver, and diffuse steatosis of hepatocytes, appeared in the livers of mice inoculated with DMEM, Ad5-GFP, Ad5-Gc or Ad5-Gn-Gc. In addition, there were no obvious abnormalities in the spleens of the Ad5-Gn mice. However, the red pulp and white pulp of the spleen in the other four groups were not clearly defined, with white pulp atrophy, apoptotic lymphocyte infiltration and multiple areas of local necrosis.

Anti-Gn antibodies are crucial for immune protection against SFTSV infection

To investigate the precise protective role of VNA against SFTSV infection, serum specific for SFTSV Gn or Gc was collected from immunized mice. However, the VNA titers of the mice immunized with Ad5-Gc in this study were too low to protect against SFTSV infection. To detect the protective effect of Gc VNA, Gc-specific serum was collected from AAV-immunized mice in another study by our team (data not shown), with mean titers reaching 1:256-1:384. Gc-specific serum samples were stratified into a high-titer serum (VNA titer >1:250) group and a low-titer serum (VNA titer ≤1:250) group. Gn- or Gc-specific serum was transferred i.p. into naive IFNAR Ab mice 12 hours before the SFTSV challenge (Fig. 8a). All the mice that received Gn-specific serum and were challenged with a lethal dose of SFTSV exhibited only slight weight loss and mild symptoms, after which they recovered to a normal state, and all the mice survived. Notably, 50% of the mice that received high-titer Gc-specific serum survived 21 d after SFTSV infection, whereas all the mice that received low-titer Gc-specific serum died within 7 d after challenge, similar to the results of the mock group (Fig. 8b, c, d). Collectively, these results suggested that anti-Gn VNA is sufficient for protective immunity against SFTSV infection and that high-titer Gc-specific serum partly protected IFNAR Ab mice from SFTSV challenge.

Fig. 8. Passive transfer of anti-Gn antibodies protected recipient IFNAR Ab mice from SFTSV challenge.

a Schematic representation of the passive transfer of serum from vaccinated to naive IFNAR Ab mice. Wild-type 6- to 8-week-old female BALB/c mice (n = 6 in each group) were injected with the MAR1-5A3 mAb 24 h prior to SFTSV infection. Twelve hours following the injection of the MAR1-5A3 mAb, naive IFNAR Ab mice were injected i.p. with serum specific for SFTSV Gn, high-titer (VNA titers >1: 250) or low-titer (VNA titers ≤ 1:250) serum specific for SFTSV Gc, or DMEM. The IFNAR Ab recipient mice were challenged with 1 × 10^5.25TCID₅₀ of SFTSV. The body weights (b), survival rates (c) and clinical symptom scores (d) of the recipient IFNAR Ab-treated mice were monitored daily for 21 d after the SFTSV challenge.

Discussion

SFTSV GPCs are co- and post-translationally cleaved into two mature glycoproteins (G), Gn and Gc, by a number of specific host cell proteases. Gn and Gc are responsible for receptor binding and membrane fusion and are the main targets of neutralizing antibodies. SFTSV DNA vaccine²⁰ and live attenuated recombinant vesicular stomatitis virus-based vaccine²¹ expressing SFTSV G have demonstrated robust activation of humoral and cellular immunity, leading to effective protection against lethal SFTSV infection in immunocompromised animal models. Our previous study demonstrated that immunization with Ad5-G-Gn (co-expressing RABV G and SFTSV Gn) could significantly reduce the splenic SFTS viral load in wild-type C57BL/6 mice²². However, existing studies have focused mostly on the immunogenicity of full-length G or Gn alone, and the precise role of Gn or Gc in the immunogenicity of SFTSV G has remained unclear. In this study, for the first time, we reported that Gn, rather than Gc, is the predominant antigenic region for SFTSV, and a challenge model with immunocompromised mice was also constructed to provide an important model for evaluating the protection rate of the SFTSV vaccine. Additionally, treatment with Gn-induced antibodies provides better protection in SFTSV-infected mice than treatment with Gc-induced antibodies, suggesting a promising role for Gn-induced antibodies in SFTS treatment.

