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Neuraminidase resistant sialosides for the detection of influenza viruses Yun He, Yang Yang, and Suri S. Iyer Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00150 • Publication Date (Web): 03 May 2016 Downloaded from http://pubs.acs.org on May 9, 2016

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Bioconjugate Chemistry

Neuraminidase resistant sialosides for the detection of influenza viruses Yun He, Yang Yang and Suri S. Iyer* 788 Petit Science Center, Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia, USA - 30302. Email: [email protected]

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Abstract: We report the synthesis of influenza virus neuraminidase (NA) resistant sialosides that include different glycoside linkages (C-, S- and triazole). These unnatural sialosides were printed onto glass slides to generate a small focused microarray. We evaluated the binding affinity of multiple lectins and compared the stability of these sialosides with O-linked sialosides towards influenza virus neuraminidase and intact virus. We demonstrated the ability of these molecules to capture eight different strains of influenza virus at ambient temperature without the addition of NA inhibitors. The glycans capture extremely low, clinically relevant concentrations of viruses and each strain gives rise to a specific “fingerprint” binding pattern, which could potentially be used in rapid diagnostic tests.


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Introduction:

Influenza virus is an acute respiratory pathogen that causes considerable harm to humans and livestock.1 In addition to seasonal influenza that leads to approximately 200,000 hospitalizations and 36,000 deaths per year in the United States alone, catastrophic pandemics such as the 1918 Spanish flu (H1N1) can be devastating.2 Most antivirals are highly efficacious if administered within 24-48 hours within onset of infection or earlier, especially in people with developing or weakened immune systems.3,4 In addition, influenza virus spreads extremely rapidly. For example, the 2009 H1N1 influenza "swine" strain spread rapidly across the globe within ten days, resulting in extraordinary measures5,6 and overwhelming public health care facilities.7 Most public hospitals requested patients to stop coming to the emergency room during that time; the WHO reported infections in 206 countries within weeks of the initial outbreak.8 Clearly, rapid and accurate diagnosis is important in our fight against influenza viruses.

There are a host of commercial detection technologies approved by public health officials for the detection of influenza viruses. Among these, nucleic acid based technologies are highly accurate and considered the "gold standard", but are cost prohibitive and limited in scope for testing large numbers of patients, especially in a primary care setting. RT-PCR tests also require appropriate primers, sample preparation, trained personnel and equipment and therefore, are best suited for centrally located clinical laboratories. Alternatives to nucleic acid tests include antibody based tests. These rapid diagnostic tests with a test to result time of less than 30 minutes are plagued with a variety of issues that include sensitivity, antibody degradation at ambient temperature, cross reactivity and procurement issues during an emergency. 9,10 It typically takes a long time to generate antibodies for an emerging strain. Purification, incorporation into lateral flow assays, testing and validation takes additional time and resources. In addition, commercial antibody tests are quite variable in their ability to accurately detect and differentiate between different strains; the test is only as good as the antibody, which is problematic as rules for antibody standardization is still a work in progress.11 Also, antibody based tests are expensive. Due to the myriad of concerns, the CDC (Centers for Disease Control, Atlanta, GA) recommends that they be used for surveillance purposes or in conjunction with other tests. (http://www.cdc.gov/flu)

An alternate approach towards the detection of influenza has come from the recognition that influenza virus surface glycoproteins, Hemagluttinin (HA) and Neuraminidase (NA) binds to the terminal residues ACS Paragon Plus Environment


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of N-acetyl neuraminic acid (or sialic acid) of glycoproteins and glycolipids of the host cell. Therefore, sialic acid analogues, natural or synthetic, could be used as recognition molecules in biosensors to capture the virus and potentially identify the strain.1,12,13 This receptor based approach is highly advantageous to the detection of influenza viruses as the virus needs sialic acid to gain entry into the cell. Therefore, all strains are expected to a panel of sialosides leading to a "pattern of recognition". In contrast, an antibody based approach is highly dependent on the epitope the antibody recognizes. If an emerging strain modifies the epitope, the antibody produced in previous years against existing strains will not be able to recognize the new strain. Given the high mutation rate, it is not surprising that antibody based tests are not suitable for emerging strains or for that matter, mutated seasonal strains.

