Glycan Antigens Research Articles

Reduction in Xenogeneic Epitopes on Porcine Endothelial Cells by Periodate Oxidation.

Patterns of humoral immune responses represent a major hurdle in terms of pig-to-human xenotransplantation approaches. The best-known xenogeneic glycan antigens present in pigs are the αGal (Galili antigen) and the non-human sialic acid Neu5Gc. As there are further differences between porcine and human cellular surface glycosylation, a much broader range of glycan epitopes with xeno-reactive relevance can be anticipated. Therefore, we set out to chemically modify porcine cellular surface glycans in a global approach by applying sodium periodate (NaIO4) oxidation. Porcine endothelial cells were exposed to oxidation with 1 to 5 mM NaIO4 for different time periods at 37 °C or 4 °C and under static or dynamic conditions. The impact on cellular survival was determined by applying live/dead assays. Oxidation of αGal-epitopes was assessed by fluorescence microscopy-based quantification of isolectin-B4 (IL-B4) staining. Overall immunogenicity of porcine cells was determined by human serum antibody binding. Treatment of porcine endothelial cells and tissues with NaIO4 led to reduced binding of the αGal-specific IL-B4 and/or human serum antibodies. NaIO4 was revealed to be cytotoxic when performed at elevated temperatures and for a prolonged time. However, by applying 2 mM NaIO4 for 60 min at 4 °C, a high extent of cellular viability and a relevant reduction in detectable αGal epitope were observed. No differences were detected irrespectively on whether the cells were oxidized under static or flow conditions. Glycan epitopes on living cells can be oxidized with NaIO4 while maintaining their viability. Accordingly, this strategy holds promise to prevent immune reactions mediated by preformed anti-glycan antibodies.

Deciphering immunogenic diversity via glycan antigen characterization in genetically modified pigs for xenotransplantation

Deciphering immunogenic diversity via glycan antigen characterization in genetically modified pigs for xenotransplantation

Analysis of the clinical efficacy of laparoscopic middle pancreatectomy in the treatment of benign or low-grade malignant tumors of the pancreas.

The aim of this study was to investigate the clinical efficacy of laparoscopic middle pancreatectomy in the treatment of benign and junctional tumors of the pancreas. Retrospective analysis of basic data, tumor diameter, statistical analysis, and evaluation of efficacy-related indicators such as operative time, intraoperative bleeding, pathological findings, postoperative hospital stay, postoperative pancreatic fistula incidence, and pancreatic endocrine function was carried out on 17 patients diagnosed with benign or low-grade malignant tumors of the pancreas and laparoscopic middle pancreatic resection from January 2018 to January 2023 at the First Affiliated Hospital of Hunan Normal University. A total of 17 patients were screened. There were eight males and nine females; mean age was 42.8 ± 17.4 years (range: 15-69 years); BMI was 22.6 ± 2.5 kg/m2 (range: 18.4-27.5 kg/m2), and the tumor size was 3.4 ± 1.2cm (range: 1.5-5.5 cm). Preoperative glycan antigen CA19-9 was negative and CA125 was negative. Surgical time was 393.2 ± 57.9min; intraoperative bleeding was 211.7 ± 113.9ml; tumor diameter size was 3.4 ± 1.2cm; postoperative admission time was 19.4 ± 7.6 days; postoperative pancreatic fistula (POPF) grading was 17 cases, including nine cases of A-grade fistula, three cases of B-grade fistula, and none of C-grade fistula; postoperative pathology results were five cases of plasmacytoma, three cases of mucinous cystadenoma, four cases of SPN (solid pseudopapillary neoplasm), one case of Intraductal Papillary Mucinous Neoplasm (IPMN), three cases of pancreatic Neuroendocrine Neoplasm (pNEN), one case of inflammatory myofibroblastic osteoblastoma. All cases did not develop pancreatic origin diabetes or exacerbation of previous diabetes, and no cases presented symptoms of exocrine insufficiency such as dyspepsia and diarrhea. Laparoscopic middle pancreatectomy is safe and feasible in the treatment of benign or low-grade malignant tumors in the body of the pancreatic neck and is not accompanied by increased risk of intraoperative and postoperative complications and endocrine dysfunction of the pancreas.

Design and testing of a humanized porcine donor for xenotransplantation

Recent human decedent model studies1,2 and compassionate xenograft use3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.