Compared with those in the Mock group, Ad5-GFP, Ad5-Gn, Ad5-Gc, and Ad5-Gn-Gc showed no pathogenicity in wild-type mice. No significant changes were found in body weight among the five groups. VNA is the key indicator of the protective effect of a vaccine in vivo. Although the VNA titer threshold required for protection against SFTSV infection is unknown, we found that Ad5-Gn immunization induced greater levels of neutralizing antibodies than did Ad5-Gc and Ad5-Gn-Gc. Our data were consistent with those of a previous neutralizing assay in SFTSV-infected patients, indicating functional humoral response deficiency in fatal patients and revealing potential roles for Gn-specific antibodies in the survival of SFTS patients²³. Gn-specific VNA may provide better immunity against SFTSV infection. DCs are potent antigen-presenting cells in the immune system that connect innate immunity and adaptive immunity and play an important role in vaccine immunogenicity¹⁸. In this study, more DCs were recruited and activated in the Ad5-Gn group than in the control group at three-time points. DCs highly expressing MHC class I factors can follow the classic MHC class I pathway to present endogenous antigens and activate cytotoxic T cells (CTLs), prompting B cells to produce antibodies. Mature DCs that highly express MHC class II molecules can form peptide-MHC complexes and present them to T cells, thereby initiating MHC class II-restricted Th1 responses. DC activation induces antigen presentation to CD4⁺ T cells via MHC II, after which B cells are stimulated to generate antigen-specific antibodies²⁴. Our results showed that Ad5-Gn immunization induced increased SFTSV-specific neutralizing antibody production to protect the mice more effectively, proving that Ad5-Gn immunization promoted an early immune response partly through the activation of DCs in the lymph nodes.

Song et al. reported that SFTS fatality is associated with the absence of virus-specific B-cell immunity and a low IgG antibody titer²³. Angela Park et al. further demonstrated that SFTSV infection may inhibit high-affinity antibody maturation and the secretion of plasma B cells and suppress VNA production²⁵. In our research, mice immunized with Ad5-Gn exhibited significant activation of CD19⁺ CD40⁺ B cells both in the peripheral blood and in the lymph nodes. CD19 is a widespread marker of B cells, and CD40 is located on the surface of mature B cells. Mature B cells can synthesize plasma cells and secrete antibodies after protein stimulation²⁶, thereby inducing strong humoral immunity and protecting against SFTSV infection. ELISpot experiments showed that the Ad5-Gn group produced more specific T cells secreting IFN-γ and IL-4 in the spleen than did the other four groups, illustrating that Ad5-Gn could promote Th1 and Th2 immune responses and contribute to the generation of CD8⁺ T cells. Furthermore, IgG isotype analysis further revealed that the Ad5-Gn group rapidly induced stronger SFTSV-specific Th1 responses. Our findings demonstrated that Ad5-Gn immunization can effectively induce humoral immunity and cellular immunity against SFTSV.

Adult wild-type mice with normal immune responses to SFTSV infection show no severe clinical symptoms or death and are mainly used for evaluating the safety and efficacy of candidate vaccines and related therapies²⁷. Previous studies have shown that the type I IFN signaling pathway is significant in SFTSV infection²⁸. Several lethal animal models for SFTSV infection have been established, such as aged ferrets²⁹ and immunocompromised mouse models (IFNAR^−/−18 and STAT2^−/− mice³⁰, etal). The IFNAR^−/− mouse model is the most widely used SFTSV small animal model for evaluating the efficacy of human-specific virus infection due to its high susceptibility to SFTSV and severe attack characteristics^31,32. However, IFNAR^−/− mice cannot effectively replicate the interactions of viral infection with the innate immune system due to a lack of type I IFN-mediated antiviral responses. Moreover, IFNAR^−/− mice generally succumb to an acute disease course, failing to recapitulate the features of human cases fully. To better elucidate the effects of Ad5-Gn, Ad5-Gc and Ad5-Gn-Gc against the SFTSV challenge, we utilized an IFNAR antibody to inhibit IFN signaling temporarily. The remarkable advantages of this mouse model include easy availability and manipulation, temporary immune deficiency, more natural disease progression, prolonged survival intervals, increased virus replication, and close similarity to IFNAR^−/− mice³³.

IFNAR Ab mice in the Ad5-Gn group experienced mild symptoms after SFTSV infection, but symptoms were restored, and all the mice survived. Almost no SFTSV RNA was detected in the spleen, liver, brain, or lung tissues from the Ad5-Gn group. All IFNAR Ab-treated mice in the Mock, Ad5-GFP, and Ad5-Gc groups died, and only 1 mouse in the Ad5-Gn-Gc group survived. The deceased mice exhibited high viral loads in various tissues. Histopathological observations of the spleen and liver revealed no obvious histological changes in the Ad5-Gn group, while tissues from the other four groups exhibited severe pathological changes. These results suggested that Ad5-Gn could effectively protect IFNAR Ab mice from SFTSV infection. Passive immunization with Gn-specific serum prior to SFTSV infection could provide complete protection, and high-titer Gc-specific serum presented suboptimal protective effects, whereas low-titer Gc-specific serum had no protective effects against SFTSV challenge. VNA, which targets Gn, was shown to potently inhibit SFTSV infection and prevent thrombocytopenia in a humanized mouse model³⁴. Given the above evidence, Gn-specific neutralizing antibodies may be promising therapeutic agents for the treatment of SFTSV infection, providing novel insights into its use in SFTSV treatment and prevention.

