Humanized Major Histocompatibility Complex Transgenic Mouse Model Can Play a Potent Role in SARS-CoV-2 Human Leukocyte Antigen-Restricted T Cell Epitope Screening

Abstract

Background: COVID-19, caused by SARS-CoV-2, poses a significant threat to human health. Vaccines designed for T-cell epitopes play an important role in eliminating the virus. However, T cell epitope screening often requires the use of a large number of peripheral blood mononuclear cells (PBMCs) from infected or convalescent patients, and if MHC humanized mice can be used for epitope screening, they will not have to wait for enough PBMCs to be available to screen for epitopes, thus buying time for epitope confirmation and vaccine design. Methods: In this study, we used SARS-CoV-2 BA.5 to infect HLA-A11/DR1, C57BL/6, hACE2 mice, and detected body weight changes, viral load, and pathological changes after infection. Fourteen days after the HLA-A11/DR1 and C57BL/6 mice were immunized against inactivated viruses, IgG antibodies were detected in mouse serum using ELISA, and IFN-γ produced by peptide stimulation of splenocytes was detected by ELISpot. Results: There is no obvious pathogenic phenotype of SARS-CoV-2 infection in HLA-A11/DR1 mice. Specific IgG antibodies were detected in serum after immunization of inactivated virus in both HLA-A11/DR1 and C57BL/6 mice, but specific IFN-γ was detected in splenocytes of HLA-A11/DR1 mice. Conclusions: Although HLA-A11/DR1 mice are unable to replicate the virus effectively in vivo, they are able to generate cellular immune responses after immunization inactivated viruses. Therefore, it can be used as a tool to substitute for human PBMCs in epitope screening, thus shortening the timeliness of T cell epitope screening and obtaining the immunogenicity information of new epitopes in a timely manner.

Keywords: SARS-CoV-2; epitope; human leukocyte antigen (HLA) complex; immunological evaluation; mouse model.

Figure 1

Weight changes and viral load in HLA-A11/DR1 mice infected with SARS-CoV-2. Mice were divided into three groups—HLA-A11/DR1, C57BL/6, and hACE2—and intranasally infected with 1 × 10⁴ TCID₅₀ SARS-CoV-2 (BA.5 strain). Mice were weighed every 2 days over 14 days, and weight changes were expressed as percentages. On dpi 3, lung tissues were collected: the lung for histopathology, SARS-CoV-2 nucleocapsid protein detection and for viral load detection. (A) Weight changes in the three mouse groups after intranasal inoculation with the BA.5 virus strain. (B) Viral load detection in lung tissues of the three groups post-infection. (C) **** p < 0.0001.

Figure 2

Histological and viral antigen detection in mouse lung tissues after SARS-CoV-2 infection. Mice were euthanized on dpi 3. The lung was fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and stained with H&E for pathological scoring. Lung tissue histology (original magnification: 10×, scale bar: 200 μm) (A). Distribution of SARS-CoV-2 nucleocapsid protein on lung tissue surfaces (original magnification: 10×, scale bar: 200 μm) (B).

Figure 3

The titer of specific antibodies in the serum of HLA-A11/DR1 mice 14 days post-vaccination with inactivated SARS-CoV-2 (A). The titer of specific antibodies in the serum of C57BL/6 mice 14 days after vaccination with inactivated SARS-CoV-2 (B). * p < 0.05, ** p < 0.01.

Figure 4

Screening for HLA-restricted positive epitope peptides. We selected 14 HLA-restricted epitope peptides from the novel coronavirus epitope library established by our research group, including nine HLA-A*11:01-restricted epitope peptides (represented in purple): pA1: GVYFASTEK [27,28,29], pA2: GVYYHKNNK [28,30], pA3: CTLKSFTVEK [28], pA4: TLKSFTVEK [30], pA5: ASVYAWNRK [30], pA6: NSASFSTFK [28], pA7: KCYGVSPTK [31], pA8: RLFRKSNLK [29], pA9: KSTNLVKNK [32]. It also included five HLA-DRB1*01:01-restricted epitope peptides (represented in blue): pB1: FKIYSKHTPINLVRD [33], pB2: TRFQTLLALHRSYLT [33,34], pB3: LKPFERDISTEIYQA [35], pB4: YQPYRVVVLSFELLHAPATV [36], pB5: LSFELLHAPATVCGPKKSTN [36].

Figure 5

The cell-mediated immune response of HLA-A11/DR1 transgenic mice was tested using positive epitope peptides. Four HLA-A11/DR1 mice and four C57BL/6 mice were intramuscularly injected with inactivated SARS-CoV-2 virus. Fourteen days after vaccination, the mice were euthanized and their spleen tissues were collected. The spleen cells were stimulated with 14 selected HLA-restricted epitope peptides, and the production of IFN-γ in the spleen cells was detected by ELISpot. The study was analyzed by the Mabtech IRIS FluoroSpot/ELISpot reader (Apex 1.1), using RAWspot technology for multiplexing at the single-cell level. * p < 0.05, ** p < 0.01, “ns” means no significance.