Without any doubt, vaccines are the most effective measures to prevent infectious diseases and promote individual and public health. Since more than 15 years, our group developed and optimized gene-based vaccines, such as DNA, RNA and viral vector vaccines for several viral pathogens, including HIV, RSV and Influenza A Virus. During the recent SARS-CoV-2 pandemic, the enormous potential of mRNA or adenoviral vector vaccines to induce broadly protective immunity was demonstrated also for the first time in humans. The licensed COVID19 vaccines are applied intramuscularly and provide excellent protection from severe disease, hospitalization and deaths, but do not effectively prevent breakthrough infection in and transmission among vaccinated people. These findings are line with our previous knowledge from viral respiratory tract infections in animal models, which guided our research interest to the development of vaccines able to induce immunity at the site of viral entry, the mucosa of the respiratory tract. In the following, some highlights of our recent research activities and on-going projects will be shortly summarized.
Along the newly emerged SARS-CoV-2, established pathogens such as Influenza A Viruses (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. 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 can be induced by primary infection with H1N1 and could provide heterosubtypic immunity against other IAV strains like H3N2. Since these 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 immunomodulatory cytokines, e.g. IFN-γ (Fig.1).
Since intramuscularly applied vaccines do not induce this kind of mucosal immune response, our experiments revealed that local antigen presentation is needed for the induction and imprinting of lung TRM (Lapuente et al., 2018b). We evaluated different platforms, including PEI-complexed DNA or replication-deficient adenoviral vectors for their potential to induce local antibody and T-cell responses. Concerning the pre-existing immunity to the commonly used adenoviral vector based on the serotype Ad5, we specifically evaluated the rare human serotype Ad19a as alternative mucosal vaccine vector in mouse models for IAV, RSV and SARS-CoV-2 (Lapuente et al, 2018a; Lapuente el al., 2021b). These studies proved that the rare serotype Ad19a can be used as a vaccine vector in mucosal immunizations and is capable to induce humoral and cellular responses, although slightly less immunogenic than Ad5. Thus, Ad19a expands the spectrum of available replication-defective vectors that are suitable for human vaccination.
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.2).
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.
One way to improve the immunogenicity of vaccines is the use of adjuvants. To allow temporal and spatial co-delivery of the adjuvants with the antigen, we investigated adenoviral vectors encoding various chemokines or cytokines as potential adjuvants for the induction of mucosal immunity. So far, the co-delivery of an adenoviral vector encoding IL-1ß dramatically increased the numbers of IAV-NP-specific CD103+ CD69+ TRM in the lung, which translated in superior protection against divergent IAV strains, such as pH1N1, H3N2, and H7N7 (Lapuente et al., 2018b, Fig. 3).
In the context of RSV, the Ad-IL-1β adjuvanted, adenoviral vector vaccine conferred even superior protection against secondary RSV infection than natural immunity by primary RSV-infection in mice (Maier et al, 2022).
Most recently, in a collaborative study with the Pirbright Institute, we could confirm the adjuvant effect of Ad-IL-1β on the induction of cross-reactive TRM in the highly relevant large animal model of pigs. Interestingly, the increased number of porcine, NP-specific TRM cells had no significant impact on a heterologous IAV infection. In follow-up project, we would like to decipher potential differences in the correlates of heterosubtypic immunity between different animal models (Schmidt et al, 2023).
Furthermore, in an on-going PhD project within the RTG2504, we evaluate several other adenoviral vectors encoding chemokines involved in the T-cell migration or TRM imprinting as alternative adjuvants to sharpen the TRM profile of our vaccine-induced T-cells.
Breast cancer is the most prevalent cancer worldwide with 2.3 million diagnoses each year accounting for 25% of all malignancies in women. An early diagnosis followed by a timely excision of the primary lesion yields a relatively good prognosis (5-year survival of 99%). However, this prognosis worsens significantly if distant metastases are present (5-year survival 29%). One major site of breast cancer metastasis is the lung. 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 tumor-associated antigens (gp70 AH1, Mage-b). Depending on the route of vaccine administration, the vaccine establishes either robust systemic immunity after intramuscular delivery or pronounced tissue-resident memory T-cell (TRM) responses after intranasal delivery (Fig. 4 A+B). Vaccine-induced TRM display a prototypical CD69+CD103+ phenotype (Fig. 4 C) and remain unstained upon in vivo intravenous antibody staining (a method to distinguish circulating from resident immune cells; by Anderson et al. 2014). In a prophylactic setting, in which the vaccine is given prior the induction of pulmonary metastases (Fig. 4 D), the mucosal vaccination inhibited the tumor growth more efficiently than a systemic immunization highlighting the importance of mucosal immunity at barrier sites (Fig. 4 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 like immune checkpoint blockade or chemotherapy. In a collaboration with Prof. Udo Gaipl, Dr. Benjamin Frey and Dr. Michael Rückert (Radiation Oncology, University Hospital Erlangen), we are investigating a combination of radiotherapy and mucosal immunization against lung-metastasized breast cancer.
The importance of lung-resident memory T-cells (TRM) 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 highly 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. 5) and the differential transcriptional programming will be analysed by single cell RNA sequencing. Finally, we will particular address the impact of secondary inflammatory events, such as unrelated bacterial or viral infections, on the fate of pre-existing, virus-specific lung TRM.
In the early phase of the pandemic, we established serological assays to analyse antibody responses to SARS-CoV-2 after infections and at later stages after vaccinations. A flow cytometric-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 analysed 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).
In our recent SARS-CoV-2 serology studies, 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 neutralising 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, like antibody-mediated cellular cytotoxicity (ADCC) or phagocytosis (ADCP), and is considered as anti-inflammatory (Fig.6). Up to now, only a small amount of research in the context of viral infectious diseases has been carried out on these antibodies, because they are quite rare. This exciting discovery in the field of immunology thus raises new questions about antibody maturation. The second important question is what are the consequence of a strong IgG4 responses in preventing viral infection or disease progression, especially if they have reduced neutralizing capacity which would be a likely scenario in case of newly emerging variants. In our recent projects, we want to address these questions in appropriate small animal models.