Upon APC-targeted DNA vaccination, transfected cells secrete fusion proteins with targeting units specific for surface molecules on APC. In this study, we have tested several different targeting units for their ability to influence the magnitude and subclass of Ab responses to hemagglutinin from influenza A virus. The experiments employed bivalent homodimeric Ig-based molecules (vaccibodies). The overall efficiency in BALB/c mice depended on the targeting units in the following order: aMHC

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... II . aCD11c . aCD40 . Xcl-1 = MIP-1a . FliC . GM-CSF . Flt-3L . aDEC205. GM-CSF induced mainly IgG1, whereas Xcl1, MIP-1a, aCD40, and aDEC205 induced predominantly IgG2a. A more balanced mixture of IgG1 and IgG2a was observed with aCD11c, aMHC class II, Flt-3L, and FliC. Similar results of IgG subclass-skewing were obtained in Th1-prone C57BL/6 mice with a more limited panel of vaccines. IgG1 responses in BALB/c occurred early after immunization but declined relatively rapidly over time. IgG2a responses appeared later but lasted longer (.252 d) than IgG1 responses. The most efficient targeting units elicited short-and long-term protection against PR8 influenza (H1N1) virus in BALB/c mice. The results suggest that targeting of Xcr1 + conventional type 1 dendritic cells preferentially induces IgG2a responses, whereas simultaneous targeting of several dendritic cell subtypes also induces IgG1 responses. The induction of distinct subclass profiles by different surface molecules supports the APC-B cell synapse hypothesis. The results may contribute to generation of more potent DNA vaccines that elicit high levels of Abs with desired biologic effector functions. ImmunoHorizons, 2018, 2: 38-53.