Absence of Neu5Gc and Presence of Anti-Neu5Gc Antibodies in Humans-An Evolutionary Perspective.

The glycocalyx of human cells differs from that of many other mammals by the lack of the sialic acid N-glycolylneuraminic acid (Neu5Gc) and increased abundance of its precursor N-acetylneuraminic acid (Neu5Ac). Most humans also have circulating antibodies specifically targeting the non-human sialic acid Neu5Gc. Recently, several additional mammalian species have been found to also lack Neu5Gc. In all cases, loss-of-function mutations in the gene encoding the sialic acid-modifying enzyme CMAH are responsible for the drastic change in these species. Unlike other glycan antigens, Neu5Gc apparently cannot be produced by microbes, raising the question about the origin of these antibodies in humans. Dietary exposure and presentation on bacteria coating themselves with Neu5Gc from the diet are distinct possibilities. However, the majority of the non-human species that lack Neu5Gc do not consume diets rich in Neu5Gc, making it unlikely that they will have been immunized against this sialic acid. A notable exception are mustelids (ferrets, martens and their relatives) known for preying on various small mammal species rich in Neu5Gc. No studies exist on levels of anti-Neu5Gc antibodies in non-human species. Evolutionary scenarios for the repeated, independent fixation of CMAH loss-of-function mutations at various time points in the past include strong selection by parasites, especially enveloped viruses, stochastic effects of genetic drift, and directional selection via female immunity to paternal Neu5Gc. Convergent evolution of losses of the vertebrate-specific self-glycan Neu5Gc are puzzling and may represent a prominent way in which glycans become agents of evolutionary change in their own right. Such change may include the reconfiguration of innate immune lectins that use self-sialic acids as recognition patterns

Biogenesis of Influenza A Virus Hemagglutinin Cross-Protective Stem Epitopes

Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4°C and well at 37°C. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from N-linked glycans.</p

Glycocalyx crowding with mucin mimetics strengthens binding of soluble and virus-associated lectins to host cell glycan receptors

Membrane-associated mucins protect epithelial cell surfaces against pathogenic threats by serving as nonproductive decoys that capture infectious agents and clear them from the cell surface and by erecting a physical barrier that restricts their access to target receptors on host cells. However, the mechanisms through which mucins function are still poorly defined because of a limited repertoire of tools available for tailoring their structure and composition in living cells with molecular precision. Using synthetic glycopolymer mimetics of mucins, we modeled the mucosal glycocalyx on red blood cells (RBCs) and evaluated its influence on lectin (SNA) and virus (H1N1) adhesion to endogenous sialic acid receptors. The glycocalyx inhibited the rate of SNA and H1N1 adhesion in a size- and density-dependent manner, consistent with the current view of mucins as providing a protective shield against pathogens. Counterintuitively, increasing the density of the mucin mimetics enhanced the retention of bound lectins and viruses. Careful characterization of SNA behavior at the RBC surface using a range of biophysical and imaging techniques revealed lectin-induced crowding and reorganization of the glycocalyx with concomitant enhancement in lectin clustering, presumably through the formation of a more extensive glycan receptor patch at the cell membrane. Our findings indicate that glycan-targeting pathogens may exploit the biophysical and biomechanical properties of mucins to overcome the mucosal glycocalyx barrier

Aptamer-Nanoparticle Assembly for Logic-Based Detection

In this work, gold nanoparticles perform Boolean logic operations in response to two proangiogenic targets important in cancer diagnosis and treatment: PDGF and VEGF. In the absence of protein target, gold nanoparticles are initially dispersed as a red solution; the addition of target proteins causes nanoparticle aggregation, turning the solution blue, as well as the release of dye-labeled aptamer probes, which causes an increase in fluorescence. These outputs constitute an AND or OR gate for simultaneous protein detection. We believe this logic-gate-based detection system will become the basis for novel rapid, cheap, and reliable sensors for diagnostic applications

Mucin-mimetic glycan arrays integrating machine learning for analyzing receptor pattern recognition by influenza A viruses.