Serum components influence antibody reactivity to glycan and DNA antigens

We previously generated three types of anti-glycan monoclonal IgM antibodies that react with certain structures on the glycans of glycosphingolipids and glycoproteins. As the nucleotide sequences for the variable regions of these IgM antibodies showed homology with those of anti-DNA antibodies deposited in public databases, we analyzed the reactivity of the anti-glycan IgM antibodies to DNA by ELISA. We found that anti-α2,6-sialyl LacNAc IgM in the supernatant of a hybridoma culture cross-reacted with DNA, and after purification of the IgM by zirconia column chromatography, the highly purified IgM showed increased cross-reactivity to DNA. As most of the contaminating bovine serum proteins in the culture supernatant were removed by the purification process, it is likely that a part of the removed components influences antibody reactivity to DNA. Purified anti-DNA antibodies prepared from lupus model NZB/W F1 and MRL/lpr mouse sera and normal human serum were then analyzed, and similar results showing increased reactivity to DNA were obtained. Furthermore, ELISA using these purified antibodies and various carbohydrate antigens showed that the antigen-binding specificity of these antibodies was altered by the purification process from serum-containing antibody preparations. Our results indicate that mammalian serum contains components that strongly influence antibody reactivity to carbohydrate antigens, including DNA.

Antibody attributes, Fc receptor expression, gestation and maternal SARS-CoV-2 infection modulate HSV IgG placental transfer

Antibody-dependent cellular cytotoxicity (ADCC) is associated with protection against neonatal herpes. We hypothesized that placental transfer of ADCC-mediating herpes simplex virus (HSV) immunoglobulin G (IgG) is influenced by antigenic target, function, glycans, gestational age, and maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Maternal and cord blood were collected from HSV-seropositive (HSV+) mothers pre-COVID and HSV+/SARS-CoV-2+ mothers during the pandemic. Transfer of HSV neutralizing IgG was significantly lower in preterm versus term dyads (transfer ratio [TR] 0.84 vs. 2.44) whereas the TR of ADCC-mediating IgG was <1.0 in both term and preterm pre-COVID dyads. Anti-glycoprotein D IgG, which had only neutralizing activity, and anti-glycoprotein B (gB) IgG, which displayed neutralizing and ADCC activity, exhibited different relative affinities for the neonatal Fc receptor (FcRn) and expressed different glycans. The transfer of ADCC-mediating IgG increased significantly in term SARS-CoV-2+ dyads. This was associated with greater placental colocalization of FcRn with FcγRIIIa. These findings have implications for strategies to prevent neonatal herpes.

Self-Assembled Proteinaceous Nanoparticles for Co-Delivery of Antigens and Cytosine Phosphoguanine (CpG) Adjuvants: Implications for Nanovaccines.

Nanotechnology has developed rapidly, giving rise to "nanovaccinology". In particular, protein-based nanocarriers have gained widespread attention because of their excellent biocompatibility. As the development of flexible and rapid vaccines is challenging, modular extensible nanoparticles are urgently needed. In this study, a multifunctional nanocarrier capable of delivering various biomolecules (including polysaccharides, proteins, and nucleic acids) was designed by fusing the cholera toxin B subunit with streptavidin. Then, the nanocarrier was used to prepare a bioconjugate nanovaccine against S. flexneri by co-delivery of antigens and CpG adjuvants. Subsequent experimental results indicated that the nanovaccine with multiple components could stimulate both adaptive and innate immunity. Moreover, combining nanocarriers and CpG adjuvants with glycan antigens could improve the survival of vaccinated mice during the interval of two vaccination injections. The multifunctional nanocarrier and the design strategy demonstrated in this study could be utilized in the development of many other nanovaccines against infectious diseases.

Mechanism of Antigen Presentation and Specificity of Antibody Cross-Reactivity Elicited by an Oligosaccharide-Conjugate Cancer Vaccine.

Polysaccharides have been successfully used as immunogens for the development of vaccines against bacterial infection; however, there are no oligosaccharide-based vaccines available to date and no previous studies of their processing and presentation. We reported here the intracellular enzymatic processing and antigen presentation of an oligosaccharide-conjugate cancer vaccine prepared from the glycan of Globo-H (GH), a globo-series glycosphingolipid (GSL). This oligosaccharide-conjugate vaccine was shown to elicit antibodies against the glycan moieties of all three globo-series GSLs that are exclusively expressed on many types of cancer and their stem cells. To understand the specificity and origin of cross-reactivity of the antibodies elicited by the vaccine, we found that the vaccine is first processed by fucosidase 1 in the early endosome of dendritic cells to generate a common glycan antigen of the GSLs along with GH for MHC class II presentation. This work represents the first study of oligosaccharide processing and presentation and is expected to facilitate the design and development of glycoconjugate vaccines based on oligosaccharide antigens.

