Screening and Identification of an H-2Kb-Restricted CTL Epitope within the Glycoprotein of Hantaan Virus

Abstract

The cytotoxic T lymphocyte (CTL) response plays a key role in controlling viral infection, but only a few epitopes within the HTNV glycoprotein (GP) that are recognized by CTLs have been reported. In this study, we identified one murine HTNV GP-derived H2-K^b-restricted CTL epitope in C57BL/6 mice, which could be used to design preclinical studies of vaccines for HTNV infection. First, 15 8-mer peptides were selected from the HTNV GP amino acid sequence based on a percentile rank of <=1% by IEDB which is the most comprehensive collection of epitope prediction and analysis tool. A lower percentile rank indicates higher affinity and higher immune response. In the case of the consensus method, we also evaluated the binding score of peptide-binding affinity by the BIMAS software to confirm that all peptides were able to bind H2-K^b. Second, one novel GP-derived CTL epitope, GP6 aa456-aa463 (ITSLFSLL), was identified in the splenocytes of HTNV-infected mice using the IFN-γ ELISPOT assay. Third, a single peptide vaccine was administered to C57BL/6 mice to evaluate the immunogenic potential of the identified peptides. ELISPOT and cell-mediated cytotoxicity assays showed that this peptide vaccine induced a strong IFN-γ response and potent cytotoxicity in immunized mice. Last, we demonstrated that the peptide-vaccinated mice had partial protection from challenge with HTNV. In conclusion, we identified an H2-K^b-restricted CTL epitope with involvement in the host immune response to HTNV infection.

Keywords: HTNV; IFN-γ; epitope; glycoprotein; infection.

Figure 1

Identification of an HTNV GP-specific CD8⁺ T cell epitope by IFN-γ ELISPOT assay. (A) IFN-γ ELISPOT analysis of splenocytes obtained 7 d after HTNV infection (n = 6). Cells were stimulated with 15 8-mer peptides corresponding to HTNV glycoprotein. (B) IFN-γ ELISPOT analysis of splenocytes obtained 7 d after infection (n = 6) with the depletion of CD4⁺ T cells (CD4 depleted SP) and CD8⁺ T cells (CD8 depleted SP) or without depletion (Total SP). ^***P < 0.0001.

Figure 2

Cytotoxic activity of splenocytes derived from HTNV-infected mice in vitro. The primary antiviral CTL responses were first detected. Splenocytes from C57BL/6 mice infected with HTNV (1 × 10⁵ pfu/head) were used as effector cells. As targets, EL-4 cells were pulsed with GP6 (A) and NP1 (B) at a concentration of 10 μg/mL. Splenocytes that were restimulated with GP6 (C) and NP1 (D) were used as effector cells in the second round of the CTL assay. Macrophages infected with HTNV (■) and peptide-pulsed EL-4 cells (•) were used as target cells. P815 cells (▴) were used as negative controls. (E) Evaluation of the mean lysis percentage of naïve CD8+T cells to kill no peptide-pulsed target cells. Macrophages (■), EL-4 cells (•), and P815 cells (▴) were served as target cells, respectively. Data are expressed as the mean ± SEM (n = 6). ^**P < 0.001, ^***P < 0.0001.

Figure 3

The rapid secretion of IFN-γ after recognition of the HTNV-specific peptide. (A) Representative flow cytometric plots of IFN-γ production by peptide-specific CD8⁺ T cells. The upper lane (A) shows the results 4 h after peptide restimulation and both negative and positive controls, and the lower lane (B) shows the frequency of IFN-γ production in CD8⁺ T cell populations. Data are expressed as the mean ± SEM (n = 6). ^***P < 0.0001.

Figure 4

The proliferative capacity of HTNV peptide-specific CD8⁺ T cells. Representative flow cytometric histogram of the expansion percentage of CD8⁺ T cells stimulated by the GP6 (A) and NP1 (B). Anti-mouse CD3 (Biolegend) (D) stimulation or no peptide (C) stimulation served as positive or negative controls, respectively. (E) The statistical data of the proliferation assay. Data are expressed as the mean percentage ± SEM (n = 6). ^*P < 0.05.

Figure 5

The enhanced cellular immune responses in peptide-immune C57BL/6 and the timeline of immunization. (A) Scheme of peptide immunization. Mice were primed and boosted with GP6 and NP1 packaged with Freud's adjuvants (Pep Immun group) or PBS (Mock group). Ten days after the final boost, splenocytes from mice were harvested for subsequent assays. (B) IFN-γ ELISPOT analysis of splenocytes obtained from HTNV peptide-immunized mice. Cells were stimulated with purified HTNV NP. (C) Splenocytes from immunized mice, restimulated with relative peptide were used as effector cells. As targets, EL-4 cells were pulsed with GP6 or NP1 at a concentration of 10 μg/ml. (■) represents NP1 immunization; (•) represents GP6 immunization; (▴) represents a positive control from the HTNV-challenged mice; (□) represents vaccine control; (◦) represents PBS control; (▾) represents adjuvant control. Data are expressed as the mean ± SEM (n = 6). ^***P < 0.0001.

Figure 6

Detection of HTNV-specific antigens from peptide-immunized mice by ELISA. ELISA was used to detect HTNV antigens in the tissues of each group including the cerebrum (A), heart (B), liver (C), spleen (D), lung (E), and kidney (F). (•) represents the GP6 peptide-immunized group; (■) represents the NP1 peptide-immunized group; (▴) represents the inactivated Hantavirus vaccine control group; (◦) represents the PBS control group; (♦) represents the adjuvant control group. Data are expressed as the mean ± SEM (n = 4). ^*P < 0.05, ^**P < 0.001.

Figure 7

qRT-PCR results of HTNV-specific nucleic acids in organs of peptide-immunized mice. This method detected HTNV nucleic acid in the tissues of each group including the cerebrum (A), heart (B), liver (C), spleen (D), lung (E), and kidney (F). The viral nucleic acids are distributed mainly in the spleen and liver, but to a lesser degree in the kidney. Data are expressed as the mean ± SEM (n = 6). ^**P < 0.001, ^***P < 0.0001.

Figure 8

Histopathological analysis of the spleens from peptide-immunized mice after challenge with HTNV by H&E staining. (A) The GP6 peptide-immunized group. (B) The NP1 peptide-immunized group. (C) The inactivated Hantavirus vaccine group. (D) The PBS control group. (E) The adjuvant control. Red arrows show diffused lymphocyte infiltration, diffused hemorrhaging and increased white pulp in the spleens of two control groups.