N-glycosylation of intrinsic and engineered N-X-S/T motifs by <em>Pichia pastoris</em> can be exploited to ligate the mannose receptor but reveals no gain in immunogenicity per se

Vaccination has the power to eradicate viral diseases and is a promising approach to cure cancer. However, modern vaccination strategies repeatedly fail in inducing a robust cytotoxic CD8+ T cell response, which is needed to eradicate virus-infected or malignantly transformed cells. One way to improve this induction is the targeting of C-type lectin receptors on dendritic cells that lead to the presentation of antigens on MHC class I molecules to CD8+ T cells in a process termed cross-presentation. In addition, certain C-type lectin receptors have the capacity to induce DC maturation, which provides the second crucial signal to induce cytotoxic T cell activation. Yeasts such as Pichia pastoris produce N-glycans that are able to ligate such C-type lectin receptors and are thus generally considered to be immunostimulatory. N-glycosylation by P. pastoris might hence be exploited in vaccine strategies to promote both MHC I-restricted antigen presentation and DC maturation. However, the benefit of such a vaccination approach remains elusive since the particular effect of P. pastoris-derived N-glycans on a cellular and humoral response in vivo has not been investigated so far. Here we tested whether it is possible to introduce N-glycans on proteins that are not glycosylated in their native state by recombinant expression in P. pastoris in order to target C-type lectin receptors and increase antigen cross-presentation. For this purpose we expressed β-galactosidase (β-gal), a cytosolic Escherichia coli protein bearing several potential glycosylation sites, and a GFP-derivative with an engineered glycosylation site (NST-GFP) in P. pastoris. We show that both intrinsic and artificially designed N-glycosylation motifs are readily glycosylated after secretion by P. pastoris. We demonstrate that the attached N-glycans ligate the calcium-dependet carbohydrate recognition domains of the mannose receptor (MR), a C-type lectin receptor that mediates cross-presentation of the model antigen ovalbumin (OVA). Antigens internalized by bone marrow-derived dendritic cells (BM-DCs) were consistently routed to OVA-positive compartments related to cross-presentation. However, subsequent in vitro analysis revealed that P. pastoris-derived N-glycans had no immunostimulatory capacity on BM-DCs per se. To elucidate the impact of such enforced N-glycosylation in vivo we subcutaneously immunized mice with N-glycosylated or enzymatically deglycosylated β-gal or NST-GFP. Surprisingly, the effect of N-glycosylation on in vivo cross-presentation proved to be dependent on the nature of the antigen. The presence of N-glycans increased the in vivo cytotoxicity against β-gal, whereas a decrease was observed against NST-GFP fused to an immunodominant OVA-epitope. Importain vivo ntly, for both antigens tested we observed a reduction of the humoral immune response in the presence of N-glycans as indicated by decreased serum IgG in comparison to the group immunized with deglycosylated proteins. Our data demonstrate that P. pastoris can be used to tag non-glycoproteins with N-glycans that target the MR. However, a beneficial effect of such N-glycans on in vivo cross-presentation was dependent on the antigen, and the presence of N-glycans consistently decreased a humoral response. These findings have important implications on recombinant vaccines using Pichia pastoris as an expression system

Cross-Presentation: How to Get there – or How to Get the ER

Antigen cross-presentation enables dendritic cells (DCs) to present extracellular antigens on major histocompatibility complex (MHC) I molecules, a process that plays an important role in the induction of immune responses against viruses and tumors and in the induction of peripheral tolerance. In order to allow intracellular processing for cross-presentation, internalized antigens are targeted by distinct endocytic receptors toward specific endosomal compartments, where they are protected from rapid lysosomal degradation. From these compartments, antigens are processed for loading onto MHC I molecules. Such processing generally includes antigen transport into the cytoplasm, a process that is regulated by members of the ER-associated degradation (ERAD) machinery. After proteasomal degradation in the cytoplasm, antigen-derived peptides have been shown to be re-imported into the same endosomal compartment by endosomal transporter associated with antigen processing, another ER protein, which is recruited toward the endosomes after DC maturation. In our review, we highlight the recent advances on the molecular mechanisms of cross-presentation. We focus on the necessity of such antigen storage compartments and point out important parallels to MHC I-restricted presentation of endogenous antigens. We discuss the composition of such endosomes and the targeting of extracellular antigens into this compartment by specific endocytic receptors. Finally, we highlight recent advances on the recruitment of the cross-presentation machinery, like the members of the MHC I loading complex and the ERAD machinery, from the ER toward these storage compartments, a process that can be induced by antigen encounter or by activation of the dendritic cell after contact with endotoxins

CARs derived from broadly neutralizing, human monoclonal antibodies identified by single B cell sorting target hepatitis B virus-positive cells

To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs

Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2.

