Recognition of carbohydrates by antibodies can be affected by antigen composition and density. This had been investigated in a variety of controllable multivalent systems using synthetic carbohydrate antigens, yet such effects on anticarbohydrate antibodies in circulating human serum have not been fully addressed thus far. All humans develop a polyclonal and diverse response against carbohydrates containing a nonhuman sialic acid form, N-glycolylneuraminic acid (Neu5Gc). This red meat-derived monosaccharide is incorporated into a diverse collection of human glycans resulting in circulating anti-Neu5Gc antibodies in human sera. Such antibodies can cause exacerbation of diseases mediated by chronic inflammation such as cancer and atherosclerosis. We aimed to evaluate how different presentation modes of Neu5Gc-glycans can affect the detection of anti-Neu5Gc IgGs in human serum. Here, we compare serum IgG recognition of Neu5Gc-containing glycoproteins, glycopeptides, and synthetic glycans. First, Neu5Gc-positive or Neu5Gc-deficient mouse strains were used to generate glycopeptides from serum glycoproteins. Then we developed a reproducible ELISA to screen human sera against Neu5Gc-positive glycopeptides for detection of human serum anti-Neu5Gc IgGs. Finally, we evaluated ELISA screens against glycopeptides in comparison with glycoproteins, as well as against elaborated arrays displaying synthetic Neu5Gc-glycans. Our results demonstrate that the presentation mode and diversity of Neu5Gc-glycans are critical for detection of the full collection of human serum anti-Neu5Gc IgGs.

Polyclonal antibodies

Glycobiology

10.1021/acs.bioconjchem.8b00817

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Citations (26)

Assembly of the TLR2/6 Transmembrane Domains Is Essential for Activation and Is a Target for Prevention of Sepsis

The Journal of Immunology (2013)

Avner Fink Eliran Moshe Reuven Christopher J. Arnusch Liraz Shmuel-Galia Niv Antonovsky

TLR2, together with TLR1 and TLR6, is essential for detecting lipopeptides and bacterial cell wall components such as lipoteichoic acid from Gram-positive bacteria. In this study, we report that transmembrane domain (TMD)-derived peptides from TLR2 and TLR6 specifically inhibit TLR2 activation. Secretion of the cytokines TNF-α and IL-6 by cultured macrophages (RAW264.7 cell line) was inhibited by these peptides in response to TLR2 activation by lipoteichoic acid (TLR2/6 activator) or palmitoyl (3)-Cys-Ser-Lys(4)-OH (TLR2/1 activator) but not by LPS (TLR4 activator). Extensive biophysical and biochemical assays, combined with GALLEX experiments, show that these peptides heterodimerize with their complementary TMDs on their reciprocal protein. These results suggest that TLR2/6/1 TMD assembly is essential for activating this complex. Importantly, when administered to mice inflicted by TLR2, but not TLR4-driven lethal inflammation, a selected peptide rescued 60% of these septic mice, showing potent in vivo inhibition of TNF-α and IL-6 secretion. Furthermore, this peptide also showed high protection in a whole bacteria model. Owing to the importance of TLR2 regulation under a variety of pathological conditions, compounds that can fine-tune this activity are of great importance.

Lipoteichoic acid

Lipopeptide

10.4049/jimmunol.1202033

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Citations (26)

The role of antibody responses against glycans in bioprosthetic heart valve calcification and deterioration

Nature Medicine (2022)

