• The B2M encodes a serum protein found in association with the major histocompatibility complex (MHC) class I heavy chain on the surface of nearly all nucleated cells. The protein has a predominantly beta-pleated sheet structure that can form amyloid fibrils in some pathological conditions. HLA-A belongs to the HLA class I heavy chain paralogues. The heavy chain is anchored in the membrane. Class I molecules play a central role in the immune system by presenting peptides derived from the endoplasmic reticulum lumen so that they can be recognized by cytotoxic T cells. They are expressed in nearly all cells. HLA-DRA is one of the HLA class II alpha chain paralogues. HLA-DRB1 belongs to the HLA class II beta chain paralogs. This class II molecule is a heterodimer consisting of an alpha (DRA) and a beta (DRB) chain, both anchored in the membrane. It plays a central role in the immune system by presenting peptides derived from extracellular proteins. Class II molecules are expressed in antigen presenting cells (APC: B lymphocytes, dendritic cells, macrophages). The alpha chain is approximately 33-35 kDa and its gene contains 5 exons. The beta chain is approximately 26-28 kDa and its gene contains 6 exons. DRA does not have polymorphisms in the peptide binding part and acts as the sole alpha chain for DRB1, DRB3, DRB4 and DRB5.
• The B2m gene (Exon1 to Exon3) of mouse was replaced by the sequence encompassing the human B2M CDS and HLA-A*0201 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the murine H-2D^b gene containing the α3, transmembrane and cytoplasmic domains. The exon 2 of mouse I-Ea gene that encodes the extracellular domain of I-Ea was replaced by human HLA-DRA encoding the α1 domain in B-HLA-A2.1/HLA-DRB1*1.1 mice. The exon 2 of mouse I-Eb1 gene that encodes the extracellular domain of I-Eb1 was replaced by human HLA-DRB encoding the β1 domain in B-HLA-A2.1/HLA-DRB1*1.1 mice. The  mouse I-Aa, I-Ab1 and I-Eb2 were knocked out in B-HLA-A2.1/HLA-DRB1*1.1 mice.
• Human B2M, HLA-A2.1, and HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.
• B-HLA-A2.1/HLA-DRB1*1.1 mice provide a powerful preclinical model for in vivo evaluation of vaccines.
• Application: For example, this product is used for pharmacodynamics and safety evaluation of vaccines for cancers.

Gene targeting strategy for B-HLA-A2.1/HLA-DRB1*1.1 mice.

The B2m gene (Exon1 to Exon3) of mouse was replaced by the sequence encompassing the human B2M CDS and HLA-A*0201 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the murine H-2D^b gene containing the α3, transmembrane and cytoplasmic domains.

The exon 2 of mouse I-Ea gene that encodes the extracellular domain of I-Ea was replaced by human HLA-DRA encoding the α1 domain in B-HLA-A2.1/HLA-DRB1*1.1 mice. The exon 2 of mouse I-Eb1 gene that encodes the extracellular domain of I-Eb1 was replaced by human HLA-DRB encoding the β1 domain in B-HLA-A2.1/HLA-DRB1*1.1 mice. The  mouse I-Aa, I-Ab1 and I-Eb2 were knocked out in B-HLA-A2.1/HLA-DRB1*1.1 mice.

Strain specific B2M and HLA expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice and homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, and analyzed by flow cytometry with species-specific anti-mouse B2M antibody (BD, 744802), anti-human B2M (Biolegend, 395712), anti-mouse H-2Db (Biolegend, 111513) and anti-human HLA-ABC (Biolegend, 311406). Mouse B2M and H-2Db were only detectable in wild-type mice. Human B2M and HLA-A2.1 were exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Blood cells were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Bone marrow cells were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Bone marrow cells were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Strain specific HLA-DRB1 expression analysis in wild-type (WT) C57BL/6JNifdc mice and homozygous (HO) B-HLA-A2.1/HLA-DRB1*1.1 mice by flow cytometry. Bone Marrow cells were collected from wild-type C57BL/6JNifdc (+/+) and homozygous humanized B-HLA-A2.1/HLA-DRB1*1.1 mice, respectively, and analyzed by flow cytometry with species-specific anti-mouse I-A/I-E antibody (Biolegend, 107607), and species-specific anti-human HLA-DR antibody (Biolegend, 307610). Human HLA-DRB1 was exclusively detectable in homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice, but not in wild-type C57BL/6JNifdc mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6JNifdc mice and homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice (male, 8-week-old, n=3). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of T cells, B cells, NK cells, dendritic cells, monocytes, macrophages, neutrophils and Tregs in B-HLA-A2.1/HLA-DRB1*1.1 mice were similar to those in C57BL/6JNifdc mice. The frequency of CD8+ T cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were higher than that in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.0001.

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood were isolated from wild-type C57BL/6JNifdc mice and homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice (male, 8-week-old, n=3). A. Flow cytometry analysis of the blood was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of T cells, NK cells, dendritic cells, monocytes, macrophages, and Tregs in B-HLA-A2.1/HLA-DRB1*1.1 mice were similar to those in C57BL/6JNifdc mice. The frequency of neutrophils in B-HLA-A2.1/HLA-DRB1*1.1 mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of B cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were higher than that in C57BL/6JNifdc mice. The frequency of CD8+ T cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were higher than that in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.0001.

Frequency of leukocyte subpopulations in lymph node by flow cytometry. Lymph node cells were isolated from wild-type C57BL/6JNifdc mice and homozygous B-HLA-A2.1/HLA-DRB1*1.1 mice (male, 8-week-old, n=3). A. Flow cytometry analysis of the lymph node was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Frequencies of NK cells, and Tregs in B-HLA-A2.1/HLA-DRB1*1.1 mice were similar to those in C57BL/6JNifdc mice. The frequency of T cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were higher than that in C57BL/6JNifdc mice. The frequency of CD8+ T cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were lower than that in C57BL/6JNifdc mice, whereas the frequency of CD4+ T cells in B-HLA-A2.1/HLA-DRB1*1.1 mice were higher than that in C57BL/6JNifdc mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.0001.

Antitumor activity of NY-ESO-1 mRNA vaccine against syngeneic tumors. (A) Experimental scheme. (B) Antitumor activity of therapeutic treatment with mRNA vaccine. B-HLA-A2.1/HLA-DRB1*1.1 mice (n=6/group) were vaccinated with PBS, LNP or LNP-mRNA. B-HLA-A2.1/HLA-DRB1*1.1 mice were implanted subcutaneously with 1×10⁶ B-HLA-A2.1/hNY-ESO-1 MC38 tumor cells in the right flank and immunized four times at 1-week intervals with PBS, LNP or mRNA vaccines via intravenous (i.v.) injection on days 0, 7, 14 and 21 after tumor volume reached approximately 100 mm³. As shown in panel B, the mRNA vaccines demonstrated robust inhibition of tumor growth and significantly prolonged overall survival compared to the control group. C. Body weight changes during tumor growth observation. These results demonstrate that B-HLA-A2.1/HLA-DRB1*1.1 mice provide a powerful preclinical model for in vivo evaluation of mRNA vaccines. Values are expressed as mean ± SEM.