Previous clinical data showed that the titers of Gn-specific IgG in recovered SFTS patients tended to increase and were negative for all deceased SFTS patients during the same period. Interestingly, none of the SFTS patient sera reacted with Gc²³. Moreover, our results showed that Ad5-Gn induced more neutralizing antibodies than Ad5-Gc in a mouse model. The differences in the antigenicity of SFTSV between Gn and Gc were the focus of our study, but the underlying mechanism remains unclear. In addition, why Ad5-Gn stimulated a stronger immune response than Ad5-Gn-Gc might be related to the higher expression level of Gn in Ad5-Gn than in Ad5-Gn-Gc. The sequence length of the inserted Gn-Gc in Ad5-Gn-Gc was longer than that of the inserted Gn in Ad5-Gn, which led to lower translation and transcriptional efficiency. Additionally, the P2A insertion between Gn and Gc in Ad5-Gn-Gc might influence the expression efficiency of Gn-Gc.

Viral neutralization sites are reportedly located on the Gn and Gc domains of SFTSV, while in our study, Gc did not provide good protection against SFTSV infection compared to Gn. The absence of Gc reactivity was also reported¹⁷. In a previous study, expression plasmids encoding SFTSV glycoprotein, SFTSV-Gn and SFTSV-Gc were constructed. The study demonstrated that the expression level of Gn is greater than that of Gc, with Gc rarely expressed. The researchers indicated that Gn was required for virion incorporation of Gc and that Gn facilitated the transport of Gc to the site of viral budding, the Golgi apparatus. The integrity of the predicted signal sequence in Gc might be required for the processing of glycoproteins into mature Gn and Gc proteins and viral infectivity. Therefore, the optimization of Gc to improve its expression and immunogenicity in vaccine design is a topic worthy of in-depth exploration. Moreover, there are many questions and challenges concerning the function and structure of SFTSV glycoproteins^16,35–37.

In summary, we successfully constructed the recombinant adenovirus vaccine candidates Ad5-Gn, Ad5-Gc and Ad5-Gn-Gc, which express the SFTSV Gn and Gc proteins, among which Ad5-Gn has no pathogenicity, good protective efficacy or immunogenicity against SFTSV infection. Ad5-Gn can induce the rapid recruitment/activation of DCs in wild-type mice, promote the activation of B cells, generate specific T cells, induce rapid production of high levels of SFTSV VNA and enhance cellular and humoral immune responses. Ad5-Gn completely protected IFNAR Ab mice against lethal SFTSV infection and protected the spleen, liver, brain, lungs and other organs against SFTSV infection-related histopathological changes. Moreover, passive immunization with Gn-specific serum prior to SFTSV infection completely protected against lethal SFTSV attack. SFTSV Gn is a potential promising target for vaccine or therapeutic antibody development. Further studies need to be performed to evaluate the safety and immunogenicity of Ad5-Gn and uncover the underlying mechanisms involved.

Methods

Cell lines and Viruses

HEK-293 and Vero cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY) and 1% penicillin/streptomycin at 37 °C with 5% CO₂. The SFTSV JS2011-013-1 strain was kindly provided by Professor Yu Xuejie (School of Public Health, Wuhan University). The replication-deficient human adenovirus type 5 (Ad5) recombinant vector was purchased from Weizhen Biotechnology Co., Ltd. (Jinan, Shandong, China).

Animal ethics statement

Six- to eight-week-old female BALB/c mice were purchased from Pengyue Animal Breeding Company (Jinan, Shandong, China) and were raised in an SPF environment with a temperature of 24–26 °C and a 12 h light/dark cycle at the Model Animal Research Center of Shandong University. This study was conducted strictly in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals, the Office of Animal Welfare, and the United States Department of Agriculture. The animal treatments and sample preparations strictly complied with the Animal Ethics Procedures and Guidelines approved by the Institutional Animal Care and Use Committee of Shandong University (LL20220603). The procedures used for euthanasia of study mice followed tenets of the ARRIVE reporting guidelines³⁸. Mice were euthanized using carbon dioxide inhalation. All procedures were performed under trained personnel and under the supervision of veterinary staff.

