Long-term evolution of human seasonal influenza virus A(H3N2) is associated with an increase in polymerase complex activity.

Since the influenza pandemic in 1968, influenza A(H3N2) viruses have become endemic. In this state, H3N2 viruses continuously evolve to overcome immune pressure as a result of prior infection or vaccination, as is evident from the accumulation of mutations in the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, phylogenetic studies have also demonstrated ongoing evolution in the influenza A(H3N2) virus RNA polymerase complex genes. The RNA polymerase complex of seasonal influenza A(H3N2) viruses produces mRNA for viral protein synthesis and replicates the negative sense viral RNA genome (vRNA) through a positive sense complementary RNA intermediate (cRNA). Presently, the consequences and selection pressures driving the evolution of the polymerase complex remain largely unknown. Here, we characterize the RNA polymerase complex of seasonal influenza A(H3N2) viruses representative of nearly 50 years of influenza A(H3N2) virus evolution. The H3N2 polymerase complex is a reassortment of human and avian influenza virus genes. We show that since 1968, influenza A(H3N2) viruses have increased the transcriptional activity of the polymerase complex while retaining a close balance between mRNA, vRNA, and cRNA levels. Interestingly, the increased polymerase complex activity did not result in increased replicative ability on differentiated human airway epithelial (HAE) cells. We hypothesize that the evolutionary increase in polymerase complex activity of influenza A(H3N2) viruses may compensate for the reduced HA receptor binding and avidity that is the result of the antigenic evolution of influenza A(H3N2) viruses.

Centralized intestinal organoid generation is a feasible and safe approach for personalized medicine as demonstrated in the HIT-CF Europe Organoid Study.

BACKGROUND: Patient-derived intestinal organoids (PDIOs) show great potential as in vitro drug testing platform for personalised medicine in Cystic Fibrosis and oncology. PDIOs can be generated by culturing adult stem cells obtained through rectal forceps biopsy or suction biopsy, but the safety of these procedures and the success rates of generating organoids after shipment to a centralized lab using these procedures has not been studied in this context. We here report the safety and success rates of both biopsy procedures and the subsequent generation of PDIOs in the international multicentre HIT-CF Organoid Study. METHODS: 502 biopsy procedures were conducted, on 489 adult people with Cystic Fibrosis from 33 different hospitals across 12 countries. Depending on the preference of the hospital, either rectal forceps biopsies or suction biopsies were obtained and internationally shipped to a central laboratory for organoid generation. RESULTS: No adverse events were reported for 280 forceps biopsy procedures, while 222 rectal suction biopsy procedures resulted in 2 adverse events, namely continued bleeding and a probably nonrelated gastroenteritis. The success rate of organoid generation from all biopsies was 95%, and the main reason for failure was insufficient sample viability (3.2%). CONCLUSION: Our results indicate that both rectal suction biopsy and forceps biopsy procedures are safe procedures. The high success rates of PDIO generation from the obtained tissue samples demonstrate the feasibility of the organoid technology for personalised in vitro testing in an international setting.

Repeatability and reproducibility of the Forskolin-induced swelling (FIS) assay on intestinal organoids from people with Cystic Fibrosis.

BACKGROUND: The forskolin-induced swelling (FIS) assay measures CFTR function on patient-derived intestinal organoids (PDIOs) and may guide treatment selection for individuals with Cystic Fibrosis (CF). The aim of this study is to demonstrate the repeatability and reproducibility of the FIS assay following a detailed Standard Operating Procedure (SOP), thus advancing the validation of the assay for precision medicine (theranostic) applications. METHODS: Over a 2-year period, FIS responses to CFTR modulators were measured in four European labs. PDIOs from six subjects with CF carrying different CFTR genotypes were used to assess the repeatability and reproducibility across the dynamic range of the assay. RESULTS: Technical, intra-assay repeatability was high (Lin's concordance correlation coefficient (CCC) 0.95-0.98). Experimental, within-subject repeatability was also high within each lab (CCCs all >0.9). Longer-term repeatability (>1 year) showed more variability (CCCs from 0.67 to 0.95). The reproducibility between labs was also high (CCC ranging from 0.92 to 0.97). Exploratory analysis also found that between-lab percentage of agreement of dichotomized CFTR modulator outcomes for predefined FIS thresholds ranged between 78 and 100 %. CONCLUSIONS: The observed repeatability and reproducibility of the FIS assay within and across different labs is high and support the use of FIS as biomarker of CFTR function in the presence or absence of CFTR modulators.

