Karoline Droebner

Antiviral activity of the MEK-inhibitor U0126 against pandemic H1N1v and highly pathogenic avian influenza virus in vitro and in vivo

The emergence of the 2009 H1N1 pandemic swine influenza A virus is a good example of how this viral infection can impact health systems around the world in a very short time. The continuous zoonotic circulation and reassortment potential of influenza A viruses (IAV) in nature represents an enormous public health threat to humans. Beside vaccination antivirals are needed to efficiently control spreading of the disease. In the present work we investigated whether the MEK inhibitor U0126, targeting the intracellular Raf/MEK/ERK signaling pathway, is able to suppress propagation of the 2009 pandemic IV H1N1v (v=variant) as well as highly pathogenic avian influenza viruses (HPAIV) in cell culture and also in vivo in the mouse lung. U0126 showed antiviral activity in cell culture against all tested IAV strains including oseltamivir resistant variants. Furthermore, we were able to demonstrate that treatment of mice with U0126 via the aerosol route led to (i) inhibition of MEK activation in the lung (ii) reduction of progeny IAV titers compared to untreated controls (iii) protection of IAV infected mice against a 100× lethal viral challenge. Moreover, no adverse effects of U0126 were found in cell culture or in the mouse. Thus, we conclude that U0126, by inhibiting the cellular target MEK, has an antiviral potential not only in vitro in cell culture, but also in vivo in the mouse model.

Role of Hypercytokinemia in NF-κB p50-Deficient Mice after H5N1 Influenza A Virus Infection

ABSTRACT During H5N1 influenza virus infection, proinflammatory cytokines are markedly elevated in the lungs of infected hosts. The significance of this dysregulated cytokine response in H5N1-mediated pathogenesis remains to be determined. To investigate the influence of hypercytokinemia, or “cytokine storm,” a transgenic mouse technology was used. The classical NF-κB pathway regulates the induction of most proinflammatory cytokines. Deletion of the p50 subunit leads to a markedly reduced expression of the NF-κB-regulated cytokines and chemokines. Here we show that H5N1 influenza virus infection of this transgenic mouse model resulted in a lack of hypercytokinemia but not in altered pathogenesis.

Antibodies and CD4+ T-cells mediate cross-protection against H5N1 influenza virus infection in mice after vaccination with a low pathogenic H5N2 strain

A H5N2 low pathogenic avian influenza virus (LPAIV) was isolated from a natural reservoir in Bavaria during a routine screen and was used as a vaccine strain to scrutinize the immune response involved in cross-protection after challenge infection with a H5N1 highly pathogenic avian influenza virus (HPAIV). The challenge virus was also isolated from a natural reservoir in Bavaria. Wild type, antibody deficient (muMT), CD4(-/-) and CD8(-/-) mice were infected with the apathogenic H5N2 vaccine strain and challenge infection with a 100-fold MLD(50) of the H5N1 strain was performed 80 days later. While 100% of the wild type and 100% of the CD8(-/-) mice stayed healthy, only 50% of the CD4(-/-) and none of the antibody deficient mice were protected. These results support the view that the humoral immune response and to certain extends the CD4(+) T helper cells are a prerequisite for cross-protective immunity against H5 influenza virus.

Low-Dose Interferon Type I Treatment Is Effective Against H5N1 and Swine-Origin H1N1 Influenza A Viruses In Vitro and In Vivo

The recent emergence of pandemic swine-origin influenza virus (H1N1) and the severe outbreaks of highly pathogenic avian influenza virus of the H5N1 subtype leading to death in humans is a reminder that influenza remains a frightening foe throughout the world. Besides vaccination, there is an urgent need for new antiviral strategies to protect against influenza. The innate immune response to influenza viruses involves production of interferon alpha and beta (IFN-α/β), which plays a crucial role in virus clearance during the initial stage of infection. We examined the effect of IFN-α on the replication of H5N1 and H1N1 in vitro and in vivo. A single pretreatment with low-dose IFN-α reduced lung virus titers up to 1.4 log(10) pfu. The antiviral effect increased after multiple pretreatments. Low-dose IFN-α protected mice against lethal H5N1 viral infection. Further, IFN-α was also effective against H1N1 in vitro and in the mouse model. These results indicate that low-dose IFN-α treatment leads to the induction of antiviral cytokines that are involved in the reduction of influenza virus titers in the lung. Moreover, it might be possible that a medical application during pandemic outbreak could help contain fulminant infections.

The NF-kappaB inhibitor SC75741 protects mice against highly pathogenic avian influenza A virus.