Construction of the Recombinant Adenovirus

Referring to the M fragment of the genome sequence of the SFTSV JS2011-013-1 strain (GenBank NO. KC505127.1), the open reading frame (ORF) sequences of the SFTSV Gn (19-1704 bp) and Gc (1707-3240 bp) genes (Fig. 1a) were selected, cloned and inserted into the Ad5 vector by digestion with the restriction enzymes SgfI and MluI, followed by ligation with T4 DNA ligase as previously described³⁹. The target sequences of SFTSV Gn, Gc, and Gn-Gc (linked by the P2A gene) were generated in the Ad5 vector (Fig. 1b). The recombinant adenovirus was constructed as previously described⁴⁰. An adenovirus carrying the green fluorescence protein-encoding gene (Ad5-GFP) served as a control. Ad5-Gn, Ad5-Gc, and Ad5-Gn-Gc were propagated in HEK-293 cells, purified and stored at −80 °C. The insertion results were confirmed by PCR and sequencing. Viral titers (plaque-forming units/ml, PFU/ml) were determined in HEK-293 cells using the GFP-labeling method. HEK-293 cells were seeded in 96-well plates at a density of 2 × 10⁴ cells/well. The recombinant adenovirus samples were serially tenfold diluted from 10⁻¹ to 10⁻⁸ with a final diluent volume of 400 μl. The culture medium of the HEK-293 cells in the 96-well plate was discarded, and 100 μl of each adenovirus dilution was added to the corresponding wells in triplicate. The cells were cultured for 24 h at 37 °C. The titers of adenovirus were observed and recorded under a fluorescence microscope. The number of fluorescent cells in the last two wells where fluorescence could be observed was recorded. The method for calculating the adenovirus titer was as follows: adenovirus titer (PFU/ml) = (A + B × 10) × 1000/2/(virus volume (μl) in A). A represents the average number of fluorescent cells in the last wells, and B represents the average number of fluorescent cells in the second to last wells.

Western blotting

The monolayers of HEK-293 cells were infected with recombinant adenovirus Ad5-Gn, Ad5-Gc, Ad5-Gn-Gc and Ad5-GFP (MOI = 10) or mock-infected with the same volume of DMEM for 72 h. Cells infected with SFTSV were used as a positive control. After infection, infected cells were lysed with RIPA buffer containing 1 mM PMSF (Beijing Solarbio Science & Technology Co., Ltd.). The lysates were centrifuged (16,000 rpm for 10 min) to remove precipitates. Equal amounts of protein were loaded onto a 10% SDS‒PAGE gel and transferred to PVDF membranes (0.2 μm, Millipore, Darmstadt, Germany). After blocking with 5% skim milk, the membranes were incubated with mouse anti-Gn monoclonal antibody (1:2000) or rabbit anti-Gc polyclonal antibody (1:1000) overnight at 4 °C. Subsequently, the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (1:5000, Abcam, Cambridge, United Kingdom) for 1 h at room temperature. The positive signals were detected with enhanced chemiluminescence (ECL) substrate (Thermo, USA) with a Western Blotting Imaging System (General Electric Co., USA).

Immunofluorescence assay

The recombinant adenoviruses Ad5-Gn, Ad5-Gc, Ad5-Gn-Gc and Ad5-GFP or Mock inoculated with DMEM were inoculated for 48 h in Vero cells in 96-well plates (4 replicate wells for each group). Then, the cells were washed and fixed in precooled 80% acetone overnight at −20 °C. Mouse anti-Gn monoclonal antibody (donated by Professor Yu Xuejie from Wuhan University) and mouse anti-Gc polyclonal antibody (synthesized by Nanjing GenScript, China) were added to the corresponding wells, incubated at 37 °C for 1 h, and washed 3 times with PBS. Alexa Fluor 488-conjugated goat anti-mouse or Alexa Fluor 594-conjugated goat anti-mouse secondary antibodies (Abcam, Cambridge, United Kingdom) were added and incubated for 1 h, after which the nuclei were stained with DAPI and washed 3 times with PBS. Fluorescence microscopy (Olympus, IX71) was used to observe specific fluorescence.

