Presentation Mode of Glycans Affect Recognition of Human Serum anti-Neu5Gc IgG Antibodies

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

Characterization of immunogenic Neu5Gc in bioprosthetic heart valves

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 Siaa2-3> Siaa2-6 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 Siaa2-3> Siaa2-6 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/mu g 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

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

<div><p>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 <i>in vitro</i> and <i>in vivo</i> 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 <i>in vivo</i> 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.</p></div

The gp41 TMD reduces serum cytokine levels in vivo and protects from consequential spleen damage.

<p>(<b>A–B</b>) Blood serum TNFα and IL-6 levels of PBS, gp41 TMD and gp41 mutant treated mice estimated 90 minutes and 210 minutes after LTA minutes and 210 minutes after LTA minutes after LTA/GLN injection (n = 9, ** - p<0.005, ***-p<0.0005). (<b>C</b>) H&E staining of spleen sections of control (top), LTA and no peptide (middle) and LTA and gp41 TMD treated (bottom) mice. Apoptotic foci in the white pulp are indicated in circles and red pulp as squares. Scale bars = 200 µm and 400 µm for left and right panels, respectively.</p

The gp41 TMD inhibits TLR2 induced signaling through ERK1,2 and cytokine secretion.

<p>(<b>A</b>) ERK1,2 phosphorylation in RAW264.7 cells was detected before and after LTA exposure. Cells were either pre-treated with indicated peptides for 2 hours prior to LTA addition or untreated (center). GAPDH shows equal loading. Here shown representative data of 3 typical experiments. (<b>B</b>) Quantification of the band intensity was determined for the p-ERK levels after 20 minutes LTA exposure. The WT peptide shows over 50 minutes LTA exposure. The WT peptide shows over 50% decrease in p-ERK levels while the mutant peptide shows approximately 15%. Statistical analysis was done by student T-test, (n = 4, ** p<0.01). (<b>C–D</b>) mRNA expression levels of MCP-1 (C) and TNFα (D) detected by real-time PCR. Expression levels were normalized to the expression of non-activated cells. The data is shown as the calculated 2^∧−dCT in the relativity of each gene to HPRT. (The figure is an average of three samples, * - p<0.05).</p

The gp41 TMD rescues mice in a LTA/GLN sepsis model and reduces liver damage.

<p>(<b>A</b>) Mice were inoculated with a lethal dosage of LTA/GLN and simultaneously treated with either the gp41 TMD peptide (▪) the gp41 mutant (♦) or PBS as control (•). Peptides were administrated at 5 mg mg/kg. (<b>B</b>) H&E staining of liver sections. Apoptotic foci are indicated in circles and arrows indicate hemorrhage. Cell death is multi focal as expected as a result of GLN treatment. Scale bar = 100 µm.</p

ENV TMD inhibits secretion of cytokines and chemokines induced by TLR2.

<p>(<b>A–C</b>) RAW cells were incubated with/without the indicated peptides for 2 hours, then washed twice and activated with 500 ng hours, then washed twice and activated with 500 ng ng/ml of purified LTA. Media was collected after 5 hours (TNFα) or 24 hours (MCP-1 and IL-6). ELISA was performed using standard kits. (n = 3, ± represent SD) (<b>D</b>) THP-1 human macrophages were incubated using similar conditions as described in A, and TNFα levels were measured (* - p<0.05).</p

Biomimetic Glyconanoparticle Vaccine for Cancer Immunotherapy

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