Additional advantages to a glycan based approach is that glycans can be synthesized in appreciable yields economically, are highly robust under a variety of conditions and do not require refrigeration.14-16 Multiple groups have used this approach to capture HA and intact viruses using microarrays of natural Osialosides, synthesized from enzymatic and chemo-enzymatic methods.17,18 This approach has advanced our understanding of some of the fundamental aspects of influenza virus-receptor interactions; glycan microarrays constructed using O-sialosides have been used (in conjunction with other methods) to explain why certain virus strains prefer 2,3 linkages, in contrast to other strains which prefer 2,6 linkages.17-20 Additionally, the CDC has been using customized glycan microarrays consisting of O-sialosides from the Consortium for Functional Glycomics (CFG, http://www.functionalglycomics.org/)

21,22

to test the HA

binding specificities of various strains using recombinant HAs.(18) While these microarrays using natural O-sialosides have found considerable use in defining receptor preferences, steps towards translational point of care devices can be difficult, because NA cleaves O-glycoside linkages rapidly.23 Indeed, most publications report assays with influenza viruses at low temperatures (4 0C) and/or use NA inhibitors to avoid issues with NA cleaving the natural O-sialosides.24-26 In a recent publication, Air et al. demonstrated that seasonal and pandemic H1N1 strains (or its recombinant NA) isolated from patients cleave natural occurring O-sialosides from the glycan microarray, albeit inefficiently, to expose the residual glycans at ambient temperatures.27 Some strains cleave more efficiently, other strains are less efficient. The same group demonstrated that parainfluenza strains cleave O-sialosides present on microarrays completely, presumably because the HA and NA are fused and presented as Hemagluttinin-Neuraminidase (HN) protein.28 The cleavage efficiency depends on the viral strain, assay conditions, the structure, density and presentation of the sialoside; in general, 2-3 linkages are more susceptible to cleavage than 2-6 linkages. These cleavage results are not entirely unexpected, because the two main antivirals, Zanamivir and


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Oseltamivir, block the action of viral NA which has been shown to cleave O-sialosides present on the residual host cells.29,30

Our long term goal is to use sialic acid analogues to capture influenza viruses in a point of care setting. As a first step, we designed and produced C-, S- and triazole linked sialosides that are insensitive to NA cleavage. These sialosides were printed onto glass slides to develop a small focused sialoside microarray. We demonstrate that these synthetic analogues can capture different influenza strains at ambient temperature and without NA inhibitors. The analytical limit of detection falls within the clinical limit of detection, 31 suggesting that these molecules could indeed be used in a clinical setting after further development. We also demonstrate the capture of eight different strains of influenza viruses to give rise to unique "fingerprint pattern of recognition" for each strain.

Results and Discussion:

1. Synthesis of glycans:

Our approach towards the construction of influenza NA insensitive glycans was to synthesize S-, Cand triazole linked sialoside linkages (Figure 1, SC 1-7) as reports have demonstrated that 2,3 linked Osialosides are cleaved within minutes.23 We choose to develop these sialosides as replacing the O linkage by C, S or triazole could inhibit the NA from cleaving the unnatural bond. Indeed, we and others have reported that non hydrolysable linked sialosides inhibit influenza viruses with IC50 values ranging from low micromolar to high nanomolar values.32,33 For comparison assays, we used commercial O-linked sialosides in some of the assays. The synthesis of one of the glycoconjugates, SC2, a 2,3 S-linked sialoside, which has a six carbon spacer, is shown in Scheme 1. We choose to use a six carbon spacer because our previous binding studies with recombinant influenza HA suggest that a six carbon spacer may be optimal.34 Our approach was to install the thiol group at the 3 position on the galactose acceptor and react it with β chloro-N-acetylneuraminic acid 8 via a SN2 type reaction to obtain the desired disaccharide as the alternate strategy of introducing the thiol group on the N-acetylneuraminic acid and displacing a good leaving group on the galactoside acceptor leads to side reactions, especially if the leaving group is on a secondary carbon.35,36 Briefly, the known β-galatoside derivative, 134 was converted to the appropriately protected guloside 6 through a number of protecting/deprotecting group conversions. First, the 3-OH group in 1 was protected with a paramethoxybenzyl group, which can be readily removed using ACS Paragon Plus Environment


