Comparison of model applicability between HLA-A2.1Tg/+huHSC-reconstituted mice and Cynomolgus monkeys in mRNA drug immunogenicity evaluation
10.13200/j.cnki.cjb.004741
- VernacularTitle:HLA-A2.1Tg/+huHSC免疫重建小鼠与食蟹猴在mRNA药物免疫原性评价中模型适用性的比较
- Author:
Qiyao LIU
- Publication Type:Journal Article
- Keywords:
mRNA medicines;
Immunogenicity;
Ovalbumin(OVA);
HLA-A2.1~(Tg/+)huHSC-reconstituted mice;
Cynomolgus monkeys
- From:
Chinese Journal of Biologicals
2026;39(07):777-787
- CountryChina
- Language:Chinese
-
Abstract:
ObjectiveTo compare the model applicability of HLA-A2.1~(Tg/+)huHSC immune reconstitution mice and Cynomolgus monkeys in the immunogenicity evaluation of mRNA medicines, and to explore the possibility of immune reconstitution mice as a supplementary or alternative model of non-human primate(NHP) models with C57 BL/6 mice as a reference, in order to provide experimental basis for the model selection of preclinical immunogenicity evaluation of mRNA medicines.MethodsUsing ovalbumin(OVA)-mRNA as a model drug, four female SPF-grade C57 BL/6 mice(10 ??g/mouse), four female HLA-A2.1~(Tg/+)huHSC-reconstituted mice(10 ??g/mouse) and three female Cynomolgus monkeys(30 ??g/mouse) were immunized by intramuscular injection, once a week for three consecutive doses. Blood was collected from the orbit of two kinds of mice to separate serum, and the mouse spleen was aseptically taken to prepare a spleen cell suspension 22 days(D22) after the first administration. Blood was collected from the hind limb vein of Cynomolgus monkeys before administration and on D7, D15, and D22 after administration, and peripheral blood mononuclear cells(PBMCs) were prepared from peripheral blood by Ficoll density gradient centrifugation on D22. Flow cytometry was employed to detect the proportions of T cells(CD3+,CD4+, CD8+activated subsets and effector subsets) and B cells(B cells, activated B cells, memory B cells, plasma cells) in mouse spleen cells and Cynomolgus monkey whole blood. ELISpot was applied to measure the number of IFNγ spots secreted by mouse spleen cells and Cynomolgus monkey PBMCs after stimulation with OVA peptides(OVA_(257-264)and OVA_(323-339)). ELISA was used to determine the levels of OVA-specific antibodies(IgG/IgM) in the serum of mice and Cynomolgus monkeys.ResultsCompared with C57 BL/6 mice, the proportions of CD3+ and total CD8+ T cells in the spleen of HLA-A2.1~(Tg/+)huHSCreconstituted mice significantly decreased(F = 4. 046 and 5. 290, respectively, P < 0. 01), while the proportion of total CD4+T cells significantly increased(F = 6. 231, P < 0. 01); the proportions of both activated CD4+ and CD8+ T cells were significantly elevated(F = 42. 280 and 130. 100, respectively, each P < 0. 01), while the proportions of effector CD4+ and CD8+ T cells were significantly reduced(F = 8. 067 and 5. 160, respectively, P < 0. 01 and < 0. 05, respectively). The numbers of IFNγ spots in immunologically reconstituted mice after stimulation with OVA_(257-264)and OVA_(323-339)peptides were significantly lower than those in C57 BL/6 mice(each P < 0. 01). The proportions of B cells and memory B cells in immunologically reconstituted mice were higher than those in C57 BL/6 mice(F = 4. 578 and 1. 469, P < 0. 01 and P < 0. 05, respectively), but the difference in the proportion of plasma cells was not statistically significant(F = 1. 178, P > 0. 05). The level of OVA-specific antibodies in the serum of C57 BL/6 mice was significantly higher than that of immundogically reconstituted mice(P < 0. 05).The proportions of CD3+ and total CD8+ T cells in Cynomolgus monkeys in the OVA-mRNA group showed an increasing trend after administration, with the highest increases of(20. 07 ± 4. 33)% and(34. 37 ± 2. 61)%, respectively(F = 4. 260 and2. 865, respectively, each P = 0. 05), and there was no significant difference in the proportions of total CD4+, activated CD4+,activated CD8+, effector CD4+ and effector CD8+ T cells(F = 0. 359-3. 516, each P > 0. 05), compared with those before administration. Compared with the PBS group, the proportions of activated B cells in Cynomolgus monkeys of the OVAmRNA group significantly increased on D15 and D22 after administration(F = 3. 514, P = 0. 05), while the proportion of plasma cells was significantly reduced on D22 after administration(F = 7. 000, P < 0. 05); there was no significant difference in the numbers of IFNγ spots after stimulation with OVA peptides( F = 1. 480 and 3. 129, respectively, each P > 0. 05), but the levels of OVA-specific antibody in serum increased significantly(P < 0. 05).ConclusionAlthough HLA-A2.1~(Tg/+)huHSCreconstituted mice can capture early T cell activation signals, they cannot form complete cellular and humoral immune effects; although Cynomolgus monkeys did not produce obvious cellular immune response, they could produce significant OVA-specific humoral immune response, which still possesses unique advantages in humoral immune evaluation. Immune reconstitution mice can be used as an important supplement to conventional animal models, especially for capturing early human-specific cellular immune events, but they cannot completely replace NHP models such as Cynomolgus monkeys. The combined application of the two can provide more comprehensive immunogenicity evaluation data.