50% tissue culture infective dose (TCID₅₀) of SFTSV

Monolayers of Vero cells were inoculated with SFTSV. The virus culture medium was collected when the percentage of cell lesions exceeded 80%, and the supernatants were collected by centrifugation at 4 °C and 3500 rpm for 10 min and stored at −80 °C. The viruses were serially diluted tenfold and inoculated into Vero cells, which reached 80% confluence, in 96-well plates with 4 parallel controls. The 96-well plates were cultured at 37 °C in a 5% CO₂ incubator for 3 d. Then, the culture media were discarded, and the cells were washed 3 times with PBS and incubated at 37 °C with rabbit anti-SFTSV Gn/Gc polyclonal antibody (1:400) followed by secondary goat anti-rabbit IgG H&L antibody (1:800). The results were observed and recorded under a fluorescence microscope. The viral titer (TCID₅₀) was calculated using the Karber method: lgTCID₅₀ = L + d(S-0.5)⁴¹. L represents the logarithm of the highest dilution, d represents the difference between the logarithms of the dilutions, and S represents the sum of the ratios of positive wells.

Immunization and pathogenicity evaluation

Six- to eight-week-old wild-type female BALB/c mice were randomly divided into 5 groups (n = 8 in each group). Then, 100 μl of DMEM or Ad5-GFP, Ad5-Gn, Ad5-Gc or Ad5-Gn-Gc virus dilutions (equivalent to 1 × 10⁸ PFU/ml) were injected intramuscularly (i.m.) into the corresponding mice. After immunization, the body weights and physical states of the mice were continuously monitored for 21 d.

Virus-neutralizing antibodies

At 2, 4, and 8 weeks after immunization, blood samples (n = 6 in each group at each time point) were collected to determine SFTSV virus neutralizing antibody (VNA) levels using a fluorescent reduction neutralization test (FRNT) as previously described²². Serum samples were separated and serially diluted two-fold with DMEM from 1:8 to 1:2048 and incubated with 100 TCID₅₀ of the SFTSV JS-2011-013-1 strain for 1 h at 37 °C in a 5% CO₂ incubator. When the Vero cells in the 96-well plate were 90% confluent, the above mixture of serum and virus or the control was added, and the cells were cultured for 48 h at 37 °C. After the cells were fixed with precooled 80% acetone, they were incubated with a mouse anti-Gc polyclonal antibody and then with an Alexa Fluor-488-labeled goat anti-mouse secondary antibody. The number of fluorescent spots was observed under a fluorescence microscope. When the number of fluorescent spots in the sample wells was reduced by 50% compared to that in the virus control wells, the corresponding serum dilution was considered the neutralizing antibody titer.

Flow cytometry

At 3, 6, and 9 d post-immunization (dpi), mice (n = 5 in each group at each time point) were sacrificed for humanitarian reasons, and single immunocyte suspensions of inguinal lymph nodes or peripheral blood were prepared and stained separately with antibodies (BD Biosciences, Franklin, TN, United States) against DCs (CD80⁺, MHC I⁺, MHC II⁺, CD86 and CD11c⁺) or B cells (CD40⁺ and CD19⁺). The flow cytometry procedure was performed with a BD FACSCelesta flow cytometer, and the data were analyzed using FlowJo software.

Enzyme-Linked Immune Spot Assay (ELISpot)

At 28 dpi, the spleens of the immunized mice (n = 6 in each group at each time point) were collected. Lymphocytes in the spleen were dissociated and stimulated with 2 μg of purified SFTSV or Gn protein for 20 h in a 37 °C incubator with 5% CO₂. Mouse IFN-γ/IL-4 ELISpot kits (Dakewe Biotech Co. Ltd., China) were used to determine the number of lymphocytes that secreted IFN-γ or IL-4 according to the manufacturer’s instructions. The number of spot-forming cells (SFCs) was calculated by eliminating background responses with an ELISpot plate reader (AID‘ ELISPOT reader-iSpot, AID GmbH, Germany).

Serum-specific IgG subtypes

IgG typing data were identified by ELISA. At 2, 4, and 8 weeks after immunization, the serum levels of IgG1 and IgG2a antibodies against SFTSV in the Ad5-Gn, Ad5-Gc and Ad5-Gn-Gc groups were detected. A total of 600 ng of intact SFTSV protein per well was precoated in 96-well plates, and the titers of IgG1 and IgG2a were detected by ELISA, ensuring that the antibody levels in the serum of immunized mice specific for SFTSV Gn or Gc could be detected. After washing and blocking, 100 μl of 10-fold serially diluted sera from the Ad5-Gn, Ad-Gc and Ad5-Gn-Gc groups were added and incubated overnight at 4 °C. The plates were washed and then incubated at room temperature for 3 h with 100 μl of HRP-conjugated goat anti-mouse IgG1 and IgG2a at a dilution of 1:500. ABTS was added, and the plates were incubated at 37 °C for 15 min after washing. The absorbance at 405 nm was measured using a microplate reader (Mutishan MK3, Thermo Scientific). Serum samples from mock-immunized mice were used as negative controls. Sample titers were considered to be positive if the absorbance reading was at least 2.1 times that of the mock group. The positive antibody end-point titers are expressed as the reciprocals of the highest dilution of serum⁴².

