Influenza neuraminidase active site proximity assay (NASPA) for rapid profiling of inhibitory antibodies and antigenic drift

Abstract

An influenza virus neuraminidase (NA) active site proximity assay (NASPA) for detecting and / or quantifying NAI antibodies in biological samples is described. NASPA can also be used, for example, to evaluate NA antigenicity and profile anti-NA antibody responses in humans and non-human animals. NASPA is based on the ability to detect the binding of a large NA active site inhibitor in the presence of anti-NA antibodies with a small reporter substrate. The assay is performed by sequentially adding test sera to a defined amount of NA (or influenza virus containing NA), followed by addition of the NA active site inhibitor and then the small substrate for measuring NA activity. In view of the flexibility of the modular approach and the ease of automation, the disclosed methods can be used to efficiently collect NA antigenic data and profile complex anti-NA antibody responses.

Description

[0001]9531-111023-03INFLUENZA NEURAMINIDASE ACTIVE SITE PROXIMITY ASSAY (NASPA) FOR RAPID PROFILING OF INHIBITORY ANTIBODIES AND ANTIGENIC DRIFT CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.63 / 596,874, filed November 7, 2023, which is herein incorporated by reference in its entirety. FIELD This disclosure concerns improved methods for detecting influenza virus neuraminidase (NA) inhibitory antibodies in biological samples, the results of which can be used to evaluate influenza virus antigenic drift, guide strain selection for influenza virus vaccines, and to measure antibody responses to influenza virus vaccines. INCORPORATION OF ELECTRONIC SEQUENCE LISTING The electronic sequence listing, submitted herewith as an XML file named 9531-111023-03.xml (1,904 bytes), created on October 31, 2024, is herein incorporated by reference in its entirety. BACKGROUND Influenza virus infections are largely orchestrated by the viral envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA). The more abundant HA glycoprotein facilitates cell entry by binding to host cell surface receptors containing terminal sialic acid residues and triggering fusion of the viral and host endosomal membrane following endocytosis (Kosik and Yewdell, Viruses 11(4):346, 2019). In contrast, NA is a Ca2+-dependent enzyme that promotes the movement of the virus by removing local sialic acid residues that enable persistent HA binding (Wang et al., Nat Microbiol 4:2565-2577, 2019; Dou et al., Front Immunol 9:1581, 2018). NA catalyzes the removal by hydrolyzing the sialyl glycosidic linkage that is connected to the underlying sugar molecules, which are often galactosides (Gottschalk, Biochim Biophys Acta 23:645-646, 1957; Anonymous, In Varki et al. (ed), Essentials of Glycobiology, 4th ed, doi:10.1101 / 9781621824213, Cold Spring Harbor (NY), 2022). This enzymatic function of NA has been one of the primary targets for influenza antiviral (e.g. oseltamivir, zanamivir and peramivir) development (Batool et al., Microorganisms 11(1):183, 2023), whereas influenza vaccines have historically focused on eliciting antibodies that inhibit the receptor binding function of HA (Wei et al., Nat Rev Drug Discov 19:239-252, 2020). Recently many different vaccine strategies have been pursued with the goal of improving the breadth and efficacy of current seasonal vaccines (Morens et al., Cell Host Microbe 31:146-157, 2023; CDC: cdc.gov / flu / vaccines-work / past-seasons-estimates, 2022; Wu and Ellebedy, Trends Immunol 45:11- 19, 2024; Rak et al., Vaccines (Basel) 11(12):1747, 2023; He et al., Vaccines (Basel) 11(10):1573, 2023). One of these involves developing approaches for eliciting antibody responses against NA in addition to HA9531-111023-03(Strohmeier et al., mBio 12:e0224121, 2021; Gao et al., PLoS Pathog 17:e1009171, 2021; McMahon et al., Proc Natl Acad Sci USA 119:e2206333119, 2022; Martinez et al., Vaccine 41:4302-4312, 2023; Cheung et al., NPJ Vaccines 8:150, 2023; Kang et al., iScience 27:110038, 2024; Sia et al., Cell Rep Med 5:101433, 2024; Raha et al., Antiviral Res 225:105877, 2024). This strategy is supported by numerous studies that show NA antigens from a variety of sources can elicit anti-NA antibodies which are capable of reducing the severity of influenza infections in both humans (Murphy et al., N Engl J Med 286:1329-1332, 1972; Couch et al., J Infect Dis 129:411-420, 1974) and animal models (Strohmeier et al., mBio 12:e0224121, 2021; Martinez et al., Vaccine 41:4302-4312, 2023; Kang et al., iScience 27:110038, 2024; Rott et al., J Gen Virol 22:35-41, 1974; Webster et al., Virology 164:230-237, 1988; Wohlbold et al., MBio 6:e02556, 2015; Skarlupka et al., J Virol 95:e0075921, 2021). Furthermore, several clinical studies have indicated that NA activity inhibitory (NAI) antibody responses may correlate with protection in humans (Ng et al., Nat Med 25:962-967, 2019; Memoli et al., mBio 7:e00417-16, 2016; Monto et al., J Infect Dis 212:1191-9, 2015), although it is not clear if this correlation applies to steric or enzymatic NAI antibodies, or both. In addition, HA and NA antigenic drift has been shown to be discordant (Sandbulte et al., Proc Natl Acad Sci USA 108:20748-53, 2011). Together, these findings imply that anti-NA antibody responses may help to increase the breadth of protection provided by influenza vaccines (Wu and Ellebedy, Trends Immunol 45:11-19, 2024; Eichelberger and Monto et al., J Infect Dis 219:S75-S80, 2019; Krammer et al., mBio 9(2):e02332, 2018). However, a strain selection framework is currently only in place for HA antigens, making it difficult to identify appropriate NAs for new vaccine strategies. Vaccine strains for HA are selected based on a combination of genetic and antigenic data from circulating strains along with their prevalence and location (Russell et al., Vaccine 26(Suppl 4):D31-D34, 2008). During this selection process, NA genetic data is also often obtained, but little to no antigenic data is collected. HA antigenicity is commonly assessed using ferret antisera in hemagglutination inhibition (HI) assays or high-content imaging-based neutralization tests (HINT). A similar approach has been taken for NA that uses ferret antisera to monitor changes in NAI antibody titers (Gao et al., MBio 10(2):e00307, 2019; Gao et al., NPJ Vaccines 7:79, 2022; Catani et al., Elife 12:RP90782, 2024). However, these NAI titers have generally been obtained from a fetuin-based enzyme-linked lectin assay (ELLA) that is susceptible to interference from antibodies against HA (Kosik et al., Virology 500:178-183, 2017; Kosik et al., J Exp Med 216:304-316, 2019; Chen et al., J Virol 93(4):e01526-18, 2019). To minimize this issue, several ELLA modifications have been implemented, including using test viruses with HA subtypes that should not be recognized by serum antibodies (Couzens et al., J Virol Methods 210:7-14, 2014; Gao et al., J Vis Exp doi:10.3791 / 54573, 2016), detergent treated viruses (Cate et al., Vaccine 28:2076-2079, 2010), and recombinant NA (Fritz et al., J Infect Dis 205:28-34, 2012). However, these approaches further increase the time, labor and complexity, making it expensive and difficult to implement ELLA for each season. The lack of approaches that can be easily implemented to identify suitable vaccine strains for NA antigens remains a major barrier for the development of new vaccines containing NA.9531-111023-03SUMMARY Disclosed herein is the development of an NA active site proximity assay (NASPA) for measuring NAI antibodies and its application for evaluating NA antigenicity and profiling anti-NA antibody responses in humans (FIG.22). NASPA is based on the ability to detect the binding of a large ‘bulky’ NA active site inhibitor in the presence of anti-NA antibodies with a small reporter substrate, such as 4-methylumbelliferyl- N-acetyl-α-D-neuraminic acid (MUNANA) (Potier et al., Anal Biochem 94:287-96, 1979). Consequently, the assay can be performed by sequentially adding test sera to a set amount of NA, followed by the large bulky NA active site inhibitor and then the small substrate for measuring NA activity. The flexibility of the modular approach combined with the ease of automation, indicate NASPA can be used to efficiently collect NA antigenic data and profile complex anti-NA antibody responses in humans. Provided herein is a method for detecting the presence or absence of influenza virus neuraminidase (NA) inhibitory (NAI) antibodies in a biological sample. An NAI inhibitory antibody is an antibody that inhibits the enzymatic function of NA. In some aspects, the method includes incubating the biological sample in a reaction vessel that includes recombinant NA protein or includes influenza virus containing NA protein, under conditions sufficient for any NAI antibodies present in the biological sample to bind the recombinant NA protein or the NA protein of the influenza virus; adding a NA inhibitor (NAi) to the reaction vessel, wherein the NAi binds to the NA active site in the absence of NAI antibodies in the biological sample; adding a NA reporter substrate to the reaction vessel, wherein the NA reporter substrate includes a reporter molecule and sialic acid or a sialic acid derivative, and the reporter molecule is detectable upon cleavage of the sialic acid; and detecting the presence or absence of detectable reporter molecule. An increase in the detectable reporter molecule compared to a control sample indicates the biological sample contains NAI antibodies. No change in the detectable reporter molecule compared to a control sample indicates the biological sample does not contain NAI antibodies. A decrease in the detectable reporter molecule compared to a control sample indicates the biological sample contains NAI antibodies that bind to the NA active site. In some aspects, the method further includes serially diluting the biological sample prior to adding the biological sample to the reaction vessel. In some aspects, the method further includes diluting the recombinant NA protein or the influenza virus containing NA protein prior to adding to the reaction vessel. In some aspects, the method further includes calculating the NAI antibody titer in the biological sample. In some aspects of the method, the reporter molecule is a fluorescent molecule (such as 4-methyl umbelliferone), a colorimetric molecule, or a chemiluminescent molecule. In some examples, the reporter substrate is 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid (MUNANA). In some aspects of the method, the NAi is a monoclonal antibody. In other aspects, the NAi is a synthetic molecule, or a synthetic molecule and a protein.9531-111023-03The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS FIGS.1A-1F: Large NA substrates are needed to detect steric NA inhibitory (NAI) antibodies. (FIGS.1A and 1B) Diagrams showing the detection of NA catalytic activity by the small monovalent reporter substrate MUNANA (FIG.1A) and an ELLA analysis of multivalent glycoproteins (FIG.1B). MUNANA cleavage is monitored by fluorescent detection of the released umbelliferone and cleavage of the sialylated glycans on the glycoprotein are measured by binding of peanut agglutinin to the exposed terminal galactose residue. (FIGS.1C and 1D) NA activity inhibition by ferret and mouse antisera was measured with a H1N1 / CA09 virus by ELLA using the glycoprotein fetuin (FIG.1C) and MUNANA (FIG.1D). Ferret and mouse antisera were generated by intranasal infection with a reassortant virus (H6N1 / CA09) carrying a mismatched HA (H6) and a matching NA (N1 / CA09). (FIG.1E) H1N1 / CA09 virus was mixed with serial dilutions of H1 MAbs that inhibit the receptor binding function of its HA (Table 1) and the NA activity was measured by ELLA. Purified H1 MAbs were diluted as indicated from an initial concentration of ~1 mg / ml. (FIG.1F) NA activity inhibition by ferret and mouse antisera was measured by ELLA with the same virus (H6N1 / CA09) that was used to generate the antisera. FIGS.2A-2E: NASPA can measure steric NAI antibodies. (FIG.2A) Schematic of NASPA showing the expected outcomes of the three steps in the presence or absence of steric NAI antibodies. Following (Step 1) a sera titration with a fixed amount of NA, (step 2) a large ‘bulky’ NA inhibitor (NAi) is added, and (Step 3) the ability of steric NAI antibodies to block the large NA inhibitor is detected by MUNANA. (FIG.2B) NA titration was performed by serially diluting H1N1 / CA09 virus in the absence or presence of TX100 (0.025% final concentration) and measuring the NA activity for 10 minutes after MUNANA addition. (FIG.2C) Inhibition curves are displayed for the NAi-MAb (FNI-9) with untreated and TX100-treated H1N1 / CA09 virus. The NAi-MAb was incubated 3 hours prior to measuring the activity by MUNANA. (FIGS.2D and 2E) NA activity detected by NASPA that was performed using untreated and TX100-treated H1N1 / CA09 virus with the indicated ferret antisera (FIG.2D) or HA MAbs (FIG.2E) that inhibit the receptor binding function of the HA in the H1N1 / CA09 virus. Assays were performed in duplicate using NAi-MAb amounts approximating the IC80 and are displayed as the mean ± the standard deviation (SD). NA activities were read for 10 minutes after MUNANA addition. Grey regions indicate the mean (dashed line) ± 3 SDs (dotted line) that were determined using negative control ferret sera (FIG.2D) or multiple wells containing PBS (FIG.2E). FIGS.3A-3D: NASPA vaccine strain testing with HA mismatched and matched ferret antisera. (FIG.3A) Schematic of the NA and HA antigens in the type A (H1N1 and H3N2) and type B (HBNB) vaccine strains and the corresponding H6 reassortant viruses that were analyzed by NASPA. (FIG.3B) Graph displaying changes in the NAi-MAb (FNI-9) IC50 with respect to the incubation time with the indicated vaccine viruses in the absence and presence of TX100. Results are displayed as the mean ± SD9531-111023-03from two independent experiments. (FIG.3C) NASPA results for the three vaccine strains in the absence (upper panels) or presence (lower panels) of TX100 and the indicated ferret antisera that were generated against the vaccine strain NAs using H6 reassortant viruses. Negative ferret serum was included as a control. (FIG.3D) NASPA results for the three H6 reassortant viruses used to generate the vaccine strain NA ferret antisera. NASPA was performed in the absence (upper panels) or presence (lower panels) of TX100 with the indicated ferret antisera that matched the HAs in all the test viruses. Negative ferret serum was included as a control. All NASPA assays were performed in duplicate using NAi-MAb concentrations corresponding to the IC80and are shown as the mean ± SD of the NA activities measured for 10 minutes after MUNANA addition. Grey