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Although intramuscular (i.m.) administration is the most commonly used route for licensed vaccines, subcutaneous (s.c.) delivery is being explored for several new vaccines under development. Here, we use rhesus macaques, physiologically relevant to humans, to identify the anatomical compartments and early immune processes engaged in the response to immunization via the two routes. Administration of fluorescently labeled HIV-1 envelope glycoprotein trimers displayed on liposomes enables visualization of targeted cells and tissues. Both s.c. and i.m. routes induce efficient immune cell infiltration, activation, and antigen uptake, functions that are tightly restricted to the skin and muscle, respectively. Antigen is also transported to different lymph nodes depending on route. However, these early differences do not translate into significant differences in the magnitude or quality of antigen-specific cellular and …