Influenza A viruses (IAVs) exploit host glycans in airway mucosa for entry and infection. Detection of changes in IAV glycan-binding phenotype can provide early indication of transmissibility and infection potential. While zoonotic viruses are monitored for mutations, the influence of host glycan presentation on viral specificity remains obscured. Here, we describe an array platform which uses synthetic mimetics of mucin glycoproteins to model how receptor presentation and density in the mucinous glycocalyx may impact IAV recognition. H1N1 and H3N2 binding in arrays of α2,3- and α2,6-sialyllactose receptors confirmed their known sialic acid-binding specificities and revealed their different sensitivities to receptor presentation. Further, the transition of H1N1 from avian to mammalian cell culture improved the ability of the virus to recognize mucin-like displays of α2,6-sialic acid receptors. Support vector machine (SVM) learning efficiently characterized this shift in binding preference and may prove useful to study viral evolution to a new host

Mucin-mimetic glycan arrays integrating machine learning for analyzing receptor pattern recognition by influenza A viruses

Influenza A viruses (IAVs) exploit host glycans in airway mucosa for entry and infection. Detection of changes in IAV glycan-binding phenotype can provide early indication of transmissibility and infection potential. While zoonotic viruses are monitored for mutations, the influence of host glycan presentation on viral specificity remains obscured. Here, we describe an array platform which uses synthetic mimetics of mucin glycoproteins to model how receptor presentation and density in the mucinous glycocalyx may impact IAV recognition. H1N1 and H3N2 binding in arrays of α2,3- and α2,6-sialyllactose receptors confirmed their known sialic acid-binding specificities and revealed their different sensitivities to receptor presentation. Further, the transition of H1N1 from avian to mammalian cell culture improved the ability of the virus to recognize mucin-like displays of α2,6-sialic acid receptors. Support vector machine (SVM) learning efficiently characterized this shift in binding preference and may prove useful to study viral evolution to a new host

Aptamer-Nanoparticle Assembly for Logic-Based Detection

In this work, gold nanoparticles perform Boolean logic operations in response to two proangiogenic targets important in cancer diagnosis and treatment: PDGF and VEGF. In the absence of protein target, gold nanoparticles are initially dispersed as a red solution; the addition of target proteins causes nanoparticle aggregation, turning the solution blue, as well as the release of dye-labeled aptamer probes, which causes an increase in fluorescence. These outputs constitute an AND or OR gate for simultaneous protein detection. We believe this logic-gate-based detection system will become the basis for novel rapid, cheap, and reliable sensors for diagnostic applications

Human Influenza A Virus Hemagglutinin Glycan Evolution Follows a Temporal Pattern to a Glycan Limit

Frequent mutation of its major antibody target, the glycoprotein hemagglutinin, ensures that the influenza virus is perennially both a rapidly emerging virus and a major threat to public health. One type of mutation escapes immunity by adding a glycan onto an area of hemagglutinin that many antibodies recognize. This study revealed that these glycan changes follow a simple temporal pattern. Every 5 to 7 years, hemagglutinin adds a new glycan, up to a limit. After this limit is reached, no net additions of glycans occur. Instead, glycans are swapped or lost at longer intervals. Eventually, a pandemic replaces the terminally glycosylated hemagglutinin with a minimally glycosylated one from the animal reservoir, restarting the cycle. This pattern suggests the following: (i) some hemagglutinins are evolved for this decades-long process, which is both defined by and limited by successive glycan addition; and (ii) hemagglutinin's antibody dominance and its capacity for mutations are highly adapted features that allow influenza to outpace our antibody-based immunity.Human antibody-based immunity to influenza A virus is limited by antigenic drift resulting from amino acid substitutions in the hemagglutinin (HA) head domain. Glycan addition can cause large antigenic changes but is limited by fitness costs to viral replication. Here, we report that glycans are added to H1 and H3 HAs at discrete 5-to-7-year intervals, until they reach a functional glycan limit, after which glycans are swapped at approximately 2-fold-longer intervals. Consistent with this pattern, 2009 pandemic H1N1 added a glycan at residue N162 over the 2015–2016 season, an addition that required two epistatic HA head mutations for complete glycosylation. These strains rapidly replaced H1N1 strains globally, by 2017 dominating H3N2 and influenza B virus strains for the season. The pattern of glycan modulation that we outline should aid efforts for tracing the epidemic potential of evolving human IAV strains

Biogenesis of Influenza A Virus Hemagglutinin Cross-Protective Stem Epitopes

<div><p>Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several <i>N</i>-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as <i>N</i>-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4°C and well at 37°C. A <i>de novo</i> designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of <i>N</i>-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple <i>N</i>-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that <i>N</i>-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from <i>N</i>-linked glycans.</p></div