Hsa_circ_0079598 acts as a potential diagnostic and prognostic biomarker for gastric cancer

Abstract Objectives The work discussed the expression level circular RNA (circRNA) hsa_circ_0079598 for gastric cancer (GC) patients as well as clinicopathological significance. Methods qRT-PCR was utilized for determining the expression level of hsa_circ_0079598 in GC cell lines, 40 GC tissue pairs and their paracancerous tissue specimens, 60 pairs of preoperative and postoperative plasma specimens of GC patients, 20 plasma specimens of patients with gastric polyps, and 60 plasma specimens of the healthy control group. Besides, the correlation with clinicopathological parameters was analyzed. Tumor indices glycan antigen 199 (CA199), glycan antigen 724 (CA724), and pepsinogen I (PGI) were combined to evaluate their auxiliary diagnostic value. The values of hsa_circ_0079598 in the prognosis of GC patients were analyzed using survival curves. Results The relative expression of hsa_circ_0079598 in the cancerous tissues of GC patients was lower than that of their paracancerous tissues (p=0.004). The relative expression of plasma hsa_circ_0079598 in GC patients was lower than that of the healthy control group (p&lt;0.001) and patients with gastric polyps. Moreover, the relative expression of human gastric epithelial cell lines (GES-1) (p&lt;0.05) was higher than that of hsa_circ_0079598 of HGC-27, AGS, and MGC-803 cancer cell lines in GC cell lines. The total survival time of the high-expression patient group (p=0.040) was higher than that of hsa_circ_0079598 in the low-expression patient group. The expression of hsa_circ_0079598 in patients with the tissues and plasma of GC was related to the differentiated degree of tumors and TNM staging (p&lt;0.05). The auxiliary diagnostic value of hsa_circ_0079598 for GC was analyzed using ROC curves. When GC was distinguished from gastric polyps and the healthy control group, the areas under the ROC curve (AUCs) were 0.856 and 0.930, respectively. Hsa_circ_0079598 combined with tumor indices CA199, CA724, and PGI detection had higher specificity and accuracy than those of a single index. Conclusions Hsa_circ_0079598 was low-expression in cell lines, plasma, and GC tissues, which was related to the progress and prognosis of GC. Hsa_circ_0079598 combined with CA199, CA724, and PGI detection can improve the diagnosis of GC.

Unraveling cross-reactivity of anti-glycan IgG responses in filarial nematode infections.

Parasitic nematodes responsible for filarial diseases cause chronic disablement in humans worldwide. Elimination programs have substantially reduced the rate of infection in certain areas, but limitations of current diagnostics for population surveillance have been pointed out and improved assays are needed to reach the elimination targets. While serological tests detecting antibodies to parasite antigens are convenient tools, those currently available are compromised by the occurrence of antibodies cross-reactive between nematodes, as well as by the presence of residual antibodies in sera years after treatment and clearance of the infection. We recently characterized the N-linked and glycosphingolipid derived glycans of the parasitic nematode Brugia malayi and revealed the presence of various antigenic structures that triggered immunoglobulin G (IgG) responses in infected individuals. To address the specificity of IgG binding to these glycan antigens, we screened microarrays containing Brugia malayi glycans with plasma from uninfected individuals and from individuals infected with Loa loa, Onchocerca volvulus, Mansonella perstans and Wuchereria bancrofti, four closely related filarial nematodes. IgG to a restricted subset of cross-reactive glycans was observed in infection plasmas from all four species. In plasma from Onchocerca volvulus and Mansonella perstans infected individuals, IgG binding to many more glycans was additionally detected, resulting in total IgG responses similar to the ones of Brugia malayi infected individuals. For these infection groups, Brugia malayi, Onchocerca volvulus and Mansonella perstans, we further studied the different IgG subclasses to Brugia malayi glycans. In all three infections, IgG1 and IgG2 appeared to be the major subclasses involved in response to glycan antigens. Interestingly, in Brugia malayi infected individuals, we observed a marked reduction in particular in IgG2 to parasite glycans post-treatment with anthelminthic, suggesting a promising potential for diagnostic applications. Thus, we compared the IgG response to a broad repertoire of Brugia malayi glycans in individuals infected with various filarial nematodes. We identified broadly cross-reactive and more specific glycan targets, extending the currently scarce knowledge of filarial nematode glycosylation and host anti-glycan antibody response. We believe that our initial findings could be further exploited to develop disease-specific diagnostics as part of an integrated approach for filarial disease control.