regions indicate the mean (dashed line) ± 3 SDs (dotted line) from the negative control sera. FIGS.4A-4D: NASPA and ELLA results from MAbs against N1, N2 and type B NA. (FIG. 4A) Correlation plot of ELLA and NASPA titers obtained using panels of MAbs against NAs from the vaccines strains A / Victoria / 19 (H1N1), A / Darwin / 21 (H3N2) and B / Austria / 21. Pearson’s correlation coefficient (R2) for the linear regression (dotted line) of the 27 MAbs is shown. Negative (enzymatic inhibitory) NASPA titers were treated as positive for the correlation and values below the limit of detection were assigned 0. (FIGS.4B-4D) NASPA data obtained with TX100-treated B / Austria / 21 (FIG.4B), A / Victoria / 19 (H1N1) (FIG.4C), and A / Darwin / 21 (FIG.4D) vaccine strains and the indicated MAbs. Duplicate hybridoma clones were analyzed and a single representative of each is shown. Analysis of the type B and H1N1 strains used the NAI MAb FNI-9 at an ~IC80, whereas the modified NAi-MAb (FNI-9 R27D) was used at an ~IC80 for the H3N2 analysis. Endpoint NA activities (RFU) were measured after a 10-minute incubation with MUNANA. Grey regions indicate the mean (dashed line) ± 3 SDs (dotted line) from control reactions containing serially diluted hybridoma medium. FIGS.5A-5B: Antigenic analysis of NAs from recent H1N1 and type B vaccine strains by NASPA. (FIG.5A) NASPA results for each TX100-treated H1N1 vaccine strain with the indicated NA ferret antisera are shown. The H1N1 strains A / Brisbane / 2007 (BR07), A / California / 2009 (CA09), A / Michigan / 2015 (MI15), A / Brisbane / 2018 (BR18) and A / Victoria / 2019 (Vic19) were analyzed. (FIG.5B) NASPA results for each TX100-treated type B vaccine strain with the indicated NA ferret antisera are displayed. The B Yamagata lineage strain B / Phuket / 2013 (Phu13) and the B Victoria lineage strains B / Brisbane / 2008 (BR08), B / Colorado / 2017 (CO17), B / Washington / 2019 (WA19) and B / Austria / 2021 (Austria21) were tested. All analyses were performed in duplicate using NAi-MAb concentrations corresponding to the IC80 and are shown as the mean ± SD of the NA activities measured for 10 minutes after MUNANA addition. Grey regions indicate the mean (dashed line) ± 3 SDs (dotted line) of the control wells. FIGS.6A-6D: NASPA analysis of human NA responses pre- and post-challenge with H1N1 / CA09. (FIG.6A) Endpoint titers determined with NASPA and a MUNANA activity assay are displayed for the indicated patients pre-challenge (upper panel) and 2 months post-challenge (lower panel) along with the ELLA titers previously reported for these samples (Memoli et al., mBio 7:e00417-16, 2016). Data was obtained using a TX100-treated H6 reassortant virus grown in eggs that contained the NA from the H1N1 / CA09 challenge strain (FIG.6B) Pre-challenge (upper panel) and post-challenge (lower panel)9531-111023-03correlation plots showing the steric and enzymatic NAI endpoint titers that were obtained from each serum by NASPA and the MUNANA activity assay, respectively. Limits of detection (LODs) for the two assays are shown by dotted lines. Values below the LOD were assigned 0. (FIGS.6C and 6D) Post-challenge sera that showed strong steric (FIG.6C) or enzymatic (FIG.6D) NAI inhibition were immunoglobulin depleted with protein A and G agarose or treated with control agarose beads prior to performing NASPA or MUNANA analyses. Results are from one of two independent analysis. Assays were run with the TX100- treated H6 reassortant virus using NAi-MAb concentrations corresponding to the IC80. NA endpoint activities were measured 2 hours after MUNANA addition. Grey regions indicate the mean (dashed line) ± 3 SDs (dotted line) of the control wells. FIGS.7A-7F: Benefits of using a bulky synthetic NA inhibitor for NASPA. (FIG.7A) Diagram showing the novel chemoenzymatically synthesized biotinylated NA inhibitor (NAi: 4-guanidino- Neu5Acα2–3Galβ1–4GlcNAc–βProNH-PEG4-Biotin) that was combined with streptavidin (Sa) for use in NASPA. (FIG.7B) Schematic showing how the synthetic NA inhibitor bound to streptavidin can detect steric NAI antibodies in a NASPA approach. (FIG.7C) IC50 values of the NAi, NAi+Sa, NAi-MAb (FNI-9) and zanamivir for the TX100-treated H1N1 / BR18 virus after the indicated incubation times are displayed. (FIG.7D) Streptavidin binding to the NAi is required for NASPA. NA activity data is displayed from NASPA that was performed using TX100-treated H1N1 / BR18 virus with the NAi, NAi+Sa or Sa and the indicated NA ferret antisera. Assays were performed in duplicate using amounts approximating the IC65 and are displayed as the mean ± the SD. Grey region indicates the mean (dashed line) ± 3 SDs (dotted line) of the mock control wells. (FIG.7E) IC50 values of the NAi, NAi+Sa, and the NAi-MAbs (FNI-9 and FNI-9 R27D) for the indicated H3N2 vaccine strains are displayed. Each assay was run twice using either a 30- minute incubation (NAi and NAi+Sa) or a 3-hour incubation (NAi-MAbs). (FIG.7F) Representative NA activity data set is shown from NASPA that was performed using TX100-treated H3N2 / Dar21 virus with the NAi+Sa mixture and the indicated purified N2 MAbs. The N2 MAb were diluted as indicated from a starting concentration of ~20 µg / ml. The assay was performed using NAi+Sa amounts approximating the IC80. Grey region indicates the mean (dashed line) ± 3 SDs (dotted line) of the mock control wells. FIGS.8A-8C: NASPA results with different monoclonal antibodies that bind the NA active site. (FIG.8A) Inhibition curves for the NAi-MAbs FNI-19 and 1G01 with untreated and TX100-treated H1N1 / CA09 virus are shown. The indicated NAiMAb amounts were incubated with the virus for 3 hours prior to measuring NA activity by MUNANA. (FIGS.8B and 8C) NA activity data is displayed from NASPA that was performed using the NAi-MAbs FNI-19 (FIG.8B) and 1G01 (FIG.8C) with untreated and TX100-treated H1N1 / CA09 virus and the indicated ferret antisera. Assays were performed in duplicate using NAi-MAb amounts approximating the IC80 and are displayed as the mean ± SD. NA activities were read for 10 minutes after MUNANA addition. Grey regions indicate the mean (dashed line) ± 3 SDs (dotted line) that were determined using negative control ferret sera. FIGS.9A-9C: Time-dependent variation in the NAi-MAb (FNI-9) binding to NAs.9531-111023-03(FIGS.9A-9C) NAi-MAb (FNI-9) inhibition curves obtained after the indicated incubation times at 37℃ with the vaccine viruses H1N1 / BR18 (FIG.9A), H3N2 / HK14 (FIG.9B), and B / Austria21 (FIG.9C) are displayed. Measurements were performed using virus (upper panels) and TX100-treated virus (lower panels). Assay were run in duplicate and displayed as the mean ± SD. NA activities were measured for 10 minutes after MUNANA addition. FIGS.10A-10C: NASPA results from vaccine viruses with different ferret antisera incubation times. (FIGS.10A-10C) NASPA results of the untreated and TX100 treated vaccine viruses H1N1 / BR18 (FIG.10A), H3N2 / HK14 (FIG.10B), and B / Austria21 (FIG.10C) incubated with ferret antisera for the indicated times at 37℃ are shown. Assays were performed in duplicate using NAi-MAb amounts approximating the IC80 and are displayed as the mean ± SD. NA activities were measured for 10 minutes after MUNANA addition. FIGS.11A-11C: Influence of TX100 on NASPA results from ferret antisera that recognize the HA in the test virus. (FIGS.11A-11C) NASPA was performed with H6 reassortant viruses carrying NAs from the vaccine strains H1N1 / BR18 (FIG.11A), H3N2 / HK14 (FIG.11B), and B / Austria21 (FIG.11C) in the presence of increasing TX100 concentrations (values correspond to the final concentrations in the 100 µl reaction) and the indicated ferret antisera. Assays were performed in duplicate using NAi-MAb amounts approximating the IC80 and are displayed as the mean ± SD. NA activities were measured for 10 minutes after MUNANA addition. Percent NA activities were calculated using control wells that did not receive the NAi-MAb. FIGS.12A-12B: NASPA results comparing viral and recombinant NAs. (FIG.12A) NA activities of the indicated recombinant NA (rNA) or TX100-treated virus samples that were used for NASPA. rNAs and the viruses corresponded to the H1N1 vaccine strain A / Victoria / 2019 (N1 / Vic19) or the type B vaccine strain B / Austria / 21 (B / Austria21). (FIG.12B) NASPA results from the indicated rNAs and TX100-treated H6 reassortant viruses incubated with ferret antisera against either H6N1 / Vic19 or H6NB / Austria21 are shown. Assays were performed in duplicate using NAi-MAb amounts approximating the IC80 and are displayed as the mean ± SD. NA activities were measured for 10 minutes after MUNANA addition. Dotted lines correspond to the mean+3SD of the mock control wells for each sample. FIGS.13A-13D: Identification of optimal NAi-MAb concentration for NASPA. (FIGS.13A-13C) NASPA results are displayed for TX100 treated H6 reassortant viruses carrying NAs from the vaccine strains H1N1 / BR18 (FIG.13A), H3N2 / HK14 (FIG.13B), and B / Austria21 (FIG.13C) that were incubated with the indicated NAi-MAb (FNI-9) concentrations for 3 hours at 37℃ prior to the addition of ferret antisera. Assays were performed in duplicate and are displayed as the mean ± SD. NA activities were measured for 10 minutes after MUNANA addition. Highlighted regions for each NAi-MAb concentration corresponds to the mean (dashed line) ± 3 SDs (dotted line) of mock control wells. (FIG.13D) NASPA NAI titers obtained for each virus with the indicated inhibitory concentration (IC) of the NAi-MAb (FNI-9) are shown. NASPA titers correspond to the largest dilution factor that was above (positive titers) or below (negative titers) 3SDs of the control wells.9531-111023-03FIGS.14A-14F: Influence of NA amounts and substrate incubation times on NASPA results. (FIGS.14A-14C) Results from NASPA performed with TX100-treated H6N1 / BR18 (FIG.14A), H6N2 / HK14 (FIG.14B), and H6NB / Austria21 (FIG.14C) reassortant virus samples with the indicated NA activities and ferret antisera are displayed. Data were collected after a 2-hour incubation with MUNANA at 37℃. Dotted lines correspond to the mean+3SD of the mock control wells for the different NA amounts. (FIGS.14D-14F) NASPA titers determined using the TX100-treated H6N1 / BR18 (FIG.14D), H6N2 / HK14 (FIG.14E), and H6NB / Austria21 (FIG.14F) reassortant virus samples with the indicated NA activities and matching ferret antisera are displayed. Titers were collected for each sample by measuring NA activities for 10 minutes after MUNANA addition (kinetic), after a 10-minute incubation with MUNANA (endpoint) or after a 2-hour incubation with MUNANA (endpoint). FIGS.15A-15F: ELISA and ELLA results for the NA MAb panel. (FIGS.15A-15C) ELISA results from the indicated NA MAb hybridoma are shown. Wells were coated with either 1 µg of purified B / Austria / 21 virus (FIG.15A), H1N1 virus A / Victoria / 19 (FIG.15B), or H3N2 virus A / Darwin / 21 (FIG. 15C). ELISA endpoint titers corresponded to the largest dilution factor that was 2-fold higher than the medium only control (dashed lined). (FIGS.15D-15F) ELLA results for the indicated NA MAb hybridoma media are shown. ELLA was performed using allantoic fluid containing either B / Austria / 21 virus (FIG. 15D), H1N1 virus A / Victoria / 19 (FIG.15E), or H3N2 virus A / Darwin / 21 (FIG.15F). ELLA titers corresponded to the dilution factor that resulted in ~50% loss of NA activity. FIG.16: Inhibition curves of the NAi-MAb FNI-9 and two modified variants with H3N2 / Darwin / 21. H3N2 / Dar21 virus treated with TX100 was incubated with the indicated amounts of the NAi-MAb FNI-9 or the two modified variants (R27D and R27E) for 3 hours at 37℃ prior to measuring the NA activity with MUNANA for 10 minutes. FIGS.17A-17C: Influence of the NA MAbs on NA enzymatic activity. (FIGS.17A-17C) NA MAb hybridoma media were serially diluted and incubated with either TX100-treated B / Austria / 21 virus (FIG. 17A), H1N1 virus A / Victoria / 19 (FIG.17B), or H6N2 / Dar21 virus (FIG.17C) for 1 hour at 37℃ prior to incubating with MUNANA and taking an endpoint reading. Grey region indicates the mean (dashed line) ± 3 SDs (dotted line) of the mock control wells with medium. FIGS.18A-18B: NAi-MAb IC50determinations for NAs from the H1N1 and type B vaccine strains. NAi-MAb (FNI-9) inhibition curves with the indicated H1N1 (FIG.18A) and type B (FIG.18B) vaccine strains are displayed. The assays were performed with TX100-treated incubated with the indicated NAi- MAb concentrations for 3 hours at 37℃ prior to measuring NA activity with MUNANA. FIGS.19A-19B: NASPA analysis of human NA responses pre- and post-challenge with H1N1 / CA09. (FIG.19A) Endpoint titers determined with NASPA and a MUNANA activity assay for the indicated patients pre-challenge (upper panel) and 2 months post-challenge (lower panel) are displayed with the ELLA titers that were previously reported for these samples (Memoli et al., mBio 7:e00417-16, 2016). Data was obtained using TX100-treated H1N1 / CA09 challenge virus grown in MDCK cells. (FIG.19B) Pre-challenge (upper panel) and post-challenge (lower panel) correlation plots showing the steric inhibition9531-111023-03and NA enzymatic inhibition endpoint titers that were obtained from each serum by NASPA and the MUNANA activity assay, respectively. Limits of detection (LODs) for the two assays are shown by dotted lines. Values below the LOD were assigned 0. FIGS.20A-20B: Ferret antiserum inhibits NA binding to sialoside-based NA inhibitors by an ELISA strategy. (FIG.20A) Schematic (generated using BioRender) of an ELISA strategy to measure NA binding to biotinylated sialosides in the absence and presence of NAI antibodies. (FIG.20B) The indicated biotinylated sialosides containing a terminal α2–3-linked 4-guanidino-Neu5Ac or NH2Neu5Ac and a negative control sialoside Galβ1–4GlcNAc–βProNH-PEG4-Biotin were incubated in 384-well neutravidin- coated plates. Plates were washed and blocked with 1% ovalbumin prior to adding recombinant N1 / BR18 (left panel) or N2 / KS17 (right panel) that was preincubated with ferret antisera raised against H6N1 / BR18 or H6N2 / KS17 viruses. NA binding was detected with a monoclonal anti-His antibody followed by an HRP- conjugated anti-mouse secondary and TMB. Samples containing no ferret antisera or no recombinant NA were used as controls. FIGS.21A-21B: Inhibition curves for the synthetic NAi ± Sa and MAb 13F10 with the indicated viruses. (FIG.21A) Inhibition curves for the NAi (left panel), NAi+Sa mixed at a 1:2 molar ratio (middle panel), and Zanamivir (right panel) after the indicated incubation times with the H1N1 virus A / Brisbane / 18 (H1N1 / BR18) at 37℃ are displayed. (FIG.21B) TX100-treated virus was incubated with the indicated N2 MAb 13F10 concentrations for 1 hour at 37℃ prior