Human in vitro generated monocyte-derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High-dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM-CSF and IL-4 stimulation combinatorically and temporally, resulting in a mode- and time-dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high-dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies

Restriction of HIV-1 Escape by a Highly Broad and Potent Neutralizing Antibody

Broadly neutralizing antibodies (bNAbs) represent a promising approach to prevent and treat HIV-1 infection. However, viral escape through mutation of the HIV-1 envelope glycoprotein (Env) limits clinical applications. Here we describe 1-18, a new V_H1-46-encoded CD4 binding site (CD4bs) bNAb with outstanding breadth (97%) and potency (GeoMean IC₅₀ = 0.048 μg/mL). Notably, 1-18 is not susceptible to typical CD4bs escape mutations and effectively overcomes HIV-1 resistance to other CD4bs bNAbs. Moreover, mutational antigenic profiling uncovered restricted pathways of HIV-1 escape. Of most promise for therapeutic use, even 1-18 alone fully suppressed viremia in HIV-1-infected humanized mice without selecting for resistant viral variants. A 2.5-Å cryo-EM structure of a 1-18-BG505_(SOSIP.664) Env complex revealed that these characteristics are likely facilitated by a heavy-chain insertion and increased inter-protomer contacts. The ability of 1-18 to effectively restrict HIV-1 escape pathways provides a new option to successfully prevent and treat HIV-1 infection

Exploiting B Cell Receptor Analyses to Inform on HIV-1 Vaccination Strategies

The human antibody repertoire is generated by the recombination of different gene segments as well as by processes of somatic mutation. Together these mechanisms result in a tremendous diversity of antibodies that are able to combat various pathogens including viruses and bacteria, or malignant cells. In this review, we summarize the opportunities and challenges that are associated with the analyses of the B cell receptor repertoire and the antigen-specific B cell response. We will discuss how recent advances have increased our understanding of the antibody response and how repertoire analyses can be exploited to inform on vaccine strategies, particularly against HIV-1

Modeling the Amplification of Immunoglobulins through Machine Learning on Sequence-Specific Features

Successful primer design for polymerase chain reaction (PCR) hinges on the ability to identify primers that efficiently amplify template sequences. Here, we generated a novel Taq PCR data set that reports the amplification status for pairs of primers and templates from a reference set of 47 immunoglobulin heavy chain variable sequences and 20 primers. Using logistic regression, we developed TMM, a model for predicting whether a primer amplifies a template given their nucleotide sequences. The model suggests that the free energy of annealing, Delta G, is the key driver of amplification (p = 7.35e-12) and that 3' mismatches should be considered in dependence on Delta G and the mismatch closest to the 3' terminus (p = 1.67e-05). We validated TMM by comparing its estimates with those from the thermodynamic model of DECIPHER (DE) and a model based solely on the free energy of annealing (FE). TMM outperformed the other approaches in terms of the area under the receiver operating characteristic curve (TMM: 0.953, FE: 0.941, DE: 0.896). TMM can improve primer design and is freely available via openPrimeR (http://openPrimeR.mpi-inf.mpg.de)

Antibody-mediated neutralization of SARS-CoV-2

Neutralizing antibodies can block infection, clear pathogens, and are essential to provide long-term immunity. Since the onset of the pandemic, SARS-CoV-2 neutralizing antibodies have been comprehensively investigated and critical information on their development, function, and potential use to prevent and treat COVID-19 have been revealed. With the emergence of SARS-CoV-2 immune escape variants, humoral immunity is being challenged, and a detailed understanding of neutralizing antibodies is essential to guide vaccine design strategies as well as antibody-mediated therapies. In this review, we summarize some of the key findings on SARS-CoV-2 neutralizing antibodies, with a focus on their clinical application

Feature Matrix for Development of Models for Estimating the Likelihood of PCR Amplification

This data set contains pairs of primers and immunoglobulin heavy chain variable sequences with annotated experimental amplification status according to gel electrophoresis. The data set tabulates the features (e.g. annealing temperature, mismatches) that determine whether a primer leads to the successful amplification of a template