Well-Defined Heparin Mimetics Can Inhibit Binding of the Trimeric Spike of SARS-CoV-2 in a Length-Dependent Manner

The emergence of new SARS-CoV-2 variants and the dangers of long-covid necessitate the development of broad-acting therapeutics that can reduce viral burden. SARS-CoV-2 employs heparan sulfate (HS) as an initial cellular attachment factor, and therefore, there is interest in developing heparin as a therapeutic for SARS-CoV-2. Its use is, however, complicated by structural heterogeneity and the risk of causing bleeding and thrombocytopenia. Here, we describe the preparation of well-defined heparin mimetics by a controlled head-to-tail assembly of HS oligosaccharides having an alkyne or azide moiety by copper-catalyzed azide-alkyne cycloaddition (CuAAC). Alkyne- and azide-containing sulfated oligosaccharides were prepared from a common precursor by modifying an anomeric linker with 4-pentynoic acid and by enzymatic extension with an N-acetyl-glucosamine having an azide moiety at C-6 (GlcNAc6N 3), respectively, followed by CuAAC. The process of enzymatic extension with GlcNAc6N 3 followed by CuAAC with the desired alkyne-containing oligosaccharides could be repeated to give compounds composed of 20 and 27 monosaccharides, respectively. The heparin mimetics could inhibit the binding of the SARS-CoV-2 spike or RBD to immobilized heparin or to Vero E6 cells. The inhibitory potency increased with increasing chain length, and a compound composed of four sulfated hexasaccharides linked by triazoles had a similar potency as unfractionated heparin. Sequence analysis and HS microarray binding studies with a wide range of RBDs of variants of concern indicate that they have maintained HS-binding capabilities and selectivities. The heparin mimetics exhibit no or reduced binding to antithrombin-III and platelet factor 4, respectively, which are associated with side effects

Revealing the Specificity of Human H1 Influenza A Viruses to Complex N-Glycans

Influenza virus infection remains a threat to human health since viral hemagglutinins are constantly drifting, escaping infection and vaccine-induced antibody responses. Viral hemag-glutinins from different viruses display variability in glycan recognition. In this context, recent H3N2 viruses have specificity for alpha 2,6 sialylated branched N-glycans with at least three N- acetyllactosamine units (tri-LacNAc). In this work, we combined glycan arrays and tissue binding analyses with nuclear magnetic resonance experiments to characterize the glycan specificity of a family of H1 variants, including the one responsible for the 2009 pandemic outbreak. We also analyzed one engineered H6N1 mutant to understand if the preference for tri-LacNAc motifs could be a general trend in human-type receptor-adapted viruses. In addition, we developed a new NMR approach to perform competition experiments between glycans with similar compositions and different lengths. Our results point out that pandemic H1 viruses differ from previous seasonal H1 viruses by a strict preference for a minimum of di-LacNAc structural motifs.R.P.d.V. is a recipient of an ERC Starting grant from the European Commission (802780) and a Beijerinck Premium of the Royal Dutch Academy of Sciences. The glycan array setup was supported by the Netherlands Organization for Scientific Research (NWO, TOP-PUNT 718.015.003 to G.-J.P.H.B.). Dr. Lin Liu (CCRC) and Dr. Margreet A . Wolfert (Utrecht University) developed, printed, and validated the glycan microarray. We would like to thank Nikoloz Nemanichvili for technical assistance. A.C. acknowledges funding from Agencia Estatal de Investigacion "Spanish Ministry of Science and Innovation" (MICINN) project PID2019-105237GB-I00. J.P.C. acknowledges funding by the Spanish MICINN, grant no. RTI2018-095588-B-I00 (co-funded by the European Regional Development Fund/European Social Fund, "Invest-ing in your future"). JJB also tha n k s funding by the European Research Council (RECGLYCANMR, Advanced grant no. 788143), the Agencia Estatal de Investigacion (Spain) for grants RTI2018-094751-B-C21 and C22 and PDI2021-1237810B-C21 and C22, and CIBERES, an initiative of the Instituto de Salud Carlos III (ISCIII), Madrid, Spain. The NMR spectra were acquired at the NMR service of CIBMargarita Salas and in the NMR faci l i t y of the UCM. We also acknowledge Prof. Robert Woods group for sending us the coordinates of a glycan-hemagglut i n i n model

