Research Group of M. Tenbusch

Virology

Head of Institute:
Prof. Dr. med. Klaus Überla

Gene-based immunization strategies

Without any doubt vaccines are the most effective measures to prevent infectious diseases and promote individual and public health. Nevertheless, there are still a number of infectious disease where no or only inadequate vaccines are available. Therefore new vaccines and alternative vaccination platforms are still urgently needed.   Currently, we address the potency of different gene-based vaccines in following projects:

Mucosal immunity against viral respiratory tract infections induced by gene-based immunizations

Influenza A Virus (IAV) and the Respiratory Syncytial Virus (RSV) are causative agents of severe respiratory tract infection especially in young children and elderly people. The global disease burden is estimated to ~ 650 million cases per year for these two viruses leading to estimated 0.5 million deaths/year worldwide. While there is no prophylactic vaccine against RSV available at the time at all, there is an annual vaccination program against influenza viruses based mainly on tri- or quadrivalent inactivated influenza vaccines (T/QIV) which is recommended for certain risk groups (i.e. elderly or people with chronic lung diseases). Nevertheless, the overall vaccine efficacy is modest and the antibody mediated protection is rather short-lived and very strain-specific providing nearly no protection against drifted IAVs.
We developed in the last years DNA and adenoviral vector vaccines encoding the viral antigens hemagglutinin and nucleoprotein from IAV and the F protein from RSV. Furthermore, we evaluated a variety of application forms and determined the vaccine efficacy in the mouse and rhesus macaque model.
The most protective vaccination strategies included at least one immunization delivered via the mucosal route highlighting the exceptional role of local immune responses. We analyzed in detail the T-cell and antibody responses in the mucosal and in the systemic compartment.
Interestingly, a combination of a systemic DNA prime immunization with an intranasal adenoviral boost was the most effective schedule. This results in huge numbers of tissue-resident memory T-cells (TRM) in the lungs which can very rapidly control infections with different heterologous IAV strains in the mouse model. Furthermore, this strategy also proved efficacy against an RSV infection in the rhesus macaque model.
In our recent work, we would like to decipher the correlates of protection for the different infection models and learn how the systemic and mucosal immune cells act together. Furthermore, we compare different viral vector systems and analyze possible synergistic effects in term of protection.

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Signal molecules of the innate immune system as genetic adjuvants

From influenza infection models, it is known that not only the local antigen expression is important for the efficient induction of adaptive immune responses but also the induction of innate immune signaling.
Therefore, we used adenoviral vectors as genetic adjuvants which encode Interferons, components of the RIG-I/MAVS pathway or the effector molecules Interleukin-1ß and IL-18 derived from the inflammasome activation pathway.
So far, we could demonstrate that the co-delivery of an adenoviral vector encoding IL-1ß (rAd-IL-1β) dramatically improved the induction of the local immune response. Mucosal delivery of rAd-IL-1β enhanced HA-specific antibody responses including strain-specific neutralizing antibodies. Nevertheless, the beneficial effects on the local T-cell responses were much more impressive reflected by increased numbers of CD103+ CD69+ TRM.
This increased immunogenicity translated into superior protection against infections with homologous and heterologous strains including H1N1, pH1N1, H3N2, and H7N7 (Figure 1). Inhibition of the egress of circulating T-cells out of the lymph nodes during the heterologous infection had no impact on the degree of protection underscoring the unique potential of TRM for the local containment of mucosal infections.
The local co-expression of IL-1β and antigen lead to the activation of critical checkpoints in the formation of TRM including activation of epithelial cells, expression of chemokines and adhesion molecules, recruitment of lung-derived CD103+ DCs, and finally local TRM imprinting.
Given the importance of TRM-mediated protection at mucosal barriers, this study has major implications for the vaccine development against viral respiratory tract infections. Furthermore, it might be a step forward to the development of a broadly effective universal flu vaccine.

Figure 1: Enhanced heterosubtypic protection after co-delivery of rAd-IL-1β mediated by increased numbers of vaccine-induced TRM. BALB/c mice were immunized with 2 × 108 particles of rAd-HA and rAd-NP plus 1 × 109 particles of either rAd-empty, rAd-IL-18, or rAd-IL-1β. Seven weeks after the immunization, mice were challenged with one of three heterologous influenza A strains, namely the pandemic pH1N1 (A), H3N2/HK68 (B, left) or H7N7/Sc35 (B, right). Weight loss is indicative for the disease progression. The viral loads in the lungs after the pH1N1 challenge were determined via qRT-PCR (A, middle) and the tissue damage were indirectly measured by the total protein content in the BALF (A, right).
#p < 0.05 vs. naive; * or the respective group sign, p < 0.05 vs. HA/NP + empty

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Evaluation of Ad19a as a novel vector for mucosal vaccination

Replication-deficient adenoviral vectors have been extensively studied as vaccine vectors. The most common used systems are based on the adenovirus serotype 5. A possible drawback of this vector is the global seroprevalence of estimated 60-90% which might dampen the overall vaccine efficacy. Therefore, more rare human serotypes or adenoviruses from different species (e.g. simian adenoviruses) were evaluated as potential alternative vector systems. In this regard, we generated HA/NP-expressing Ad19a vectors and analyzed their immunogenicity and efficacy in the direct comparison to the Ad5-based vectors in our mouse model.
The adenoviral vectors were applied intranasally and induced detectable antigen-specific T-cell responses in the lung and in the spleen as well as robust antibody responses. A prior DNA immunization significantly improved the immunogenicity of both vectors and resulted in full protection against a lethal infection with a heterologous H3N2 virus. Nevertheless, the Ad5-based vectors were slightly superior in reducing viral replication in the lung which corresponded to higher NP-specific T-cell responses measured in the lungs.
Taken together, we proved that the rare serotype Ad19a can be used as a vaccine vector in mucosal immunizations and, although slightly less immunogenic than Ad5, it is able to induce protective humoral and cellular responses. Thus, Ad19a expands the spectrum of available replication-defective vectors that are suitable for human vaccination.

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Therapeutic immunization in the allergen-induced asthma model

In our group, we wanted to combine the advantages of gene-based vaccines with the efficient antigen presentation observed after Dendritic cell (DC) targeting. We generated single chain antibodies recognizing the DC-endocytic receptor DEC205, fused it to our antigen of interest and cloned the whole expression cassette in our adenoviral vector vaccines. Although we could demonstrate an enhanced antigen presentation, our studies revealed that we rather generate a local T-cell tolerance than antigen-specific effector T-cells probably due to missing maturation signals for the antigen-presenting DCs. A possible mechanism might be the induction of antigen-specific, regulatory cells instead of effector cells by immature DCs.
Based on these findings, we evaluated if the local induction of tolerance by this strategy might be suitable to dampen the allergen-specific immune response in a murine asthma model.
In this model, mice were sensitized with the model allergen ovalbumin resulting in a Th2 biased immune response which leads to airway hyper-responsiveness after inhalation of an OVA-containing aerosol. We treated this animal after the sensitization phase with adenoviral vectors encoding DC-targeted or non-targeted OVA and characterized the impact on the immunological and functional parameters of the asthma phenotype after challenge.
Indeed, only the intranasal application of vectors encoding the DC-targeted antigens was able to dampen the Th2 T-cell response and thereby reduce the strong eosinophilic infiltration and the airway hyper- responsiveness which are characteristics for the asthmatic phenotype.
This interesting approach should be further analyzed by using different vector systems and possibly new targeting strategies to improve the long-term effects of such a treatment. It might become an attractive alternative to the specific immune therapy which is until now the only causative treatment for allergies in patients.

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