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cerium ammonium nitrate in the presence of acid labile groups, to produce 2. Next, the 4,6 and the 2 hydroxyl groups were protected in a one pot reaction using benzaldehyde dimethyl acetal and benzoyl chloride respectively to yield 3 in appreciable yield. Conversion of the chloride to the azide was achieved using sodium azide in a near quantitative yield. Selective removal of the paramethoxybenzyl group was followed by inversion of the 3 hydroxyl group from the equatorial position to the axial guloside derivative 6 using triflic anhydride to active the hydroxyl group and invert it using tetrabutyl ammonium nitrite at a slightly elevated temperature. We found that this reaction is not straightforward; specifically, the tetrabutylammonium nitrate has to be completely oxygen and moisture free, else the compound reverts to the galactoside derivative 5. Activation of the axial hydroxyl using triflic anhydride, followed by SN2 type substitution by potassium thioacetate resulted in the desired installation of the thiol group on the 3 position of the galactoside derivative 7. Selective removal of the acetate in 7 using hydrazine acetate was followed by reaction with the N-acetylneuraminic acid chloride derivative 8 in the presence of a commercial cryptand to yield the 2,3 S-linked derivative 9, in reasonable yield. The product was confirmed by NMR and mass spectroscopic analysis; the hydrogen and the carbon at the 3 position of galactose resonated at 4.60 and 101.0 ppm, respectively. The anomeric carbon of sialic acid resonated at 96.6 ppm and the equatorial H-3’e appeared at 2.61 ppm (dd, 1H, J3eq,3ax = 12.8 Hz, J3eq, 4 = 4.6 Hz) indicating α sialoside. After removal of the benzylidene group, deprotection of the acetate, benzoyl and ester functional groups in 10 under basic conditions, followed by hydrogenation of the azide yielded the desired 2,3 S-sialoside glycoconjugate SC2 with a pendant amine for conjugation to activated carboxyl acid surfaces. Details of the synthesis of the glycoconjugates, SC1-SC7 are given in supporting information. SC8-SC9, were obtained from a commercial source.

2. Lectin and HA Binding studies:

With the sialosides in hand, we tried to demonstrate the binding ability using standard ELISA plates as described by our group previously.34 Unfortunately, despite several efforts that included a variety of blocking methods and using ELISA plates from different manufacturers, we found that the non-specific binding were substantial and we were consuming significant material for a single assay. For instance, a minimum of 30 µg of compounds were required in a single well, based on the minimum coating concentration of 750 µM volume of 100 µl, and 90 µg for one target protein for performing the assay in triplicate. Therefore, we switched to glass slides prefabricated with PEG and activated carboxyl groups. We found that this approach decreased non-specific binding significantly. Thus, seven amine terminated


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S-, C- and triazole linked sialosides and two commercial 2,3 and 2,6 O linked sialosides as controls were printed onto commercial NHS ester functionalized glass slide at different concentrations. Details are provided in the Supporting information. We found that a glycan concentration of 100 µM provides an excellent signal to noise ratio and we used that concentration for all our studies. While our pilot studies with printing simple sugars such as α-mannoside and probing them with fluorescent Con A were successful, (data not shown) we were very particular in ensuring that all sialosides were printed on the surface. After the printing was complete, we tested the binding of sialosides with lectins to compare and contrast the binding preferences of these unnatural sialosides to published studies. The first lectin tested was Wheat germ agglutinin (WGA), which has been demonstrated to exhibit binding to Nacetylglucosamine and sialic acid in previous studies (Figure 2A).