Revealing the Specificity of Human H1 Influenza A Viruses to Complex N-Glycans

Influenza virus infection remains a threat to human health since viral hemagglutinins are constantly drifting, escaping infection and vaccine-induced antibody responses. Viral hemagglutinins from different viruses display variability in glycan recognition. In this context, recent H3N2 viruses have specificity for α2,6 sialylated branched N-glycans with at least three N-acetyllactosamine units (tri-LacNAc). In this work, we combined glycan arrays and tissue binding analyses with nuclear magnetic resonance experiments to characterize the glycan specificity of a family of H1 variants, including the one responsible for the 2009 pandemic outbreak. We also analyzed one engineered H6N1 mutant to understand if the preference for tri-LacNAc motifs could be a general trend in human-type receptor-adapted viruses. In addition, we developed a new NMR approach to perform competition experiments between glycans with similar compositions and different lengths. Our results point out that pandemic H1 viruses differ from previous seasonal H1 viruses by a strict preference for a minimum of di-LacNAc structural motifs

Distinct spatial arrangements of ACE2 and TMPRSS2 expression in Syrian hamster lung lobes dictates SARS-CoV-2 infection patterns

SARS-CoV-2 attaches to angiotensin-converting enzyme 2 (ACE2) to gain entry into cells after which the spike protein is cleaved by the transmembrane serine protease 2 (TMPRSS2) to facilitate viral-host membrane fusion. ACE2 and TMPRSS2 expression profiles have been analyzed at the genomic, transcriptomic, and single-cell RNAseq levels. However, transcriptomic data and actual protein validation convey conflicting information regarding the distribution of the biologically relevant protein receptor in whole tissues. To describe the organ-level architecture of receptor expression, related to the ability of ACE2 and TMPRSS2 to mediate infectivity, we performed a volumetric analysis of whole Syrian hamster lung lobes. Lung tissue of infected and control animals was stained using antibodies against ACE2 and TMPRSS2, combined with SARS-CoV-2 nucleoprotein staining. This was followed by light-sheet microscopy imaging to visualize their expression and related infection patterns. The data demonstrate that infection is restricted to sites containing both ACE2 and TMPRSS2, the latter is expressed in the primary and secondary bronchi whereas ACE2 is predominantly observed in the bronchioles and alveoli. Conversely, infection completely overlaps where ACE2 and TMPRSS2 co-localize in the tertiary bronchi, bronchioles, and alveoli

Drivers of recombinant soluble influenza A virus hemagglutinin and neuraminidase expression in mammalian cells

Recombinant soluble trimeric influenza A virus hemagglutinins (HA) and tetrameric neuraminidases (NAs) have proven to be excellent tools to decipher biological properties. Receptor binding and sialic acid cleavage by recombinant proteins correlate satisfactorily compared to whole viruses. Expression of HA and NA can be achieved in a plethora of different laboratory hosts. For immunological and receptor interaction studies however, insect and mammalian cell expressed proteins are preferred due to the presence of N-linked glycosylation and disulfide bond formation. Because mammalian-cell expression is widely applied, an increased expression yield is an important goal. Here we report that using codon-optimized genes and sfGFP fusions, the expression yield of HA can be significantly improved. sfGFP also significantly increased expression yields when fused to the N-terminus of NA. In this study, a suite of different hemagglutinin and neuraminidase constructs are described, which can be valuable tools to study a wide array of different HAs, NAs and their mutants

The Microbiota Contributes to the Control of Highly Pathogenic H5N9 Influenza Virus Replication in Ducks