to measuring NA activity with MUNANA. FIG.22: Schematic of a rapid and universal influenza neuraminidase inhibitory (NAI) antibody assay, referred to herein as NASPA. In the absence of an NA inhibitor, NA cleaves a reporter substrate that can be monitored by changes in fluorescence or color. In the presence of a chemical inhibitor or protein that mimics an inhibitor, such as an inhibitory influenza NA antibody, cleavage of the substrate is blocked, resulting in a lack of signal, such as fluorescence or color. When the assay is performed with serum samples containing NAI antibodies, the NA inhibitor can no longer block NA-induced cleavage of the reporter substrate. Thus, the presence of signal (such as fluorescence or color) indicates that the serum sample contains NAI antibodies. Any type of NA inhibitor that possesses sufficient molecular size naturally or by modification to provide a steric sensing capacity for NAI antibodies that bind near the NA active site can be used in this assay. FIG.23: Structures of N-acetylneuraminic acid (Neu5Ac, 1); 2,3-dehydro-3-deoxy-Neu5Ac (Neu5Ac2en, 2) and its 4-N-substituted derivatives (3–6); 4-amino-Neu5Ac (4NH2Neu5Ac, 7), α2–3 and α2–6-linked Neu5Ac-terminated α-sialosides (Neu5AcαOR, 8a / b) (R = GalβpNP) and their derivatives containing a 4-N-substituted Neu5Ac (9a / b–12a / b); and α2–3-linked 4-O-acetyl Neu5Ac-terminated α- sialoside (Neu4,5Ac2αOR, 13a). FIG.24: Chemical synthesis of 4NH2Neu5Ac (7) and 4-N-substituted Neu5Ac2en including 4NH2Neu5Ac2en (3), 4N3Neu5Ac2en (5), and 4NAcNeu5Ac2en (6) from commercially available Neu5Ac.9531-111023-03FIG.25: Chemoenzymatic synthesis of 4NH2Neu5Acα2–3GalβpNP (9a) and 4NH2Neu5Acα2– 6GalβpNP (9b) and other 4-N-derivatives including 4-guanidino-Neu5Acα2–3 / 6GalβpNP (10a / b), 4N3Neu5Acα2–3 / 6GalβpNP (11a / b), and 4NAcNeu5Acα2–3 / 6GalβpNP (12a / b). FIGS.26A-26D: Sialidase substrate specificity studies using sialyl GalβpNPs with low (30 minutes) (FIGS.26A-26B) and high (1 hour) (FIGS.26C-26D) enzyme concentrations. Sialidase amounts for the low-enzyme-concentration assay conditions were standardized using the substrate Neu5Acα2– 3GalβpNP. Sialidases in the high-enzyme-concentration assays were 10-fold higher than those in the low- enzyme-concentration assays except for Vc sialidase which was used at 4 × of the low-enzyme- concentration assay conditions. Abbreviations: h, human; Bi, Bifidobacterium infantis; Au, Arthrobacter ureafaciens; Cp, Clostridium perfringens; Vc, Vibrio cholerae; Sp, Streptococcus pneumoniae; PmST1, the sialidase activity of Pasteurella multocida α2–3-sialyltransfearse 1 in the presence of CMP (0.4 mM). FIG.27: 4NH2Neu5Acα2–3Gal / LacNAcβpProNH-PEG4-Biotin (9c / d) and 4-guanidino- Neu5Acα2–3Gal / LacNAcβpProNH-PEG4-Biotin (10c / d) are suitable affinity ligands for recombinant NAs from both IAVs and IBVs as demonstrated by ELISA using neutravidin-coated 384-well plates. Black bars, GalβProNH-PEG4-Biotin (19) and the corresponding α2–3-linked (by default, not labeled on the x-axis) or α2–6-linked (Neu5Ac only as controls, labeled on the x-axis) sialosides; gray bars, LacNAcβProNH-PEG4- Biotin (20), and the corresponding sialosides. FIGS.28A-28B: BLI assays for the binding of NAs from (FIG.28A) IAVs and (FIG.28B) IBVs to sialyl LacNAc-glycosides. FIGS.29A-29B: KDvalues determination by real-time BLI assays for (FIG.29A) N1-BR18 and (FIG.29B) NB-Phu134 with NH2Neu5Acα2–3LacNAcβProNH-PEG4-Biotin (9d) or 4-guanidino- Neu5Acα2–3LacNAcβProNH-PEG4-Biotin (10d). FIGS.30A-30D: 4-Guanidino-Neu5Acα2–3LacNAcβProNH-PEG4-Biotin (10d) is an effective affinity ligand for detecting and purifying H1N1 virus as demonstrated by (FIG.30A) ELISA using streptavidin-coated 96-well plates and (FIGS.30B-30D) streptavidin-Dynabeads with LacNAcβProNH- PEG4-Biotin (20) as a negative control ligand. (FIG.30A) Binding of H1N1 / BR18 virus in egg allantoic fluid (input) to streptavidin-coated wells incubated with the indicated concentration of 10d or 20 was detected with a monoclonal HA antibody. Data are the mean ± SD of an assay run in duplicate; (FIG.30B) Isolation of H1N1 / BR18 virus by streptavidin-Dynabeads containing 10d or 20 was monitored by measuring HA titers in the input, unbound (U), and elution (E) fractions. (FIG.30C) Purity of the isolated H1N1 / BR18 virus was analyzed by SDS-PAGE and Coomassie staining of the input, U and E fractions obtained with 10d and the negative control 20. E fraction from 10d was resolved without dithiothreitol (DTT) and bands corresponding to the viral hemagglutinin (HA), nucleoprotein (NP) and matrix 1 (M1) proteins are shown. (FIG.30D) Diameter and polydispersity (Pd) of the H1N1 / BR18 virus isolated with 10d was measured by dynamic light scattering.9531-111023-03FIG.31: Structures of sialosides synthesized and used for sialidase substrate specificity and inhibition assays. FIGS.32A-32B: Structures of Neu5Acα2–3GalβMU (18) and biotinylated glycans (19–20, 8c–f, 9c–d, 10c–d, 11c–d, and 12c–d). FIG.33: Illustration (generated using BioRender) of the ELISA using neutravidin-coated 384-well plates and biotinylated-glycans. FIG.34: BLI assays using IAV NA N2-Kan17 showed better binding of sialylated LacNAc- glycosides compared to sialylated Gal-glycosides. FIGS.35A-35B: Steady state analysis of sialoside binding to (FIG.35A) N1-BR18 and (FIG.35B) NB-Phu13 with NH2Neu5Acα2–3LacNAcβProNH-PEG4-Biotin (9d) or 4-guanidino-Neu5Acα2– 3LacNAcβProNH-PEG4-Biotin (10d) by real-time BLI assays. SEQUENCE LISTING The amino acid sequence listed in the accompanying sequence listing is shown using single letter code for amino acids, as defined in 37 C.F.R.1.822. In the accompanying sequence listing: SEQ ID NO: 1 is the amino acid sequence of the murine IgG heavy chain signal peptide. DETAILED DESCRIPTION I. Introduction Approaches to aid in the selection of vaccine strains for the influenza neuraminidase (NA) antigen are currently needed to assist in the development of vaccines containing NA. Disclosed herein a rapid and cost-effective solution-based NA active site proximity assay (NASPA) for measuring NA activity inhibitory (NAI) antibodies. The simplified assay is based on the concept of using large ‘bulky’ NA active site-binding inhibitors to replace the sialylated glycoprotein substrates used for NA enzyme-linked lectin assay (ELLA) approaches. The results disclosed herein show a strong correlation between NASPA and ELLA titers from ferret antisera and monoclonal antibodies against vaccine strain NAs and that NASPA provides similar results with viral and recombinant NA antigens. In different applications, NASPA revealed incremental antigenic changes in the NAs from recent influenza B virus Victoria lineage vaccine strains, and that steric and enzymatic NAI antibodies are common in adult human sera. Finally, it is demonstrated herein that NASPA can be modified or improved by incorporating novel NA substrate-analog-based inhibitors. Together, these results indicate that NASPA can aid in the development of vaccines containing NA by helping to select suitable vaccine strains and profile anti-NA antibody responses. The disclosed NASPA assay is superior to currently available methods (e.g., ELLA) for measuring NAI antibody titers at least because it is rapid (less than 1 day to complete compared to 3-5 days for currently available assays), avoids the complication of HA interference, is universal to any NA, can use NA from any source (virus or recombinant protein), requires only two reagents (making the assay simple and9531-111023-03inexpensive), requires no specialized equipment, has improved reproducibility, lower error rate, and is automatable for high throughput. II. Abbreviations BLI bio-layer interferometry ELLA enzyme-linked lectin assay HA hemagglutinin HINT high content imaging-based neutralization test IAV influenza A virus IBV influenza B virus IC inhibitory concentration LOD limit of detection M1 matrix 1 MAbs monoclonal antibodies MUNANA 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid NA neuraminidase NAi neuraminidase inhibitor NAI neuraminidase activity inhibitory (antibodies) NASPA neuraminidase active site proximity assay Sa streptavidin III. Summary of Terms Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “a fusion protein” includes singular or plural fusion proteins and can be considered equivalent to the phrase “at least one fusion protein.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided: Biological sample: A sample obtained from a subject (such as a human or veterinary subject). Biological samples, include, for example, fluid, cell and / or tissue samples. In some aspects herein, the9531-111023-03biological sample is a fluid sample. Fluid samples include, but are not limited to, whole blood, serum, plasma, urine, feces, saliva, breast mile, tears, cerebral spinal fluid (CSF), and bronchoalveolar lavage (BAL) fluid. In some examples herein, the biological sample is a blood or serum sample. Biological samples can also refer to cells or tissue samples, such as biopsy samples, tissue sections, or isolated cells. Contacting: Placement in direct physical association; includes both in solid and liquid form. “Contacting” is often used interchangeably with “exposed.” For example, contacting can occur in vitro with NA protein and a biological sample (such as a sample including influenza virus antibodies) in solution. Control: A reference standard, for example a positive control or negative control. A positive control is known to provide a positive test result (e.g., known to include influenza virus NAI antibodies). A negative control is known to provide a negative test result (e.g., known to not include influenza virus NAI antibodies). However, the reference standard can be a theoretical or computed result, for example a result obtained in a population. Detergent: A surfactant or a mixture of surfactants, which decrease the surface tension of interfacial tension between two liquids. In the context of the present disclosure, the detergent is capable of breaking up lipid membranes. In some examples, the detergent is Triton X-100 (C14H22O(C2H4O)n), a nonionic surfactant that has a hydrophilic polyethylene oxide side chain and an aromatic hydrocarbon lipophilic or hydrophobic groups. Fluorophore: A chemical compound, which when excited by exposure to a particular wavelength of light, emits light (fluoresces), for example at a different wavelength than that to which it was exposed. In some aspects herein, the NA reporter substrate includes a fluorophore, such as 4-methyl umbelliferone. Other exemplary fluorophores include, but are not limited to, fluorescein, 6-carboxyfluorescein (FAM), tetrachlorofluorescein (TET), tetramethylrhodamine (TMR), hexachlorofluorescein (HEX), JOE, 6- carboxy-X-rhodamine (ROX), CAL FluorTM, PulsarTM, QuasarTM, Texas RedTM, CyTM3 and CyTM5. Other examples of fluorophores are described in U.S. Patent No.5,866,366. These include: 4-acetamido-4'- isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl)amino-naphthalene-1-sulfonic acid (EDANS), 4-amino-N-[3- vinylsulfonyl)phenyl]-naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)- maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4- methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5', 5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5- [dimethyl-amino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethyl- aminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5- (4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein9531-111023-03(JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron® Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other fluorophores include thiol-reactive europium chelates that emit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999). Fluorophores such as cyanine, merocyanine, stryl, and oxonyl compounds, for example, those disclosed in U.S. Patent Nos.5,627,027; 5,486,616; 5,569,587; and 5,569,766, can also be used in various detection methods. Other fluorophores include GFP, Lissamine™, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Patent No.5,800,996) and derivatives thereof. Numerous fluorophores are commercially available from known sources. Hemagglutinin (HA): An influenza virus surface glycoprotein. HA mediates binding of the virus particle to host cells and subsequent entry of the virus into the host cell. HA also causes red blood cells to agglutinate. HA (along with NA) is one of the two major influenza virus antigenic determinants. Influenza virus: A segmented, negative-strand RNA virus that belongs to the Orthomyxoviridae family. Influenza viruses are enveloped viruses. There are four types of influenza viruses, A, B, C and D. Influenza A virus (IAV): A negative-sense, single-stranded, segmented RNA virus, which has eight RNA segments (PB2, PB1, PA, NP, M, NS, HA and NA) that code for 11 or more proteins, including RNA-directed RNA polymerase proteins (PB2, PB1 and PA), nucleoprotein (NP), neuraminidase (NA), hemagglutinin (subunits HA1 and HA2), the matrix proteins (M1 and M2) and the non-structural proteins (NS1 and NS2). This virus is prone to rapid evolution by error-protein polymerases and by segment reassortment. The host range of IAV is quite diverse, and includes humans, birds (e.g., chickens and aquatic birds), horses, marine mammals, pigs, bats, mice, ferrets, cats, tigers, leopards, and dogs. Animals infected with IAV often act as a reservoir for the influenza viruses and certain subtypes have been shown to cross the species barrier to humans. Influenza A viruses can be classified into subtypes based on allelic variations in antigenic regions of two genes that encode surface glycoproteins, namely hemagglutinin (HA) and neuraminidase (NA), which are required for viral attachment and mobility. There are currently 18 different IAV HA antigenic subtypes (H1 to H18) and 11 different IAV NA antigenic subtypes (N1 to N11). H1-H16 and N1-N9 are found in wild bird hosts and may be a pandemic threat to humans. H17-H18 and N10-N11 have been described in bat hosts and are not currently thought to be a pandemic threat to humans.9531-111023-03Specific examples of IAV include but are not limited to: H1N1 (such as 1918 H1N1), H1N2, H1N7, H2N2 (such as 1957 H2N2), H2N1, H3N1, H3N2, H3N8, H4N8, H5N1, H5N2, H5N8, H5N9, H6N1, H6N2, H6N5, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H8N4, H9N2, H10N1, H10N7, H10N8, H11N1, H11N6, H12N5, H13N6, and H14N5. In animals, most IAVs cause self-limited localized infections of the respiratory tract in mammals and / or the intestinal tract in birds. However, highly pathogenic IAV strains, such as H5N1, cause systemic infections in poultry in which mortality may reach 100%. In 2009, H1N1 influenza was the most common cause of human influenza. A new strain of swine-origin H1N1 