Mice IFNAR Ab treatment and virus challenge

Six- to eight-week-old female BALB/c mice were randomly divided into 5 groups (n = 8 in each group). The immunization schedule is shown in Fig. 6a. 100 μL of DMEM, Ad5-GFP, Ad5-Gn, Ad5-Gc or Ad5-Gn-Gc virus dilution (equivalent to 1 × 10⁸ PFU/ml) was injected i.m. into the corresponding mice. At 27 dpi, a MAR1-5A3 (mouse anti-mouse IFNAR, IgG1) monoclonal antibody (mAb) (Leinco Technologies, United States) was intraperitoneally (i.p.) injected at a dose of 1.25 mg per mouse 1 d before SFTSV infection to block type I IFN signaling¹⁹. At 28 dpi, IFNAR Ab-treated mice were challenged with SFTSV at a dose of 1 × 10^5.25TCID₅₀. Body weights and clinical manifestations were continuously recorded for 21 d after the virus challenge. We established a scoring standard based on diet, sleep, behavior, body weight, and hair to evaluate the health status of infected mice in detail (Table 1). Total RNA from the spleen, liver, lung, and brain tissues of IFNAR Ab mice on the 7th d post-SFTSV challenge was collected with an RNAprep Pure Tissue Kit (TIANGEN Biotech, Beijing Co. Ltd., China) for real-time PCR detection (DaAnGene Co. Ltd., China) of SFTSV viral loads following the manufacturer’s instructions.

Table 1.

Clinical symptom scoring standard

Clinical symptoms	Scores	Scoring criteria
Health	0	Smooth hair, spirit, good diet, no significant change in weight
Mild	1	Mild weight loss, a little eye closure
Moderate	2	Rapid weight loss, closed eyes, ruffled fur, anorexia
Severe	3	Sustained weight loss, eyes closed, ruffled fur, hunched back, depression
Death	4	Death

Histopathology

The spleens and livers of IFNAR Ab-treated mice were collected on the 21st d or before death after the SFTSV challenge and fixed with 4% paraformaldehyde for 24 hours. After gradient alcohol dehydration and clearing, the tissues were paraffin-embedded and sectioned into 5 μm slices. The slices were stained with hematoxylin and eosin for histological observation.

Passive transfer of serum from vaccinated mice to IFNAR Ab-immunized mice

Wild-type 6- to 8-week-old female BALB/c mice (n = 6 in each group) were injected with the MAR1-5A3 mAb 24 h prior to SFTSV infection, as described in section “Mice IFNAR Ab Treatment and Virus Challenge”. The immunization schedule is shown in Fig. 8a. Twelve hours following the injection of the MAR1-5A3 mAb, naive IFNAR Ab mice were injected intraperitoneally with 200 μl of serum specific for SFTSV Gn, high-titer (VNA titers > 1:250) or low-titer (VNA titers ≤1:250) serum specific for SFTSV Gc, or DMEM. IFNAR Ab mice with passive transfer of serum were challenged with 1 × 10^5.25 TCID₅₀ of SFTSV. Afterwards, body weights and clinical manifestations were monitored for 21 d.

Statistical analysis

The experiments were repeated, and the data were collected at least 3 times. The data were analyzed using SPSS 26.0 statistical software (SPSS Inc., Chicago, IL, United States) and are presented as the mean ± standard deviation. Statistical differences among the groups were identified through one-way ANOVA. P < 0.05 was considered to indicate statistical significance. Graphs were drawn with GraphPad Prism 8 statistical software. *p < 0.05; ** p < 0.01; *** p < 0.001.

Author contributions

X.Z. designed the study. H.Q., L.T., W.L., L.L., M.L., Z.Z., X.L., Z.Z. conducted the experiments. H.Q., L.T., W.Z., and X.Z. analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.

Data availability

All data generated or analyzed during this study are included in this published article.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41541-024-00993-y.