China is one of the main epidemic areas for hemorrhagic fever with renal syndrome (HFRS). Currently, there is no human antibody specific to Hantaan virus (HTNV) for the emergency prevention and treatment of HFRS. To prepare human antibodies with neutralizing activity, we established an anti-HTNV phage antibody library using phage display technology by transforming peripheral blood mononuclear cells (PBMCs) of patients with HFRS into B lymphoblastoid cell lines (BLCLs) and extracting cDNA from BLCLs that secreted neutralizing antibodies. Based on the phage antibody library, we screened HTNV-specific Fab antibodies with neutralizing activities. Our study provides a potential way forward for the emergency prevention of HTNV and specific treatment of HFRS.
Hantaan virus (HTNV) infections can cause severe hemorrhagic fever with renal syndrome (HFRS) in humans, which is associated with high fatality rates. Cytotoxic T cell (CTL) responses contribute to virus elimination; however, to date, HLA class I allele-restricted HTNV glycoprotein (GP) epitopes recognized by CTLs have not been reported, limiting our understanding of CTL responses against HTNV infection in humans. In this study, 34 HTNV GP nine-mer epitopes that may bind to HLA-A*0201 molecules were predicted using the BIMAS and SYFPEITHI database. Seven of the epitopes were demonstrated to bind to HLA-A*0201 molecules with high affinity via the T2 cell binding assay and were successfully used to synthesize peptide/HLA-A*0201 tetramers. The results of tetramer staining showed that the frequencies of each epitope-specific CTL were higher in patients with milder HFRS, which indicated that the epitopes may induce protective CTL responses after HTNV infection. IFN-γ-enzyme-linked immunospot analysis further confirmed the immunoreactivity of epitopes by eliciting epitope-specific IFN-γ-producing CTL responses. In an HTNV challenge trial, significant inhibition of HTNV replication characterized by lower levels of antigens and RNA loads was observed in major target organs (liver, spleen, and kidneys) of HLA-A2.1/Kb transgenic mice pre-vaccinated with nonapeptides VV9 (aa8-aa16, VMASLVWPV), SL9 (aa996-aa1004, SLTECPTFL) and LL9 (aa358-aa366, LIWTGMIDL). Importantly, LL9 exhibited the best ability to induce protective CTL responses and showed a prominent effect on the kidneys, potentially preventing kidney injury after HTNV infection. Taken together, our results highlight that HTNV GP-derived HLA-A*0201-restricted epitopes could elicit protective CTL responses against the virus, and that epitope LL9 functions as an immunodominant protective epitope that may advance the design of safe and effective CTL-based HTNV peptide vaccines for humans.
Xianyang city is one of the main hemorrhagic fever with renal syndrome (HFRS) epidemic areas in northwest China. Although the HFRS immunity program has been provided in this city, HFRS is still occurred every year. In order to implement the vaccination program effectively and to control HFRS, the analysis of antibody responses specific to Hantaan virus (HTNV) in individuals after vaccination is essential. In this study, a total of 100 subjects were divided into 5 groups: unvaccinated, 1, 3, 29 and 33 months after boost vaccination. The levels and the positive rates of HTNV-NP-specific IgM and IgG antibodies as well as HTNV neutralizing antibodies were significantly increased in the serum of the vaccinated individuals. The positive rates and levels of HTNV-NP-specific IgG and HTNV neutralizing antibody reached their highest values at 3 months respectively and could be sustained up to 33 months after vaccination. Moreover, the titres of HTNV-NP-specific IgM or IgG antibody and the titres of HTNV neutralizing antibody at 1 month after vaccination have a positive correlation. The level of HTNV-NP-specific IgG antibody was much higher than that of HTNV-NP-specific IgM antibody or HTNV neutralizing antibody. In addition, the strongest responses of antibody-secreting cells were observed at 3 months after vaccination, which was consistent with the serum results. Therefore, the HFRS immunization program is effective to induce humoral immunity in the population of northwest China.