emerged in 2009 and was declared pandemic by the World Health Organization. This strain was referred to as "swine flu." H1N1 IAVs were also responsible for the Spanish flu pandemic in 1918, the Fort Dix outbreak in 1976, and the Russian flu epidemic in 1977-1978. Influenza B virus (IBV): A negative-sense, single-stranded, RNA virus, which has eight RNA segments. IBV has eight RNA segments (PB1, PB2, PA, HA, NP, NA, M1 and NS1) that code for 11 or more proteins, including RNA-directed RNA polymerase proteins (PB1, PB2 and PA), nucleoprotein (NP), neuraminidase (NA), hemagglutinin (subunits HA1 and HA2), matrix protein (M1), non-structural proteins (NS1 and NS2) and ion channel proteins (NB and BM2). IBV is less prone to evolution than IAV, but it mutates enough such that lasting immunity has not been achieved. The host range of IBV is narrower than IAV and is only known to infect humans and seals. Influenza B viruses are not divided into subtypes but can be further broken down into lineages and strains. Specific examples of IBV include, but are not limited to: B / Yamagata, B / Victoria, B / Shanghai / 361 / 2002 and B / Hong Kong / 330 / 2001. Neuraminidase (NA): An influenza virus membrane glycoprotein. NA is involved in the destruction of the cellular receptor for the viral HA by cleaving terminal sialic acid residues from carbohydrate moieties on the surfaces of infected cells. NA also cleaves sialic acid residues from viral proteins, preventing aggregation of viruses. NA (along with HA) is one of the two major influenza virus antigenic determinants. Neuraminidase inhibitor (NAi): A molecule, agent or compound that is a NA active site inhibitor. As used herein, a “universal NAi” is an NAi that blocks the activity of substantially all known NA proteins, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of all known NA proteins. In some aspects, the NAi is a synthetic molecule, such as 4-guanidino-Neu5Acα2–3Galβ1–4GlcNAc–βProNH-PEG4-Biotin, 4-guanidino- Neu5Acα2–3GalβProNH-PEG4-Biotin, 4-guanidino-Neu5Acα2–3Galβ1–4GlcNAcβ1–3Galβ1– 4GlcNAcβProNH-PEG4-Biotin, 4-guanidino-Neu5Acα2–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcβProNH- PEG4-Biotin, 4-guanidino-Neu5Acα2–6Galβ1–4GlcNAcβProNH-PEG4-Biotin, or other 4-guanidino- Neu5AcαOR compounds. Synthetic NAi molecules also include any NA inhibitor, substrate or ligand that is attached to a large molecule (to block the NA active site). In other aspects, the NAi is a monoclonal antibody that specifically binds influenza virus NA from substantially all known influenza viruses. Exemplary NAi antibodies include those described in Momont et al. (Nature 618(7965):590-597, 2023), such as FNI-9, FNI-19 and 1G01.9531-111023-03Neuraminidase inhibitory (NAI) antibody: An antibody that inhibits the enzymatic activity of influenza virus NA. In some aspects herein, NAI antibodies in a serum sample obtained from a subject who has been infected with an influenza virus are detected and / or quantified using NASPA. Neuraminidase (NA) reporter substrate: A small (e.g., molecular weight less than 1500 Daltons) compound that enables detection of NA activity. In some aspects herein, the NA reporter substrate includes a reporter molecule (such as a fluorescent molecule) covalently linked to sialic acid or a sialic acid derivative. In the presence of active NA, the sialic acid (or sialic acid derivative) is cleaved, leading to the release of the reporter molecule which can then be detected and / or quantified. In some examples, the NA reporter substate is 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid (MUNANA). Reaction vessel: Any container suitable for carrying out the NASPA assay disclosed herein. The reaction vessel has a volume sufficient to allow for the combination of the biological sample, influenza virus (or recombinant NA protein), NAi and NA reporter substrate. Exemplary reaction vessels include, but are not limited to, multi-well plates (or a well of a multi-well plate) or microcentrifuge tubes. Recombinant: A recombinant protein, nucleic acid, vector, or virus is one that has a sequence that is not naturally occurring or has a sequence that is artificially produced using genetic engineering. For example, recombinant NA protein includes NA protein that is synthesized in a laboratory, such as NA protein produced by infecting Sf9 insect cells with recombinant baculoviruses encoding NA. Reporter molecule: A molecule that produces a detectable signal. In some aspects herein, the reporter molecule is a fluorescent molecule, a colorimetric molecule, or a chemiluminescent molecule. In some examples, the reporter molecule is the fluorescent molecule 4-methyl umbelliferone. Sialic acid: A class of alpha-keto acid monosaccharides with a nine-carbon backbone. Sialic acids are typically found to be terminating branches of N-glycans, O-glycans and glycosphingolipids. The most common sialic acid is N-acetylneuraminic acid (Neu5Ac). The NA reporter substrates described herein include a reporter molecule covalently linked to sialic acid or a sialic acid derivative. Exemplary sialic acid derivatives include, for example, N-glycolylneuraminic acid (Neu5Gc), N-azidoacetylneuraminic acid (Neu5AcN3), 9-O-acetyl-N-acetylneurmainic acid (Neu5,9Ac2), 9-N-acetyl-9-deoxy-N-acetylneurmainic acid (Neu5Ac9NAc), 9-azido-9-deoxy-N-acetylneurmainic acid (Neu5Ac9N3), 9-amino-9-deoxy-N- acetylneurmainic acid (Neu5Ac9NH2), 9-O-acetyl-N-acetylneurmainic acid (Neu5,9Ac2), 8-amino-8-deoxy- N-acetylneurmainic acid (Neu5Ac8NH2), 7-N-acetyl-7-deoxy-N-acetylneurmainic acid (Neu5Ac7NAc), 7- azido-7-deoxy-N-acetylneurmainic acid (Neu5Ac7N3), 7-amino-7-deoxy-N-acetylneurmainic acid (Neu5Ac7NH2), 4-O-acetyl-N-acetylneurmainic acid (Neu4,5Ac2), and 4-azido-4-deoxy-N-acetylneurmainic acid (Neu5Ac4N3). Influenza virus uses sialic acid as a cellular receptor. Subject: Living multi-cellular vertebrate organisms, a category that includes both human and non- human mammals, such as non-human primates. In some aspects, a subject is one that can be infected with an influenza virus, such as humans, birds (e.g., chickens, ducks, turkeys), horses, pigs, bats, mice, ferrets, cats, tigers, leopards, seals, and dogs. In some aspects, the subject is one who has been infected with influenza virus or who is suspected of being infected (or previously infected) with an influenza virus.9531-111023-03Synthetic: Produced by artificial means in a laboratory, for example a chemically synthetic NAi. Exemplary synthetic NAi molecules include, but are not limited to, 4-guanidino-Neu5Acα2–3Galβ1– 4GlcNAc–βProNH-PEG4-Biotin, 4-guanidino-Neu5Acα2–3GalβProNH-PEG4-Biotin, 4-guanidino- Neu5Acα2–3Galβ1–4GlcβProNH-PEG4-Biotin, 4-guanidino-Neu5Acα2–3Galβ1–4GlcNAcβ1–3Galβ1– 4GlcNAcβProNH-PEG4-Biotin, 4-guanidino-Neu5Acα2–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1– 3Galβ1–4GlcNAcβProNH-PEG4-Biotin, 4-guanidino-Neu5Acα2–3Galβ1–4GlcNAcβ1–3Galβ1– 4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβProNH-PEG4-Biotin, 4-guanidino-Neu5Acα2– 3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1– 4GlcNAcβProNH-PEG4-Biotin. IV. Neuraminidase Inhibitory (NAI) Antibody Titer Assay Described herein is a rapid influenza NAI antibody titer assay, referred to herein as a neuraminidase active site proximity assay (NASPA). In addition to detecting NAI antibodies in biological samples, NASPA can be used to evaluate antigenic drift in circulating field strains and protective efficacy following administration of a NA vaccine. The evaluation of NA antigenic drift is important for seasonal strain selection analysis to identify which NAs should be incorporated into a seasonal vaccine. Antibody responses against the influenza virus surface antigens hemagglutinin (HA) and neuraminidase (NA) have both been shown to be protective in humans and multiple animal models. However, influenza vaccines continue to be manufactured and regulated solely by the HA antigen properties due to the lack of simple approaches for (1) identifying strains with the most appropriate NA antigens for seasonal vaccines; and (2) producing sufficient quantities of these NA antigens that are immunogenic. Currently, HA antigens in the annual vaccine are determined by a combinatorial approach that examines the antigenicity of HA in circulating field strains with the temporal frequency and location of particular HAs based on sequencing. The antigenicity of HAs in isolated field strains is determined by a hemagglutination inhibition (HAI) assay or high content imaging-based neutralization test (HINT) that is carried out by numerous World Health Organization (WHO) collaborating centers (CCs) around the world. Although a similar framework could be performed for NA antigens, one has not been established primarily because prior to the present disclosure, a simple assay for determining NA antigenicity in field strains had not been available. The HAI assay uses the loss of viral hemagglutination to detect and measure antibodies that bind epitopes near the HA receptor binding domain, implying this region is crucial for determining HA antigenicity and ultimately vaccine efficacy. An analogous NA inhibition antibody assay was previously developed for detecting and measuring antibodies that bind epitopes near the NA enzymatic pocket and inhibit its ability to cleave terminal sialic acid residues on the N-linked glycans of a glycoprotein. Over time, this enzyme-linked lectin assay (ELLA) for detecting and measuring NAI antibodies was improved and standardized to enable its use for evaluating the antigenicity of NA in circulating field strains. However,9531-111023-03WHO CCs have not instituted the current ELLA for strain selection because it remains a HA-dependent NA activity assay that consists of multiple reagents and is labor and time (at least 3 days) intensive. To solve this unmet need, the present disclosure describes the development of a NA-dependent NAI antibody assay (NASPA) that is based on the existence of NA inhibitors that are capable of binding NAs from virtually all known influenza A and B strains and the observation that NAI antibodies generally cannot block small synthetic reporter substrates from entering the NA active site and being cleaved (see FIGS.2A and 22). The underlying concept is that by attaching a large enough molecule to an NA inhibitor, antibodies that bind around the NA active site would sterically hinder the ability of the large inhibitor from binding and thereby prevent the cleavage of a small synthetic substrate. To test this concept, a NA inhibitory (NAi) monoclonal antibody was used that universally binds and inhibits the activity of NAs from all nine influenza A subtypes and the two influenza B lineages by mimicking the interactions of oseltamivir with NA. The assay disclosed herein uses two reagents – a large NA inhibitor (NAi) and a small synthetic NA reporter substrate. In some aspects, the large NAi is a monoclonal antibody that binds substantially all NA proteins, such as a monoclonal antibody described in Momont et al. (Nature 618(7965):590-597, 2023). In other aspects, the NAi is a synthetic molecule that is a large ‘bulky’ NA active site-binding inhibitor. In some aspects, the small synthetic NA reporter substrate includes a reporter molecule (such as a fluorophore or colorimetric molecule) linked to any derivative of sialic acid with or without additional linked sugars (such as galactose), which is susceptible to cleavage by NA such that in the presence of NA, the reporter molecule is released from the substrate and produces a signal, such as fluorescence or color. In some aspects, the reporter substrate is 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid (MUNANA) and / or the reporter molecule is 4-methyl umbelliferone. The assay can be applied to NA from any source (field strains, recombinant viruses, recombinant protein). In some aspects, it is carried out by calculating the amount of each NA for the assay by using the small synthetic reporter substrate; determining the large NA inhibitor IC50 concentration for each NA that will be analyzed; and measuring the NAI titer by incubating a serial dilution of any sera with the established NA amount and then adding the inhibitor at a concentration corresponding to the IC50 or higher (e.g., IC95) and then the small synthetic NA reporter substrate. Following substrate addition, NA activity is read over time (kinetically) or after a defined time period (endpoint). The presence of NAI antibodies and the NAI titer can be determined by plotting the activity with respect to the sera dilution and identifying the dilution factors where NA activity increases compared to the largest dilution factors or a mock sera control. Changes in the IC50 between NAs of the same or different subtypes or types can be used to provide an initial indication of antigenic changes near the active site of the two NAs being compared. The assay can be made HA-independent by either using recombinant NA protein or adding a detergent such as TX-100 to the virus at a final concentration in the total reaction that is above the critical micelle concentration (CMC) to separate NA from HA if field strains, vaccine strains, or recombinant viruses are used. The assay is amenable to high throughput and can be performed start to finish in under 1 day, making it applicable for NA strain selection analysis and for monitoring NA efficacy in clinical trials similar to the current HAI assay.9531-111023-03The disclosed NAI titer assay has several superior features compared to currently available assays (such as ELLA), including: it is rapid (less than 1 day to complete compared to 3-5 days for currently available assays), avoids the complication of HA interference, is universal to any NA, requires only two reagents making the assay simple and inexpensive, requires no specialized equipment, has improved reproducibility, has a low error rate, and is automatable for high throughput. Provided herein are methods for detecting the presence or absence of influenza virus neuraminidase (NA) inhibitory (NAI) antibodies in a biological sample. In some aspects, the method includes incubating the biological sample in a reaction vessel that includes recombinant NA protein or includes influenza virus containing NA protein, under conditions sufficient for any NAI antibodies present in the biological sample to bind the recombinant NA protein or the NA protein of the influenza virus; adding a NA inhibitor (NAi) to the reaction vessel, wherein the NAi binds to the NA active site in the absence of NAI antibodies in the biological sample; adding a NA reporter substrate to the reaction vessel, wherein the NA reporter substrate includes a reporter molecule and sialic acid or a sialic acid derivative, and the reporter molecule is detectable upon