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Human immunodeficiency virus (HIV) vaccines have not been successful in clinical trials. Dimeric IgA (dIgA) in the form of secretory IgA is the most abundant antibody class in mucosal tissues, making dIgA a prime candidate for potential HIV vaccines. We coupled Positron Emission Tomography (PET) imaging and fluorescent microscopy of 64Cu-labeled, photoactivatable-GFP HIV (PA-GFP-BaL) and fluorescently labeled dIgA to determine how dIgA antibodies influence virus interaction with mucosal barriers and viral penetration in colorectal tissue. Our results show that HIV virions rapidly disseminate throughout the colon two hours after exposure. The presence of dIgA resulted in an increase in virions and penetration depth in the transverse colon. Moreover, virions were found in the mesenteric lymph nodes two hours after viral exposure, and the presence of dIgA led to an increase in virions in mesenteric lymph nodes. Taken together, these technologies enable in vivo and in situ visualization of antibody-virus interactions and detailed investigations of early events in HIV infection., Competing Interests: The authors have declared that no competing interests exist. Author Beth Goins was unable to confirm their authorship contributions. On their behalf, the corresponding author has reported their contributions to the best of their knowledge.