Chemoenzymatic Synthesis of Tri-antennary N-Glycans Terminating in Sialyl-Lewisx Reveals the Importance of Glycan Complexity for Influenza A Virus Receptor Binding.

Sialyl-Lewisx (SLex) is involved in immune regulation, human fertilization, cancer, and bacterial and viral diseases. The influence of the complex glycan structures, which can present SLex epitopes, on binding is largely unknown. We report here a chemoenzymatic strategy for the preparation of a panel of twenty-two isomeric asymmetrical tri-antennary N-glycans presenting SLex-Lex epitopes on either the MGAT4 or MGAT5 arm that include putative high-affinity ligands for E-selectin. The N-glycans were prepared starting from a sialoglycopeptide isolated from egg yolk powder and took advantage of inherent substrate preferences of glycosyltransferases and the use of 5'-diphospho-N-trifluoracetylglucosamine (UDP-GlcNHTFA) that can be transferred by branching N-acetylglucosaminyltransferases to give, after base treatment, GlcNH2-containing glycans that temporarily disable an antenna from enzymatic modification. Glycan microarray binding studies showed that E-selectin bound equally well to linear glycans and tri-antennary N-glycans presenting SLex-Lex. On the other hand, it was found that hemagglutinins (HA) of H5 influenza A viruses (IAV) preferentially bound the tri-antennary N-glycans. Furthermore, several H5 HAs preferentially bound to N-glycan presenting SLex on the MGAT4 arm. SLex is displayed in the respiratory tract of several avian species, demonstrating the relevance of investigating the binding of, among others IAVs, to complex N-glycans presenting SLex.

Probing altered receptor specificities of antigenically drifting human H3N2 viruses by chemoenzymatic synthesis, NMR, and modeling.

Prototypic receptors for human influenza viruses are N-glycans carrying α2,6-linked sialosides. Due to immune pressure, A/H3N2 influenza viruses have emerged with altered receptor specificities that bind α2,6-linked sialosides presented on extended N-acetyl-lactosamine (LacNAc) chains. Here, binding modes of such drifted hemagglutinin's (HAs) are examined by chemoenzymatic synthesis of N-glycans having 13C-labeled monosaccharides at strategic positions. The labeled glycans are employed in 2D STD-1H by 13C-HSQC NMR experiments to pinpoint which monosaccharides of the extended LacNAc chain engage with evolutionarily distinct HAs. The NMR data in combination with computation and mutagenesis demonstrate that mutations distal to the receptor binding domain of recent HAs create an extended binding site that accommodates with the extended LacNAc chain. A fluorine containing sialoside is used as NMR probe to derive relative binding affinities and confirms the contribution of the extended LacNAc chain for binding.

Asymmetrical Biantennary Glycans Prepared by a Stop-and-Go Strategy Reveal Receptor Binding Evolution of Human Influenza A Viruses.

Glycan binding properties of respiratory viruses have been difficult to probe due to a lack of biologically relevant glycans for binding studies. Here, a stop-and-go chemoenzymatic methodology is presented that gave access to a panel of 32 asymmetrical biantennary N-glycans having various numbers of N-acetyl lactosamine (LacNAc) repeating units capped by α2,3- or α2,6-sialosides resembling structures found in airway tissues. It exploits that the branching enzymes MGAT1 and MGAT2 can utilize unnatural UDP-2-deoxy-2-trifluoro-N-acetamido-glucose (UDP-GlcNTFA) as donor. The TFA moiety of the resulting glycans can be hydrolyzed to give GlcNH2 at one of the antennae, which temporarily blocks extension by glycosyl transferases. The N-glycans were printed as a microarray that was probed for receptor binding specificities of the evolutionary distinct human A(H3N2) and A(H1N1)pdm09 viruses. It was found that not only the sialoside type but also the length of the LacNAc chain and presentation at the α1,3-antenna of N-glycans are critical for binding. Early A(H3N2) viruses bound to 2,6-sialosides at a single LacNAc moiety at the α1,3-antenna whereas later viruses required the sialoside to be presented at a tri-LacNAc moiety. Surprisingly, most of the A(H3N2) viruses that appeared after 2021 regained binding capacity to sialosides presented at a di-LacNAc moiety. As a result, these viruses again agglutinate erythrocytes, commonly employed for antigenic characterization of influenza viruses. Human A(H1N1)pdm09 viruses have similar receptor binding properties as recent A(H3N2) viruses. The data indicate that an asymmetric N-glycan having 2,6-sialoside at a di-LacNAc moiety is a commonly employed receptor by human influenza A viruses.