cleavage of the sialic acid; and detecting the presence or absence of the detectable reporter molecule. An increase in the detectable reporter molecule compared to a control sample indicates the biological sample contains NAI antibodies. No change in the detectable reporter molecule compared to a control sample indicates the biological sample does not contain NAI antibodies. A decrease in the detectable reporter molecule compared to a control sample indicates the biological sample contains NAI antibodies that bind to the NA active site. The biological sample can be any fluid, cell or tissue sample that may contain influenza virus- specific antibodies. In some aspect, the biological sample is a blood or serum sample. For example, the biological sample can be obtained from a subject who has been infected with an influenza virus (such as in the previous week, the previous two weeks, the previous month, the previous three months, the previous six months, the previous nine months, or the previous year), currently has an influenza virus infection, or is suspected of having an influenza virus infection. In some aspects, the method further includes serially diluting the biological sample prior to adding the biological sample to the reaction vessel. In some examples, at least two, at least three, at least four, or at least five dilutions of the biological sample are incubated in separate wells of the reaction vessel. Serial dilution of biological samples is described in Example 1. In some aspects, the method further includes diluting the recombinant NA protein or the influenza virus containing NA protein prior to adding to the reaction vessel (see Example 1). In some examples, the NA protein or the influenza virus containing NA protein is diluted such that the resulting NA activity is about 0.01-1.0 pmol / second, about 0.05-0.9 pmol / second, about 0.10-0.8 pmol / second, about 0.15-0.7 pmol / second, about 0.20-0.6 pmol / second, or about 0.25-0.5 pmol / second of substrate. In some examples, the influenza virus containing NA protein is diluted such that the resulting NA activity is 0.25-0.5 pmol / second of substrate.9531-111023-03In some aspects, the method further includes calculating the NAI antibody titer in the biological sample. Methods for determining antibody titer are well-known and exemplary methods are described in Example 1. In some aspects, the biological sample is incubated with the recombinant NA protein or the influenza virus containing NA protein for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, or about 90 minutes. For example, the recombinant NA protein or the influenza virus containing NA protein can be incubated with the biological sample for about 10 to about 90 minutes, about 20 to about 80 minutes, about 30 to about 70 minutes, about 40 to about 60 minutes, or about 50 minutes. In some examples, the recombinant NA protein or the influenza virus containing NA protein is incubated with the biological sample for about 60 minutes, such as about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 minutes. In some aspects, the NAi is incubated with the biological sample and the recombinant NA protein or the influenza virus containing NA protein for about 20 minutes to about 4 hours, such as about 30 minutes to about 3.5 hours, about 45 minutes to about 3 hours, about 60 minutes to about 2.5 hours, or about 90 minutes to about 1 hour. In some examples, the NAi is incubated with the biological sample and the recombinant NA protein or the influenza virus containing NA protein for about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, or about 4 hours. In some examples, the incubation time is about 3 hours. In specific examples, when the NAi is a monoclonal antibody, the incubation time is about 3 hours. In other specific examples, when the NAi is a synthetic molecule (such as 4-guanidino-Neu5Acα2–3Galβ1–4GlcNAc–βProNH-PEG4-Biotin), the incubation time is about 30 minutes. In some aspects of the method, the presence or absence of the reporter molecule is detected at multiple timepoints after addition of the NA reporter substrate (a kinetic assay). In some examples, the multiple timepoints include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least 10 timepoints. In some examples, detection is performed every one minute, every two minutes, every three minutes, every four minutes or every five minutes. In specific non- limiting examples, detection is performed at multiple timepoints between 1 and 20 minutes after addition of the NA reporter substrate. In other aspects of the method, the presence or absence of the reporter molecule is detected at a single timepoint following addition of the NA reporter substrate (an endpoint assay). In some examples, detection is performed at about 2 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes or about 30 minutes after addition of the NA reporter substrate. In some examples, a stop solution is added prior to detecting the presence or absence of the reporter molecule. In9531-111023-03specific non-limiting examples, the stop solution includes 0.133 mM glycine, 0.06 M NaCl, and 0.083N Na2CO3at pH10.7. In some aspects of the method, the reporter molecule (of the NA reporter substrate) is a fluorescent molecule, a colorimetric molecule, or a chemiluminescent molecule. In some examples, the fluorescent molecule is 4-methyl umbelliferone. In some examples, the colorimetric molecule is para-nitrophenol (pNP)-based sialic acid. The reporter molecule, following its release from the reporter substrate, can be detected using any suitable means, such as by using a plate reader to detect fluorescence, color or chemiluminescence. In some aspects of the method, the NA reporter substrate is 4-methylumbelliferyl-N-acetyl-α-D- neuraminic acid (MUNANA). In some aspects of the method, the NAi is a monoclonal antibody, such as a universal NA-specific monoclonal antibody that binds all, or substantially all, influenza virus NA proteins. Exemplary NA- specific monoclonal antibodies suitable for the disclosed method are described in Momont et al. (Nature 618(7965):590-597, 2023), such as the FNI-9, FNI-19 and 1G01 antibodies. In other aspects of the method, the NAi is a synthetic molecule. The synthetic molecule can be any molecule, agent, or compound that is a NA active site inhibitor, such as any NA inhibitor, substrate or ligand that is attached to a large molecule (to block the NA active site), such as streptavidin, human serum albumin or bovine serum albumin. In some examples, the NAi is 4-guanidino-Neu5Acα2–3Galβ1–4GlcNAc– βProNH-PEG4-Biotin, 4-guanidino-Neu5Acα2–3GalβProNH-PEG4-Biotin, 4-guanidino-Neu5Acα2– 3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβProNH-PEG4-Biotin, 4-guanidino-Neu5Acα2–3Galβ1– 4GlcNAcβ1–3Galβ1–4GlcβProNH-PEG4-Biotin, 4-guanidino-Neu5Acα2–6Galβ1–4GlcNAcβProNH- PEG4-Biotin, or another 4-guanidino-Neu5AcαOR compound, coupled to a large molecule, such as streptavidin. Any suitable reaction vessel can be used to perform the disclosed methods. In some aspects, the reaction vessel is a multi-well plate. In some aspects of the method, the reaction vessel further includes detergent, such as when influenza virus containing NA protein is used in the method, which spatially separates NA and HA present in the virus envelope. In some examples, the detergent is Triton-X100. However, a skilled person can select an appropriate detergent to use in the method. EXAMPLES The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.9531-111023-03Example 1: Materials and Methods This example describes the materials and experimental procedures for the studies described in Examples 2-11. Viruses Vaccine strains obtained from the WHO were propagated in 10-day-old specific pathogen-free (SPF) embryonated chicken eggs for 3 days at 33°C. These included H1N1 strains: A / Brisbane / 59 / 2007 (BR07), A / California / 07 / 2009 (CA09), A / Michigan / 45 / 2015 (MI15), A / Brisbane / 02 / 2018 (BR18) and A / Victoria / 2570 / 2019 (Vic19); H3N2 strains: A / Hong Kong / 4801 / 2014 (HK14), A / Singapore / INFIMH-16- 0019 / 2016 (SG16), A / Cambodia / e0826360 / 2020 (Cam20), and A / Darwin / 9 / 2021 (Dar21); and influenza B strains from the Yamagata lineage (B / Phuket / 3072 / 2013 (Phu13)) and the Victoria lineage strains: B / Brisbane / 60 / 2008 (BR08), B / Colorado / 06 / 2017 (CO17), B / Washington / 02 / 2019 (WA19) and B / Austria / 1359417 / 2021 (Austria21). The following reassortant viruses containing HA from the H6N2 strain A / turkey / Massachusetts / 3740 / 1965 and the NA from the indicated vaccine strain and the six internal gene segments from the PR8 strain A / PR / 8 / 1934 were generated by reverse genetics as described previously (Malik et al., ACS Synth Biol 12:432-445, 2023; Hoffmann et al., Proc Natl Acad Sci USA 97:6108-6113, 2000) and propagated in SPF eggs: H6N1 / BR07, H6N1 / CA09, H6N1 / MI15, H6N1 / BR18, H6N2 / HK14, H6N2 / Dar21, H6NB / Austria21, and H6NB / Phu13. NAs in the reassortant viruses for the two B vaccine strains were chimeras that were generated by replacing the coding region for residues 36-437 in the N1 / BR07 reverse genetics plasmid with the coding region for residues 34-466 of the indicated B vaccine strains. Generation of ferret and mouse antisera Antisera was obtained using intranasal inoculations. Inoculations were performed on 14-week-old male ferrets using allantoic fluid containing the indicated reassortant virus at an infectious titer of 106-108TCID50 / ml on MDCK cells. The inoculum (~0.5 ml) was equally dispensed between each nostril using a 1 ml syringe equipped with a 20 Gauge feeding needle. Body temperatures and weight were recorded daily for two weeks. Sera were harvested at 3 weeks, aliquoted and stored at -80°C. Mouse antisera were generated by inoculating anesthetized 8-week-old female DBA / 2J mice with allantoic fluid (~0.1 ml) containing the indicated reassortant virus diluted in PBS pH 7.2 to an infectious titer of 102-104TCID50 / ml on MDCK cells. Body temperatures and body weight were recorded daily for two weeks and sera were harvested at 3 weeks, aliquoted and stored at -80°C. Enzyme-Linked Lectin Assay (ELLA) titer determination ELLA was performed on 96-well Maxisorp plates coated with 2.5 µg / well of bovine fetuin as previously described (Couzens et al., J Virol Methods 210:7-14, 2014; Gao et al., J Vis Exp doi:10.3791 / 54573, 2016). Test virus concentrations were first determined by adding 2-fold serial dilutions9531-111023-03of virus in ELLA buffer (2-(N-morpholino)ethanesulfonic acid (MES) 25 mM pH 6.5, 150 mM NaCl, 20 mM CaCl2, 1% bovine serum albumin) containing 0.5% Tween 20 to the fetuin-coated plates and incubating at 37°C overnight. Plates were washed with 3 × 200 µl / well PBS pH 7.4 containing 0.05% Tween 20 (PBST) and 100 µl / well of horseradish peroxidase-linked peanut agglutinin diluted in ELLA buffer (~1 µg / ml) was added. Plates were incubated 2 hours at room temperature, washed 3 x 200 µl / well with PBST and OPD substrate was added. After a 10-minute incubation at 37°C, reactions were stopped with 100 µl / well 1N sulfuric acid and absorbance (Abs) at 490 nm was measured on a Cytation 5 (Biotek) plate reader. Test virus dilutions with an Abs 490 nm ~2.0 were used for the following assay. Antisera or antibodies were serially diluted in ELLA buffer containing 0.5% Tween 20, transferred (50 µl / well) to a fetuin-coated plate and the test virus diluted in the same buffer was added (50 µl / well). Plates were incubated at 37°C overnight and processed as described above. Abs 490 nm results were plotted with respect to antisera / antibody dilution factor in GraphPad Prism 8.0 and ELLA titers corresponding to the half- maximal inhibitory concentration (IC50) were determined by a variable slope four-parameter least squares regression analysis. Recombinant NAi-MAb expression NAi-MAbs were all produced as recombinant murine IgG1 chimeras by transfection of XPi CHO cells and purification by protein A Sepharose (Genscript). Briefly, variable regions of the heavy- (VH) and light chain (VL) from the reported human antibodies FNI-9, FNI-19, 1G01 (Momont et al., Nature 618:590- 597, 2023) were inserted into the murine IgG1 heavy chain (CH1 + Hinge + CH2 + CH3 regions) and the light chain (kappa), respectively, and both included the murine IgG heavy chain signal peptide (MGWSCIILFLVATATGVHS; SEQ ID NO: 1) on the N-terminus. The two genes were codon optimized for CHO cell expression and inserted into pcDNA3.4 using EcoR1 and Hind III sites following synthesis. Plasmids were then propagated in E. coli and used for the CHO cell transfection. The FNI-9 antibody carrying the substitutions R27D or R27E were designed based on computational analysis (PDB: 8G3O) that indicated R27 in the light chain of FNI9 would likely be repelled by the E244K substitution in the NAs from recent H3N2 strains. Recombinant NA insect cell expression and purification Recombinant NA (rNA) proteins were produced by infecting Sf9 insect cells with recombinant baculoviruses (BVs) that were generated using pFastBac vectors encoding the secreted rNA constructs. The His-tagged rN1 / BR18 and rN2 / KS17 were designed and purified as previously described (Kang et al., J Chromatogr B Analyt Technol Biomed Life Sci 1232:123975, 2023; Kooner et al., ACS Infect Dis 9:33-41, 2023). The secreted rN1 / Vic19, rN2 / Dar21 and rNB / Austria21 constructs were all designed similar with a cleavable azurocidin signal peptide followed by a Strep-Tag, the tetrabrachion tetramerization domain (rN1 / Vic19 and rN2 / Dar21) or the vasodilator-stimulated phosphoprotein tetramerization domain (rNB / Austria21) and residues 35-469 of the NAs from A / Victoria / 2570 / 2019 (H1N1) or A / Darwin / 9 / 20219531-111023-03(H3N2), or residues 39-466 from B / Austria / 1359417 / 2021. Briefly, Sf9 cells were harvested 72 hours post- infection, clarified by sequential sedimentations (10 minutes; 4000 × g and 30 minutes; 10^000 × g), concentrated 6-fold by tangential flow filtration (TFF) using a 30 kDa molecular weight cutoff (MWCO) Sartocon Slice 200 PES membrane (Sartorius), and diafiltrated using 5 volumes of binding buffer (30 mM HEPES pH 7.0, 150 mM NaCl, 1 mM CaCl2). Diafiltrated samples were purified by Strep-Tactin XT affinity chromatography (Cytiva) according to the manufacturer’s recommendations. Purified rNAs were diafiltrated by TFF using 30 kDa MWCO PES membrane with binding buffer, concentrations were determined by a BCA assay (Pierce) and rNAs were adjusted to ∼0.5–1.0 mg / ml prior to aliquoting and storage at -80°C. NA monoclonal antibody hybridomas and antibody purification All hybridomas were contracted out to Genscript. Briefly, mice were immunized three times with rN1 / Vic19, rN2 / Dar21 or rNB / Austria21 in