However, the apparent difference in the type of humoral immune response was not reflected by the cellular immune responses since the S protein-encoding vector and Engerix-B GANT61 induced the same level of S protein-specific IFN-c-secreting PBMC. The PreS1-specific antibody titers detected in some animals immunized with S protein-encoding vectors, though at a low level, was unexpected. These antibodies might be cross-reactive with low specificity for the PreS1 protein, although sequence alignments revealed no similarity between the PreS1 and S protein amino acid sequence. In this context it is puzzling that the S protein-specific antibodies induced by Engerix-B were not cross-reactive. S protein expressed from DNA in immunized animals differs from recombinant yeast-derived S protein and antigen presentation after DNA and protein immunization might deviate as well which potentially influences the development of cross-reactive antibodies. In humans, antibody levels wane over time after vaccination with recombinant HBsAg protein vaccines. Accordingly, we have observed a decline in antibody levels within 6 months after the third immunization of pigs. Though the low number of animals allows no definitive conclusion, the decline seemed less pronounced in pigs immunized with S protein-encoding MIDGETh1 vectors compared to pigs immunized with Engerix-B. Prolonged presence of the MIDGE-Th1 vector in skin and draining lymph nodes resulting in prolonged antigen expression and immune stimulation may account for this effect. Another possible explanation is that the DNA vaccine induced more long-lived antibody-secreting plasma cells maintaining specific antibody levels in the absence of antigen. From our studies in mice and pigs we conclude that the SAINT18-formulated MIDGE-Th1 vector encoding the S protein of HBsAg is the most promising vaccine candidate at present. Although immunogenicity in humans remains to be determined, our data indicate that the generation of total S-specific antibodies, the hallmark for licensing of HBV vaccines, can approach that of conventional vaccines after immunization with this vaccine candidate. Although the L protein-encoding vector elicited high PreS1-specific antibody titers in pigs, the level of S-specific antibodies does not suggest that this vaccine candidate can confer protection in humans. Thus, further optimization of the L protein antigen sequence, e.g. allowing for efficient secretion of the L protein, would be required prior to clinical testing. Taken together, we established efficacy of our SAINT-18formulated MIDGE-Th1 DNA vaccine for prophylaxis of HBV infection in a large animal model. With this relevant proof of concept, the application of SAINT-18 formulated MIDGE-Th1 vectors can be expanded to the development of other prophylactic vaccines with an antibody-based mode of protection against a broad range of pathogens. Its ability to withstand desiccation, disinfection and to form biofilms on abiotic surfaces, including medical devices such as catheters and ventilators, is believed to significantly contribute to survival and persistence of A. baumannii in the clinical environment.