Viral envelope proteins fused to multiple distinct fluorescent reporters to probe receptor binding.

Enveloped viruses carry one or multiple proteins with receptor-binding functionalities. Functional receptors can be glycans, proteinaceous, or both; therefore, recombinant protein approaches are instrumental in attaining new insights regarding viral envelope protein receptor-binding properties. Visualizing and measuring receptor binding typically entails antibody detection or direct labeling, whereas direct fluorescent fusions are attractive tools in molecular biology. Here, we report a suite of distinct fluorescent fusions, both N- and C-terminal, for influenza A virus hemagglutinins and SARS-CoV-2 spike RBD. The proteins contained three or six fluorescent protein barrels and were applied directly to cells to assess receptor binding properties.

Complex N-glycans are important for interspecies transmission of H7 influenza A viruses.

Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo. We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo, the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N-glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N-glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.IMPORTANCEInfluenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N-acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N-glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N-glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection.

A Biomimetic Synthetic Strategy Can Provide Keratan Sulfate I and II Oligosaccharides with Diverse Fucosylation and Sulfation Patterns.

Keratan sulfate (KS) is a proteoglycan that is widely expressed in the extracellular matrix of various tissue types, where it performs multiple biological functions. KS is the least understood proteoglycan, which in part is due to a lack of panels of well-defined KS oligosaccharides that are needed for structure-binding studies, as analytical standards, to examine substrate specificities of keratinases, and for drug development. Here, we report a biomimetic approach that makes it possible to install, in a regioselective manner, sulfates and fucosides on oligo-N-acetyllactosamine (LacNAc) chains to provide any structural element of KS by using specific enzyme modules. It is based on the observation that α1,3-fucosides, α2,6-sialosides and C-6 sulfation of galactose (Gal6S) are mutually exclusive and cannot occur on the same LacNAc moiety. As a result, the pattern of sulfation on galactosides can be controlled by installing α1,3-fucosides or α2,6-sialosides to temporarily block certain LacNAc moieties from sulfation by keratan sulfate galactose 6-sulfotransferase (CHST1). The patterns of α1,3-fucosylation and α2,6-sialylation can be controlled by exploiting the mutual exclusivity of these modifications, which in turn controls the sites of sulfation by CHST1. Late-stage treatment with a fucosidase or sialidase to remove blocking fucosides or sialosides provides selectively sulfated KS oligosaccharides. These treatments also unmasked specific galactosides for further modification by CHST1. To showcase the potential of the enzymatic strategy, we have prepared a range of poly-LacNAc derivatives having different patterns of fucosylation and sulfation and several N-glycans decorated by specific arrangements of sulfates.

Organoids as a biomarker for personalized treatment in metastatic colorectal cancer: drug screen optimization and correlation with patient response.

BACKGROUND: The inability to predict treatment response of colorectal cancer patients results in unnecessary toxicity, decreased efficacy and survival. Response testing on patient-derived organoids (PDOs) is a promising biomarker for treatment efficacy. The aim of this study is to optimize PDO drug screening methods for correlation with patient response and explore the potential to predict responses to standard chemotherapies. METHODS: We optimized drug screen methods on 5-11 PDOs per condition of the complete set of 23 PDOs from patients treated for metastatic colorectal cancer (mCRC). PDOs were exposed to 5-fluorouracil (5-FU), irinotecan- and oxaliplatin-based chemotherapy. We compared medium with and without N-acetylcysteine (NAC), different readouts and different combination treatment set-ups to capture the strongest association with patient response. We expanded the screens using the optimized methods for all PDOs. Organoid sensitivity was correlated to the patient's response, determined by % change in the size of target lesions. We assessed organoid sensitivity in relation to prior exposure to chemotherapy, mutational status and sidedness. RESULTS: Drug screen optimization involved excluding N-acetylcysteine from the medium and biphasic curve fitting for 5-FU & oxaliplatin combination screens. CellTiter-Glo measurements were comparable with CyQUANT and did not affect the correlation with patient response. Furthermore, the correlation improved with application of growth rate metrics, when 5-FU & oxaliplatin was screened in a ratio, and 5-FU & SN-38 using a fixed dose of SN-38. Area under the curve was the most robust drug response curve metric. After optimization, organoid and patient response showed a correlation coefficient of 0.58 for 5-FU (n = 6, 95% CI -0.44,0.95), 0.61 for irinotecan- (n = 10, 95% CI -0.03,0.90) and 0.60 for oxaliplatin-based chemotherapy (n = 11, 95% CI -0.01,0.88). Median progression-free survival of patients with resistant PDOs to oxaliplatin-based chemotherapy was significantly shorter than sensitive PDOs (3.3 vs 10.9 months, p = 0.007). Increased resistance to 5-FU in patients with prior exposure to 5-FU/capecitabine was adequately reflected in PDOs (p = 0.003). CONCLUSIONS: Our study emphasizes the critical impact of the screening methods for determining correlation between PDO drug screens and mCRC patient outcomes. Our 5-step optimization strategy provides a basis for future research on the clinical utility of PDO screens.