37,38

A previous NMR binding study

showed that WGA preferred the α-2, 3 over α-2, 6 linkage when bound to sialic acid (KD = 0.73mM with α-2, 3-sialyllactose; KD = 5.3mM with α-2, 6-sialyllactose).37 In accordance with the published NMR binding study, WGA showed moderate binding to SC8 (α-O-2, 3-sialyllactoside) while very weak binding to SC9 (α-O-2, 6-sialyllactoside). A similar binding preference was also seen with the thiosialoside pair: WGA showed highest binding to SC2 (α-S-2, 3-sialylgalactoside) among all ten sialosides, whereas only moderate binding to SC3 (α-S-2, 6-sialylgalactoside). These results indicated that the replacement of Owith S- did not change the conformation of glycans to the extent that could alter the WGA binding specificity for these glycans. In contrast, this binding preference of WGA was not observed when bound to the synthetic triazole based sialosides. In fact, both SC4 and SC5 showed only weak to moderate binding to WGA, which might be resulted from the conformational change induced by the insertion of a rigid ring to replace the glycoside bond. Moderate to strong binding were observed with the two monosaccharides SC1 and SC6 and C-linked trisaccharide SC7. We also obtained the binding profile of three other lectins, including Sambucus nigra agglutinin (SNA) (Figure 2B), Maackia amurensis leukoagglutinin (MAL-I) and Maackia amurensis hemagglutinin (MAL-II) (Figure 2C, D). SNA was first reported to bind to D-galactose and N-acetylgalatosamine, and capable of recognizing terminal sialic acid, preferably with α-2,6-linkage.39 We observed the highest binding was with SC9 (α-O-2, 6-sialyllactoside) while significant binding to SC8 (α-O-2, 3-sialyllactoside) was also seen. The binding of SC3 (α-S-2, 6sialylgalactoside) was also strong, compared to its α-S-2, 3-linked analogue SC2. Similar to WGA, SNA also showed moderate binding to the triazole monosaccharides SC1, but did not bind well with triazole linked disaccharides and C-linked sialosides. MAL-I and MAL-II showed very weak binding to S- and triazole linked sialosides while weak to moderate binding to O- and C- linked sialosides. To summarize this section, these experiments using multiple lectins demonstrate that the sialosides were printed on the surface. Another observation is that the binding preferences of the C-, S- and triazole linked sialosides are ACS Paragon Plus Environment


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similar, but not identical to that of reported studies that use O- linked sialosides, a result that is not entirely unexpected, because these are "unnatural" sialosides and are subject to slightly different conformations. Specifically, sulphur is bigger than oxygen, the C-linked sialosides have one atom less than the O-linked sialosides and the triazole ring is a five membered ring; all these differences can contribute to differences in binding affinities. Also, "glycans on a solid surface" can exhibit different binding preferences when compared to glycans in solution40 and glycan cross platform comparisons are highly dependent on glycan structure, density, orientation and assay conditions.34,41