Ducks usually show little or no clinical signs following highly pathogenic avian influenza virus infection. In order to analyze whether the microbiota could contribute to the control of influenza virus replication in ducks, we used a broad-spectrum oral antibiotic treatment to deplete the microbiota before infection with a highly pathogenic H5N9 avian influenza virus. Antibiotic-treated ducks and nontreated control ducks did not show any clinical signs following H5N9 virus infection. We did not detect any significant difference in virus titers neither in the respiratory tract nor in the brain nor spleen. However, we found that antibiotic-treated H5N9 virus-infected ducks had significantly increased intestinal virus excretion at days 3 and 5 postinfection. This was associated with a significantly decreased antiviral immune response in the intestine of antibiotic-treated ducks. Our findings highlight the importance of an intact microbiota for an efficient control of avian influenza virus replication in ducks.IMPORTANCE Ducks are frequently infected with avian influenza viruses belonging to multiple subtypes. They represent an important reservoir species of avian influenza viruses, which can occasionally be transmitted to other bird species or mammals, including humans. Ducks thus have a central role in the epidemiology of influenza virus infection. Importantly, ducks usually show little or no clinical signs even following infection with a highly pathogenic avian influenza virus. We provide evidence that the microbiota contributes to the control of influenza virus replication in ducks by modulating the antiviral immune response. Ducks are able to control influenza virus replication more efficiently when they have an intact intestinal microbiota. Therefore, maintaining a healthy microbiota by limiting perturbations to its composition should contribute to the prevention of avian influenza virus spread from the duck reservoir

Drivers of recombinant soluble influenza A virus hemagglutinin and neuraminidase expression in mammalian cells

Recombinant soluble trimeric influenza A virus hemagglutinins (HA) and tetrameric neuraminidases (NAs) have proven to be excellent tools to decipher biological properties. Receptor binding and sialic acid cleavage by recombinant proteins correlate satisfactorily compared to whole viruses. Expression of HA and NA can be achieved in a plethora of different laboratory hosts. For immunological and receptor interaction studies however, insect and mammalian cell expressed proteins are preferred due to the presence of N-linked glycosylation and disulfide bond formation. Because mammalian-cell expression is widely applied, an increased expression yield is an important goal. Here we report that using codon-optimized genes and sfGFP fusions, the expression yield of HA can be significantly improved. sfGFP also significantly increased expression yields when fused to the N-terminus of NA. In this study, a suite of different hemagglutinin and neuraminidase constructs are described, which can be valuable tools to study a wide array of different HAs, NAs and their mutants

Distinct spatial arrangements of ACE2 and TMPRSS2 expression in Syrian hamster lung lobes dictates SARS-CoV-2 infection patterns

SARS-CoV-2 attaches to angiotensin-converting enzyme 2 (ACE2) to gain entry into cells after which the spike protein is cleaved by the transmembrane serine protease 2 (TMPRSS2) to facilitate viral-host membrane fusion. ACE2 and TMPRSS2 expression profiles have been analyzed at the genomic, transcriptomic, and single-cell RNAseq levels. However, transcriptomic data and actual protein validation convey conflicting information regarding the distribution of the biologically relevant protein receptor in whole tissues. To describe the organ-level architecture of receptor expression, related to the ability of ACE2 and TMPRSS2 to mediate infectivity, we performed a volumetric analysis of whole Syrian hamster lung lobes. Lung tissue of infected and control animals was stained using antibodies against ACE2 and TMPRSS2, combined with SARS-CoV-2 nucleoprotein staining. This was followed by light-sheet microscopy imaging to visualize their expression and related infection patterns. The data demonstrate that infection is restricted to sites containing both ACE2 and TMPRSS2, the latter is expressed in the primary and secondary bronchi whereas ACE2 is predominantly observed in the bronchioles and alveoli. Conversely, infection completely overlaps where ACE2 and TMPRSS2 co-localize in the tertiary bronchi, bronchioles, and alveoli

