Ps-bpb07-1: development of anti-thrombotic vaccine against human s100a9

Background & Purposes: In post-stroke patients, a decreased adherence to antiplatelet drugs is a major challenge in the prevention of recurrent stroke. Previously, we have reported that a newly designed antiplatelet vaccine against S100A9 could prevent thrombosis in the middle cerebral artery in mice by inhibiting S100A9/CD36 signaling in platelets. However, the difference in amino acid sequences in epitopes between mice and humans might be problematic for clinical use. In the present study, we redesigned the S100A9 vaccine for the common sequence in both humans and monkeys and examined its effects in cynomolgus monkeys. Methods: First, we screened several candidate epitopes in mice and the selected epitope in human S100A9, which is common in monkey S100A9, was conjugated to Keyhole limpet hemocyanin. A mixture of the vaccine and Alum adjuvant was subcutaneously injected into 4 cynomolgus monkeys, three times every two weeks. Antibody production against human S100A9 was examined by ELISA or western blotting. Anti-thrombotic effects in collagen-induced thrombosis were examined in a mixture of vaccinated serum and fresh whole blood from another cynomolgus monkey using an automated microchip flow-chamber system. Bleeding time was evaluated in two monkeys. Results: Based on results of computed epitope prediction, we first assessed whether 2 to 11, 97–106, or 104–113 amino acids, where the amino acid sequence is approximately similar between humans and monkeys, could raise antibodies against S100A9 in mice. Compared with the vaccine for the 2–11 amino acids epitopes, 97–106 amino acids and 104–113 amino acids demonstrated higher antigenicity, indicating that epitopes in the C-terminus are stronger than those in the N-terminus. Based on this result, we selected 102 to 112 amino acids as the epitope for the human S100A9 vaccine. When the monkeys were vaccinated, antibody titers against human S100A9 were significantly increased. Western blotting analysis showed that the produced antibody recognized human S100A9. Although the addition of pre-vaccinated serum into fresh whole blood did not affect thrombus formation, post-vaccination serum inhibited collagen-induced thrombosis formation in two monkeys. Anti-thrombotic effects were partially inhibited by the epitope peptide, indicating the feasibility of neutralizing anti-thrombotic effects by vaccination. Notably, prolongation of bleeding time was not observed in vaccinated monkeys. Conclusions: Anti-human S100A9 vaccine revealed anti-thrombotic effects in some monkeys without affecting bleeding time. Although further studies are necessary to increase the effects and warrant the safety aspects, this vaccine could clarify the use of an anti-thrombotic vaccine in clinical settings.