Vaccination confers protection against pathogens that is built up via active or passive immunization. This thesis is divided into two projects presenting novel approaches for passive and active vaccination. Passive immunization aims at providing instant support to the human immune system that is not able to curtail and clear infections. Past and current strategies, such as serum therapy and monoclonal antibodies, however, lack memory formation and thus require regular administration. Newer approaches of cellular vaccines, such as engineering of B cells, aim at replacing native B cell receptor sequences ex vivo with those of known neutralizing antibodies. Upon reinjection, modified B cells differentiate into long-lasting plasma cells expressing these protective antibodies. It needs to be considered, however, that amino acid changes in immunodominant epitopes of viral antigens - the main target of neutralizing antibodies - allow viruses to evade the immune system. The discovery of LAIR1-containing antibodies that bind broadly to various malaria-causing Plasmodium falciparum isolates has prompted us to establish a new type of passive vaccination, which integrates key virus-binding domains of cell entry receptors into antibodies. Antibodies containing pathogen receptor domains are expected to be difficult to be evaded by the pathogen and therefore would diminish viral immune escape. In addition, LAIR1-receptor antibodies were generated via a cell-intrinsic mechanism, likely involving the activity of activation-induced cytidine deaminase (AID). AID contributes to antibody diversity via induction of double-strand breaks in the antibody gene. In my thesis I show that CD40L plus IL-4 stimulation is the strongest inducer of AID mRNA transcripts and further enabled the successful in vitro engineering of LAIR1 insert-containing B cells, albeit at low frequency. To optimize insert modality for its subsequent integration and to quantify insertion efficiency, a flow-cytometry-based GFP-reporter assay was performed. Engineered B cells maintained 0.1 to 1% GFP expression with a diverse set of DNA substrates. Single-stranded DNA containing homology arms represented the most promising substrate design. Speaking of other pathogens than malaria, HIV concurrently lacks a working vaccine and SARS-CoV-2 appeared during my doctoral thesis as a highly relevant target. Therefore, I aimed at applying the obtained knowledge from LAIR1, integrated into antibodies, to key entry receptor domains for HIV-1 and SARS-CoV-2. While LAIR1-antibodies were designed by nature, insert-containing antibodies against HIV and SARS-CoV-2 still need to be developed. To this end, I recombinantly expressed antibodies containing pivotal cell entry receptor or nanobody domains and was able to obtain insert-mediated specific binding. Hence, such knowledge can serve as a template for functional protein domain insertions into antibodies of primary human B cells in the future. Taken together, this first project has laid the base for AID-stimulated B cell engineering. Primary human B cells can be endowed with key sites of viral entry receptors, such as immunoglobulin-like receptor domains from LAIR1, and may contribute a new strategy of passive vaccination. Active immunization involves the direct contact with pathogens that mounts adaptive immune cell responses, thereby eradicating infection and inducing long-term memory. The outbreak of COVID-19 and its impact around the globe has illustrated the need for safe and effective vaccines. Hitherto, conventional vaccine designs have not considered interactions of immunogens with the host cell receptor as a potential drawback of vaccine efficacy. We hypothesized that high-affinity binding of the SARS-CoV-2 spike to the ubiquitously expressed ACE2 receptor may on the one hand exert masking and on the other hand promote displacement of the vaccine antigen, thereby limiting immunogen availability and hampering the generation of neutralizing antibodies. Hence, we aimed for an active vaccine design strategy that by abrogation of entry receptor binding diminishes antigen displacement and preserves immunogenicity as well as accessibility to all epitopes of the receptor binding domain (RBD) of the viral spike. We therefore developed a bioinformatically residue scoring in collaboration with Prof. Dr. Simon Olsson, which was based on available data from high throughput cell receptor binding and antibody escape studies. We were able to stratify amino acids of the SARS-CoV-2 RBD into different groups based on their contribution to ACE2 receptor binding, their involvement in recognition by neutralizing antibodies as well as their respective expressibility. We identified a high scoring candidate, G502E, which displayed similar binding of neutralizing antibodies compared to WT, but lost association with ACE2. My in vitro studies confirmed higher adsorption as well as increased internalization of the SARS CoV-2 spike or RBD WT proteins compared to G502E by ACE2-expressing cells. Immunization of rabbits with the G502E spike induced higher anti-RBD antibody titers, indicating that receptor binding abrogation can focus the response towards the RBD. Furthermore, a G502E RBD mutant induced superior antibody titers and neutralization compared to WT. Taken together, this project merges information of the immunogenic nature and ACE2 binding features of amino acids of the RBD from the SARS-CoV-2 spike to design an improved protein vaccine candidate by abrogating interaction with the viral entry receptor. In future, this methodology may be applied to other vaccine platforms and to pathogens that engage cell entry receptors with high affinity and lack vaccines that elicit protective antibody responses.
