HLA-A2.1/Kb transgenic (Tg) mice, which were originally purchased from the Jackson Laboratory (BarHarbor, ME), were kindly provided by Dr. Yuzhang Wu (Third Military Medical University, Chongqing, China). All Tg mice were maintained under pathogen-free conditions with good welfare in the Animal House Facility at the Center for Laboratory Animal, Fourth Military Medical University, Xi’an, China. A chimeric gene, consisting of the α1 and α2 domains of HLA-A*0201 and the α3 transmembrane and cytoplasmic domain of H-2Kb, was represented in the Tg mice with C57BL/6 background. HLA-A*02 expression on the cell surface was detected by phycoerythrin (PE)-labelled anti-HLA-A*02 mAb BB7.2 (Biolegend, San Diego, CA, USA) in flow cytometry.
Peptide design and synthesis
Based on our previous study, the identified HLA-A*02 restricted HTNV-GP CD8+ T-cell epitope (aa8-aa16, VMASLVWPV, VV9) were selected to construct multi-epitope peptides, given that epitope VV9 has been proven to bind with HLA-A*02 with highest affinity among the seven HLA-A*02-restricted HTNV-GP CTL epitope . The Pan HLA-DR T-helper epitope (PADRE, AKXVAAWTLKAAA, X = cyclohexylalanine)  was used to connect the HTNV epitopes in series. The linear multi-epitope peptide consists of the HLA-A*02-restricted HTNV CTL epitope (VV9) in tandem with PADRE, defined as “PADRE-VV9”. The K-S-S was used as the adaptor sequence and the A-A-A in PADRE could be used as the linker sequence for the connection of the multi-epitope peptide. All the peptides were synthesized and characterized by analytical high-performance liquid chromatography (HPLC) and mass spectrometry by Synpeptide. Co. Ltd (Nanjing, China), with purity > 95%. Lyophilized peptides were resuspended in sterile phosphate-buffered saline (PBS) solution at 1 mM and stored in aliquots at − 80 °C until further use.
Vaccine and Virus
The commercial bivalent and purified HFRS inactivated vaccine (YOUERJIAN®, Zhejiang Tianyuan Bio- Pharmaceutical Co., Ltd., China) composed of a mixture of both HTNV and SEOV was provided as a positive control in the immunization of the Tg mice. The HTNV 76-118 strain was preserved and kindly provided by the Department of Microbiology of the Fourth Military Medical University.
Immunization of HLA-A2.1/Kb Tg mice with the peptide
Fifty male six-to-eight-week-old HLA-A2.1/Kb Tg mice were divided into five groups (n = 10 each) for immunization, including the single HTNV CTL epitope VV9 immunization group, HTNV linear multi-epitope peptide PADRE-VV9 immunization group, unrelated peptide HLA-B*35-restricted HTNV CTL epitope (VPILLKALY, VY9) immunization group , inactivated vaccine inoculation group and PBS injection group. The peptide immunizations of the Tg mice were carried out using an N-terminal fragment of the murine glycoprotein 96 (gp96) as a chaperone [30, 31]. The heat shock protein gp96 and its N-terminal fragment (N333) showed adjuvant effects in enhancing the peptide-specific CTL response through binding with peptide. The association of gp96 with MHC ligands may indicate its role in MHC class I peptide processing and presentation . Meanwhile, complete/incomplete Freund's adjuvants were also used for Tg mice immunization. The Mycobacteria in complete Freund′s adjuvant could attract macrophages and other cells to the injection site, which enhances the immune response. Therefore, complete Freund′s adjuvant is used for the initial injections. While incomplete Freund′s adjuvant, which lacks the Mycobacteria, is used for subsequent boost injections.
Briefly, for the first-time immunization, 50 μg peptide was mixed with 30 μg of the N-terminal fragment N333 (aa22-aa355) of murine gp96. Then, the mixture was emulsified in 1:1 ratio with complete Freund’s adjuvant (Difco), and was adjusted to 100 μL of the injection volume for each Tg mouse. The immunization was performed via subcutaneous injection at 4–5 locations on the neck and back of each mouse. The dose of each injection location was about 20–25 μL. The same immunization methods were carried out for the second- and third-round immunization of the Tg mice with the peptide and gp96 mixture emulsified in 1:1 ratio with incomplete Freund’s adjuvant (Difco). Each peptide was prepared separately using the same method. Replacing the peptide with PBS, and then immunizing Tg mice with the same method, was used as a negative control group. Subcutaneous injections with the commercialized HFRS inactivated vaccine were used as the positive control groups. Specifically, 10 μL of the injection volume of the commercialized HFRS inactivated vaccine was used for each round immunization in each Tg mouse . The inactivated vaccine immunization was performed via subcutaneous injection at one location on the neck of each mouse. The same method was then used for the second- and third-round immunization of inactivated vaccine in Tg mice. All Tg mice were injected the same number of times. Three immunization injections were administered to each mouse at intervals of two weeks. Ten days after the last immunization, half number of the mice (n = 5) in each immunized group was euthanised by cervical vertebra dislocated method. The splenocytes were isolated from each mouse using lymphocyte separation medium (Dakewe Biotech Company Ltd, Shenzhen, China).
The remaining immunized HLA-A2.1/Kb Tg mice (n = 5) in each group were subsequently challenged with intramuscular injection of the HTNV 76-118 strain (1 × 105 pfu/mouse) ten days after the final immunization booster. On day 4 post-challenge, the Tg mice were sacrificed. Tissue samples from six major organs, including cerebrum, heart, liver, spleen, lung and kidney, were harvested for viral load detection.
