New Opportunities in Glycan Engineering for Therapeutic Proteins

Glycans as sugar polymers are important metabolic, structural, and physiological regulators for cellular and biological functions. They are often classified as critical quality attributes to antibodies and recombinant fusion proteins, given their impacts on the efficacy and safety of biologics drugs. Recent reports on the conjugates of N-acetyl-galactosamine and mannose-6-phosphate for lysosomal degradation, Fab glycans for antibody diversification, as well as sialylation therapeutic modulations and O-linked applications, have been fueling the continued interest in glycoengineering. The current advancements of the human glycome and the development of a comprehensive network in glycosylation pathways have presented new opportunities in designing next-generation therapeutic proteins.

Role of EXO1 nuclease activity in genome maintenance, the immune response and tumor suppression in Exo1D173A mice

ABSTRACTDNA damage response pathways rely extensively on nuclease activity to process DNA intermediates. Exonuclease 1 (EXO1) is a pleiotropic evolutionary conserved DNA exonuclease involved in various DNA repair pathways, replication, antibody diversification, and meiosis. But, whether EXO1 facilitates these DNA metabolic processes through its enzymatic or scaffolding functions remains unclear. Here we dissect the contribution of EXO1 enzymatic versus scaffolding activity by comparing Exo1DA/DA mice expressing a proven nuclease-dead mutant form of EXO1 to entirely EXO1-deficient Exo1−/− and EXO1 wild type Exo1+/+ mice. We show that Exo1DA/DA and Exo1−/− mice are compromised in canonical DNA repair processing, suggesting that the EXO1 enzymatic role is important for error-free DNA mismatch and double-strand break repair pathways. However, in non-canonical repair pathways, EXO1 appears to have a more nuanced function. Next-generation sequencing of heavy chain V region in B cells showed the mutation spectra of Exo1DA/DA mice to be intermediate between Exo1+/+ and Exo1−/− mice, suggesting that both catalytic and scaffolding roles of EXO1 are important for somatic hypermutation. Similarly, while overall class switch recombination in Exo1DA/DA and Exo1−/− mice was comparably defective, switch-switch junction analysis suggests that EXO1 might fulfill an additional scaffolding function downstream of class switching. In contrast to Exo1−/− mice that are infertile, meiosis progressed normally in Exo1DA/DA and Exo1+/+ cohorts, indicating that a structural but not the nuclease function of EXO1 is critical for meiosis. However, both Exo1DA/DA and Exo1−/− mice displayed similar mortality and cancer predisposition profiles. Taken together, these data demonstrate that EXO1 has both scaffolding and enzymatic functions in distinct DNA repair processes and suggest a more composite and intricate role for EXO1 in DNA metabolic processes and disease.

Rarely Recognized Antibody Diversification in Covid-19 Evolution to Counteract Advanced SARS-CoV-2 Evasion Strategies, and Implications for Prophylactic Treatment

The ongoing Covid-19 pandemic underscores the importance of finding effective and safe ways to combat the virus, and to optimally understand the immune response elicited upon natural infection. This likely involves all components of the immune system, both innate and adaptive. The impetus for the rapid development of prophylactic treatment options has led to an intense focus on neutralizing antibodies (Abs), and many novel and specialized platforms have been designed to achieve that goal. B-cell immunity relies on the generation of a diverse repertoire of Abs. Their structural variation is defined in terms of amino acid composition that is encoded in the genome or acquired through somatic mutations. Yet, key examples of frequently neglected antibody diversification mechanisms involving post-translational modifications such as N- or O-linked glycosylation are present in significant portions of the population. During the last few years, these and other beyond gene sequence determined humoral immune response mechanisms have in some specific cases revealed their potent immunomodulatory effects. Nonetheless, such more unusual mechanisms have not received much attention in the context of SARS-CoV-2. Thus, with specific focus on the latter, this paper presents, (1) the rationale for considering beyond sequence determined strategies, (2) evidence for their possible involvement in Covid-19 disease evolution, (3) consequences for vaccine design exemplified by one of the vaccine candidates that is currently undergoing trial, and (4) more general implications. Based on a critical interpretation of published literature, the hypotheses developed in this study point to a crucial role of non-genetic antibody diversification mechanisms in disease evolution to counteract unique immunogenicity determinants of SARS-CoV-2 infection. The involvement of post translational mechanisms may also help explain the widely varied immune response observed, not only among different patient groups, but also in terms of their observed incompatibility with SARS-CoV-2 infection in several human cell types. The article highlights potentials and challenges of these refined humoral immune response mechanisms to most optimally target non-genetic viral evasion strategies.