3. Stability studies using NA and intact viruses:

We compared the stability of sialosides with different linkages towards NA to evaluate the feasibility of using the microarray to detect virus at ambient temperature without adding NA inhibitors. We treated the slide with H1N1 NA at rt for 2h and then incubated it with either fluorescent labeled WGA or HA (A/Brisbane/59/2007). For comparison, we also performed the assay without NA cleavage. As seen in Figure 3A, WGA binds to all the expected glycans without addition of NA. However, after subjecting it to NA and reporting with WGA results in only a minor signal loss of all glycans, including SC8. We believe we observe a signal for SC8 in Figure 3A because WGA binds to the residual galactose on the microarray after the NA cleaves the terminal O-linked sialic acid. Since these results were inconclusive, we used a different reporter, namely HA (A/Brisbane/59/2007) to demonstrate that soluble NA does cleave the O-linked sialosides. As seen Figure 3B, when this HA is used without pre-incubation with NA, it binds to SC8 and SC9 and not to any of the other sialosides. After subjecting the slide to H1N1 NA, and reporting with the same HA, we observe that the signals due to SC9 remain, but the signal of SC8, the α 2,3 O-linked sialoside, is completely lost, which is a clear indication that the viral NA cleaves α 2,3 Olinkages very efficiently when present on a solid surface. The signal intensity of the α2,6 O-linked sialoside, SC9 decreases slightly, but is not completely lost as seen with α2,3 O-linked sialoside SC8. Increasing the NA incubation time doesn’t produce a different result, we observed similar fluorescent readings (data not shown), indicating that the α 2,3 O-linked sialoside is cleaved more rapidly than α2,6 O-linked sialoside. A recent NA substrate specificity study also shows a similar result. 27 In that study, Li et al. screened the substrate specificity of 37 NAs (N1-N9), including both human and avian viral NA using a colorimetric assay with 1h of hydrolysis.42 They found that α-2, 3-linked sialosides were hydrolyzed more efficient than α-2, 6-linked sialosides in solution toward all NAs. The longer hydrolysis


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time of 2,6 O-linked sialosides could potentially be exploited in the development of multivalent inhibitors of certain influenza types, which prefer 2,6 O-linkages.

Next, we wanted to demonstrate the stability of the synthetic sialosides toward intact influenza viruses because intact viruses are quite different from recombinant NAs. Our initial approach was to incubate the glycan microarray with the virus, wash the slide to remove unbound virus and test it with a suitable reporter specific for the virus. We anticipated that the unnatural sialosides will show similar binding with or without pre-incubation of the virus, whereas the O-sialosides, especially SC8, the 2,3 linked Osialoside, will show no binding after virus incubation because the virus will cleave the O-sialosides and will be removed in the subsequent washing step. Unfortunately, we observed binding to SC8, presumably because the cleavage is not efficient. Air et al. have seen similar results and have demonstrated that the cleavage by intact viruses is not as efficient as soluble NA and is dependent on the strain, structure of the glycan and assay conditions.27 The NA in intact viruses is present as a tetramer and may not be able to access sialosides with a shorter linker present on a solid surface. Since the cleavage is inefficient, we designed experiments to detect the remaining residue after hydrolysis. For direct comparison, we included amine functionalized N-acetylgalactoamine as a positive control on the printed slide as well as SC7 (Clinkage) and SC8 (O-linkage). We used only three glycans for this study. First, we tested the binding of the sialosides with no virus incubation, but with biotinylated RCA120 (Ricinus Communis Agglutinin120), followed by labeled streptavidin. We observe no binding of the RCA120 with SC7 or SC8 but good binding with the positive control, N-acetylgalactosamine as RCA120 has been demonstrated to bind to galactoside and N-acetylgalactosamine.43 Next, we incubated the printed slide with influenza (H1N1: A/Solomon Island/3/06 or H3N2 A/Hongkong/8/68) at pH 5.5, 37oC for 4 h to ensure complete cleavage. (At ambient temperature and pH 7.4, cleavage of SC8 occurs, but at a slower rate.) After extensive washing, we incubated the virus-treated slide with biotinylated RCA120 followed by rhodamine labeled streptavidin (Figure 3C). When the H1N1 (A/Solomon Island/3/06) strain is used, SC8, shows significant binding, similar to the positive control. This is because, after removal of the terminal O linked sialic acid in SC8, the remaining glycan has a terminal galactoside, which can be readily detected by RCA120. In contrast, SC7, the synthetic glycan with the unnatural C-sialoside, shows minimal binding as the sialoside is not cleaved. A similar result is observed when the H3N2 A/Hongkong/8/68 strain is used; the binding signal of SC8 to RCA120 after treated with H3N2 virus is weaker, indicating that the hydrolysis is not complete. These experiments demonstrate that O-sialosides are cleaved by viral NA and or intact virus,



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however, the cleaving efficiency are dependent on the strain, the structure of the sialosides (sialosides with longer spacers are cleaved more efficiently 27, the density and assay conditions.