the presence of adjuvant. Sera were tested for reactivity with the rNA antigen versus the two that were not used for immunization. Hybridomas from positive mice were generated and screened with the rNA antigen versus the two that were not used for immunization. Positive hybridomas were rescreened by ELLA and ELISA binding using viruses pre and post purification, respectively. Hybridomas that were positive in the rescreen for either ELISA alone or both ELISA and ELLA were selected, 2 clones of each were expanded and the hybridoma media from the expansion were used in this study along with the antibodies purified from each selected clone with Protein A Sepharose. Chemical synthesis of sialoside based NA inhibitor and streptavidin coupling Synthesis of the biotinylated sialoside-based NA inhibitor 4-guanidino-Neu5Acα2–3Galβ1– 4GlcNAc–βProNH-PEG4-Biotin is described in Examples 12-16 The inhibitor was coupled to streptavidin (NAi+Sa) by mixing in a 1:2 molar ratio (0.159 mM inhibitor and 16.8 mg / ml streptavidin) and incubating 1 hour at 37°C prior to use. NA titration using MUNANA All MUNANA assays were performed in 96-well low protein binding black clear bottom plates in duplicate. A 4-methyl umbelliferone (UM) standard was diluted to 0.2 mM in reaction buffer (25 mM MES pH 7, 150 mM NaCl, 1 mM CaCl2, 1% BSA) with or without 0.025% TX100 as indicated for the samples and used to make seven additional two-fold serial dilutions in reaction buffer. The eight standards were transferred to a column (25 μl / well) and mixed with 37°C reaction buffer (75 μl / well). Test viruses in allantoic fluid or recombinant NA were serially diluted 1:2 in 37°C reaction buffer with or without 0.025% TX100 as indicated and added to the same plate (25 μl / well). Samples were mixed with 37°C reaction buffer (50 μl / well) to a volume of 75 μl, reactions were initiated by adding 37°C reaction buffer containing 0.4 mM MUNANA (25 μl / well) and the fluorescence (Ex λ: 355 nm, Em λ: 450 nm) was measured on a Cytation 5 (Biotek) plate reader at 37°C for 10 minutes using 30 second intervals. Activities were determined by dividing the slope of the linear region of the emission (RFU) versus time graph by the slope of the RFU9531-111023-03versus UM pmol / well standard and the dilutions that cleaved ~0.1-0.4 pmol / s of MUNANA were used for all subsequent analysis. For endpoint analysis all wells received 100 μl of stop solution (0.133 mM glycine, 0.06M NaCl, 0.083N Na2CO3, pH10.7) after the indicated incubation time and the fluorescence was measured. Generation and analysis of NA inhibition curves Purified NAi-MAbs, synthesized NAi alone and the NAi coupled to Streptavidin (NAi+Sa) were serially diluted in reaction buffer with or without 0.025% TX100 and added (50 µl / well) to a 96-well plate in duplicate. Test viruses diluted to the target NA activity levels in reaction buffer with or without 0.025% TX100 were added (25 µl / well) to all wells mixed and incubated for 3 hours unless specified otherwise at 37ºC. Reactions were initiated by adding (25 µl / well) of reaction buffer with or without 0.025% TX100 and 0.4 mM MUNANA and the fluorescence was generally read by a kinetic and 10 minute endpoint analysis. NA activity measurements were normalized by the NA activity in control wells without inhibitor which was set to 100% and concentrations are with respect to the final 100 µl reaction. NA activity measurements were plotted with respect to the inhibitor concentration in GraphPad Prism 8.0 and a variable slope four-parameter least squares regression analysis was performed to determine the half-maximal inhibitory concentration (IC50) and the Hill slope (H). Concentrations corresponding to the IC80 were calculated based on the following formula where F corresponds to the desired inhibitory concentration: ICF = IC50×(F / (100-F))(1 / H). NA Active Site Proximity Assay (NASPA) and titer determination Sera, hybridoma media or purified MAbs were serially diluted in reaction buffer with or without 0.025% TX100 and added (25 µl / well) to a 96-well plate in duplicate. Control wells containing serial dilutions of hybridoma medium, negative sera or buffer and the UM standard were included. Test viruses were diluted to the target NA activity levels in reaction buffer with or without 0.025% TX100 and added (25 µl / well) to all wells except the standard and the plate was incubated for 1 hour at 37ºC. The NAi-MAb, NAi or NAi+Sa were diluted to an ~IC80in reaction buffer with or without 0.025% TX100 and added (25 µl / well) to all wells except the standard. The plate was incubated for 3 hours at 37ºC after which reactions were initiated by adding (25 µl / well) of 0.4 mM MUNANA in reaction buffer with or without 0.025% TX100 that was prewarmed to 37ºC and the fluorescence was read directly by a kinetic or by endpoint analysis after incubation for 10 minutes or 2 hours at 37ºC and the addition of stop solution (100 µl / well). The mean activity and standard deviation (SD) of the control wells were determined and titers corresponded to the largest dilution factor where both samples showed an activity level above the mean plus three SDs of the control wells (steric inhibition) or below the mean minus three SDs (enzymatic inhibition). NA titer determination by MUNANA The set up for the assay was identical to NASPA except the NAi-MAb was omitted. Briefly, sera or hybridoma medium was serially diluted in reaction buffer with 0.025% TX100 and added to the plate (259531-111023-03µl / well). Control wells were included that contained serial dilutions of hybridoma medium or buffer and the UM standard. Test viruses diluted in reaction buffer with 0.025% TX100 were added (25 µl / well) and incubated for 1 hour at 37ºC. Reactions were initiated by adding 50 µl / well of 0.2 mM MUNANA in reaction buffer containing 0.025% TX100 that was prewarmed to 37ºC and the fluorescence was read by a kinetic or endpoint analysis after incubation for 10 minutes or 2 hours at 37ºC and the addition of stop solution. The mean activity and standard deviation (SD) of the control wells were determined and titers corresponded to the largest dilution factor where both samples showed activity levels below the control mean minus three SDs. Enzyme linked immunosorbent assay (ELISA) titer determination Immunol 2HB 96-well plates were coated (100 µl / well) overnight at 4℃ with purified egg- propagated B / Austria / 21, A / Victoria / 2019 (H1N1) or A / Darwin / 9 / 21 (H3N2) viruses diluted (10 µg / ml) in 1X coating buffer. Plates were washed 3 × 200 µl / well with PBS pH 7.4 and blocked for 1 hour at 37℃ with PBS pH 7.4 containing 1% BSA (200 µl / well). Plates were washed 3 × 200 µl / well with PBS pH 7.4, clarified hybridoma medium serially diluted in PBST with 0.1% BSA was added to each well (100 µl / well), and the plate was incubated 1 hour at 37℃. Plates were washed 3 × 200 µl / well with PBST and an HRP- conjugated goat-anti-mouse IgG secondary antibody diluted (1:20000) in PBST containing 0.1% BSA was added (100 µl / well). Plates were incubated 1 hour at 37℃, washed 4 × 200 µl / well with PBST and developed with OPD for 10 minute at 37℃. Reactions were stopped by adding 50 µl / well of 2N sulfuric acid and the Abs at 490 nm was read using a Cytation 5 (Biotek) plate reader. Human sera analysis All human sera were from a previous healthy volunteer challenge study that was performed at the NIH Clinical Center after participants signed an informed consent form (Memoli et al., mBio 7:e00417-16, 2016). Human sera were heat inactivated by incubating at 55℃ for 30 minutes prior to being serially diluted in reaction buffer containing 0.025% TX100. The dilution series were then used in the NASPA and MUNANA activity assay, both with a 2-hour substrate (MUNANA) incubation time at 37℃ and an endpoint analysis following the addition of stop solution. For protein A / G depletions, 10 ml of a 20% slurry consisting of 10% Protein A Agarose beads and 10% Protein G Agarose beads or Agarose beads alone were washed 3 × 10 ml with reaction buffer containing 0.025% TX100 and each one was finally resuspended in 4 ml reaction buffer containing 0.025% TX100 to create a 50% slurry. Serum diluted 1:20 in reaction buffer (200 µl per sample) was added to tubes containing 200 µl of the Protein A / G Agarose bead slurry or the control Agarose bead slurry and rotated overnight at 37℃. Beads were sedimented (4,000 × g; 5 minutes) and the supernatant was serially diluted in reaction buffer containing 0.025% TX100. Serial dilutions were then used in the NASPA and MUNANA activity assay as described above. Assays were performed with clarified H6N1 / CA09 virus grown in SPF eggs and the H1N1 / CA09 challenge virus grown in MDCK cells.9531-111023-03ELLA titers for the human sera presented herein were from an analysis performed in a previous study (Memoli et al., mBio 7:e00417-16, 2016). Statistical analysis Statistical analysis was performed with Excel (mean and standard deviation) and GraphPad Prism 8 software. Example 2: Substrate requirements for measuring NAI antibodies NA activity is often measured using small monovalent reporter substrates like MUNANA or more biologically relevant multivalent substrates such as the glycoprotein fetuin. With MUNANA (FIG.1A), NA activity is readily measured by fluorescent detection of the released 4-methyl umbelliferone, whereas sialic acid removal from glycoproteins requires more complex assays for detection like ELLA (FIG.1B). Many NAI antibodies sterically hinder the NA active site from binding sialic acid residues on large N-linked glycans, but not on small molecules (Couzens et al., J Virol Methods 210:7-14, 2014; Wan et al., Nat Commun 6:6114, 2015; Zhu et al., Cell Host Microbe 26:729-738.e4, 2019; Gilchuk et al., Cell Host Microbe 26:715-728.e8, 2019; Yasuhara et al., Nat Microbiol 4:1024-1034, 2019). Therefore, serum NAI antibodies are commonly measured with an ELLA approach using the glycoprotein fetuin (FIG.1C) instead of the smaller substrate MUNANA (FIG.1D), which is better suited for identifying antibodies or small molecules that bind the NA active site. Example 3: HA influences on viral NA activity measured with multivalent substrates Bovine fetuin contains up to three sialylated N-linked glycans and several sialylated O-linked glycans (Lin et al., J Proteome Res 17:2861-2869, 2018), making it capable of supporting HA binding (FIG. 1B). In the context of a virus, HA binding to fetuin localizes the neighboring NA proteins near the sialylated glycan substrates, increasing the apparent enzymatic activity (Klenow et al., J Biol Chem 299:102891, 2023; Lai et al., Emerg Microbes Infect 8:327-338, 2019). Consequently, monoclonal antibodies (MAbs) or antisera that inhibit the receptor binding of HA in a test virus (Table 1) can also reduce the NA activity measured by ELLA (FIGS.1E and 1F), resulting in false positive or inflated NAI antibody measurements. This issue of interference from antibodies against HA is quite common with ELLA and has been reported with other HA antibodies (Kosik et al., Virology 500:178-183, 2017; Kosik et al., J Exp Med 216:304-316, 2019; Chen et al., J Virol 93(4):e01526-18, 2019), making it difficult to establish NAI correlations (Ng et al., Nat Med 25:962-967, 2019) or to use it for monitoring NA antigenic drift in circulating viruses. Table 1. HI sera and monoclonal antibody titers against H1N1 / CA09 and H6N1 / CA09 viruses Antisera / MAb H1N1 / CA09 H6N1 / CA09 9531-111023-03Antisera / MAb H1N1 / CA09 H6N1 / CA09 Mouse1 aH6N1 / CA09 0 160 l Example 4: Development and pilot testing of an NA active site proximity assay (NASPA) An ideal NAI assay should function with the different NAs from circulating viruses and should not be influenced by HA antibodies. It was posited that this could be achieved by creating a NA active site proximity assay (NASPA) that uses a large chemical NA active-site-binding inhibitor (NAi), or monoclonal antibody, to replace the function of the sialylated glycans on fetuin that serve as NA substrates in ELLA (FIG.2A). With this approach, it was hypothesized that sera containing steric NAI antibodies (FIG.2A, step 1) would block the large NAi from binding the active site (FIG.2A, step 2), resulting in NA activity increases that could be measured using the small substrate MUNANA (FIG.2A, step 3). In addition, it is expected that sera containing NAI antibodies that bind the active site could also block MUNANA binding and cleavage, resulting in NA activity decreases that could be confirmed in a MUNANA only assay. This concept was initially tested with the H1N1 strain A / California / 07 / 09 (H1N1 / CA09) and several recently identified NA inhibiting monoclonal antibodies (NAi-MAbs) FNI-9, FNI-19 and 1G01, which inhibit a broad range of NAs from type A and type B viruses by binding to the active site (Momont et al., Nature 618:590-597, 2023; Stadlbauer et al., Science 366:499-504, 2019). The pilot test was also performed in the absence and presence of the detergent Triton X-100 (TX100) at a final concentration of 0.025% to compare results from intact virions to spatially separated HA and NA antigens. First, virus was titrated by measuring NA activity with MUNANA to identify amounts that cleaved ~0.25–0.5 pmol / sec of substrate (FIG.2B). Next, the half-maximal inhibitory concentration (IC50) of the NAi-MAbs (FNI-9, FNI-19 and 1G01) was measured with and without TX100 using the determined virus amounts (FIGS.2C and 8A). The same virus amounts were then incubated with serial dilutions of ferret antisera raised against H1N1 / CA09 or a H6N1 / CA09 reassortant virus to investigate if the sera possess detectable steric or enzymatic NAI antibodies by NASPA and the influence of anti-HA antibodies against the test virus. Each well was then incubated with the NAi-MAb at an inhibitory concentration of ~80% (IC80) prior to measuring NA activity using a MUNANA assay. Using this approach, both ferret sera showed NA activities above the negative control ferret sera with all three NAi-MAbs that decreased to the control levels as the sera was diluted (FIGS.2D, 8B and 8C). NASPA titers were determined for each serum by identifying the largest dilution factor that showed NA activities more than 3 standard deviations (SDs) above the mean NA activity from the negative control9531-111023-03sera. The results from all three NAi-MAbs showed that TX100 reduced the NASPA titers from the ferret antisera against H1N1 / CA09 that possessed antibodies against the HA in the H1N1 / CA09 test virus (Table 1). TX100 treatment also eliminated false positive NA activity increases that were observed when NASPA was performed with MAbs that recognize the HA in H1N1 / CA09 (FIG.2E and Table 2). Together, these results suggested that NASPA could