Purtscher, A. reading frame was significantly lower than that obtained previously from an influenza virus vector expressing GFP from the NS1 ORF, the bicistronic vector appeared to be replication qualified in mice and showed outstanding genetic stability. All viral isolates derived from mouse lungs at 10 days postinfection were still capable of expressing GFP in infected cells. Utilizing this bicistronic approach, we constructed another recombinant influenza virus, allowing the secretion of biologically active human interleukin-2 (IL-2). Although this virus also replicated to high titers in mouse lungs, it did not display any mortality rate in infected animals, in contrast to control viruses. Moreover, the IL-2-expressing virus showed an enhanced CD8+ response to viral antigens in mice after a single intranasal immunization. These results indicate that influenza viruses could be engineered for the expression of biologically active molecules such as cytokines for immune modulation purposes. The generation of viral vectors for the delivery of ASP6432 foreign proteins and biologically active molecules remains an attractive approach for gene therapy, the treatment of cancer, and the prevention of infectious diseases. Since reverse genetic methods were developed, influenza viruses have also been considered potential vaccine vectors (7, 8, 10, 28, 29, 33, 34). Recently, cold-adapted intranasal influenza vaccines have been licensed for children and adults (4, 13). In theory, the viral strains comprising the live influenza vaccine could be further modified for the delivery and expression of additional proteins. In contrast to other vectors such as adenoviruses or retroviruses, influenza virus does not form a DNA intermediate during its replication cycle and is not able to integrate into the host’s chromosomes, making it attractive in terms of safety. There are several options for how to manipulate the influenza virus genome, depending on the desired aims and possibilities, to produce recombinant viruses. These strategies include the insertion of foreign proteins into the surface glycoproteins NA and HA (24, 29), the creation of additional genomic fragments (10, 34), and the manipulation of the nonstructural NS1 protein (8, 33). The influenza virus NS1 protein has several advantages as a target for engineering since it does not presumably interfere with the structure of the virions but is usually synthesized in ASP6432 large quantities in infected cells and tolerates long insertions of up to several hundred nucleotides. Additionally, because NS1 ASP6432 is not incorporated into virions, alterations of this protein would not change the antigenicity of the influenza virus itself. Furthermore, the attenuation mechanism of the currently used cold-adapted influenza vaccine is not based on the NS gene, implying the ability to integrate the recombinant NS gene into RGS13 live influenza virus vaccine strains (15). Despite these advantages, due to the intracellular localization of NS1, the development of the immune response to the NS1 protein or to the proteins fused to NS1 is limited mainly to the induction of CD8+ T-cell immunity (7, 33). Obviously, for the induction of a B-cell response or for the expression of biologically active molecules, efficient delivery of the recombinant protein to the cell surface is required. This could be achieved by constructing an additional reading frame within the NS gene and by supplementation of the foreign proteins with secretory signal sequences. Several approaches have been used to create bicistronic mRNAs for influenza viruses, including the incorporation of an internal ribosome entry site element (11) and a doubling of influenza virus promoter sequences (20). For the present work, we exploited ASP6432 a simple bicistronic strategy analogous to the influenza B virus M gene (14) in order to create an additional reading frame within the NS gene of influenza A virus. The stop-start cassette UAAUG was inserted into the influenza A virus NS1 coding sequence corresponding to amino acid (aa) position 125, followed by the insertion of the green fluorescent protein (GFP) sequence. As expected, the expression of GFP by this virus was diminished compared to that by a previously obtained vector (NS1-GFP) which expresses GFP from the NS1 reading frame (16). Nevertheless, in contrast to the latter vector, the bicistronic expression vector could replicate to high titers in mouse lungs without losing its ability to express the foreign sequence. We concluded that bicistronic influenza virus NS vectors could be suitable for the expression of biologically active molecules such as cytokines, which are effective even in small quantities. To prove this hypothesis, we created an influenza virus expressing.