Hantaan virus (HTNV) can cause endothelium injury in hemorrhagic fever with renal syndrome (HFRS) patients. Bystander activation of CD8+ T cells by virus infection has been shown that was involved in host injury, but it is unclear during HTNV infection. This project aimed to study the effect of bystander-activated CD8+ T cell responses in HTNV infection.The in vitro infection model was established to imitate the injury of endothelium in HFRS patients. Flow cytometry was performed to detect the expression of markers of tetramer+ CD8+ T cells and human umbilical vein endothelial cells (HUVECs). The levels of interleukin-15 (IL-15) in serum and supermanant were detected using ELISA kit. The expression of MICA of HUVECs was respectively determined by flow cytometry and western blot. The cytotoxicity of CD8+ T cells was assessed through the cytotoxicity assay and antibody blocking assay.EBV or CMV-specific CD8+ T cells were bystander activated after HTNV infection in HFRS patients. HTNV-infected HUVECs in vitro could produce high levels of IL-15, which was positively correlated with disease severity and the expression of NKG2D on bystander-activated CD8+ T cells. Moreover, the elevated IL-15 could induce activation of CD122 (IL-15Rβ)+NKG2D+ EBV/CMV-specific CD8+ T cells. The expression of IL-15Rα and ligand for NKG2D were upregulated on HTNV-infected HUVECs. Bystander-activated CD8+ T cells could exert cytotoxicity effects against HTNV-infected HUVECs, which could be enhanced by IL-15 stimulation and blocked by NKG2D antibody.IL-15 induced bystander activation of CD8+ T cells through NKG2D, which may mediate endothelium injury during HTNV infection in HFRS patients.
Abstract Hantaan virus is a major agent causing hemorrhagic fever with renal syndrome, a high-mortality-rate disease. However, the pathogenesis of HFRS remains to be elucidated. We found that CXCL10 expresses highly in the HFRS-patients’ sera and the increased CXCL10 is positively correlated with the severity of HFRS. To clarify the molecular mechanisms of HTNV infection inducing CXCL10 expression, the HTNV-infected human umbilical vein endothelial cells model has been used. The results indicated that the dsRNA, produced in the process of HTNV replication, could activate TLR3, RIG-I, and MDA5 pathways, which facilitated the translocation of IRF7 and NF-κB. Afterwards, the two transcription factors bound directly to the CXCL10 promoter region and promoted the transcription and expression of CXCL10. Furthermore, we found that the expression levels of CXCR3, the receptor of CXCL10, increased on the surface of monocytes at the early stage of HFRS. It suggested that CXCL10 may recruit monocytes, which may be involved in the cytokine storm and the pathogenesis of HFRS. Further studies are still needed to reveal the function of CXCL10 in the pathogenesis of HFRS. In conclusion, we reported that the increased CXCL10 level was associated with the state of the HFRS. The activation of innate immunity induced by HTNV replication may be of importance in the regulation of CXCL10 expression and the abundant CXCL10 may play an important role in the pathogenesis of HFRS.
Hantavirus infections cause severe emerging diseases in humans and are associated with high mortality rates; therefore, they have become a global public health concern. Our previous study showed that the CD8(+) T-cell epitope aa129-aa137 (FVVPILLKA, FA9) of the Hantaan virus (HTNV) nucleoprotein (NP), restricted by human leukocyte antigen (HLA)-A*02, induced specific CD8(+) T-cell responses that controlled HTNV infection in humans. However, the in vivo immunogenicity of peptide FA9 and the effect of FA9-specific CD8(+) T-cell immunity remain unclear. Here, based on a detailed structural analysis of the peptide FA9/HLA-A*0201 complex and functional investigations using HLA-A2.1/K(b) transgenic (Tg) mice, we found that the overall structure of the peptide FA9/HLA-A*0201 complex displayed a typical MHC class I fold with Val2 and Ala9 as primary anchor residues and Val3 and Leu7 as secondary anchor residues that allow peptide FA9 to bind tightly with an HLA-A*0201 molecule. Residues in the middle portion of peptide FA9 extruding out of the binding groove may be the sites that allow for recognition by T-cell receptors. Immunization with peptide FA9 in HLA-A2.1/K(b) Tg mice induced FA9-specific cytotoxic T-cell responses characterized by the induction of high expression levels of interferon-γ, tumor necrosis factor-α, granzyme B, and CD107a. In an HTNV challenge trial, significant reductions in the levels of both the antigens and the HTNV RNA loads were observed in the liver, spleen, and kidneys of Tg mice pre-vaccinated with peptide FA9. Thus, our findings highlight the ability of HTNV epitope-specific CD8(+) T-cell immunity to control HTNV and support the possibility that the HTNV-NP FA9 peptide, naturally processed in vivo in an HLA-A*02-restriction manner, may be a good candidate for the development HTNV peptide vaccines.