4. Virus binding and limit of detection. Next, we wanted to test the capturing ability of influenza viruses using these unnatural sialosides. Without premixing with NA inhibitors, we introduced BLP (betapropiolactone) inactivated influenza virus H1N1 A/Puerto Rico/8/34 (from BEI resources) at different concentrations to the printed slides, incubated at rt for 1 h, followed by incubation of primary and fluorescently labeled rhodamine secondary antibody. This was followed by scanning the slides using a Genepix scanner at 635 nm. We found nonspecific binding is negligent to the control PEG ligand. We observe differential binding to the different glycans, the triazole linked sialosides seems to bind with a higher affinity than the C- or S- linked sialosides (Figure 4A). The images then were analyzed by GenePix® Pro 6.0 software, with which the fluorescent intensity of each spot was measured, the mean values as well as standard deviations were calculated from spots of 3 replicates. Details can be found in the supporting information. We determined the binding pattern of a different influenza strain A/Brisbane/59/2007 (BEI resources). As seen in Figure 4C, the binding pattern is markedly different for this strain. Finally, we wanted to find the limit of detection by using different concentrations of the virus (Figure 4B). We choose SC5 and we were excited to observe that we can detect the virus at a concentration as low as 35 CEID50 (Chicken Embryo Infectious Dose).44 Next, we expanded the scope of this assay to include other strains. Here, we used active viruses cultured from MDCK cell lines instead of the BLP inactivated viruses originated from chicken embryo to eliminate the possible interference of BLP in the binding pattern. To this end, we grew eight strains, six H1N1 and two H3N2 strains, and quantified the each strain titer using plaque forming assay.33 The titer of these strains was adjusted to 3 X 104 PFU/ml (Plaque Forming Units) before incubation with the printed slide (Figure 5A-H). We observed a unique binding pattern for each strain, which reinforces our previous statement that every virus strain exhibits differences in binding to different sialoside structures glycans leading to a “fingerprint” pattern of recognition. These assays were performed in triplicate on three different days and we obtained fairly reproducible results. Figure 6 is a visual representation of Figure 5 to show that a fingerprint pattern of recognition can be obtained for different viral strains, the circles


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represents the fluorescence intensity, a bigger circle indicates higher fluorescence intensity and a smaller circle represents a lower intensity.

Conclusions:

In this study, we report the synthesis and fabrication of a focused sialoside microarray comprising of C-, S- and triazole linkages. These compounds could be used to probe the binding affinities of other sialic acid binding proteins and hydrolyses. We obtained the binding profiles of multiple lectins using this focused microarray and there are some binding similarities with O-linked sialosides. We demonstrated the stability of these sialosides towards viral NA and intact viruses and demonstrated the ability of these sialosides to capture viral particles, presumably through binding to both HA and NA, (as recent reports have shown that NAs can also bind to glycan microarrays(45,

46)

at ambient temperature and without

addition of inhibitors. Unique fingerprint patterns can be seen for a specific strain. In previous studies, we have demonstrated that glycans are highly robust(16) and therefore these glycans could be potentially translated to a rapid diagnostic lateral flow assay. These glycans could also be incorporated into existing or emerging biosensor platforms. The slides, once printed could be stored in a desiccator for days without any loss in capturing efficiency. These experiments demonstrate the robustness of the sialosides. For example, these small molecules could find potential use in nanobiosensors where the application requirement is minimal change in the nanoparticle size after conjugation to the recognition element. Unlike antibodies or aptamers, nanoparticle-glycan conjugates do not perturb the overall size of the nanoparticle.(47) Glycans can also be used in PCR based technologies in the sample preparation step; indeed, glycans have been attached to magnetic beads or nanoparticles to capture analytes.(16, 48) Finally, since a plethora of glycans can be synthesized with new advances in synthetic methodologies, a ‘fingerprint’ pattern of recognition can be obtained for every strain.

