Lab mouse
Investigators designed an antiplatelet vaccine they say could be a novel therapy to prevent recurrent ischemic stroke.
The vaccine inhibits S100A9 (S100 calcium-binding protein A9)/CD36 (cluster of differentiation 36) signaling in platelets, which is a key signal in arterial thrombosis but not hemostasis.
Mice immunized with the vaccine experienced an antithrombotic effect that lasted 84 days.
The vaccine inhibited thrombus formation without the risk of bleeding or adverse autoimmune responses.
The researchers say the vaccine worked as well as clopidogrel.
“Many stroke patients don’t take their blood thinning drugs as prescribed, which makes it more likely they will have another stroke,” said Hironori Nakagami, MD, of Osaka University Graduate School of Medicine in Japan.
“This vaccine might one day help solve this issue since it would only need to be injected periodically.”
Dr. Nakagami and his colleagues described the vaccine in Hypertension.
S100A9 had previously been reported to be a key molecule in the regulation of thrombus formation. The investigators therefore hypothesized that neutralizing S100A9 with a vaccine would provide sustained antithrombotic effects without increasing the risk of bleeding.
First, the researchers selected a region in S100A9 as a candidate target for the vaccine and conjugated it to keyhole limpet hemocyanin (KLH) as a carrier protein.
They also designed a S100A8 peptide vaccine. S100A8 forms a heterodimer with S100A9 but was not known to play a key role in thrombus formation.
The investigators then randomly assigned male C57BL/6J mice to receive either vaccine or KLH only as control.
The researchers vaccinated mice with S100A9 on days 0, 14, 28, and 126 with either 20 µg or 50 µg of S100A9 peptide. Other mice were vaccinated with S100A8 at a dose of 20 µg on days 0 and 14.
The vaccinated mice developed antibodies against either S100A8 or S100A9, the latter at the 50 µg dose of vaccine. The antibodies recognized the recombinant S100A8 and S100A9 without cross reactivity, which suggested specific binding of each induced antibody.
Assessing thrombus formation
The researchers examined the antithrombotic effects of S100A8 or S100A9 in mouse models of thrombotic middle cerebral artery (MCA) occlusion.
Twenty-one days after the final vaccination, mice immunized with S100A9 had significantly delayed MCA occlusion, with a greater antithrombotic effect in the 50 µg group.
This antithrombotic effect, the investigators said, was comparable to that observed in mice treated with clopidogrel at a dose of 6 mg/kg for 2 days.
The S100A8 vaccine did not prevent MCA occlusion, so the researchers focused on the S100A9 vaccine in subsequent experiments.
They studied the vaccine’s antithrombotic effects in a model of common carotid artery (CCA) occlusion and observed results similar to those seen in the MCA model.
Specifically, there was no significant difference in the CCA occlusion time between mice vaccinated with S100A9 and mice treated with clopidogrel.
The investigators also found the antithrombotic effect of the S100A9 vaccine lasted more than 2 months.
Post-stroke model
Dr. Nakagami and his colleagues then examined the antithrombotic effect of S100A9 in mice after a transient MCA stroke.
The researchers administered the S100A9 vaccine every 2 weeks, starting 1 week after the stroke, for a total of three times.
The vaccine induced anti-S100A9 antibodies, and the mice had significantly prolonged CCA occlusion times compared to KLH control mice. (The investigators said they assessed CCA occlusion because it was impossible to measure the regional cerebral blood flow after a transient MCA occlusion due to post-surgical cranial skin adhesions.)
This result suggested that S100A9 could prevent thrombus formation in mice that had already had a stroke.
Hemostatic parameters and safety
Additional experiments indicated that the S100A9 vaccine had no effect on hemostatic parameters as indicated by tail bleeding time, platelet count, activated partial thromboplastin time, and prothrombin time.
The researchers also evaluated the vaccine’s safety by examining immunologic responses.
Histological analysis at day 147 after the first immunization revealed no pathological changes, such as CD4+ T-cell infiltration or activation of F4/80+ microglia/macrophages.
The immunized mice had predominantly IgG1 responses. This indicated that the S100A9 vaccine selectively induced primary T helper 2 responses.
“We are continuing our research in hopes of being able to start clinical trials between 5 and 10 years from now, but there are differences between mice and humans in how the vaccine will be recognized by the immune system,” Dr. Nakagami explained.
“We should be able to overcome such problems and believe this vaccine provides a very promising strategy in secondary prevention of stroke.”
This research was funded by the Mochida Memorial Foundation for Medical and Pharmaceutical Research, the Japan Cardiovascular Research Foundation, the SENSHIN Medical Research Foundation, and the Japan Agency for Medical Research and Development.
Dr. Nakagami and five of the eleven authors have applied for a patent for the S100A9 vaccine.
Dr. Nakagami and five other authors are affiliated with departments at Osaka University that receive financial support from AnGes, DAICEL, FunPep, Novartis, Shionogi, Boehringer, and Rohto. One author is a founder, stockholder, and former board member of AnGes.