Hedda Wardemann

Long-term in vivo production of therapeutic proteins and development of vaccines that elicit protective levels of broadly neutralizing antibodies (bNAbs) against major pathogens face challenges. In this study, we report on an alternative gene editing approach using small numbers of hematopoietic stem and progenitor cells (HSPCs) to direct long-term, high-level expression of antibodies or cargo proteins. In mice, edited B lymphocytes derived from transplanted HSPCs were activated by cognate antigen, underwent clonal expansion, and developed into specific antibody-synthesizing or cargo protein–synthesizing plasma cells. These cells produced long-lasting, therapeutic levels of serum antibody against HIV-1, malaria, or an anti-influenza virus bNAb that mediated universal protection from heterologous lethal challenge. Our data provide a paradigm for cell therapy approaches to prevent or treat disease using self-amplifying B cell protein factories.

To enable the analysis of B cell and T cell immune responses in mouse lymph nodes, spleen, and bone marrow, including different B cell and T cell subsets and their activation status, as well as the antigen-reactivity and isotype of B cells, a novel 31-parameter spectral flow cytometry panel was developed.

Long-term in vivo production of therapeutic proteins and development of vaccines that elicit protective levels of broadly neutralizing antibodies (bNAbs) against major pathogens face challenges. Here we report on an alternative gene-editing approach using small numbers of hematopoietic stem and progenitor cells (HSPCs) to direct long-term, high-level expression of antibodies or cargo proteins. Edited B lymphocyte offspring can be activated by cognate antigen to undergo clonal expansion and develop into specific antibody or cargo protein-synthesizing plasma cells. These cells produce long-lasting, therapeutic levels of serum antibody against HIV-1 or malaria and an anti-influenza virus bNAb that mediated universal protection from heterologous lethal challenge. Our data provide a paradigm for cell therapy approaches to prevent or treat disease using self-amplifying B cell protein factories.

Immunoglobulin (Ig) A is the main antibody isotype found on mucosal surfaces in mammals, where it is predominantly present as a dimer. Here we provide an easy, scalable, efficient, and broadly applicable method to produce and purify monoclonal mouse dimeric IgA from single B cell Ig transcripts to study mucosal antibody responses at single-cell level.

Graphical To enable the analysis of B cell and T cell immune responses in mouse lymph nodes, spleen, and bone marrow, including different B cell and T cell subsets and their activation status, as well as the antigen-reactivity and isotype of B cells and level of T cell exhaustion, a novel 31-parameter spectral flow cytometry panel was developed. Abstract Figure: Created in https://BioRender.com

Plasmodium falciparum malaria remains a significant global health challenge. Current vaccines elicit antibody responses against circumsporozoite protein (PfCSP) that prevent the infection of hepatocytes but offer only moderate protection. Cellular immunity has emerged as a critical component of preerythrocytic protection that might be leveraged to develop improved PfCSP vaccines. Here, we characterized the clonality, molecular features, epitope specificity, and HLA restrictions of the human PfCSP-specific CD4+ and CD8+ T cell response to vaccination with an adjuvanted PfCSP vaccine, FMP013/ALFQ. Using TCR expression cloning, we identified novel conserved CD4+ T cell epitopes in the PfCSP N terminus and showed that the C-terminal CS.T3 epitope was targeted by CD4+ and rare CD8+ T cells, which recognized this epitope co-receptor independently presented on a class II HLA. Our findings provide insights into the utility of these epitopes as targets for strain-transcending immunity compared with the immunodominant but highly polymorphic epitopes in the PfCSP C terminus, offering guidance for the design of improved malaria vaccines.

Plasmodium falciparum malaria remains a significant global health challenge. Current vaccines elicit antibody responses against circumsporozoite protein (PfCSP) that prevent the infection of hepatocytes but offer only moderate protection. Cellular immunity has emerged as a critical component of pre-erythrocytic protection that might be leveraged to develop improved PfCSP vaccines. Here, we characterized the clonality, molecular features, epitope specificity, and HLA-restrictions of the human PfCSP-specific CD4 ⁺ and CD8 ⁺ T cell response to vaccination with an adjuvanted PfCSP vaccine, FMP013/ALFQ. Using TCR expression cloning, we identified novel conserved CD4 ⁺ T cell epitopes in the PfCSP N terminus and show that the C-terminal CS.T3 epitope was targeted by CD4 ⁺ and rare CD8 ⁺ T cells, which recognized this epitope co-receptor-independently presented on a class II HLA. Our findings provide insights into the utility of these epitopes as targets for strain-transcending immunity compared to the immunodominant but highly polymorphic epitopes in the PfCSP C terminus, offering guidance for the design of improved malaria vaccines.

The majority of lymphomas originate from B cells at the germinal center stage. Preferential selection of B-cell clones by a limited set of antigens has been suggested to drive lymphoma development. While recent studies in B-cell chronic lymphocytic leukemia (CLL) have shown that self-reactive B-cell receptors (BCR) can generate cell-autonomous signaling and proliferation, our knowledge about the role of BCRs for the development or survival of other lymphomas remains limited. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows for unbiased characterization of the human antibody repertoire on single-cell level through the generation of recombinant monoclonal antibodies from primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells to the RT-PCR-based amplification of the expressed immunoglobulin (Ig) transcripts (IGH, IGK, and IGL) and their subsequent cloning into expression vectors for the in vitro production of recombinant monoclonal antibodies. The strategy may be used to obtain information about the clonal evolution of B-cell lymphomas by single-cell sequencing of Ig transcripts and on the antibody reactivity of human lymphoma B cells.

The protective capacity of antibodies targeting circumsporozoite protein on sporozoites of the malaria parasite Plasmodium falciparum (PfCSP) is linked to high affinity and cross-reactivity with the PfCSP central repeat domain and N-terminal junction. However, the role of somatic hypermutation (SHM) in the development of such antibodies remains unclear. Here we define the contributions of SHM to the high affinity and strong repeat and N-junction cross-reactivity of the potent anti-PfCSP monoclonal antibody (mAb) 4493 and of similar antibodies with shared SHM and affinity maturation trajectories. Molecular dynamics simulations reveal that SHM reduces the flexibility of the unbound mAb 4493 but increases the flexibility of the antigen-bound complex, thereby lowering the entropic cost for antigen binding. Furthermore, we identify an inverse relation between antibody affinity and serum stability, which limits the protective capacity of these antibodies. Our study provides molecular level evidence for the different roles that the SHM process plays in increasing VH3-49+Vκ3-20 antibody affinity and cross-reactivity and demonstrates how antibody affinity maturation can negatively impact antibody stability and thereby function.