(49-52)

We are currently involved in

expanding the scope of this work by synthesizing a larger library of robust sialosides and developing a ‘fingerprint pattern of recognition” for more influenza virus strains.

Associated content Supporting Information ACS Paragon Plus Environment


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Details of the synthesis and the assays is available free of charge via the Internet at http://pubs.acs.org. Author information Corresponding Author [email protected]

Acknowledgment We are grateful for NIH-NIAID (R01-AI089450) for funding. We also thank BEI Resources, NIAID, NIH for the inactivated virus reagents and the antibodies. We also thank Dr. Binghe Wang, GSU for use of the microarray printing facilities.

References: (1) (2)

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(10)

von Itzstein, M. (2007) The war against influenza: discovery and development of sialidase inhibitors. Nat Rev Drug Discov 6, 967-74. Fiore, A. E., Shay, D. K., Broder, K., Iskander, J. K., Uyeki, T. M., Mootrey, G., Bresee, J. S., and Cox, N. S. (2008) Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 57, 1-60. Fiore, A. E., Shay, D. K., Broder, K., Iskander, J. K., Uyeki, T. M., Mootrey, G., Bresee, J. S., and Cox, N. S. (2008) Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP), 2008. MMWR Recomm Rep 57, 1-60. Aoki, F. Y., Macleod, M. D., Paggiaro, P., Carewicz, O., El Sawy, A., Wat, C., Griffiths, M., Waalberg, E., Ward, P., and Group, I. S. (2003) Early administration of oral oseltamivir increases the benefits of influenza treatment. J Antimicrob Chemother 51, 123-9. O'Dowd, A. (2009) WHO raises level of alert on flu pandemic. British Medical Journal 338, b1777. New influenza A (H1N1) virus: WHO guidance on public health measures, J. (2009) New influenza A (H1N1) virus: WHO guidance on public health measures, 11 June 2009. Wkly Epidemiol Rec 84, 261-4. Sills, M. R., Hall, M., Simon, H. K., Fieldston, E. S., Walter, N., Levin, J. E., Brogan, T. V., Hain, P. D., Goodman, D. M., Fritch-Levens, D. D., et al. Resource burden at children's hospitals experiencing surge volumes during the spring 2009 H1N1 influenza pandemic. Acad Emerg Med 18, 158-66. (2009), World Health Organization. Pandemic (H1N1) 2009 - update 75. Available from: http://www.who.int/csr/don/2009_11_20a/en/index.html. Rodriguez, W. J., Schwartz, H. R., and Thorne, M. M. (2002) Evaluation of diagnostic tests for influenza in a pediatric practice. The Pediatric Infectious Disease Journal 21, 193-196. Balish, A., Warnes, C. M., Wu, K., Barnes, N., Emery, S., Berman, L., Shu, B., Lindstrom, S., Xu, X., Uyeki, T., et al. (2009) Evaluation of rapid influenza diagnostic tests for


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(11)

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Figure 1. Structures of sialosides used to construct glycan microarray.


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Figure 2. Lectin binding profile on sialoside microarray. (A) Rhodamine labeled WGA (100 µg/ml). (B) Biotinylated SNA (100 µg/ml), detected by rhodamine labeled streptavidin (0.4 µg/ml). (C) Biotinylated MAL-I (100 µg/ml), detected by rhodamine labeled streptavidin (0.4 µg/ml). (D) Biotinylated MAL-II (100 µg/ml), detected by rhodamine labeled streptavidin (0.4 µg/ml).



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Figure 3. Stability comparison of sialosides with S-, N-, C- and O-linkages towards recombinant NA and intact influenza viruses. (A) Rhodamine labeled WAG (100µg/ml) (Green); First NA (N1, 3.3 U,


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1U=1,000 pmol/min), then rhodamine labeled WAG (100µg/ml) (Red). (B) H1 (100 µg/ml) premixed with anti-H1 rabbit polyclonal antibody and Alexa Fluor®633 labeled goat anti rabbit secondary antibody (4:2:1)(Green); First NA (N1, 3.3 U, 1U=1,000 pmol/min), then H1-antibody complex (Red). (C) Printed slide was first incubated with influenza virus (A/Solomon Island/3/2006 2X103 PFU or A/Hongkong/8/68 8X102 PFU) at 37oC, pH 5.5 for 4h, then biotinylated RCA120 (20 µg/ml) and finally rhodamine labeled streptavidin (0.4 µg/ml). Error bars represent mean ±standard error of three independent experiments.