The appearance of pandemic H1N1 and highly pathogenic avian H5N1 viruses in humans as well as the emergence of seasonal H1N1 variants resistant against neuraminidase inhibitors highlight the urgent need for new and amply available antiviral drugs. We and others have demonstrated that influenza virus misuses the cellular IKK/NF-kappaB signaling pathway for efficient replication suggesting that this module may be a suitable target for antiviral intervention. Here, we show that the novel NF-kappaB inhibitor SC75741 significantly protects mice against infection with highly pathogenic avian influenza A viruses of the H5N1 and H7N7 subtypes. Treatment was efficient when SC75741 was given intravenously in a concentration of 5mg/kg/day. In addition, application of SC75741 via the intraperitoneal route resulted in a high bioavailability and was also efficient against influenza when given 15 mg/kg/day or 7.5 mg/kg/twice a day. Protection was achieved when SC75741 was given for seven consecutive days either prior to infection or as late as four days after infection. SC75741 treatment showed no adverse effects in the concentrations required to protect mice against influenza virus infection. Although more pre-clinical studies are needed SC75741 might be a promising candidate for a novel antiviral drug against influenza viruses that targets the host cell rather than the virus itself.

Highly Pathogenic Influenza Virus Infection of the Thymus Interferes with T Lymphocyte Development

Highly pathogenic avian influenza viruses (HPAIVs) cause severe disease in humans. Still, the basis for their increased pathogenesis remains unclear. Additionally, the high morbidity in the younger population stays inexplicable, and the recent pandemic H1N1v outbreak in 2009 demonstrated the urgent need for a better understanding about influenza virus infection. In the present study, we demonstrated that HPAIV infection of mice not only led to lung destruction but also to functional damage of the thymus. Moreover, respiratory dendritic cells in the lung functioned as targets for HPAIV infection being able to transport infectious virus from the lung into the thymus. The pandemic H1N1 influenza virus was able to infect respiratory dendritic cells without a proper transport to the thymus. The strong interference of HPAIV with the immune system is especially devastating for the host and can lead to lymphopenia. In summary, from our data, we conclude that highly pathogenic influenza viruses are able to reach the thymus via dendritic cells and to interfere with T lymphocyte development. Moreover, this exceptional mechanism might not only be found in influenza virus infection, but also might be the reason for the increased immune evasion of some new emerging pathogens.

Influenza virus H5N1 hemagglutinin (HA) T-cell epitope conjugates: design, synthesis and immunogenicity.

The influenza virus, major surface glycoprotein hemagglutinin (HA) is one of the principal targets for the development of protective immunity. Aiming at contributing to the development of a vaccine that remains the first choice for prophylactic intervention, a reconstituted model of HA, mimicking its antigenic properties was designed, synthesized and tested in mice for the induction of protective immunity. Four helper T lymphocyte [HTL (T1, T3, T7 and T8)] and four cytotoxic lymphocyte [CTL (T2, T4, T5 and T6)] epitopes were coupled in two copies each to an artificial carrier, SOC4, which was formed by the repeating tripeptide Lys‐Aib‐Gly. The helical conformation of the SOC4‐conjugates preserves the initial topology of the attached epitopes, which is critical for their immunogenic properties. Survival of immunized animals, ranged from 30 to 50%, points out the induction of protective immunity by using the SOC4‐conjugates. Copyright

The Alternative NF-κB Signalling Pathway is a Prerequisite for an Appropriate Immune Response Against Lymphocytic Choriomeningitis Virus Infection

Two major nuclear factor-kappaB (NF-kappaB) signalling pathways are involved in the regulation of the immune response. While the classical NF-kappaB pathway is responsible for regulation of genes encoding components of the innate immune response, the alternative NF-kappaB signalling pathway mediates processes of the adaptive immune system. To evaluate the role of the NF-kappaB signalling pathways in the control of viral infection, we have used lymphocytic choriomeningitis virus (LCMV) infection of mice, which is known to be an excellent model for studying antiviral immune responses. Via the use of mice that were deficient in NF-kappaB subunits from either the classical (p50(-/-) mice) or the alternative NF-kappaB pathway (p52(-/-) mice), we were able to demonstrate that the alternative NF-kappaB pathway is required for the T-cell-mediated immune response against LCMV. Mice that were deficient in the alternative NF-kappaB pathway subunit p52 showed an impaired T-cell response against LCMV infection. Furthermore, these mice also showed an impaired T-cell-dependent humoral immune response against vesicular stomatitis virus (VSV) infection. Adoptive transfer experiments revealed that impaired priming, but not the T-cell response itself, was responsible for the defective cellular immune response against LCMV infection. Our data demonstrate that a functional alternative NF-kappaB signalling pathway is required to assure an adequate immune response after viral infection.