Advancing novel vaccines: Preclinical evaluation in HLA-A2.1/HLA-DRB1*1.1 humanized mice

The human leukocyte antigen (HLA) system plays a central role in orchestrating adaptive immune responses. While existing single-allele humanized models have been valuable, there is a growing need for preclinical platforms that more fully recapitulate human HLA class I and II restricted immunity to evaluate complex immunotherapies. To address this, we developed a novel MHC I/II dual-humanized mouse model, the B-HLA-A2.1/HLA-DRB1*1.1 mouse.The B-HLA-A2.1/HLA-DRB1*1.1 model was generated by co-integrating the human HLA-A02:01 and HLA-DRB1*01:01 genes into the C57BL/6 background. Human HLA-A2.1 and HLA-DRB1 protein expression was confirmed by flow cytometry on spleen, peripheral blood, and bone marrow leukocytes. Comprehensive immune cell profiling was performed to characterize T and B cell populations. The model's functionality was validated in a therapeutic tumor vaccination study using a human epitope-targeting vaccine.Flow cytometric analysis confirmed stable and robust cell surface expression of both HLA-A2.1 and HLA-DRB1 proteins in homozygous mice. The frequency of CD8+ T cells in the spleen, blood, and lymph node was significantly decreased, while the frequency of CD4+ T cells was significantly increased, demonstrating that the introduction of HLA-A02:01 and HLA-DRB1*01:01 affected the development of T cells. Additional analyses, including hematology, serum biochemistry, and histopathological examination (H&E staining) of major organs, indicated no spontaneous pathology, confirming the overall health and physiological stability of the model. In a vaccine efficacy study, immunization with a human antigen-specific vaccine significantly inhibited tumor growth in the dual-humanized mice compared to controls, demonstrating the model's capability to mount a functional HLA-restricted immune response.We have successfully developed and preliminarily validated the novel B-HLA-A2.1/HLA-DRB1*1.1 dual-humanized mouse model. It represents a powerful and physiologically relevant preclinical tool for investigating combination immunotherapies, including vaccines and immune checkpoint inhibitors, and for studying anti-tumor and infectious disease immunity within a more complete human-like context.