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Figure 4. Binding studies of β-propiolactone inactivated influenza virus from chicken embryo. (A) Finger print binding pattern of virus A/Puerto Rico/8/34 of 3.5 X 103 CEID50. (B) SC5 binds to virus of different titers. (C) Finger print binding pattern of influenza A/Brisbane/59/2007 of 3.1 X103 CEID50. The binding


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virus was detected by ferret hyperimmune sera to influenza A/Brisbane/59/2007 and rhodamine labeled goat anti-ferret IgG (0.4 µg/ml).



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Figure 5. Binding profiles of different strains of MDCK cell adapted influenza viruses. The titers of all virus strains were adjusted to 3 X 104 PFU before incubation. Error bars represent mean ±standard error of three independent experiments.


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Figure 6. “Fingerprint” of different strains of influenza viruses. The area of circle corresponds to the average fluorescence intensity produced by the binding of virus to each glycan. A: A/Puerto Rico/8/1934; B: A/Brisbane/59/2007; C: A/New Caledonia/20/1999; D: A/California/07/2009; E: A/Hong Kong/8/1968; F: A/Solomon Island/03/2006; G: A/Aichi/2/1968; H: A/Wisconsin/67/2005.



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Ph OH OH

OH OH

a

O O

HO

O

Cl 6

OH

O

PMBO OH

2

1

O

b

O O

PMBO

Cl 6

O OBz

Cl 6

3 c

O

O

O

e O O OBz

OH

Ph

Ph

Ph

N3

O

O O

HO OBz

6

6

O

d

O O O OBz

PMBO

N3 6

N3 6

4

5

f AcO

Ph O

AcHN AcO

O O

AcS

7

OAc OAc

O OBz

N3 6

Cl O

OAc OAc

AcO COOMe

AcHN AcO

8

COOMe O

O S

g

Ph O O O OBz

9

N3 6

h HO

OH OH

COOH OH OH O

AcHN HO

i-j O

S

O OH

NH2 6

AcO

OAc OAc O

AcHN AcO

COOMe OH OH O S O OBz

N3 6

10

SC2

Scheme 1. Synthesis of SC2. Reagents and conditions: (a) (i) Bu2SnO, MeOH, reflux, 8h, (ii) PMBCl, Bu4NBr, toluene, 70 0C, 8h, 80%; (b) (i) BDA, p-TSA, MeCN, rt, 5 min, (ii) BzCl, py, rt, 8h, 70%; (c) NaN3, DMF, 60 0C, 95%; (d) CAN, MeCN/H2O (16:1), rt, 30 min, 68%; (e) (i) Tf2O, py, DCM, -25 0C, 25 min, (ii) Bu4NNO2, MeCN, 500C, overnight, 70%; (f) (i) Tf2O, py, DCM, -25 0C to rt, 90 min, (ii) KSAc, DMF, 60 0C, overnight, 65%; (g) (i) H2NNH2.AcOH, DMF, rt, 2h, (ii) THF, DMF, Kryptofix 21, rt; α only, no β anomer was observed. (h) 80% AcOH(aq), 450C, overnight, 32% over 3 steps; (i) (i) 0.5M NaOMe, MeOH, rt, overnight, (ii) 0.25M NaOH(aq), 8h; (j) H2, Pd(OH)2/C, EtOH, 48h, 70%.


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Scheme 2. Illustration of our stepwise approach to test the sialoside microarray for influenza virus binding.



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Table of contents figure:


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Neuraminidase Resistant Sialosides for the ... - ACS Publications

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