be used to measure NAI antibodies and that NA and HA should be separated to avoid any potential interference from antibodies against HA. Table 2. NASPA antibody titers against H1N1 / CA09 ± Triton X-100 with different NAi-MAbs NAi-MAb (FNI-9) NAi-MAb (FNI-19)* NAi-MAb (1G0)* - TX100 +TX100 -TX100 +TX100 -TX100 +TX100 Example 5: Compatibility and optimization of NASPA with the different NAs from influenza vaccines Circulating influenza viruses generally include two type A subtypes (H1N1 and H3N2) and type B viruses that currently are from the Victoria lineage. Therefore, a more thorough analysis of NASPA was performed using different vaccine strains and the NAi-MAb FNI-9 because it was reported to possess broad NA binding properties (Momont et al., Nature 618:590-597, 2023). For the vaccine strains, H1N1 (A / Brisbane / 02 / 2018), H3N2 (A / Hong Kong / 4801 / 2014) and a type B (B / Austria / 1359417 / 2021) virus were used, as well as H6 reassortant viruses that carried the NAs from the vaccine strains (FIG.3A). After titrating the vaccine viruses, the IC50 of the NAi-MAb was measured for each virus after different incubation times in the absence and presence of TX100 (FIG.9). A temporal plot of this data showed that binding of the NAi-MAb required ~180 minutes at 37°C to reach equilibrium for all three NAs (FIG.3B). Some NA-dependent variation in the equilibration times and TX100 effects were noted. Next, different ferret sera incubation times were tested and consistent results were observed with each vaccine virus after 30 minutes (FIG.10). Finally, these parameters were used to analyze each vaccine virus in the absence and presence of TX100 with a panel of NA ferret antisera that were generated using H6 reassortant viruses to avoid recognition of HA in the vaccine strains (FIG.3C). The H1N1 and the type B vaccine viruses were only recognized by matching NA ferret antiserum and the data did not change when TX100 was present (FIG.3C, left and right panels). All three NA ferret antisera and the control sera caused NA activity increases with the H3N2 vaccine virus without TX100 (FIG.3C, upper middle panel). TX100 addition minimized these nonspecific interactions (FIG.3C, lower middle panel), suggesting that binding of9531-111023-03the H3N2 virus to serum components, presumably through HA, may block the access of the NAi-MAb to NA. Example 6: Minimizing anti-HA antibody interference during NASPA To more directly examine how NASPA performs with different NAs in the presence of anti-HA antibodies, the same H6 reassortant viruses carrying the vaccine strain NAs (FIG.3A) were tested with the ferret antisera they were used to generate. In the presence of TX100, all three HA matched ferret antisera showed NA activity increases that varied with each virus (FIG.3D, upper panels). When TX100 was present, only the ferret antisera that matched the test virus blocked the NAi-MAb and significantly increased the NA activity (FIG.3D, lower panels), indicating that the NAi-MAb binding to NA in a virus is susceptible to steric interference from anti-HA antibodies. Based on this conclusion, the TX100 amount was titrated with the same viruses to determine how the results varied with TX100. Final concentrations between 0.025–0.1% TX100 (e.g. ~2–10× the TX100 CMC) limited the interference from the anti-H6 antibodies and produced similar results (FIG.11). Using these parameters, NASPA was performed with viral and recombinant NAs, which produced similar results, further supporting that these conditions for NASPA limit potential interference from anti-HA antibodies (FIG.12). Example 7: Optimization of NAi-MAb concentrations, NA amounts and substrate incubation times NASPA titers are dependent on the ability of sera antibodies to block the binding of the NAi-MAb. Therefore, NASPA titers were measured for the three vaccine NAs in the presence of increasing NAi-MAb concentrations to determine the functional range for the inhibitor. Although different NAi-MAb concentrations were needed to obtain NASPA titers for each NA, consistent titers were obtained with NAi- MAb amounts equivalent to the IC65 or higher for all three NAs (FIG.13). These results indicated that NASPA titers are relatively insensitive to changes in the NAi-MAb concentration when it is used at an IC65 or higher. Decreasing the NA amounts in NASPA can potentially increase the sensitivity for samples with low anti-NA antibody quantities. Therefore, NASPA titers were also measured with decreasing amounts of the three vaccine NAs after different incubation times with MUNANA. Kinetic measurements were taken for 10 minutes after MUNANA addition like the earlier data and endpoint measurements were also taken 10 minutes and 2 hours after MUNANA addition (FIG.14). NASPA titers from the kinetic and 10-minute endpoint readings were very similar and the ability to detect titers was lost with decreasing amounts of NA (FIGS.14D-14F). As expected, NASPA titers from the 2-hour endpoint were more consistent across a much wider range of NA amounts, indicating longer substrate incubation times with lower NA amounts (e.g., < 0.25 pmol / sec) can increase the consistency and sensitivity of NASPA.9531-111023-03Example 8: NASPA and ELLA comparison using NA MAbs against N1, N2 and type B NA NAI titers are commonly measured with a fetuin-based ELLA approach (Couzens et al., J Virol Methods 210:7-14, 2014; Gao et al., J Vis Exp doi:10.3791 / 54573, 2016; Bernard et al., Front Immunol 13:909297, 2022). Therefore, NAI titers from NASPA and ELLA were compared using a large panel of MAbs that were validated by ELISA for binding against N1, N2 and type B NA from recent vaccine strains (FIG.15). Although NASPA titers were generally lower, a good correlation was observed with the ELLA titers for 24 out of the 27 MAbs that were tested and the three that did not correlate produced low ELLA titers (FIG.4A and Table 3). For the NA (N2 / Dar21) from the H3N2 vaccine strain A / Darwin / 9 / 2021, the IC50of the NAi-MAb (FNI-9) was not detected as it was well above 4 mg / ml (FIG.16). To perform NASPA with this NA, a structure-guided substitution (R27D in the FNI-9 heavy chain) was introduced that increased the NAi-MAb binding affinity likely by complementing the charge of residue 344K in N2 / Dar21 that was generally a negatively charged residue (E) in NAs from earlier H3N2 strains. Table 3. NASPA, ELLA and ELISA titers of MAbs against type B NA (NB), N1 and N2 Assay Virus MAb ELISA ELLA NASPA Titer1Titer2Titer3 9531-111023-03Assay Virus MAb ELISA ELLA NASPA Titer1Titer2Titer3GS.15A-15C). 3NASPA titers correspond to the lowest dilution above or below (negative values) the mean control ±3SD. Displayed enzymatic (*) inhibition or (**) activation with MUNANA (FIG.16) Contrary to ELLA, which only shows NA activity decreases (FIGS.15D-15F), NASPA displayed two distinct patterns of activity changes (e.g., increases and decreases) with the MAbs against type B NA (FIG.4B). While activity increases were expected due to steric inhibition of the NAi-MAb, activity decreases suggested these MAbs may inhibit NA enzymatic activity by binding the active site. Supporting this conclusion, each MAb that showed decreased activity in NASPA also displayed the ability to reduce NA activity measured by MUNANA alone (FIG.17). Together, this data shows that NASPA provides similar data with individual antibodies as ELLA and that it can also differentiate steric and enzymatic NAI antibodies and be performed in a shorter amount of time. However, the potential need to modify the NAi- MAb for different virus strains can create a substantial lag time for performing the assay. Example 9: NASPA can provide antigenic data for NAs from type A H1N1 and Type B strains Monitoring NA antigenic drift in circulating strains is an ideal application for NASPA. To test this possibility, a panel of recent H1N1 vaccine strains from 2007 to 2019 that contained several strains previously analyzed by ELLA (Gao et al., NPJ Vaccines 7:79, 2022) were assayed. After titrating the viruses with MUNANA and determining the NAi-MAb IC50for each strain (FIG.18A), plates were set up to individually analyze each strain with the five matching NA ferret antisera generated with H6 reassortant viruses. The NASPA graphs indicated that the NA from the H1N1 strain A / Brisbane / 59 / 2007 is antigenically distinct from the NAs of post-pandemic H1N1s with slight similarity to the NA from the original 2009 H1N1 pandemic strain A / California / 09 (FIG.5A). In contrast, NAs from the H1N1 strains A / Michigan / 45 / 2015, A / Brisbane / 02 / 2018 and A / Victoria / 2570 / 2019 all showed high antigenic similarity that was slightly related to the NA from A / California / 09. This conclusion was supported by the NASPA titer analysis which showed trends similar to the previously reported ELLA titers with the same reagents (Table 4 and Gao et al., NPJ Vaccines 7:79, 2022).9531-111023-03Table 4. NASPA and previously determined ELLA titers for H1N1 vaccine viruses αH6N1 / BR07 αH6N1 / CA09 αH6N1 / MI15 αH6N1 / BR18 αH6N1 / Vic19 H1N1 / BR07 1280 80 <40 <40 <40 reciprocal old. ELLA titers in parenthesis are from a previous study using the same ferret sera (Gao et al., NPJ Vaccines 7:79, 2022). NAs from type B vaccine strains have received little attention due to the difficulties in generating ELLA reagents (e.g., antisera that do not recognize HA in the test viruses) and can benefit from characterization by NASPA. Therefore, several type B vaccine strains were also analyzed using both matched and unmatched ferret antisera. After determining the NAi-MAb IC50 for each strain (FIG.18B), the analysis was performed with NASPA. The results indicated that the NA in the Yamagata-lineage strain (B / Phuket / 2013) was antigenically distinct from the four Victoria-lineage strains that displayed incremental antigenic changes from B / Brisbane / 60 / 2008-like to B / Austria1359417 / 2021-like (FIG.5B). NASPA titers also supported these conclusions (Table 5). Together, these results indicated that NASPA could be implemented to assess NA antigenicity in circulating H1N1 and type B influenza strains. Table 5. NASPA titers of type B vaccine viruses αH6NB / Phu13αB / BR08αH6NB / Austria21 B / Phu13 320 <40 <40 N the control mean +3SD. Example 10: NASPA human sera profiling indicates enzymatic NAI antibodies are prevalent in adults NASPA can potentially help to measure and characterize NAI antibody responses from a NA vaccine in humans. Therefore, previously analyzed sera samples from an H1N1 human challenge study (Memoli et al., mBio 7:e00417-16, 2016) were obtained and NASPA and a MUNANA analysis were performed in a blinded fashion using the challenge virus and a H6 reassortant carrying the same NA. Results with both test viruses indicated the presence of steric and enzymatic NAI antibodies with the latter being more common (FIGS.6A and 19). Unblinding revealed that the steric and enzymatic NAI titer aligned well9531-111023-03with the previously obtained ELLA titers and that the enzymatic NAI titer pattern was more similar (Memoli et al., mBio 7:e00417-16, 2016). Interestingly, some individuals possessed either measurable steric or enzymatic NAI antibodies whereas others possessed both and these profiles showed some changes post- challenge (FIGS.6A and 6B). The high frequency of enzymatic NAI antibodies was unexpected, and NA activity can be reduced by other factors than antibody binding as it is a labile and Ca2+sensitive enzyme. Therefore, an immunoglobulin depletion was also performed from the strongest post-challenge sera samples with a combination of protein A and G agarose and the NASPA and MUNANA analyses were repeated. In all cases, the activity changes measured by NASPA and MUNANA were reduced following protein A and G incubation and in most they were completely lost, confirming the results were antibody mediated. It was also observed that the enzymatic NA inhibition in some sera was somewhat reduced after incubation with the control agarose beads, suggesting some reduction may be due to non-specific binding. These results indicate that NAI antibody responses in adults are complex and function by two different mechanisms of NA inhibition that can be differentiated by an approach involving NASPA and a simple MUNANA assay. Example 11: NASPA is a flexible platform that can utilize different large ‘bulky’ NA inhibitors Although NASPA with the NAi-MAb is more time efficient than ELLA, additional studies were performed to improve it further by replacing the NAi-MAb with a large bulky synthetic inhibitor, which can potentially bind different NAs with similar affinities. This analysis used a designed and synthesized biotinylated NA sialoside-based inhibitor (4-guanidino-Neu5Acα2–3Galβ1–4GlcNAc–βProNH-PEG4- Biotin) that binds NAs with nanomolar affinity (see Examples 12-16) and the binding was found to be inhibited by ferret antisera in an ELISA format (FIG.20). Based on these results, it was reasoned that the size of the synthesized NAi could be increased by mixing it with streptavidin (FIG.7A) and the mixture could be incorporated into NASPA to detect steric NAI antibodies (FIG.7B). The synthesized NAi exhibited a higher nanomolar IC50for N1 that slightly increased after premixing with streptavidin (1:2 molar ratio, NAi+Sa) and equilibrium was reached within 10-30 minutes (FIGS.7C and 21A), whereas the NAi-Mab required ~ 3 hours. The compound was then tested in NASPA using the H1N1 / BR18 virus with NA matching and non-matching ferret antisera. The expected NA activity increase was observed with the NA matching H6N1 / BR18 ferret antisera and the mixture of the NAi+Sa mixture (FIG.7D), indicating the biotinylated sialoside-streptavidin complex is suitable for NASPA. It was previously necessary to modify the NAi-MAb (FNI-9) to improve the affinity for a NA from a recent H3N2 virus, thus the IC50of the synthesized NAi was measured with and without streptavidin for several recent H3N2 viruses. In contrast to the NAi-MAb (FNI-9), both the NAi and NAi+Sa mixture showed consistent IC50 values, suggesting a set concentration of the synthetic NAi+Sa complex could be used for NASPA (FIG.7E). Finally, the synthetic NAi was tested using H3N2 A / Darwin / 9 / 2021 and the purified N2 MAbs. Steric inhibition was observed with the three MAbs that were detected from the hybridoma media with the modified NAi-MAb, although the activity increases for two of them were less9531-111023-03pronounced, likely due to the different spatial footprints of the NAi+Sa and the NAi-MAb (FIGS.7F and 4D). Additionally, an activity loss was observed with another MAb indicative of enzymatic inhibition, which was confirmed with a MUNANA assay (FIG.21B). This purified Ab, which could also serve as an NAi- MAb in