Influenza A virus is a negative-strand RNA virus that causes significant morbidity and mortality yearly in humans and other species (33
). Protective neutralizing antibodies are able to prevent reinfection with the same strain, but due to the high rate of mutation of influenza viruses, the immunogenic epitopes in the HA and NA proteins change very rapidly, and neutralizing antibodies are not efficient at blocking reinfection with the mutated drifted viruses (reviewed in reference 46
). Cellular immunity is crucial for clearance of the virus from the lungs, and both CD4 and CD8 T cells appear to play an important role. If viruses are able to avoid elements of innate immunity, such as the IFN-α/β response, then they have a great advantage, since adaptive immunity requires more time to generate. One efficient way to reduce the effects of the immune response on virus replication is to block both arms of immunity, i.e., IFN-α/β production and DC activation, interfering with T-cell priming by DCs. Although it is known that influenza A viruses induce T-cell responses in their hosts, it is not clear whether this induction is optimal or whether mechanisms are used by these viruses to prevent the onset of a very strong T-cell response that would otherwise limit viral replication.
Using a human in vitro system, we analyzed the ability of influenza A virus to induce DC activation and examined the CD4 T-cell priming capacity of the infected DCs as an indicator of their ability to initiate Th1 immunity. We observed that the PR8 influenza virus failed to induce a strong Th1 immune response in allospecific cultures of infected DCs and naïve CD4 T cells (Fig. ). Influenza virus PR8, as well as more recent influenza virus isolates, failed to induce DC activation, as measured by the upregulation of costimulatory molecules, cytokine secretion, and the upregulation of the expression of several genes involved in the IFN-α/β response and in DC activation (Fig. , , , and ).
Among the 11 proteins encoded by influenza virus, the NS1 protein has been shown to block the production of IFN-β in infected cells, including epithelial cells and DCs (22
). This feature of influenza virus allows it to evade innate immunity. In our studies, we have analyzed the effects of the NS1 protein, not only on IFN-α/β production but also on virus-induced maturation of human myeloid DCs and their ability to prime CD4 T cells towards Th1 immunity. If the viruses used to infect DCs expressed influenza virus NS1 (influenza viruses PR8, Moscow, and Texas and NDVB1-NS1), the cells failed to mature fully in vitro (by upregulation of costimulatory molecules and release of proinflammatory cytokines) (Fig. , , , , and ). The infection of DCs with viruses lacking the NS1 protein of influenza virus (NDVB1 and the influenza virus DeltaNS1) resulted in the induction of strong DC maturation, as shown by flow cytometry (Fig. ) and ELISA (Fig. , , and ). The inhibitory effect of the NS1 protein on DC maturation could be seen even more clearly at the level of gene transcription (Fig. , , and ; see Fig. S1 in the supplemental material). The genes affected by the NS1 protein included those encoding mediators of antiviral immunity, antiviral cytokines, inflammatory chemokines, and the chemokine receptors necessary for DC migration to lymph nodes. Recently, it was shown that influenza virus infection induces a very highly coordinated chemokine response in different subsets of human DCs (35
), indicating the importance of the pattern of chemokine secretion by DCs in the generation of immunity to influenza virus. Importantly, the NS1 effect also alters the functionality of DCs by preventing the efficient activation of T cells. Thus, T cells proliferated normally but were insufficiently polarized to produce IFN-γ when incubated with DCs infected with an NS1-expressing virus (Fig. and ).
This clear inhibitory effect by the influenza virus NS1 protein on human DC maturation and function is neither a global effect nor simply the result of inhibition of IFN-α/β release and signaling. The addition of antibodies to IFN-β at the time of infection of human DCs with the PR8 influenza virus affected the levels of expression of IFN-inducible genes, while IFN-independent genes were not affected (data not shown). There were numerous genes not affected or moderately affected by the presence of the NS1 protein in infected cells (Fig. , , and ). Additionally, this gene-specific effect was very reproducible among several donors and could be observed regardless of the background virus used to express NS1 (Fig. ; see Fig. S2 in the supplemental material). The absolute values of gene expression between different donors could differ, but the effect of NS1 was maintained among multiple donors tested.
