Balb ca

Specific pathogen-free female DBA/2J, ICR, C57BL/6J, and BALB/cA mice (6 weeks old) were purchased from CLEA Japan, Inc. (Tokyo, Japan), and A/JJms, C3H/HeJYok and ddY mice (6 weeks old) were obtained from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan). The animals were housed plastic cages in an air-conditioned room at 23 ± 2°C with a relative humidity of 55 ± 10% under a 12-hour light/dark cycle, fed a standard laboratory diet and given water ad libitum. Animal experiments were approved by the Institutional Animal Care and Use Committee for Kitasato University and performed in accordance with Guide for the Care and Use of Laboratory Animals in the Kitasato University and the National Research Council Guide for the Care and Use of Laboratory Animals in Japan.

All experiments were approved by the Ethical Committee for Animal Experiments of the Osaka University Graduate School of Medicine. TLR4 KO, MyD88 KO, TLR9 KO, and TRIF KO C57BL/6 mice were purchased from OrientalBioService, Inc. Six‐week‐old female BALB/cA and male C57BL/6 mice were purchased from CLEA Japan, Inc. Six‐week old male DIS/Eis (Dahl‐Iwai S) rats were purchased from Japan SLC, Inc and housed in a temperature‐ and light cycle‐controlled facility with free access to food and water. AJP001, AJP001‐Ang II, or AJP001‐DPP4‐1 were dissolved in saline and were injected intracutaneously on days 0, 14, and 28. Serum was collected from the tail vein, and antibody titers against immunized peptides were determined by ELISA. Immunization Study of AJP001‐DPP4‐2, 3, 4 were conducted at KAC Co., Ltd and complied with the guidelines on animal care of the local Ethics Committee on the Use of Animals. AJP001‐DPP4 vaccines were immunized intracutaneously at 2 weeks interval and boosted at 9 weeks after first immunization. The peptide sequences were shown in Table S1.

BALB/cA and C.B-17/Icr-scid/scid (SCID) mice were purchased from CLEA Japan, Inc (Tokyo, Japan). Fetuses were obtained from pregnant mice at day 15 from vaginal plugs were observed (d15). All mice were housed and maintained under specific pathogen-free conditions in Research Animal Facility of National Institute of Biomedical Innovation. All animal experiments were planned in accordance with the guidelines of animal use at the National Institutes of Biomedical Innovation, Health and Nutrition and approved by an animal experiment committee of the Institutes with permission numbers DS19-88 and DS20-59.