Establishment of a human tonsil organoid platform for the investigation of vaccine mechanisms of action

Principle Investigator

Scientific interest within the context of the graduate college:

Vaccines constitute one of the most effective public health interventions and play a crucial role in the control and prevention of infectious diseases.¹ Their value to human health was recently demonstrated during the COVID-19 pandemic, when the rollout of vaccines played a pivotal role in lowering the case-fatality and thus ending the pandemic state.² The versatility of vaccines is evident not only in their widespread use for prevention of disease from infections, but they can also lower morbidity and mortality from non-communicable diseases, such as myocardial infarction. Moreover, vaccines are explored as promising immunotherapeutics against cancer. Hence, the purposeful induction of protective immunity by means of vaccination has been shown to preserve health and to prevent disease.
Given that preventive vaccines are administered to healthy individuals with the goal of mobilizing health-preserving pathways, studying their exact mechanisms of action provides insights into protective immunity and yields important information for the targeted design of safe and effective vaccines. Pre-clinical development is usually carried out in animals, such as mice, to obtain information on immunogenicity and safety. However, significant differences in the immune system of rodents and humans, including differences in the immune cell receptor repertoires and T cell polarization (e.g. TFH cells) exist that may hamper translation for some vaccine candidates and underscore the need to establish human preclinical models of vaccination.^3,4

Project description:

Introduction: Investigating the mode of action of new and old vaccines in preclinical models poses a particular challenge due to the complexity of the human immune system and restrictions in studying molecular responses in lymphoid organs after vaccination in humans. There is an increasing need for models that accommodate the genetic, immunological, and environmental diversity of humans, to complement studies in animal models^3,5 and to accelerate translation into clinical development in human systems.

Highly effective vaccines possess the capacity to generate long-lasting, protective immune memory. In some cases, such as the measles vaccine, initial vaccination offers lifelong protection with estimated antibody titre half-lives of up to 200 years.^6,7

The generation of potent protective immune responses largely occurs in micro-anatomical lymphoid structures termed germinal centers. Germinal centers form within secondary lymphoid organs and are the site of antigenic selection, clonal expansion, somatic hypermutation, affinity maturation and class switch recombination of antibodies. These processes are driven by clonal competition for antigen and cognate T cell help provided by germinal center T follicular helper cells that promote the maturation of B cells towards memory B cells and antibody secreting cells. The composition of peripheral blood-derived mononuclear cells (PBMC) differs substantially from lymphocytes in secondary lymphoid organs.⁸ Consequently, blood cells, despite the relative ease of access, are not an optimal means of mapping of vaccine responses. Secondary lymphoid organs such as tonsils are a physiological source of bona fide germinal center TFH cells and B cells and they also provide antigen presenting cells and structures conducible to more complex immune responses. Human tonsils are also readily accessible following tonsillectomy.

Several recent publications describe ex vivo tonsil organoid culture platforms, which are suitable to study human vaccine responses in vitro.^9–13 The tonsillar cell composition and their described inherent capacity to reorganize and form germinal centers make them an attractive source of cells for the investigation of vaccine responses. The objective of this project is to establish a human tonsil organoid platform based on the described publications and, in a second step, the established organoid platform is used to investigate novel vaccine candidates and to compare different vaccine platforms and assess the responses over time. These longitudinal data will then serve to model and to predict vaccine responses to individual candidates.

Aim 1: The initial aim of the study is to establish and further adapt an organoid culture platform based on previously published protocols, which have already been piloted in our laboratory.^10,13 The culture of mechanically dissociated tonsil cells in the context of suitable culture supplements and culture conditions such as high cell density and in particular, cell culture plates with ultra-low cell adhesion leads to autonomous re-aggregation of the tonsil cells into tonsil-like organoids. Upon stimulation with vaccines, this re-aggregation process is increasingly observed. Alternative culture formats to be compared include culture in round well formats and tonsil explant cultures on collagen sponges and the addition of antigen via microfluidics systems, which has been shown to improve stimulation (unpublished data, personal communication).

Primary endpoints for the establishment of the organoid system include:

• High parametric spectral flow cytometry aided characterization of tonsil cells in steady state – and upon antigen stimulation.
• The antigen-specific establishment of germinal center responses, characterized by the differentiation of B cells into plasmablasts (detection by flow cytometry: CD19+ CD27++CD38++), immunofluorescence imaging and detection of antibodies by ELISA.
• Analysis of the expansion of vaccine- antigen specific B cell responses by direct staining of B cells with fluorescence tagged antigen.

Aim 2:

• Analysis of antigen-specific T cell responses using activation induced markers (AIM assay), in particular activation-dependent expression of CD40L, CD137, TNFa and IFNy and assessment of vaccine antigen specific TFH responses (CXCR5+ICOS+PD1+).
• Comparison of different vaccine types such as adjuvanted protein vaccines, mRNA vaccines, glycoconjugate vaccines and novel semisynthetic glycoconjugate vaccines (generated in collaboration with MPI for colloids and interfaces, Prof. Dr. Peter Seeberger).
• Investigate interpersonal variability in vaccine responses and comparison of steady state phenotype and composition of tonsillar cells with the in vitro vaccine response for generation of a predictive model for vaccine responses (in collaboration with Prof. Dr. Lisa Buchauer, Infectious Diseases, Charité).

References