Hantaan virus (HTNV) infection causes an epidemic of hemorrhagic fever with renal syndrome (HFRS) mainly in Asia. It is well known that T cells mediated anti-viral immune response. Although previous studies showed that double positive T (DP T) cells, a little portion of T lymphocytes, were involved in adaptive immune response during virus infection, their kinetic changes and roles in HTNV infection have not yet been explored. In this study, we characterized DP T cells from HFRS patients based on flow cytometry data combined with scRNA-seq data. We showed that HTNV infection caused the upregulation of DP T cells in the peripheral blood, which were correlated with disease stage. The scRNA-seq data clustered DP T cells, unraveled their gene expression profile, and estimated the ordering of these cells. The production of granzyme B and CD107a from DP T cells and the abundant TCR distribution indicated the anti-viral property of DP T cells. In conclusion, this study identified, for the first time, an accumulation of DP T cells in the peripheral blood of HFRS patients and suggested these DP T cells belonging to CD8+T cells lineage. The DP T cells shared the similar characteristics with cytotoxic T cells (CTL) and exerted an anti-viral role in HFRS.
Abstract Background Hantaan virus (HTNV) can cause hemorrhagic fever with renal syndrome (HFRS) in humans with severe morbidity and high mortality. Although inactivated HFRS vaccines are given annually for prevention in populations, China still has the highest number of HFRS cases and deaths worldwide. Consequently, vaccination for HFRS requires the development of novel, more effective vaccines. Epitope peptide vaccines have been developed rapidly in recent years and are considered a novel approach for the prevention of infection. Specifically, the multiple antigenic peptide (MAP) design with preferable immunogenicity can arouse a satisfactory immune response for vaccination. However, there are few reports on the design and evaluation of MAP for HTNV. Methods Three HLA-A*02-restricted 9-mer cytotoxic T lymphocyte (CTL) epitopes on HTNV glycoprotein and one HLA-A*02-restricted 9-mer CTL epitope on the HTNV nucleocapsid, which have been proven to be immunoprotective in our previous study, were selected for the design of HTNV MAP. A four-branched HTNV MAP was evaluated by the IFN-γ-secreting enzyme-linked immunospot assay and proliferation induction capacity of CD8 + T cells and compared with the single HTNV CTL epitope in 17 HLA-A*02 + patients with HFRS. The Mann–Whitney U test was used for comparison of parameters between different subject groups. Results The macromolecular HTNV MAP was designed with a polylysine core and four radially branched single CTL epitope chains. Importantly, HTNV MAP could stimulate CD8 + T cell secretion of IFN-γ in HLA-A*02 + patients with HFRS. The frequency of IFN-γ-secreting CD8 + T cells in the MAP stimulation group was significantly higher than that in the single HTNV CTL epitope stimulation groups ( P < 0.005). Meanwhile, the activity of IFN-γ-secreting CD8 + T cells in the HTNV MAP group was also higher than that of the single CTL epitope groups ( P < 0.05). Moreover, there was a much stronger ability of HTNV MAP to stimulate CD8 + T cell proliferation compared with that of a single HTNV CTL epitope. Conclusions The designed HTNV MAP could induce CTL responses ex vivo and may be considered a candidate for the design and development of novel HTNV peptide vaccines.
Abstract Background: An effective vaccine that prevents disease caused by hantaviruses is a global public health priority, but up to now, no vaccine has been approved for worldwide use. Therefore, novel vaccines with high prophylaxis efficacy are urgently needed. Methods: Herein, we designed and synthesized Hantaan virus (HTNV) linear multi-epitope peptide consisting of HLA-A*02-restricted HTNV cytotoxic T cell (CTL) epitope and pan HLA-DR-binding epitope (PADRE), and evaluated the immunogenicity, as well as effectiveness, of multi-epitope peptides in HLA-A2.1/K b transgenic mice with interferon (IFN)-γ enzyme-linked immunospot assay, cytotoxic mediator detection, proliferation assay and HTNV-challenge test. Results: The results showed that a much higher frequency of specific IFN-γ-secreting CTLs, high levels of granzyme B production, and a strong proliferation capacity of specific CTLs were observed in splenocytes of mice immunized with multi-epitope peptide than in those of a single CTL epitope. Moreover, pre-immunization of multi-epitope peptide could reduce the levels of HTNV RNA loads in the liver, spleen and kidneys of mice, indicating that specific CTL responses induced by multi-epitope peptide could reduce HTNV RNA loads in vivo. Conclusions: This study may provide an important foundation for the development of novel peptide vaccines for HTNV prophylaxis.
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