Reverse genetics systems, developed in the 1990s, still remain a challenging and promising technology, enabling the creation of influenza viruses with modified replicative, pathogenic, antigenic, and immunogenic properties (20
). We and others have recently shown that it is possible to generate replication-deficient and thus safe, but still highly immunogenic, influenza A viruses by genetic engineering of the NS gene segment (15
). The NS genome segment encodes two proteins: the NS1 and NEP, both carrying out essential functions in the viral life cycle. The NS1 protein, which is about 230 amino acids long, was identified as a type I IFN antagonist and possesses several domains and sites for the binding of the diverse proteins required, e.g., for the processing and cellular transport of cellular mRNAs and the translation of viral mRNAs (22
). Targeted modification of its specific domains resulted in decreased, or even absent, virus replication in vivo, mainly due to the abrogation of the NS1 protein's function to suppress the IFN response machinery or other innate signaling pathways within the infected cells (6
). Moreover, infected cells produced high levels of type I IFNs, chemokines, and other immunomodulating cytokines, thus stimulating the development of the innate and adaptive immune responses (18
In the present work, we addressed the question of whether the efficacy of a cold-adapted influenza virus vaccine can be improved by generating a virus vector encoding human IL-2 from the NS gene segment. IL-2 plays a crucial role in the stimulation and maturation of the immune system and is frequently used as a vaccine supplement or genetic adjuvant (1
). For this reason, we compared the immunogenicities of the attenuated influenza ca
Sing virus and a modified variant which expresses biologically active IL-2 in young adult and aged mice.
Due to their ca phenotypes, both viruses were highly attenuated in mice, since the maximal viral load detected at day 2 p.i. did not exceed 2 × 102 PFU/g tissue. We assume that the truncation of the NS1 protein and thus alteration of its biological functions resulted in an additional attenuation of the ca Sing-IL-2 virus (titer of 1.1 × 102 TCID50s/g tissue) compared with that of the ca Sing virus (titer of 2 × 102 × TCID50s/g tissue).
We found that coexpression of human IL-2 markedly improved the immunogenicity of the ca
Sing virus. The ca
Sing-IL-2 virus induced significantly higher numbers of virus-specific CD8+
T cells and mucosal IgA in young adult and aged mice. It is noteworthy that the stimulation capacity of the ca
Sing-IL-2 virus was observed in the cellular and mucosal arms of the immune system compared with the parent ca
Sing virus but not in the humoral systemic compartment. Both viruses induced almost identical virus-specific serum IgG1 and IgG2 responses in mice, irrespective of age. Interestingly, when young adult and aged mice were primed with the ca
Sing-IL-2 virus, increased virus-specific mucosal IgA was also detected at a site distal to the site of immunization, the urogenital tract. This observation supports the concept of the common mucosal immune system playing a critical role in protecting hosts against mucosal pathogens (40
). The central role of secretory IgA in the protection of the upper respiratory tract from influenza infection has been repeatedly reported elsewhere (11
). Since the mucosal respiratory tissue provides the first barrier to influenza virus penetration, an induction of enhanced secretory IgA levels by new-generation vaccines is highly desirable.
Interestingly, ca Sing-IL-2 induces the local mucosal IgA and CD8+ T-cell immune response to markedly higher magnitudes than does the ca Sing virus in both aged and young adult mice. At distant sites devoid of the virus-encoded IL-2 production, e.g., the urogenital tract (mucosal IgA) or the spleen (systemic CD8+ response), this immune response was clearly weaker, especially for aged mice.
These observations are most probably related to the decline of immune system functions repeatedly observed in aged hosts. Impairments in humoral and cellular immunities in aged hosts include, e.g., the development of suboptimal immunogen-specific mucosal IgA titers; decreased immunogen-specific IgG avidity; disorders in the proliferation of memory cells, which are unable to secrete IL-2 on stimulation by antigens; and lack of the CD28-costimulatory molecules on expanded oligoclonal populations of activated T cells, etc. (14
). Nevertheless, by priming with the ca
Sing-IL-2 virus, it was possible to restore the distant immune response of aged mice almost to the levels in young adult mice immunized with ca
Sing. These findings may partly explain the suboptimal efficacy of vaccines and the dramatically increased mortality in the influenza virus-infected elderly compared to those in young adults (41
) but show simultaneously the high immunogenic potential of the IL-2-expressing influenza virus as a vaccine candidate especially suitable for the elderly.
It has to be noted that the ca Sing-IL-2 virus also positively stimulates the development of immunological memory. We observed a rapid onset of the recall CD8+ T-cell response 60 h postchallenge in lymph nodes draining the respiratory tracts of young adult mice primed with the ca Sing-IL-2 virus, surpassing the numbers of CD8+ T cells detected in the group of mice immunized with the ca Sing virus. Most importantly, young adult mice primed with the ca Sing-IL-2 virus were completely protected from a homotypic WT virus challenge, in contrast to ca Sing virus-primed animals.
This study demonstrates a reverse-genetics-based modeling approach to improve the immunogenicity of live influenza virus vaccines in adult hosts and especially in aged hosts who do not respond well to vaccination. Since the attenuation phenotype of existing live ca influenza vaccines has been shown to be determined by mutations in the polymerase genome segments but not in the NS gene segment, the modified NS gene encoding human IL-2 (responsible also for the cellular production of other immunomodulating cytokines) could be introduced, in theory, into any ca vaccine viral genome, thus replacing the vaccine virus's own NS gene. The partial truncation of the recombinant NS protein in the context of a live ca influenza virus master strain will ensure an enhanced local immunogenicity and, at the same time, an additional attenuation resulting in a high safety standard of live ca influenza vaccine strains. This technology also seems to be especially suitable for the development of more-efficient vaccines directed against new potential pandemic viruses, such as avian H5N1 influenza viruses. Moreover, this approach also eliminates the potential side effects observed upon systemic administration of IL-2 to hosts. Thus, we hope that this study provides the basis for the future development of safe and highly immunogenic live influenza virus vaccines.