Novel evidence that the ABO blood group shapes erythropoiesis and results in higher hematocrit for blood group B carriers

The ABO blood group (BG) system is of great importance for blood transfusion and organ transplantation. Since the same transcription factors (TFs) and microRNAs (miRNAs) govern the expression of ABO BG antigens and regulate erythropoiesis, we hypothesized functional connections between both processes. We found significantly higher hemoglobin and hematocrit values in BG B blood donors compared to BG A. Furthermore, we observed that erythropoiesis in BG B hematopoietic stem/progenitor cells (HSPCs) was accelerated compared to BG A HSPCs. Specifically, BG B HSPCs yielded more lineage-specific progenitors in a shorter time (B: 31.3 ± 2.2% vs. A: 22.5 ± 3.0%). Moreover, non-BG A individuals exhibited more terminally differentiated RBCs with higher enucleation rates containing more hemoglobin compared to BG A. Additionally, we detected increased levels of miRNA-215-5p and -182-5p and decreased expression of their target TFs RUNX1 and HES-1 mRNAs in erythroid BG B precursor cells compared to BG A. This highlights the important roles of these factors for the disappearance of differentiation-specific glycan antigens and the appearance of cancer-specific glycan antigens. Our work contributes to a deeper understanding of erythropoiesis gene regulatory networks and identifies its interference with BG-specific gene expression regulations particularly in diseases, where ABO BGs determine treatment susceptibility and disease progression.

Genetic, Functional, and Immunogenic Analyses of the O-Linked Protein Glycosylation System in Neisseria meningitidis Serogroup A ST-7 Isolates.

Neisseria meningitidis exhibits a general O-linked protein glycosylation system in which pili and other extracytoplasmic proteins are glycosylated. To investigate glycan antigenicity in humans and the significance of high glycan diversity on immune escape mechanisms, we exploited serogroup A meningococcal strains and serum samples obtained from laboratory-confirmed Ethiopian patients with meningococcal disease. The 37 meningococcal isolates were sequenced, and their protein glycosylation (pgl) genotypes and protein glycosylation phenotypes were investigated in detail. An insertion sequence (IS1655) element in pglH reduced glycan variability in the majority of isolates, while phase variation strengthened glycan variability and microheterogeneity. Homologous recombination events within the pgl genes were identified in eight of the 37 isolates, and the phenotypic consequences ranged from none detected to altered glycoforms in two of the isolates in which the whole pgl locus was exchanged. Immunoblotting of sera against a complete panel of glycan-expressing mutant strains demonstrated that most of these patient sera had IgG antibodies against various neisserial protein glycan antigens. Furthermore, using a bactericidal assay comparing a wild-type meningococcal A strain and a glycosylation-null variant strain, we showed that these protein glycan antigens interfere with bactericidal killing by antibodies in patient sera. Altogether, we were largely able to link pgl genotype with glycosylation phenotype. Our study reveals that protein glycans seem to contribute to the ability of N. meningitidis to resist the bactericidal activity of human serum, possibly by masking protein epitopes important for bactericidal killing and thus protection against meningococcal disease. IMPORTANCE Bacterial meningitis is a serious global health problem, and one of the major causative organisms is Neisseria meningitidis. Extensive variability in protein glycan structure and antigenicity is due to phase variation of protein glycosylation genes and polymorphic gene content and function. The exact role(s) of glycosylation in Neisseria remains to be determined, but increasing evidence, supported by this study, suggests that glycan variability can be a strategy to escape the human immune system. The complexity of the O-linked protein glycosylation system requires further studies to fully comprehend how these bacteria utilize variation in pgl genes to produce such high glycoform diversity and to evade the human immune response.

The effect of a methyl group on structure and function: Serine vs. threonine glycosylation and phosphorylation.

