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Social media have become a common way for people to express their personal viewpoints, including sentiments about health topics. We present the results of an opinion mining analysis on vaccination performed on Twitter from September 2016 to August 2017 in Italy. Vaccine-related tweets were automatically classified as against, in favor or neutral in respect of the vaccination topic by means of supervised machine-learning techniques. During this period, we found an increasing trend in the number of tweets on this topic. According to the overall analysis by category, 60% of tweets were classified as neutral, 23% against vaccination, and 17% in favor of vaccination. Vaccine-related events appeared able to influence the number and the opinion polarity of tweets. In particular, the approval of the decree introducing mandatory immunization for selected childhood diseases produced a prominent effect in the social discussion in terms of number of tweets. Opinion mining analysis based on Twitter showed to be a potentially useful and timely sentinel system to assess the orientation of public opinion toward vaccination and, in future, it may effectively contribute to the development of appropriate communication and information strategies.