Thomas Sénage Anu Paul Thierry Le Tourneau Imen Fellah‐Hebia M Vadori

Bioprosthetic heart valves (BHVs) are commonly used to replace severely diseased heart valves but their susceptibility to structural valve degeneration (SVD) limits their use in young patients. We hypothesized that antibodies against immunogenic glycans present on BHVs, particularly antibodies against the xenoantigens galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc), could mediate their deterioration through calcification. We established a large longitudinal prospective international cohort of patients (n = 1668, 34 ± 43 months of follow-up (0.1-182); 4,998 blood samples) to investigate the hemodynamics and immune responses associated with BHVs up to 15 years after aortic valve replacement. Early signs of SVD appeared in <5% of BHV recipients within 2 years. The levels of both anti-αGal and anti-Neu5Gc IgGs significantly increased one month after BHV implantation. The levels of these IgGs declined thereafter but anti-αGal IgG levels declined significantly faster in control patients compared to BHV recipients. Neu5Gc, anti-Neu5Gc IgG and complement deposition were found in calcified BHVs at much higher levels than in calcified native aortic valves. Moreover, in mice, anti-Neu5Gc antibodies were unable to promote calcium deposition on subcutaneously implanted BHV tissue engineered to lack αGal and Neu5Gc antigens. These results indicate that BHVs manufactured using donor tissues deficient in αGal and Neu5Gc could be less prone to immune-mediated deterioration and have improved durability.

10.1038/s41591-022-01682-w

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The HIV-1 Envelope Transmembrane Domain Binds TLR2 through a Distinct Dimerization Motif and Inhibits TLR2-Mediated Responses

PLoS Pathogens (2014)

Eliran Moshe Reuven Mohammad Ali Etai Rotem Roland Schwarzter Andrea Gramatica

HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.

10.1371/journal.ppat.1004248

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Citations (32)

Additional file 2 of Association between Neu5Gc carbohydrate and serum antibodies against it provides the molecular link to cancer: French NutriNet-Santé study

Figshare (2020)

Salam Bashir Léopold Fézeu Shani Leviatan Ben‐Arye Sharon Yehuda Eliran Moshe Reuven

Additional file 2: Supplementary data file S1. National world meat and cancer.

10.6084/m9.figshare.12991756

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Characterization of immunogenic Neu5Gc in bioprosthetic heart valves

Xenotransplantation (2016)

Eliran Moshe Reuven Shani Leviatan Ben‐Arye Tal Marshanski Michael E. Breimer Hai Yu

Abstract Background The two common sialic acids (Sias) in mammals are N ‐acetylneuraminic acid (Neu5Ac) and its hydroxylated form N ‐glycolylneuraminic acid (Neu5Gc). Unlike most mammals, humans cannot synthesize Neu5Gc that is considered foreign and recognized by circulating antibodies. Thus, Neu5Gc is a potential xenogenic carbohydrate antigen in bioprosthetic heart valves ( BHV ) that tend to deteriorate in time within human patients. Methods We investigated Neu5Gc expression in non‐engineered animal‐derived cardiac tissues and in clinically used commercial BHV , and evaluated Neu5Gc immunogenicity on BHV through recognition by human anti‐Neu5Gc IgG. Results Neu5Gc was detected by immunohistochemistry in porcine aortic valves and in porcine and bovine pericardium. Qualitative analysis of Sia linkages revealed Siaα2‐3>Siaα2‐6 on porcine/bovine pericardium while the opposite in porcine aortic/pulmonary valve cusps. Similarly, six commercial BHV containing either porcine aortic valve or porcine/bovine/equine pericardium revealed Siaα2‐3>Siaα2‐6 expression. Quantitative analysis of Sia by HPLC showed porcine/bovine pericardium express 4‐fold higher Neu5Gc levels compared to the porcine aortic/pulmonary valves, with Neu5Ac at 6‐fold over Neu5Gc. Likewise, Neu5Gc was expressed on commercial BHV (186.3±16.9 pmol Sia/μg protein), with Neu5Ac at 8‐fold over Neu5Gc. Affinity‐purified human anti‐Neu5Gc IgG showing high specificity toward Neu5Gc‐glycans (with no binding to Neu5Ac‐glycans) on a glycan microarray, strongly bound to all tested commercial BHV , demonstrating Neu5Gc immune recognition in cardiac xenografts. Conclusions We conclusively demonstrated Neu5Gc expression in native cardiac tissues, as well as in six commercial BHV . These Neu5Gc xeno‐antigens were recognized by human anti‐Neu5Gc IgG, supporting their immunogenicity. Altogether, these findings suggest BHV ‐Neu5Gc/anti‐Neu5Gc may play a role in valve deterioration warranting further investigation.

