Clin. of influenza infections is essential to limit the strain placed on health care systems by influenza epidemics and global pandemics. Intensive research has led to the discovery of therapeutic interventions to combat influenza infections; however, due to the virus’s error-prone polymerase, the hemagglutinin (HA) and neuraminidase (NA) influenza viral proteins are subject to point mutations, known as antigenic or genetic drift (12), that allow the computer virus to escape host immune responses or result in some types SPP1 of drug resistance (13). Vaccination is one of the most effective means of preventing influenza-associated morbidity and mortality. Currently, inactivated vaccines are widely used for the prevention of influenza; however, inactivated influenza vaccines provide short protection periods and limited efficacy, especially in young children and the elderly (3, 17). In addition, due to the failure to effectively elicit cell-mediated immunity, inactivated vaccines are generally less immunogenic, and hence less potent, than live attenuated vaccines, which are approved for use in a limited quantity of countries such as the Unites States. Intranasally administered live attenuated viruses are considered superior to inactivated vaccines for children because they elicit strong mucosal immunity and humoral and cellular immune responses coupled with long-lasting protective efficacy (1). However, live attenuated vaccines are currently licensed only for individuals aged 2 through 49 who lack chronic medical conditions and who are not pregnant or immunocompromised, even though licensed live attenuated influenza viruses are considered safe and stable with respect to the underlying risk of the emergence of revertant viruses. Hence, the development of improved influenza vaccines is critical to control future outbreaks. Recently, our group genetically designed PB2-knockout (PB2-KO) influenza viruses that are able to harbor a reporter gene, such as the green fluorescent protein (GFP) gene, between the packaging signals (the noncoding and coding 120 bases at both the 5 and 3 ends) of the PB2 gene (16). Growth of such PB2-KO viruses is restricted to a cell collection stably expressing the PB2 protein and yields high FTI 276 computer virus titers ( 108 PFU/ml). The HA and NA genes of a heterologous influenza computer virus could be accommodated in the PB2-KO computer virus (16). Furthermore, the recombinant PB2/reporter gene was stably incorporated into progeny PB2-KO virions and was retained through sequential passages. Therefore, the PB2-KO computer virus can be tailored to encode not only desirable combinations of the main influenza computer virus antigens, namely, HA and NA, but also non-influenza computer virus antigens, suggesting that this PB2-KO computer virus could be used as a multivalent vaccine. Here, we tested the vaccine potential of the PB2-KO computer virus by immunizing mice and examining antibody responses and protective efficacy. MATERIALS AND METHODS Cells. 293 and 293T (a derivative of the 293 collection into which the gene FTI 276 for simian computer virus 40 T antigen was inserted [2]) human embryonic kidney cells were managed in Dulbecco’s altered Eagle medium (Lonza, Basel, Switzerland) supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA). Madin-Darby canine kidney (MDCK) cells were maintained in minimum essential medium (MEM) (Invitrogen) supplemented with FTI 276 5% newborn calf serum (NCS) (Sigma, St. Louis, MO). AX4 cells, which are an MDCK-derived cell collection with enhanced expression of human-type receptors for influenza computer virus and were produced by stable transfection of a plasmid expressing the human -2,6-sialyltransferase (8), were managed in 5% NCSCMEM supplemented with puromycin (2 g/ml). AX4/PB2 cells (AX4 cells stably expressing the PB2 protein derived from A/Puerto Rico/8/34 [H1N1, PR8], established by transduction with a retroviral vector [16]) were managed in 5% NCSCMEM supplemented with puromycin (2 g/ml) and blasticidin (10 g/ml). All cells were maintained in a humidified incubator at 37C in 5% CO2. Plasmid-driven reverse genetics. The wild-type PR8 and PB2-KO viruses.