Research Group of M. Tenbusch/D. Lapuente

Along the newly emerged SARS-CoV-2, established pathogens such as IAV and 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. Due to the short incubation period of respiratory viral infections, vaccines need to induce immune responses yielding an immediate antiviral response upon exposure. Secretory IgA and tissue-resident memory T-cells (TRM) at mucosal surfaces have been identified as major players involved in the protection against pulmonary virus infections. Specifically, lung TRM cells directed against the conserved nucleoprotein of IAV provide heterosubtypic immunity against divergent IAV strains. Since TRM cells reside in the target tissue, they locally respond to a secondary infection, restrict immediate viral replication and recruit further immune cells by the secretion of inflammatory cytokines, e.g. IFN-γ.

Fitting to the observation that intramuscularly applied vaccines do not induce mucosal  T-cells, our research confirmed local antigen presentation and inflammation induced by mucosal vaccines as prerequisite for the induction and imprinting of lung TRM (Lapuente et al., 2018b). We evaluated different vaccine platforms, including DNA, RNA or replication-deficient adenoviral vectors (Ad5/Ad19a) for their potential to induce local antibody and T-cell responses and to provide protection from IAV, RSV and SARS-CoV-2 in mice (Lapuente et al, 2018a; Lapuente el al., 2021b).

However, these studies also confirmed that a prior systemic immunization with either DNA or RNA based vaccines encoding the same viral antigens maximizes the immunogenicity of the mucosally applied adenoviral vector vaccines. An intramuscular DNA immunization followed by an mucosal adenoviral boost resulted in mucosal immunity, including antiviral IgA antibodies in the BAL and TRM responses, and provided efficient protection against Influenza and RSV in mice and NHP, respectively (Lapuente et al, 2018a; Grunwald et al., 2014). A confirmatory, multi-center non-human primate study as a next step to translate this approach into a clinical trial is currently on-going.

Most recently, we utilized the licensed mRNA vaccine Comirnaty® as systemic prime immunization followed by intranasal vaccinations with adenovirus 5 and 19a vectored vaccines encoding the SARS-CoV-2 spike protein (Lapuente el al., 2021b). In contrast to two intramuscular applications of the mRNA vaccine, intranasal boosts with adenoviral vectors induced high levels of mucosal IgA and lung-TRM. The mucosal neutralization of virus variants of concern was also enhanced.

The mRNA prime provokes a comprehensive T-cell response consisting of circulating and lung TRM after the boost, while the repeated intramuscular immunization induced almost exclusively circulating, non-resident memory T-cells. Concomitantly, the intranasal boost strategies lead to complete protection against a SARS-CoV-2 infection in mice (Fig.1).

Having established this efficient combination of a systemic prime-mucosal boost vaccination schedule, we are now interested in how we can further optimize the mucosal booster to induce broad and long-lived mucosal immunity. This project is designed and will be undertaken as part of a Bavarian research consortium named FOR-COVID.

The importance of lung-resident memory T-cells for the rapid control of viral respiratory tract infections has been well documented. The programming of antigen-specific T-cells to TRM seems to happen early during the priming phase and depend very much on the local inflammatory environment and antigen presentation. In contrast, the mechanisms of the maintenance of TRM in this immune privileged tissue are not yet fully understood. We determine whether the differential induction of the TRM immunity by a primary infection or by the mucosal application of gene-based vaccines might play a role in this context. In longitudinal studies, phenotypic and functional TRM responses are assessed in mice. Furthermore, the structural organization within the tissue will be examined via immunohistology (Fig. 2) and the differential transcriptional programming will be analyzed by single cell RNA sequencing. Finally, we will particularly address the impact of secondary inflammatory events, such as unrelated bacterial or viral infections, on the fate of pre-existing, virus-specific TRM.

Breast cancer is the most prevalent cancer worldwide with 2.3 million diagnoses each year accounting for 25% of all malignancies in women. In most cases, an early diagnosis yields a relatively good prognosis (5-year survival of 99%). The presence of metastases in distant organs including the lung worsens the prognosis significantly (5-year survival 29%). Our goal is to develop a mucosal vaccination strategy to inhibit the formation and growth of pulmonary breast cancer metastases.

We have designed an adenoviral vector vaccine in the preclinical 4T1 breast cancer model for the induction of CD8⁺ T-cell responses directed against two endogenous tumor-associated antigens (gp70 AH1, Mage-b). Depending on the route of vaccine administration, the vaccine establishes either robust systemic immunity after intramuscular or pronounced TRM responses after intranasal delivery (Fig. 3 A+B). Vaccine-induced TRM display a prototypical CD69⁺CD103⁺ phenotype (Fig. 3 C) and remain unstained upon in vivo intravenous antibody staining (a method to distinguish circulating from resident immune cells). In a prophylactic setting (Fig. 3 D), the mucosal vaccination inhibited the tumor growth more efficiently than a systemic immunization highlighting the importance of mucosal immunity at barrier sites (Fig. 3 E+F). Currently, we are characterizing this vaccine approach in a therapeutic setting. Moreover, we want to understand synergistic effects of therapeutic vaccination and clinically relevant treatment options such as immune checkpoint blockade or chemotherapy. Together with Prof. Udo Gaipl (Radiation Oncology, University Hospital Erlangen), we are also investigating a combination of radiotherapy and mucosal immunization.

In the early phase of the pandemic, we established serological assays to analyze antibody responses to SARS-CoV-2 after infections. A flow cytometry-based assay allows us to quantify antibodies of the different Ig isotypes (IgM, IgA, IgG) binding to SARS-CoV-2 spike (S) protein (Lapuente et al., 2021a). In several studies, we analyzed the SARS-CoV-2 specific immune responses for example in cohorts with different immunization schedules (Tenbusch et al, 2021; Vogel et al, 2022) or in specific cohorts of patients receiving immunomodulatory medical interventions (Simon et al, 2020; Reimann et al, 2022; Kremer et al, 2021).

Together with the groups of Prof. Thomas Winkler and PD Kilian Schober, we observed an atypical increase in vaccine-induced IgG4 antibody responses against the SARS-CoV-2 spike protein after three doses of the mRNA vaccine Comirnaty (Irrgang et al, 2023). The contribution of virus-specific antibodies in preventing infections and disease progression depends beside neutralizing activity very much on secondary effector functions mediated by binding to Fc-gamma receptors (FcgR) on various immune cells. In this regard, IgG4 represents an IgG isotype with low potential of mediating antiviral effector functions such as antibody-mediated cellular cytotoxicity (ADCC) or phagocytosis (ADCP) and is considered as anti-inflammatory (Fig.4).

In the context of viral infectious diseases not much is known about the contribution of this rare subclass. Our exciting discovery in the field of immunology raises new questions about antibody maturation and the consequence of strong IgG4 responses in preventing viral infection or disease progression. In our recent projects, we want to address these questions in appropriate small animal models.