Xenotransplantation

10.1111/xen.12260

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Citations (67)

Active cancer vaccine targeting carbohydrates for immunotherapy

The Journal of Immunology (2019)

Vered Padler‐Karavani Eliran Moshe Reuven Shani Leviatan Ben‐Arye Hai Yu Roberto Duchi

Abstract Transformed cells present cell surface neoantigens, including aberrantly expressed carbohydrates. Targeting tumor-associated carbohydrate antigens could be useful for cancer immunotherapy. N-glycolylneuraminic acid (Neu5Gc) is a non-human carbohydrate consumed in diet and accumulates on human cancer cells, leading to expression of neoantigens. Passive immunotherapy with anti-Neu5Gc antibodies in mice inhibits tumor growth. Here we describe an active cancer vaccination immunotherapy strategy to target Neu5Gc-positive tumors. We generated glyconanoparticles from engineered red blood cells to form nano-ghosts (NGs) that either express (NGpos) or lack expression (NGneg) of Neu5Gc-glycoconjugates. We then show that optimized immunization of Neu5Gc-deficient Cmah-KO mice with NGpos glyconanoparticles induce a robust, diverse and long-lasting anti- Neu5Gc IgG immune response. Anti-Neu5Gc IgG antibodies also migrated into Neu5Gc-positive tumors and inhibited tumor growth in-vivo. Using glycan microarrays we also show that the kinetics and quality of the immune responses influence the efficacy of the vaccine. Hence, immunotherapy targeting Neu5Gc-carbohydrate neoantigens is promising.

Active immunotherapy

Glycoconjugate

Cancer Immunotherapy

Cancer vaccine

10.4049/jimmunol.202.supp.70.14

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Biomimetic Glyconanoparticle Vaccine for Cancer Immunotherapy

ACS Nano (2019)

Eliran Moshe Reuven Shani Leviatan Ben‐Arye Hai Yu Roberto Duchi Andrea Perota

Cancer immunotherapy aims to harness the immune system to combat malignant processes. Transformed cells harbor diverse modifications that lead to formation of neoantigens, including aberrantly expressed cell surface carbohydrates. Targeting tumor-associated carbohydrate antigens (TACA) hold great potential for cancer immunotherapy. N-glycolylneuraminic acid (Neu5Gc) is a dietary non-human immunogenic carbohydrate that accumulates on human cancer cells, thereby generating neoantigens. In mice, passive immunotherapy with anti-Neu5Gc antibodies inhibits growth of Neu5Gc-positive tumors. Here, we designed an active cancer vaccine immunotherapy strategy to target Neu5Gc-positive tumors. We generated biomimetic glyconanoparticles using engineered αGal knockout porcine red blood cells to form nanoghosts (NGs) that either express (NGpos) or lack expression (NGneg) of Neu5Gc-glycoconjugates in their natural context. We demonstrated that optimized immunization of "human-like" Neu5Gc-deficient Cmah-/- mice with NGpos glyconanoparticles induce a strong, diverse and persistent anti-Neu5Gc IgG immune response. The resulting anti-Neu5Gc IgG antibodies were also detected within Neu5Gc-positive tumors and inhibited tumor growth in vivo. Using detailed glycan microarray analysis, we further demonstrate that the kinetics and quality of the immune responses influence the efficacy of the vaccine. These findings reinforce the potential of TACA neoantigens and the dietary non-human sialic acid Neu5Gc, in particular, as immunotherapy targets.

Cancer Immunotherapy

Cancer vaccine

10.1021/acsnano.8b07241

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Citations (47)

Peptides Derived from the Transmembrane Domains of Toll-Like Receptors (TLR) 2/6 Interfere with Hetero-Associate with the TLRs and Inhibit their Activation

Biophysical Journal (2012)

Yechiel Shai Avner Fink Christopher J. Arnusch Liraz Shmuel‐Galia Eliran Moshe Reuven

10.1016/j.bpj.2011.11.1347

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