Single cell clonal analysis identifies an AID-dependent pathway of plasma cell differentiation

Germinal centers (GC) are microstructures where B cells that have been activated by antigen can improve the affinity of their B cell receptors and differentiate into memory B cells (MBCs) or antibody secreting plasma cells. Activation Induced Deaminase (AID) initiates antibody diversification in GCs by somatic hypermutation and class switch recombination. Here we have addressed the role of AID in the terminal differentiation of GC B cells by combining single cell transcriptome and immunoglobulin clonal analysis in a mouse model that traces AID-experienced cells. We identified 8 transcriptional clusters that include dark zone and light zone GC subsets, plasmablasts/plasma cells (PB), 4 subsets of MBCs and a novel prePB subset, which shares the strongest clonal relationships with PBs. Mice lacking AID have various alterations in the size and expression profiles of these transcriptional clusters. We find that AID deficiency leads to a reduced proportion of prePB cells and severely impairs transitions between the prePB and the PB subsets. Thus, AID shapes the differentiation fate of GC B cells by enabling PB generation from a prePB state.

Characterization of DNA G-Quadruplex Structures in Human Immunoglobulin Heavy Variable (IGHV) Genes

Activation-induced deaminase (AID) is a key enzyme involved in antibody diversification by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) of the Immunoglobulin (Ig) loci. AID preferentially targets WRC (W=A/T, R=A/G) hotspot motifs and avoids SYC (S=C/G, Y=C/T) coldspots. G-quadruplex (G4) structures are four-stranded DNA secondary structures with key functions in transcription, translation and replication. In vitro studies have shown G4s to form and bind AID in Ig switch (S) regions. Alterations in the gene encoding AID can further disrupt AID-G4 binding and reduce CSR in vivo. However, it is still unclear whether G4s form in the variable (V) region, or how they may affect SHM. To assess the possibility of G4 formation in human V regions, we analyzed germline human Ig heavy chain V (IGHV) sequences, using a pre-trained deep learning model that predicts G4 potential. This revealed that many genes from the IGHV3 and IGHV4 families are predicted to have high G4 potential in the top and bottom strand, respectively. Different IGHV alleles also showed variability in G4 potential. Using a high-resolution (G4-seq) dataset of biochemically confirmed potential G4s in IGHV genes, we validated our computational predictions. G4-seq also revealed variation between S and V regions in the distribution of potential G4s, with the V region having overall reduced G4 abundance compared to the S region. The density of AGCT motifs, where two AGC hotspots overlap on both strands, was roughly 2.6-fold greater in the V region than the Constant (C) region, which does not mutate despite having predicted G4s at similar levels. However, AGCT motifs in both V and C regions were less abundant than in S regions. In silico mutagenesis experiments showed that G4 potentials were generally robust to mutation, although large deviations from germline states were found, mostly in framework regions. G4 potential is also associated with higher mutability of certain WRC hotspots on the same strand. In addition, CCC coldspots opposite a predicted G4 were shown to be targeted significantly more for mutation. Our overall assessment reveals plausible evidence of functional G4s forming in the Ig V region.

Impact of HIV-1 Vpr manipulation of the DNA repair enzyme UNG2 on B lymphocyte class switch recombination

Background: HIV-1 Vpr encodes a 14 kDa protein that has been implicated in viral pathogenesis through modulation of several host cell functions. In addition to pro-apoptotic and cytostatic properties, Vpr can redirect cellular E3 ubiquitin ligases (such as DCAF1-Cul4A E3 ligase complex) to target many host proteins and interfere with their functions. Among them, Vpr binds the uracil DNA glycosylase UNG2, which controls genome uracilation, and induces its specific degradation leading to loss of uracil removal activity in infected cells. Considering the essential role of UNG2 in antibody diversification in B-cells, we evaluated the impact of Vpr on UNG2 fate in B lymphocytes and examined the functional consequences of UNG2 modulations on class switch recombination (CSR). Methods: The impact of Vpr-induced UNG2 deregulation on CSR proficiency was evaluated by using virus-like particles able to deliver Vpr protein to target cells including the murine model CSR B-cell line CH12F3 and mouse primary B-cells. Co-culture experiments were used to re-examine the ability of Vpr to be released by HIV-1 infected cells and to effectively accumulate in bystander B-cells. Vpr-mediated UNG2 modulations were monitored by following UNG2 protein abundance and uracil removal enzymatic activity.Results: In this study we report the ability of Vpr to reduce immunoglobulin class switch recombination (CSR) in immortalized and primary mouse B-cells through the degradation of UNG2. We also emphasize that Vpr is released by producing cells and penetrates bystander B lymphocytes.Conclusions: This work therefore opens up new perspectives to study alterations of the B-cell response by using Vpr as a specific CSR blocking tool. Moreover, our results raise the question of whether extracellular HIV-1 Vpr detected in some patients may manipulate the antibody diversification process that engineers an adapted response against pathogenic intruders and thereby contribute to the intrinsic B-cell humoral defect reported in infected patients.