Activated blood-derived human primary T cells support replication of HAdV C5 and virus transmission to polarized human primary epithelial cells.

Human adenoviruses (HAdVs) cause self-limiting disease but are life-threatening to immunocompromised individuals. HAdV-C5 infects epithelial cells of the airways and eyes through aerosols, contaminated hands, or medical instruments, as well as fecal-oral contacts, gives rise to viremia, persisting in lymphoid cells of the gastrointestinal tract. Here, we show that pre-activated human primary blood-derived T cells can be infected with HAdV-C5 in vitro, upon incubation of the virus with a mixture of three distinct homotrimeric adapter proteins that target the virus to T cells. Each of the adapter proteins can bind 1 of the 12 fiber knobs of the virion through a designed ankyrin repeat protein. Two of the adapters contained a single-chain antibody fragment to T cell surface proteins CD3 or CD28, and the third one contained the cytokine interleukin-2. These adapters mediated efficient infection of primary T cells by HAdV-C5 and infectious progeny release, albeit with donor-to-donor variability. Co-culture of well-polarized air-liquid interface human bronchial epithelial cells with infected CD3⁺ T cells gave rise to progressively increased viral titers from replicating but not from replication-defective E1-deleted HAdV-C5, notably with similar kinetics as cell-free virus infections, suggesting that progeny virus from T cells was further amplified in epithelial cells. This study provides a platform to explore interactions between epithelial and immune cells in acute and persistent HAdV-C5 infection settings.IMPORTANCEMany human adenoviruses (HAdV), including HAdV-C5, infect and propagate to high titers in epithelial cells of the airways. Virus ends up in lymphoid cells of the gastrointestinal and respiratory mucosa, where it can persist subclinically for years, restricted by memory T cells and humoral immune defense. In immunodeficient patients or newborns, however, HAdV can be fatal, coincident with lymphocytopenia and virus proliferation in epithelial cells. Here, we show that activated blood-derived human primary T lymphocytes can be productively infected with HAdV-C5 coated with trimerized adapter proteins targeting CD3, CD28, and the interleukin 2 receptors. A co-culture model of infected T cells and primary human bronchial epithelial cells in the absence of HAdV-specific immune cells showed that progeny virus from T cells was transferred to epithelial cells and led to increased progeny production compared to infected T cells alone, a situation potentially mimicking persistently infected mucosal lymphoid cells in immunosuppressed patients.