Non-clinical safety evaluations, including those of impurities, are important for vaccine development to ensure safety in humans. However, information on the mechanisms of impurity-induced adverse effects remains limited. In a repeated-dose toxicity study of our lipid nanoparticle formulated mRNA vaccine (mRNA-LNP vaccine) candidate, severe anemia was observed in rats after multiple administrations. In this study, we conducted hematological analyses and bone marrow examinations in vivo to investigate the cause and mechanism of test article-related delayed anemia. In addition, we performed in vitro mechanistic studies including antibody titer measurements and colony-forming unit assays. We found that test article-related anemia was caused by the inhibition of erythroid differentiation in the bone marrow, mediated by antibodies against erythropoietin (EPO). Furthermore, the test article was found to contain human EPO mRNA as an impurity. Lastly, the spike study showed that a minute quantity of human EPO mRNA present in mRNA-LNP vaccines as an impurity induced anemia in rats. Taken together, our data demonstrate that immune-mediated delayed anemia can be induced by impurity-oriented anti-EPO antibodies that neutralize endogenous EPO and inhibit erythroid differentiation. Our presented approach of determining the mechanism of delayed toxicity caused by impurities may be helpful in future safety evaluations.
Murata et al. (Thu,) studied this question.