Bioluminescent detection of viral surface proteins using branched multivalent protein switches.

Fast and reliable virus diagnostics is key to prevent the spread of viruses in populations. A hallmark of viruses is the presence of multivalent surface proteins, a property that can be harnessed to control conformational switching in sensor proteins. Here, we introduce a new sensor platform (dark-LUX) for the detection of viral surface proteins consisting of a general bioluminescent framework that can be post-translationally functionalized with separately expressed binding domains. The platform relies on (1) plug-and-play bioconjugation of different binding proteins via SpyTag/SpyCatcher technology to create branched protein structures, (2) an optimized turn-on bioluminescent switch based on complementation of the split-luciferase NanoBiT upon target binding and (3) straightforward exploration of the protein linker space. The influenza A virus (IAV) surface proteins hemagglutinin (HA) and neuraminidase (NA) were used as relevant multivalent targets to establish proof of principle and optimize relevant parameters such as linker properties, choice of target binding domains and the optimal combination of the competing NanoBiT components SmBiT and DarkBiT. The sensor framework allows rapid conjugation and exchange of various binding domains including scFvs, nanobodies and de novo designed binders for a variety of targets, including the construction of a heterobivalent switch that targets the head and stem region of hemagglutinin. The modularity of the platform thus allows straightforward optimization of binding domains and scaffold properties for existing viral targets, and is well suited to quickly adapt bioluminescent sensor proteins to effectively detect newly evolving viral epitopes.

Determinants of epidemic size and the impacts of lulls in seasonal influenza virus circulation.

During the COVID-19 pandemic, levels of seasonal influenza virus circulation were unprecedentedly low, leading to concerns that a lack of exposure to influenza viruses, combined with waning antibody titres, could result in larger and/or more severe post-pandemic seasonal influenza epidemics. However, in most countries the first post-pandemic influenza season was not unusually large and/or severe. Here, based on an analysis of historical influenza virus epidemic patterns from 2002 to 2019, we show that historic lulls in influenza virus circulation had relatively minor impacts on subsequent epidemic size and that epidemic size was more substantially impacted by season-specific effects unrelated to the magnitude of circulation in prior seasons. From measurements of antibody levels from serum samples collected each year from 2017 to 2021, we show that the rate of waning of antibody titres against influenza virus during the pandemic was smaller than assumed in predictive models. Taken together, these results partially explain why the re-emergence of seasonal influenza virus epidemics was less dramatic than anticipated and suggest that influenza virus epidemic dynamics are not currently amenable to multi-season prediction.

Chemo-Enzymatic Synthesis of Isomeric I-branched Polylactosamines Using Traceless Blocking Groups.

Poly-N-acetyl lactosamines (polyLacNAc) are common structural motifs of N- and O-linked glycan, glycosphingolipids and human milk oligosaccharides. They can be branched by the addition of ß1,6-linked N-acetyl-glucosamine (GlcNAc) moieties to internal galactoside (Gal) residues by the I-branching enzyme beta-1,6-N-acetylglucosaminyltransferase 2 (GCNT2). I-branching has been implicated in many biological processes and is also associated with various diseases such as cancer progression. Currently, there is a lack of methods that can install, in a regioselective manner, I-branches and allows the preparation of isomeric poly-LacNAc derivatives. Here, we described a chemo-enzymatic strategy that addresses this deficiency and is based on the enzymatic assembly of an oligo-LacNAc chain that at specific positions is modified by a GlcNTFA moiety. Replacement of the trifluoroacetyl (TFA) moiety by tert-butyloxycarbonyl (Boc) gives compounds in which the galactoside at the proximal site is blocked from modification by GCNT2. After elaboration of the antennae, the Boc group can be removed, and the resulting amine acetylated to give natural I-branched structures. It is also shown that fucosides can function as a traceless blocking group that can provide complementary I-branched structures from a single precursor. The methodology made it possible to synthesize a library of polyLacNAc chains having various topologies.