Distinct spatial arrangements of ACE2 and TMPRSS2 expression in Syrian hamster lung lobes dictates SARS-CoV-2 infection patterns

SARS-CoV-2 attaches to angiotensin-converting enzyme 2 (ACE2) to gain entry into cells after which the spike protein is cleaved by the transmembrane serine protease 2 (TMPRSS2) to facilitate viral-host membrane fusion. ACE2 and TMPRSS2 expression profiles have been analyzed at the genomic, transcriptomic, and single-cell RNAseq levels. However, transcriptomic data and actual protein validation convey conflicting information regarding the distribution of the biologically relevant protein receptor in whole tissues. To describe the organ-level architecture of receptor expression, related to the ability of ACE2 and TMPRSS2 to mediate infectivity, we performed a volumetric analysis of whole Syrian hamster lung lobes. Lung tissue of infected and control animals was stained using antibodies against ACE2 and TMPRSS2, combined with SARS-CoV-2 nucleoprotein staining. This was followed by light-sheet microscopy imaging to visualize their expression and related infection patterns. The data demonstrate that infection is restricted to sites containing both ACE2 and TMPRSS2, the latter is expressed in the primary and secondary bronchi whereas ACE2 is predominantly observed in the bronchioles and alveoli. Conversely, infection completely overlaps where ACE2 and TMPRSS2 co-localize in the tertiary bronchi, bronchioles, and alveoli

Influenza Hemagglutinins H2, H5, H6, and H11 are not Targets of Pulmonary Surfactant Protein D : T-glycan subtypes in host-pathogen interactions

Seasonal influenza carrying key hemagglutinin (HA) head region glycosylation sites can be removed from the lung by pulmonary surfactant protein D (SP-D). Little is known about HA head glycosylation of low pathogenicity A type influenza virus (LPAIV) subtypes. These can pose a pandemic threat through reassortmant and emergence in human populations. Since the presence of head region high mannose glycosites dictates SP-D activity, the ability to predict these glycosite glycan subtypes may be of value. Here we investigate the activities of two recombinant human SP-D forms against representative LPAIV including H2N1, H5N1, H6N1, H11N9, an avian H3N8 and a human seasonal H3N2 subtype. Using mass spectrometry, we determined the glycan subclasses and heterogeneities at each head glycosylation site. Sequence alignment and molecular structure analysis of the HAs were performed for LPIAV strains in comparison to seasonal H3N2 and avian H3N8. Intramolecular contacts were determined between protein backbone and glycosite glycan based on available three-dimensional structure data. We found that glycosite "N165" (H3 numbering) is occupied by high mannose glycans in H3 HA but by complex glycans in all LVIAV HAs. SP-D was not active on LPAIV but was on H3 HAs. Since SP-D affinity for influenza HA depends on the presence of high mannose glycan on the head region our data demonstrate that SP-D may not protect against virus containing these HA subtypes. Our results also demonstrate that glycan subtype can be predicted at some glycosites based on sequence comparisons and three dimensional structural analysis.Importance Low pathogenicity A type influenza virus (LPAIV) subtypes can reassort with circulating human strains and pandemic viruses can emerge in human populations as was seen in the 1957 pandemic, where an H2 virus reassorted with the circulating H1N1 to create a novel H2N2 genotype. Lung surfactant protein D (SP-D), a key factor in first line innate immunity defence, removes IAV through interaction with hemagglutinin (HA) head region high mannose glycan(s). While it is known that both H1 and H3 HAs, have a key high mannose glycosite(s) in the head region, little is known about such glycosylation of LPAIV strains H2N1, H5N1, H6N1, or H11N9, which may pose future health risks. Here, we demonstrate that the hemagglutinins of LPAIV strains do not have the required high mannose glycans, do not interact with SP-D, and that sequence analysis can predict glycan subtype thus predicting presence or absence of this virulence marker