A variety of glycan structures cover the surface of all cells and are involved in myriad biological processes, including but not limited to, cell adhesion and communication, protein quality control, signal transduction and metabolism, while also being intimately involved in innate and adaptive immune functions. Immune surveillance and responses to foreign carbohydrate antigens, such as capsular polysaccharides on bacteria and surface protein glycosylation of viruses, are the basis of microbial clearance, and most antimicrobial vaccines target these structures. In addition, aberrant glycans on tumors called Tumor-Associated Carbohydrate Antigens (TACAs) elicit immune responses to cancer, and TACAs have been used in the design of many antitumor vaccine constructs. A majority of mammalian TACAs are derived from what are referred to as mucin-type O-linked glycans on cell-surface proteins and are linked to the protein backbone through the hydroxyl group of either serine or threonine residues. A small group of structural studies that have compared mono- and oligosaccharides attached to each of these residues have shown that there are distinct differences in conformational preferences assumed by glycans attached to either "unmethylated" serine or ß-methylated threonine. This suggests that the linkage point of antigenic glycans will affect their presentation to the immune system as well as to various carbohydrate binding molecules (e.g., lectins). This short review, followed by our hypothesis, will examine this possibility and extend the concept to the presentation of glycans on surfaces and in assay systems where recognition of glycans by proteins and other binding partners can be defined by different attachment points that allow for a range of conformational presentations.

Identifying Genetic Regulators of Protein-Glycan Interactions with Genome-Wide CRISPR Screening.

Glycans are carbohydrate molecules appended to proteins and lipids on the surface of all living cells. Glycans play key roles in a wide array of biological processes, and structural changes in cell-surface glycosylation patterns have been connected to pathogenesis of several diseases. In particular, cancer cells frequently upregulate expression of glycans that bind to inhibitory receptors (lectins) on immune cells. These glycosylated antigens systematically inhibit immune activity and protect cancer cells from immune surveillance. Understanding how cancer cells generate these glycan ligands can thus lead to identification of novel druggable targets for therapeutic intervention. However, glycan ligand biosynthesis is subject to extremely complex genetic regulation, making it difficult to identify the key genes involved in production of immune-regulatory glycan antigens. In a recent publication, we described a CRISPR/Cas9 screening approach to identify genes that drive synthesis of ligands for glycan-binding immune receptors. Here, we outline a detailed, step-by-step protocol for completing this type of genome-wide screen. Our protocol produces a genome-wide atlas of all genes whose expression is required for cell-surface binding of a recombinant immune lectin. This dataset can be used both to identify novel ligands for immune lectins and annotate regulatory genes that drive changes in cancer-associated glycosylation. Our protocol serves as a general resource for researchers interested in the detailed study of cancer glyco-immunology. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Generation of a genome-wide CRISPR library using lentiviral transduction Support Protocol: Generation of dCas9KRAB-expressing K-562 cells Basic Protocol 2: Staining of genome-wide CRISPR libraries with Siglec-Fc reagents and fluorescence-activated cell sorting Basic Protocol 3: Library amplification and sequencing Basic Protocol 4: Data analysis and hit identification.