Exploiting Substrate Specificities of 6-O-Sulfotransferases to Enzymatically Synthesize Keratan Sulfate Oligosaccharides.

Keratan sulfate (KS) is a glycosaminoglycan that is widely expressed in the extracellular matrix of various tissue types, where it is involved in many biological processes. Herein, we describe a chemo-enzymatic approach to preparing well-defined KS oligosaccharides by exploiting the known and newly discovered substrate specificities of relevant sulfotransferases. The premise of the approach is that recombinant GlcNAc-6-O-sulfotransferases (CHST2) only sulfate terminal GlcNAc moieties to give GlcNAc6S that can be galactosylated by B4GalT4. Furthermore, CHST1 can modify the internal galactosides of a poly-LacNAc chain; however, it was found that a GlcNAc6S residue greatly increases the reactivity of CHST1 of a neighboring and internal galactoside. The presence of a 2,3-linked sialoside further modulates the site of modification by CHST1, and a galactoside flanked by 2,3-Neu5Ac and GlcNAc6S is preferentially sulfated over the other Gal residues. The substrate specificities of CHST1 and 2 were exploited to prepare a panel of KS oligosaccharides, including selectively sulfated N-glycans. The compounds and several other reference derivatives were used to construct a microarray that was probed for binding by several plant lectins, Siglec proteins, and hemagglutinins of influenza viruses. It was found that not only the sulfation pattern but also the presentation of epitopes as part of an O- or N-glycan determines binding properties.

Asymmetrical Bi-antennary Glycans Prepared by a Stop-and-Go Strategy Reveal Receptor Binding Evolution of Human Influenza A Viruses.

Glycan binding properties of respiratory viruses have been difficult to probe due to a lack of biological relevant glycans for binding studies. Here, a stop-and-go chemoenzymatic methodology is presented that gave access to a panel of 32 asymmetrical bi-antennary N-glycans having various numbers of N-acetyl lactosamine (LacNAc) repeating units capped by α2,3- or α2,6-sialosides resembling structures found in airway tissues. It exploits that the branching enzymes MGAT1 and MGAT2 can utilize unnatural UDP-2-deoxy-2-trifluoro-N-acetamido-glucose (UDP-GlcNTFA) as donor. The TFA moiety of the resulting glycans can be hydrolyzed to give GlcNH2 at one of the antennae that temporarily blocks extension by glycosyl transferases. The N-glycans were printed as a microarray that was probed for receptor binding specificities of evolutionary distinct human A(H3N2) and A(H1N1)pdm09 viruses. It was found that not only the sialoside type but also the length of the LacNAc chain and presentation at the α1,3-antenna of N-glycans is critical for binding. Early A(H3N2) viruses bound to 2,6-sialosides at a single LacNAc moiety at the α1,3-antenna whereas later viruses required the sialoside to be presented at a tri-LacNAc moiety. Surprisingly, most of the A(H3N2) viruses that appeared after 2021 regained binding capacity to sialosides presented at a di-LacNAc moiety. As a result, these viruses agglutinate erythrocytes again, commonly employed for antigenic characterization of influenza viruses. Human A(H1N1)pdm09 viruses have similar receptor binding properties as recent A(H3N2) viruses. The data indicates that an asymmetric N-glycan having 2,6-sialoside at a di-LacNAc moiety is a commonly employed receptor by human influenza A viruses.

Sialic acid O-acetylation patterns and glycosidic linkage type determination by ion mobility-mass spectrometry.

O-acetylation is a common modification of sialic acids that has been implicated in a multitude of biological and disease processes. A lack of analytical methods that can determine exact structures of sialic acid variants is a hurdle to determine roles of distinct O-acetylated sialosides. Here, we describe a drift tube ion mobility-mass spectrometry approach that can elucidate exact O-acetylation patterns as well as glycosidic linkage types of sialosides isolated from complex biological samples. It is based on the use of a library of synthetic O-acetylated sialosides to establish intrinsic collision cross section (CCS) values of diagnostic fragment ions. The CCS values were used to characterize O-acetylated sialosides from mucins and N-linked glycans from biologicals as well as equine tracheal and nasal tissues. It uncovered contrasting sialic acid linkage types of acetylated and non-acetylated sialic acids and provided a rationale for sialic acid binding preferences of equine H7 influenza A viruses.

