A number of approaches have been used in attempts to develop a safe and effective vaccine for respiratory syncytial virus (RSV) infection. This article describes an effective prophylactic intranasal gene transfer strategy utilizing chitosan-DNA nanospheres [the mucosal gene expression vaccine (MGXV)], containing a mixture of plasmid DNAs encoding RSV antigens. In a mouse model of RSV infection, a single administration of MGXV (25 microg/mouse) results in a significant reduction of viral titers and viral antigen load after acute RSV infection of these mice. MGXV-treated mice show no significant change in airway reactivity to methacholine and no apparent pulmonary inflammation. Together these results demonstrate the potential of MGXV against acute RSV infection.