2125. Determinants of Placental Transfer of HSV-specific Antibodies and the Impact of SARS-CoV-2 Coinfection

Abstract Background Primary versus recurrent herpes simplex virus 1 or 2 (HSV-1 or HSV-2) infection during pregnancy carries a higher risk of neonatal herpes. Murine and clinical studies demonstrate that antibody dependent cellular cytotoxicity (ADCC) provides greater protection against disseminated neonatal disease. To quantify relative transfer of HSV specific Abs with different functions and targets and whether SARS-CoV-2 coinfection modified transfer, we conducted a prospective cohort study of mother-infant dyads prior to and during COVID-19. Methods Total and HSV lysate, glycoprotein D (gD) and glycoprotein B (gB)-specific IgG, IgG1 and IgG3, nAbs and ADCC were quantified in paired 3rd trimester maternal and cord blood. IgG1 and IgG3 subclass and gD or gB-specific Abs were isolated by column purification and glycan profiles were assessed using mass spectrometry. The pre-COVID study population included 21 term and 15 preterm dyads who were HSV seropositive and the pri-COVID cohort included 25 HSV seropositive term dyads whose mothers were also SARS-CoV-2 PCR and COVID Ab positive at delivery. Results HSV-specific Ab and neutralizing Ab transfer ratio (TR) were higher in term compared to preterm pre-COVID dyads (all p&amp;lt; 0.05), but the ADCC TR was &amp;lt; 1.0 for both groups. To determine if the low ADCC TR reflected antigenic target, subclass and/or glycans, we enriched for anti-gD and anti-gB specific and IgG1 and IgG3 Abs. The anti-gD Abs were exclusively IgG, had only neutralizing activity and had glycans associated with FcRn binding. In contrast, anti-gB Abs were both IgG1 and IgG3; had both neutralizing and ADCC activity and expressed glycans associated with both FcRn and FcγRIIIa binding. There was no significant difference in HSV-specific IgG TR in pre-COVID vs COVID dyads (0.42) but the nAb TR was lower (p=0.018) and ADCC TR higher (p&amp;lt; 0.001) in COVID compared to pre-COVID patients. Placental immunohistochemistry showed an increased colocalization of FcRn and FcγRIIIA in SARS-CoV-2 positive mothers, which would favor transfer of ADCC Abs. Conclusion Defining the determinants of ADCC transfer has implications for future vaccine and monoclonal Ab strategies to prevent/treat neonatal herpes. We speculate that increasing the transfer of ADCC may be a key element in providing immune protection Disclosures Betsy Herold, MD, Viracor (Eurofins): Advisor/Consultant|X-Vax, Technologies: Advisor/Consultant|X-Vax, Technologies: Grant/Research Support|X-Vax, Technologies: Serve on Scientific Advisory Board for X-Vax, Technologies and receiving reserach funding for related worl. Betsy Herold, MD, Viracor (Eurofins): Advisor/Consultant|X-Vax, Technologies: Advisor/Consultant|X-Vax, Technologies: Grant/Research Support|X-Vax, Technologies: Serve on Scientific Advisory Board for X-Vax, Technologies and receiving reserach funding for related worl.

Semisynthetic Glycoconjugate Vaccine Candidates against Escherichia coli O25B Induce Functional IgG Antibodies in Mice.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a major health concern due to emerging antibiotic resistance. Along with O1A, O2, and O6A, E. coli O25B is a major serotype within the ExPEC group, which expresses a unique O-antigen. Clinical studies with a glycoconjugate vaccine of the above-mentioned O-types revealed O25B as the least immunogenic component, inducing relatively weak IgG titers. To evaluate the immunological properties of semisynthetic glycoconjugate vaccine candidates against E. coli O25B, we here report the chemical synthesis of an initial set of five O25B glycan antigens differing in length, from one to three repeat units, and frameshifts of the repeat unit. The oligosaccharide antigens were conjugated to the carrier protein CRM197. The resulting semisynthetic glycoconjugates induced functional IgG antibodies in mice with opsonophagocytic activity against E. coli O25B. Three of the oligosaccharide–CRM197 conjugates elicited functional IgGs in the same order of magnitude as a conventional CRM197 glycoconjugate prepared with native O25B O-antigen and therefore represent promising vaccine candidates for further investigation. Binding studies with two monoclonal antibodies (mAbs) revealed nanomolar anti-O25B IgG responses with nanomolar KD values and with varying binding epitopes. The immunogenicity and mAb binding data now allow for the rational design of additional synthetic antigens for future preclinical studies, with expected further improvements in the functional antibody responses. Moreover, acetylation of a rhamnose residue was shown to be likely dispensable for immunogenicity, as a deacylated antigen was able to elicit strong functional IgG responses. Our findings strongly support the feasibility of a semisynthetic glycoconjugate vaccine against E. coli O25B.

The respective relevance of sensitization to alloantigens and xenoantigens in pig organ xenotransplantation

BackgroundAntibody-mediated rejection is a major cause of graft injury and contributes to failure of pig xenografts in nonhuman primates (NHPs). Most ‘natural’ or elicited antibodies found in humans and NHPs are directed against pig glycan antigens, but antibodies binding to swine leukocyte antigens (SLA) have also been detected. Of clinical importance is (i) whether the presence of high levels of antibodies directed towards human leukocyte antigens (HLA) (i.e., high panel-reactive antibodies) would be detrimental to the outcome of a pig organ xenograft; and (ii) whether, in the event of sensitization to pig antigens, a subsequent allotransplant would be at increased risk of graft failure due to elicited anti-pig antibodies that cross-react with human HLA or other antigens. SummaryA literature review of pig-to-primate studies indicates that relatively few highly-HLA-sensitized humans have antibodies that cross-react with pigs, predicting that most would not be at increased risk of rejecting an organ xenograft. Furthermore, the existing evidence indicates that sensitization to pig antigens will probably not elicit increased alloantibody titers; if so, ‘bridging’ with a pig organ could be carried out without increased risk of subsequent antibody-mediated allograft failure. Key MessageThese issues have important implications for the design and conduct of clinical xenotransplantation trials.