Continued adaptation of A/H2N2 viruses during pandemic circulation in humans.

Influenza A viruses of the H2N2 subtype sparked a pandemic in 1957 and circulated in humans until 1968. Because A/H2N2 viruses still circulate in wild birds worldwide and human population immunity is low, the transmissibility of six avian A/H2N2 viruses was investigated in the ferret model. None of the avian A/H2N2 viruses was transmitted between ferrets, suggesting that their pandemic risk may be low. The transmissibility, receptor binding preference and haemagglutinin (HA) stability of human A/H2N2 viruses were also investigated. Human A/H2N2 viruses from 1957 and 1958 bound to human-type α2,6-linked sialic acid receptors, but the 1958 virus had a more stable HA, indicating adaptation to replication and spread in the new host. This increased stability was caused by a previously unknown stability substitution G205S in the 1958 H2N2 HA, which became fixed in A/H2N2 viruses after 1958. Although individual substitutions were identified that affected the HA receptor binding and stability properties, they were not found to have a substantial effect on transmissibility of A/H2N2 viruses via the air in the ferret model. Our data demonstrate that A/H2N2 viruses continued to adapt during the first year of pandemic circulation in humans, similar to what was previously shown for the A/H1N1pdm09 virus.

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection.

Recent human H3N2 influenza A viruses have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by (humanized) Madin-Darby Canine Kidney cells, which are commonly employed to propagate influenza A virus, resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases ß-1,3-N-acetylglucosaminyltransferase and ß-1,4-galactosyltransferase 1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAcs), would result in improved A/H3N2 propagation. Stable overexpression of ß-1,3-N-acetylglucosaminyltransferase and ß-1,4-galactosyltransferase 1 in Madin-Darby Canine Kidney and "humanized" Madin-Darby Canine Kidney cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the ß-1,3-N-acetylglucosaminyltransferase and/or ß-1,4-galactosyltransferase 1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on "humanized" Madin-Darby Canine Kidney-ß-1,3-N-acetylglucosaminyltransferase cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 influenza A viruses require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency.

Intestinal organoids as an in vitro platform to characterize disposition, metabolism, and safety profile of small molecules.

Intestinal organoids derived from LGR5+ adult stem cells allow for long-term culturing, more closely resemble human physiology than traditional intestinal models, like Caco-2, and have been established for several species. Here we evaluated intestinal organoids for drug disposition, metabolism, and safety applications. Enterocyte-enriched human duodenal organoids were cultured as monolayers to enable bidirectional transport studies. 3D enterocyte-enriched human duodenal and colonic organoids were incubated with probe substrates of major intestinal drug metabolizing enzymes (DMEs). To distinguish human intestinal toxic (high incidence of diarrhea in clinical trials and/or black box warning related to intestinal side effects) from non-intestinal toxic compounds, ATP-based cell viability was used as a readout, and compounds were ranked based on their IC50 values in relation to their 30-times maximal total plasma concentration (Cmax). To assess if rat and dog organoids reproduced the respective in vivo intestinal safety profiles, ATP-based viability was assessed in rat and dog organoids and compared to in vivo intestinal findings when available. Human duodenal monolayers discriminated high and low permeable compounds and demonstrated functional activity for the main efflux transporters Multi drug resistant protein 1 (MDR1, P-glycoprotein P-gp) and Breast cancer resistant protein (BCRP). Human 3D duodenal and colonic organoids also showed metabolic activity for the main intestinal phase I and II DMEs. Organoids derived from specific intestinal segments showed activity differences in line with reported DMEs expression. Undifferentiated human organoids accurately distinguished all but one compound from the test set of non-toxic and toxic drugs. Cytotoxicity in rat and dog organoids correlated with preclinical toxicity findings and observed species sensitivity differences between human, rat, and dog organoids. In conclusion, the data suggest intestinal organoids are suitable in vitro tools for drug disposition, metabolism, and intestinal toxicity endpoints. The possibility to use organoids from different species, and intestinal segment holds great potential for cross-species and regional comparisons.

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.