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Lyme borreliosis (LB) or Lyme disease is the most common vector-borne disease in the northern hemisphere and at present there is no vaccine available to prevent infections. Recent analyses showed that the number of infections in the US and Europe are largely underreported, emphasizing the need for an effective vaccine. An OspA based vaccine (LYMErix™) was previously shown to be efficacious against disease caused by the most prevalent B. burgdorferi in the US. In Europe, the majority of LB cases are caused by four different Borrelia species expressing six different OspA serotypes. Since Outer surface protein A (OspA) is one of the dominant antigens expressed by the spirochetes when present in the tick vector we have developed a vaccine for global use, consisting only of the C-terminal part of OspA which is sufficient for protection. In order to target the Borrelia species expressing the six different OspA serotypes prevalent in US and Europe, we have designed a multivalent OspA-based vaccine (VLA15), including three proteins, each containing the C-terminal half of two OspA serotypes linked to form a single fusion protein. The OspA fusion proteins were at least 85% triacylated which ensured high immunogenicity and were highly purified for further preclinical testing. Active immunization with the adjuvanted Lyme borreliosis vaccine VLA15 protected mice from a challenge with spirochetes expressing either OspA serotype 1, 2, 4, 5 or 6, using infected ticks or in vitro grown bacteria as a challenge. Further immunological analyses (ELISA, surface binding and growth inhibition) indicated that the vaccine can provide protection against the majority of human pathogenic Borrelia species, including OspA serotype 3. This rational designed VLA15 vaccine was therefore prepared for evaluation in a first-in-man study which is currently ongoing.