Analysis on the Efficacy of Bronchial Artery Chemoembolization Combined with 125I Seed Implantation in the Therapy of Advanced Non-Small-Cell Lung Cancer Based on the Medical Database.

Objective To investigate the use and the efficacy of bronchial artery chemoembolization combined with 125I seed implantation in advanced non-small-cell lung cancer (NSCLC) therapy based on the medical database. Methods A total of 102 patients with advanced NSCLC were randomly divided into two groups. The control group was treated with 125I seed implantation, and the observation group was treated with bronchial artery chemoembolization (BACE) combined with 125I seed implantation based on medical database. The clinical efficacy, carcinoembryonic antigen (CEA), cytokeratin 19 fragment antigen 21-1 (CYFRA21-1), glycan antigen 125 (CA125), peripheral blood CD3+, CD8+, CD4+/CD8+ T cells, insulin-like growth factor type 1 receptor (IGF-1R), S100 calcium-binding protein A2 (S100A2), long-term efficacy (time to disease progression, six-month survival rate, and one-year survival rate), and safety were then analyzed. Result The disease remission rate in the observation group was 62.75%, which was higher than that in the control group (41.18%). After 1 month and 3 months of treatment, the levels of serum CYFRA21-1, CEA, CA125, and IGF-1R were lower, while serum S100A2 was higher in the observation group than in the control group (P < 0.05). For safety assessment, we found that the incidences of neutropenia, thrombocytopenia, and gastrointestinal reactions had no statistical differences between two groups. The time to disease progression in the observation group was 129.85 d longer than that in the control group, 89.74 d, and the six-month survival rate and 1-year survival rate were higher in the observation group relative to the control group. Conclusion Medical database-based BACE combined with 125I seed implantation in the therapy of advanced NSCLC patients has definite efficacy with certain safety, which can enhance antitumor effect and prolong survival rate in advanced NSCLC patients.

P31: Antigenic target, antibody subclass, function and glycans and SARS‐CoV‐2 coinfection impact the placental transfer of herpes simplex virus antibodies

P31: Antigenic target, antibody subclass, function and glycans and SARS‐CoV‐2 coinfection impact the placental transfer of herpes simplex virus antibodies

Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines

BackgroundGlycoengineering, in the biotechnology workhorse bacterium, Escherichia coli, is a rapidly evolving field, particularly for the production of glycoconjugate vaccine candidates (bioconjugation). Efficient production of glycoconjugates requires the coordinated expression within the bacterial cell of three components: a carrier protein, a glycan antigen and a coupling enzyme, in a timely fashion. Thus, the choice of a suitable E. coli host cell is of paramount importance. Microbial chassis engineering has long been used to improve yields of chemicals and biopolymers, but its application to vaccine production is sparse.ResultsIn this study we have engineered a family of 11 E. coli strains by the removal and/or addition of components rationally selected for enhanced expression of Streptococcus pneumoniae capsular polysaccharides with the scope of increasing yield of pneumococcal conjugate vaccines. Importantly, all strains express a detoxified version of endotoxin, a concerning contaminant of therapeutics produced in bacterial cells. The genomic background of each strain was altered using CRISPR in an iterative fashion to generate strains without antibiotic markers or scar sequences.ConclusionsAmongst the 11 modified strains generated in this study, E. coli Falcon, Peregrine and Sparrowhawk all showed increased production of S. pneumoniae serotype 4 capsule. Eagle (a strain without enterobacterial common antigen, containing a GalNAc epimerase and PglB expressed from the chromosome) and Sparrowhawk (a strain without enterobacterial common antigen, O-antigen ligase and chain length determinant, containing a GalNAc epimerase and chain length regulators from Streptococcus pneumoniae) respectively produced an AcrA-SP4 conjugate with 4 × and 14 × more glycan than that produced in the base strain, W3110. Beyond their application to the production of pneumococcal vaccine candidates, the bank of 11 new strains will be an invaluable resource for the glycoengineering community.