The NS1 protein of influenza virus has been described as mediating multiple effects on cells after virus infection. Predominantly, it has been shown to inhibit pathways involved in IFN-β production, such as the protein kinase R (PKR) pathway (6
), and the activation of the transcription factors NF-κB, AP-1, IRF3, and IRF7 (39
). Our hypothesis is that one or more of these elements involved in IFN-β production may also be the target for the inhibitory function of NS1 on the expression of genes involved in DC maturation. PKR has been reported to be important for the production of IFN-β in response to double-stranded RNA treatment (8
); however, other groups have shown that it is not essential for IFN-β production or for DC activation after virus infection (39
). IRF3 and IRF7, two molecules involved in IFN-β production, may be targets for the NS1 protein to downregulate gene transcription. However, we have observed that DCs genetically deficient in IRF3 mature normally to negative-strand RNA viruses (data not shown), and IRF7 synthesis in conventional DCs depends on IFN signaling, which is not essential for full DC maturation in response to RNA virus infection (22
). Most likely, the inhibition of genes involved in DC maturation by NS1 is mediated by inhibition of transcription factors such as NF-κB and AP-1. These transcription factors, which were shown to be inhibited by the NS1 protein (25
), participate in both IFN-β and cytokine production. Interestingly, we found that the chemokine IL-8, which is strongly dependent on NF-κB and AP-1 for its induction (20
), is strongly inhibited by the NS1 protein in DCs (Fig. ; see Fig. S1 in the supplemental material). Although the effects of NS1 were gene specific, it is also possible that part of these effects are due to the proposed inhibition of gene expression by the NS1 protein through a block in cellular mRNA processing (29
One of the most important elements of the antiviral innate immune system is IFN-α/β. Most cell types, including respiratory epithelial cells and DCs, synthesize IFN-α/β in response to virus infection (1
). Upon release from infected cells, IFN-α/β binds to its receptor, present in the secreting cells and in adjacent cells, thus establishing an antiviral state characterized by the production of numerous proteins that inhibit the ability of viruses to replicate (reviewed in references 1
). Many viruses evade the IFN-α/β system through the expression of viral IFN antagonist proteins. In the case of influenza A virus, the NS1 protein was shown to be a potent IFN antagonist in epithelial cells through its N-terminal double-stranded RNA-binding domain, which prevents the induction of type I IFN synthesis during viral infection (9
). Our results demonstrate that influenza virus inhibits not only IFN-α/β release in human DCs but also maturation of DCs, and they suggest that the NS1 protein of influenza virus attenuates the initiation of adaptive immune responses in infected individuals. Recent reports have also shown low levels of IFN-α/β, TNF-α, and IFN-λ1,2,3 release by influenza virus-infected human DCs compared to those in Sendai virus infection and low levels of DC maturation induced by influenza virus (31
). A number of reports have documented enhanced immunogenicities of viruses with mutated or truncated NS1, consistent with an inhibitory effect of this protein on adaptive immunity (12
). Due to the attenuation properties of NS1 mutant viruses in their hosts (11
) and to the enhanced ability of NS1 mutant viruses to induce human DC maturation (this report), we suggest that these viruses are good candidates to be considered as live attenuated vaccines against influenza. Thus, a lack of NS1 function results in mutant influenza viruses with more potent adjuvant properties in DCs. Further investigation will be required to demonstrate the vaccine efficacy of NS1 mutant viruses in humans as well as to determine whether our observations might also be generalized to other viruses that carry IFN-α/β antagonists. Interestingly, it has recently been shown that the IFN-α/β antagonist genes of respiratory syncytial viruses also appear to prevent optimal stimulation of human DCs (38
Here we report an inhibitory effect of NS1 on elements from DCs, which are important for the initiation of Th1 immunity. This inhibitory effect on adaptive immunity may have been overlooked due to previous virus exposures in the human outbred population. This effect could be compensated for by the generation of a strong CD8 T-cell memory response after influenza virus infection that overcomes the weak CD4 T-cell response, as has been suggested for mouse models (48
). On the other hand, the expected decrease in the adaptive immune response due to the NS1 effect could be compensated for by the release of type I IFN or other proinflammatory cytokines from plasmacytoid DCs (pDCs) in vivo, providing an adjuvant effect in trans
on myeloid DCs, as recently suggested (4
). While a direct role for pDCs in the induction of adaptive immune responses against viruses in vivo has not been documented, a number of studies have demonstrated a requirement for type I IFN for optimal maturation of myeloid DCs (14
). Further experimentation will be required to evaluate the role of pDCs in the induction of immunity against influenza viruses.
In summary, using recombinant NDV and influenza viruses expressing the NS1 IFN antagonist of influenza virus, we have shown that the NS1 protein is able to attenuate a primary human cell system in both innate immunity and a critical element necessary for the initiation of adaptive immunity, DC maturation. This dual immune evasion strategy is likely to be utilized by other viral IFN antagonist proteins. Further characterization of this strategy, in which a single protein targets both innate and adaptive immunity, may prove to be extremely useful for the design of live virus vaccines or therapeutic agents with improved immune efficacy.