Determination of relative HTNV RNA loads with quantitative RT-PCR
The tissue samples of the six major organs from post-exposure Tg mice were preserved in a non-frozen tissue RNA preservation solution (Solarbio, CN) at − 4 °C. An RNA purification extraction kit (Tiangen Biotech, CN) was used to extract the tissue RNA of each type of organ. The extracted tissue RNA was then used as a template for reverse transcription to obtain the cDNA with PrimeScript™ RT-PCR kit (Takara). The target sequence of the HTNV S segment was detected for determination of the relative HTNV RNA loads in each organ sample using an SYBR real-time quantification PCR kit (Takara) with the following primers: HTNV forward, 5′-GATCAGTCACAGTCTAGTCA-3′; HTNV reverse, 5′-TGATTCTTCCACCATTTTGT-3′; mouse GAPDH forward, 5′-AGGCCGGTGCTGAGTATGTC-3′; mouse GAPDH reverse, 5′-TGCCTGCTTCACCACCTTCT-3′. The results were recorded as cycle time (Ct) and quantified by 2−ΔΔCt.
Ex vivo IFN-γ enzyme-linked immunospot (ELISPOT) assay
The IFN-γ ELISPOT assay (Dakewe Biotech Company Ltd, Shenzhen, China) was used for the determination of the immunogenicity of the HTNV linear multi-epitope peptide ex vivo . Briefly, the splenocytes isolated from immunized HLA-A2.1/Kb Tg mice in each group were placed on ELISPOT plates (IFN-γ mAb precoated), at 1 × 106 cells/well. Cells were stimulated with the single HTNV CTL epitope (10 μM) for 24 h at 37 °C. Phytohemagglutinin (PHA, 10 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) stimulation served as a positive control, while the irrelevant peptide and media alone served as negative controls. Spots developed after incubation for 10–30 min with 3-amino-9-ethylcarbazole (AEC) substrate in the dark and counted with an ELISPOT plate reader (Cellular Technology Limited, USA). The number of spots was expressed as adjusted spot-forming cells (SFC)/106 splenocytes after subtracting the average negative values. The positive response was defined as at least 50 SFC/106 input cells, exceeding 3-times the background response. The SFC/106 splenocytes in the unstimulated control wells never exceeded 5 spots per well.
Intracellular cytokine staining and degranulation assay
Splenocytes (2 × 106) obtained from the immunized HLA-A2.1/Kb Tg mice were restimulated with HTNV single CTL epitope (5 μM) in the presence of 1 μg/mL costimulatory molecules anti-mouse CD49d mAb (clone 9C10) and anti-mouse CD28 mAb (clone 37.51) (Biolegend, San Diego, CA, USA). One hour after peptide stimulation, 10 μg/mL brefeldin A (Sigma-Aldrich, St. Louis, MO, USA) and 0.7 μl/mL monensin (Golgistop, BD Biosciences, San Jose, CA, USA) were added, with an additional 5 h incubation at 37 °C. Cells stimulated with 0.1 μg/mL phorbol myristate acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA) and 0.05 μg/mL ionomycin (Sigma-Aldrich, St. Louis, MO, USA) or medium alone were used as positive or negative controls, respectively. The cells were washed and stained for surface markers with anti-mouse CD3-PerCP-Cy5.5 (clone 17A2) and anti-mouse CD8α-APC (clone 53-6.7) mAbs (Biolegend, San Diego, CA, USA), fixed and permeabilized using a Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit (BD Pharmingen, San Diego, CA, USA), and then stained with anti-mouse granzyme B-FITC mAb (clone GB11) (Biolegend, San Diego, CA, USA). FITC-, PerCP-Cy5.5- and APC-conjugated mouse IgG1, κ were used as isotype controls. A total of 200,000 events per sample were collected using FACSCalibur™ (BD Biosciences, San Jose, CA, USA). The analysis was performed immediately with FlowJo version 9.2 (TreeStar, Ashland, OR, USA). Splenocytes were defined as FSC/SSC, and CD8+ T-cells were defined as CD3+CD8+ events, displayed on a dot plot of CD8 versus granzyme B. The positive response was determined as being when the percentage of cytokine was greater than 0.1% after background subtraction.
Ex vivo proliferation assay
Splenocytes (2 × 107/mL) from the immunized Tg mice were labeled with 10 μM 5,6-carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes, Eugene, OR, USA) at 37 °C for 15 min, and terminated upon the addition of equivoluminal fetal bovine serum without dilution. Then, the CFSE-labeled cells were stimulated with HTNV single CTL epitope (10 μM). PHA (10 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) stimulation served as a positive control. Cells without stimulation were used as background control. After 2 days, 10% exogenous IL-2 (20 U/mL) was added. After 7 days from the peptide stimulation, the cells were harvested and stained with the anti-mouse CD3-PerCP-Cy5.5 (clone 17A2), anti-mouse CD8-APC (clone 53-6.7) mAbs (Biolegend, San Diego, CA, USA). Approximately 300,000 cells were acquired using a FACSCalibur™ (BD Biosciences, San Jose, CA, USA).
Statistical analysis was performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA) and graphing was performed using GraphPad Prism software, version 6 (GraphPad; La Jolla, CA, USA). The frequency of the CD8+ T-cells and the cytokines secreted are presented as the mean ± standard error of mean (SEM). The unpaired t test was used for comparison of the parameters between the two subject groups. A P value below 0.05 (P ≤ 0.05) was considered to be statistically significant.
HTNV 76-118 strain glycoprotein GenBank accession number: P08668.1; HTNV 76-118 strain nucleoprotein GenBank accession number: M14626.
The research was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Health and Medical Research Council of China. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Fourth Military Medical University with the license number XJYYLL-2014437. All procedures were performed under sodium pentobarbital anesthesia, with the best effort to minimize animal suffering.