Targeting Subtype-Independent Immune Responses Against Influenza A Virus

Influenza A virus (IAV) is a major burden for public health. Besides seasonal, human IAV, which circulates during the cold seasons and is responsible for hundreds of thousands of deaths each year, its potential to reassort with avian IAV fuels the fear of future pandemics. Currently licensed vaccines are standardized to elicit antibodies directed against the head domain of the hemagglutinin (HA) on the viral surface and binding of these can effectively neutralize virus particles. However, the antigenic plasticity of HA in consecutive IAV seasons and the above-mentioned potential for upcoming reassorted viruses make it necessary to update IAV vaccines on an annual basis and still, these vaccines cannot protect against outbreaks of zoonotic origin. It is therefore a major objective to develop new subtype-independent approaches against IAV via immunization as a first line of defense or via antiviral drugs. This thesis is divided into two different projects. The first one investigates the protection against heterologous challenge after vaccination with single or combined IAV proteins and the underlying humoral immune response. The second project aims at characterizing the effect of ADAR1 (adenosine deaminase acting on RNA 1), a potential target for antiviral compounds, on IAV. In the first project, the internal proteins NP and M1 derived from the live-attenuated influenza vaccine were investigated regarding their potential to elicit protective immunity against heterologous virus challenge and compared to currently used approaches based on the stem region of HA (HAstem) or the membrane-integral M2 protein. Furthermore, underlying immune responses were characterized. We demonstrate that VSV-vectored immunization with the internal protein NP and M2, but also H3stem can remarkably reduce IAV-induced disease in a heterologous manner and that this effect is independent of detectable T cell responses. Analysis of humoral immunity revealed high IgG antibodies, distinct IgG subclass profiles against different viruses, and most importantly, activation of the murine FcγRIV, known to mediate antibody-dependent cell-mediated cytotoxicity and –phagocytosis via alveolar macrophages. Furthermore, we showed that the absence of humoral immunity after viral vector-based immunization with M1 correlated with the lack of protection against IAV challenge in mice, which suggests absence of protective, but undetected, T cell responses, and strengthens our assumption of antibody-mediated protection. Humoral immunity against internal proteins of IAV have first been described decades ago but are often considered inferior to those directed against surface antigens. The main reason for this is the localization of internal proteins, as it raises questions about the capability to provide protection through humoral immune responses. Therefore, they were often neglected or ignored in the past. While these antibodies do not mediate neutralization, they can in fact activate Fc-mediated effector functions and protect from homologous as well as heterologous disease. Our results add further insights in these mechanisms and correlate with previously described capability of NP and M2 to induce protective antibodies. Our results therefore imply that these immune responses should not be ignored in the rational design of future IAV vaccines. In the second project, we used two different cell culture systems to investigate the effect of different ADAR1 isoforms on the replication of IAV, in order to consider its potential as a target for antiviral therapies. We demonstrated that ADAR1p150 is a proviral factor for IAV infection and is required for efficient viral protein expression in HeLa cells, which is in line with previous publications describing this in other eukaryotic cell lines. This finding consolidates the concept of proviral ADAR1p150 for IAV, as it was described for other RNA viruses. Furthermore, we generated MDCK cells deficient for ADAR1p150 or complete ADAR1 using a CRISPR/Cas9n system and confirmed the proviral effect of ADAR1p150 on viral replication. We showed that absence of ADAR1 resulted in strikingly decreased IAVinduced cell death, which may be an essential factor for targeting it with antiviral compounds. Individuals passing away because of an IAV infection show severe damage of lung tissue, resulting from exaggerated innate immune responses, a proinflammatory milieu, and the subsequent collapse of the epithelial barrier in the lung. Our results in HeLa cells indicate that a potential knock-down or inhibition of ADAR1 does not necessarily lead to activated innate immunity pathways, as it could be expected from other RNA viruses, and in combination with the above-mentioned decreased cell death in infected MDCK ADAR1KO cells builds a promising basis for the further investigation of ADAR1 as a target for anti-IAV treatment therapies.