NASPA, was not previously detected with the NAi-MAb due to low concentrations in the hybridoma media. Together, these data demonstrate that NASPA is an efficient and cost-effective platform that can utilize different large ‘bulky’ NA active site inhibitors to profile NA antigenicity and antibody responses, making it well suited to aid in the development of new influenza vaccines containing NA. CONCLUSION Currently influenza vaccine strains are only recommended for the HA antigens due to the absence of a simple NA antigenic assay that can be implemented to help select appropriate vaccine strains for the NA antigens. The present disclosure describes the development of a simple and cost-effective NA active site proximity assay (NASPA) for measuring NAI antibody responses and antigenicity. The assay is performed in a 96-well plate format and is based on the concept of replacing the large sialylated glycans that are used for NA substrates in ELLA with a large ‘bulky’ NA inhibitor. The entire assay can be run in a single day as it only requires two up front titrations steps to determine the NA and inhibitor amounts prior to its execution, which involves sequentially adding the NA, sera, inhibitor, and substrate. Some applications of this easily automatable assay were also demonstrated by profiling NA antigenicity in different vaccine strains and NAI antibody responses in humans. A major advantage of NASPA is that it is not sensitive to antibodies against the HA in the test virus, making it ideal for utilizing the reagents generated to analyze HA antigenicity in circulating strains to also analyze NA antigenicity. This was achieved by including detergent to separate the NA and HA antigens in the test virus and using a monovalent small molecule NA reporter substrate (MUNANA) for detection. These two steps eliminate potential steric interference from anti-HA antibodies (Kosik et al., Virology 500:178-183, 2017; Kosik et al., J Exp Med 216:304-316, 2019; Chen et al., J Virol 93(4):e01526-18, 2019) and HA-mediated increases in the apparent activity of NA in a virus (Klenow et al., J Biol Chem 299:102891, 2023) that can contribute to false positive and inflated NAI titer measurements by ELLA. This property is one of the reasons NASPA was used to examine NA antigenicity in type B viruses as it is difficult to develop ELLA reagents for these viruses that minimize the potential interference from anti-HA antibodies. While only a few strains were analyzed, NASPA can easily be expanded with the limiting factor being the availability of matching ferret antisera. Also described is the use of a chemoenzymatically synthesized NA inhibitor that can be coupled with streptavidin. The incubation time for this inhibitor is ~2 hours less than with NAi-Mab and it showed similar binding affinities across the NAs that were tested, shortening the assay time further and potentially eliminating the need to run an IC50 test for each NA. The surprising finding of prevalent enzymatic NAI antibodies in the human sera analyses, prompted the use of an additional Ig extraction step to ensure that the NA inhibition was indeed antibody-mediated.9531-111023-03This confirmed the unexpected observation, suggesting that adults commonly possess antibodies capable of inhibiting substrate binding or reducing the activity of NAs. Supporting this conclusion, 6 out of the 7 individuals observed with steric NAI antibodies post-challenge also possessed enzymatic NAI antibodies, indicating that adults can generate polyclonal NAI responses that function by two different mechanisms. Furthermore, multiple NA MAbs that inhibited NA enzymatic activity were isolated from mice that were immunized three times, suggesting the development of these antibodies is related to a number of exposures. Based on these results, it is speculated that pediatric populations are likely to be more biased toward steric NAI antibodies and that enzymatic NAI antibodies develop over time from multiple influenza infections. Regardless, distinguishing between these two types of responses in humans may be a factor for optimizing the design of NA vaccines and for identifying a correlate of protection for NA. Mechanistically, anti-NA antibodies can provide protection by directly inhibiting enzymatic activity (like FNI-9 and 1G01), sterically inhibiting enzymatic activity, activating antibody-dependent cellular cytotoxicity and phagocytosis via the Fcγ receptor, or a combination of these (Wan et al., Nat Commun 6:6114, 2015; Gilchuk et al., Cell Host Microbe 26:715-728.e8, 2019; Yasuhara et al., Nat Microbiol 4:1024-1034, 2019; Momont et al., Nature 618:590-597, 2023; Stadlbauer et al., Science 366:499-504, 2019; DiLillo et al., J Clin Invest 126:605-610, 2016; Job et al., mBio 10(5):e01667-19, 2019). NASPA is designed to help detect steric and enzymatic NAI antibodies, many of which are also likely to activate the Fcγ receptor, suggesting the measurements capture most of the protective antibodies. Overall, the present disclosure provides a time and cost-effective method that can be used to distinguish steric and enzymatic NAI responses in animals and humans, alleviate confounding results observed from ELLA due to anti-HA antibody interference, and help to select NA seasonal vaccine strains. Example 12: Materials and Methods This example provides the materials and methods for the studies described in Examples 13-16. Materials and general methods Chemicals were purchased and used without further purification. GalβpNP and GalβMU were from Fisher Scientific. Nuclear magnetic resonance (NMR) spectra were recorded in the NMR facility of the University of California, Davis on 600 MHz and 800 MHz Bruker Avance III-NMR spectrometers. Chemical shifts are reported in parts per million (ppm) on the δ scale. High resolution electrospray ionization (ESI) mass spectra were obtained using a Thermo Electron LTQ-Orbitrap Hybrid mass spectrometer at the mass spectrometry facility at the University of California, Davis. Column chromatography was performed using a CombiFlash® Rf 200i system with an ODS-SM (C18) column (14 g, 50 µm, 120 Å, Yamazen) or manually using columns packed with silica gel 60 Å (230–400 mesh, Sorbent Technologies). Gel filtration chromatography was performed with a column (100 cm × 2.5 cm) packed with Bio-Gel P-2 Fine resins (Bio-Rad). Thin layer chromatography (TLC) was performed on9531-111023-03silica gel plates (Sorbent Technologies) using anisaldehyde sugar stain or 5% sulfuric acid in ethanol stain for detection. Arthrobacter ureafaciens sialidase was purchased from EY laboratories, Inc. (San Mateo, CA, USA). Vibrio cholerae sialidase, Clostridium perfringens sialidase (CpNanI), and β-galactosidase from Aspergillus oryzae (≥ 8 units / mg solid) were purchased from Sigma-Aldrich. Recombinant Neisseria meningitidis CMP-sialic acid synthetase (NmCSS) (Yu et al., Bioorg. Med. Chem.12, 6427–6435, 2004), Pasteurella multocida α2–3-sialyltransferase 1 (PmST1) (Yu et al., J. Am. Chem. Soc.127, 17618–17619, 2005) and its M144D mutant (PmST1 M144D) (Sugiarto et al., ACS Chem. Biol.7, 1232–1240, 2012), Campylobacter jejuni Cst-I (CjCst-I) (Zhang et al., Molecules 28, 2753, 2023), Photobacterium sp. α2–6- sialyltransferase mutant (Psp2,6ST_A366G), human cytoplasmic sialidase hNEU2 (Li et al., Mol. Biosyst.7, 1060–1072, 2011), Streptococcus pneumoniae sialidases SpNanA (Tasnima et al., Org. Biomol. Chem.15, 160–167, 2016), SpNanB (Xiao et al., J. Org. Chem.83, 10798–10804, 2018), and SpNanC (Xiao et al., ACS Catal.8, 43–47, 2018), and Bifidobacterium infantis sialidase (BiNanH2) (Sela et al., J. Biol. Chem. 286, 11909–11918, 2011) were expressed and purified as described previously. IAV and IBV NAs were expressed in Sf9 cells using baculovirus expression systems as reported previously (Mishra et al., Angew. Chem. Int. Ed. Engl.63, e202403133, 2024). His6-tagged tetrabrachion tetramerization domain-containing N1-BR18 (82–469 aa) and N2-Kan17 (74–469 aa) were from the respective IAV strains A / Brisbane / 02 / 2018 (H1N1) or A / Kansas / 14 / 2017 (H3N2); Strep-tagged tetrabrachion tetramerization domain-containing N1- Vic19 (35–469 aa) and N2-Dar21 (74–469 aa) were from the respective strains A / Victoria / 2570 / 2019 (H1N1) and A / Darwin / 9 / 2021 (H3N2); Strep-tagged tetrabrachion tetramerization domain-containing NB- Aus21 (39–466 aa) and NB-Phu13 (39–466 aa) were from the respective strains B / Austria / 1359417 / 2021 and B / Phuket / 3073 / 2013. Pierce™ streptavidin and NeutrAvidin-coated plates were purchased from Thermo Fisher Scientific. The binding was detected using a horseradish peroxidase (HRP)-conjugated anti-His6 monoclonal antibody (Thermo Fisher Scientific) or HRP-conjugated anti-StrepTag II monoclonal antibody (Sigma-Aldrich), and a colorimetric substrate solution 3,3’,5,5’-tetramethylbenzidine (TMB) (Thermo Fisher Scientific). Biolayer interferometry (BLI) assays were performed using a Octet® RED384 instrument. Octet® high precision streptavidin (SAX) Biosensors, Octet® kinetics buffer 10 ×, and Octet® 384-well titted-bottom plates were purchased from Sartorius Corporation. Dynabeads MyOne Streptaidin T1 were from Invitrogen. Synthesis of Neu5Acα2–3GalβMU (18) GalβMU (100 mg, 0.30 mmol), CTP (234 mg, 0.44 mmol), and Neu5Ac (119 mg, 0.38 mmol) were dissolved in water in a 50 mL centrifuge tube containing Tris-HCl buffer (100 mM, pH 8.5) and MgCl2(20 mM). NmCSS (3 mg) and CjCst-I (2 mg) were then added. The reaction mixture (20 mL) was incubated at 30°C with agitation at 180 rpm. The product formation was monitored by HRMS. After 16 hours, the reaction mixture was incubated in a boiling water bath for 5 minutes to denature the enzymes, cooled down to room temperature, then centrifuged at 8000 rpm for 30 minutes at 4°C. The supernatant was concentrated9531-111023-03and purified by a preconditioned DSC-18 SPE cartridge (bed wt.10 g). After washing the cartridge with water (30 mL), a mixture solvent of methanol in water (40%) was used to elute the product. The solvent was removed by evaporation in vacuo and the pure product was obtained as a white powder (171 mg, 89% yield).1H NMR (800 MHz, D2O) δ 7.45 (d, J = 8.9 Hz, 1H), 6.97 (dd, J = 8.8, 2.5 Hz, 1H), 6.84 (d, J = 2.5 Hz, 1H), 5.98 (t, J = 1.2 Hz, 1H), 5.15 (d, J = 7.8 Hz, 1H), 4.27 (dd, J = 9.8, 3.3 Hz, 1H), 4.08 (d, J = 3.6 Hz, 1H), 3.95–3.86 (m, 4H), 3.85–3.77 (m, 3H), 3.74–3.68 (m, 2H), 3.67 (dd, J = 10.4, 2.1 Hz, 1H), 3.65– 3.60 (m, 2H), 3.17 (q, J = 7.4 Hz, 1H), 2.81 (dd, J = 12.5, 4.8 Hz, 1H), 2.23 (d, J = 1.1 Hz, 3H), 2.03 (s, 3H), 1.86 (t, J = 12.2 Hz, 1H), 1.25 (t, J = 7.4 Hz, 2H).13C NMR (200 MHz, D2O) δ 174.98, 173.84, 164.03, 159.39, 155.83, 153.43, 126.42, 114.66, 113.93, 110.94, 103.27, 99.95, 99.84, 75.51, 75.26, 72.90, 71.74, 68.78, 68.37, 68.01, 67.24, 62.46, 60.68, 59.30, 51.69, 46.63, 39.73, 22.04, 17.86, 8.21. HRMS (ESI- Orbitrap) m / z: [M-H]- calculated for C27H34NO16628.1883; found 628.1884. General procedures for synthesizing biotinylated glycan acceptors 19 and 20 GalβProN3(Yu et al., J. Am. Chem. Soc.127, 17618–17619, 2005) or LacNAcβProN3(Lau et al., Chem. Commun.46, 6066–6068, 2010) (100–150 mg) was dissolved in water-methanol solution (12 mL, 1:2 by volume), a catalytic amount of 10% palladium on charcoal was added to a 100 mL round bottom flask. The mixture was stirred under a hydrogen environment for 2 hours. The solution was passed through a syringe filter and concentrated. After lyophilization, the obtained glycosyl propylamine was dissolved in anhydrous DMF (10 mL) and was added to a solution of biotin-PEG4-CO2H (1.05 equiv.), HBTU (1.1 equiv.), and DIPEA (3.5 equiv.) in anhydrous DMF (5 mL). The reaction mixture was stirred at room temperature for 24 hours. The solvent was removed by evaporation in vacuo and the residue was purified by Bio-gel P2 to obtain the desired pure product. GalβProNH-PEG4-Biotin (19) 261 mg, 87% yield.1H NMR (600 MHz, D2O) δ 4.62 (dd, J = 7.8, 4.8 Hz, 1H), 4.43 (dd, J = 7.8, 4.2 Hz, 1H), 4.39 (d, J = 7.8 Hz, 1H), 3.97 (dt, J = 10.2, 6.0 Hz, 1H), 3.93 (d, J = 3.6 Hz, 1H), 3.86–3.58 (m, 23H), 3.52 (dd, J = 9.6, 7.8 Hz, 1H), 3.40 (t, J = 5.4 Hz, 2H), 3.36–3.28 (m, 3H), 3.01 (dd, J = 13.2, 5.4 Hz, 1H), 2.79 (d, J = 13.2 Hz, 1H), 2.52 (t, J = 6.0 Hz, 2H), 2.29 (t, J = 7.2 Hz, 2H), 1.85 (p, J = 6.6 Hz, 2H), 1.79–1.53 (m, 4H), 1.48–1.38 (m, 2H).13C NMR (150 MHz, D2O) δ 176.87, 173.88, 165.29, 102.82, 75.13, 72.77, 70.77, 69.65, 69.62, 69.60, 69.56, 69.54, 69.50, 69.41, 69.33, 68.88, 68.63, 67.66, 67.62, 66.80, 62.07, 60.97, 60.24, 55.35, 39.69, 38.89, 36.31, 36.08, 35.46, 28.45, 27.89, 27.87, 27.70, 25.14. HRMS (ESI-Orbitrap) m / z: [M+Na]+calculated for C30H54N4NaO13S 733.3306; found 733.3306. LacNAcβProNH-PEG4-Biotin (20) 222 mg, 89% yield.1H NMR (800 MHz, D2O) δ 4.60 (dd, J = 8.0, 4.8 Hz, 1H), 4.50 (d, J = 8.0 Hz, 1H), 4.46 (d, J = 8.0 Hz, 1H), 4.41 (dd, J = 8.0, 4.8 Hz, 1H), 3.97 (dd, J = 12.8, 2.4 Hz, 1H), 3.94–3.88 (m,9531-111023-032H), 3.82 (dd, J = 12.8, 5.6 Hz, 1H), 3.80–3.55 (m, 23H), 3.53 (dd, J = 9.6, 8.0 Hz, 1H), 3.38 (t, J = 6.4 Hz, 2H), 3.35–3.30 (m, 1H), 3.27 (dt, J = 14.4, 7.2 Hz, 1H), 3.18 (dt, J = 13.6, 7.2 Hz, 1H), 2.98 (dd, J = 12.8, 4.8 Hz, 1H), 2.77 (d, J = 13.6 Hz, 1H), 2.51 (t, J = 6.4 Hz, 2H), 2.27 (t, J = 7.2 Hz, 2H), 2.03 (s, 3H), 1.85– 1.53 (m, 6H), 1.50–1.36 (m, 2H).13C NMR (200 MHz, D2O) δ 176.88, 174.44, 173.83, 165.29, 102.86, 101.03, 78.46, 75.32, 74.74, 72.48, 72.40, 70.93, 69.62, 69.60, 69.58, 69.54, 69.48, 69.39, 68.85, 68.51, 67.69, 66.78, 62.05, 60.98, 60.22, 60.05, 55.33, 55.06, 39.67, 38.87, 36.22, 36.02, 35.43, 28.35, 27.86, 27.67, 25.11, 22.13. HRMS (ESI-Orbitrap) m / z: [M+H]+calculated for C38H68N5O18S 914.4280; found 914.4279. Chemical synthesis of 4-N-substituted Neu5Ac2en including 4NH2Neu5Ac2en (3), 4N3Neu5Ac2en (5), and 4NAcNeu5Ac2en (6), as well as 4NH2Neu5Ac (7), Methyl 5-acetamido-2,4,7,8,9-penta-O-acetyl- 3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate (14) To a stirred solution of Neu5Ac (1) (10.0 g, 32.4 mmol) in MeOH (100 mL), 1.5 g of H+resin was added, and the mixture was stirred at room temperature for 16 hours. The resin was filtered, and the reaction mixture was concentrated by evaporation in vacuo. Methylated Neu5Ac was dissolved in a stirred solution in pyridine (60 mL) at 0°C. Acetic anhydride (60 mL) was added, and the resulting mixture was stirred at room temperature for 18 hours. The solvent was then removed by evaporation in vacuo, and the residue was purified by silica gel column chromatography (hexane:EtOAc = 1:9, by volume) to produce the protected methyl ester (14) (15.0 g, 87% yield) as a white solid.1H NMR (800 MHz, CDCl3) δ 5.39–5.37 (m, 0.8H), 5.36-5.35 (m, 0.2H), 5.32–5.21 (m, 1.8H), 5.19 (ddd, J = 7.2, 6.4, 3.2 Hz, 0.2H), 5.07 (ddd, J = 6.4, 5.6, 2.4 Hz, 0.8H), 5.01 (ddd, J = 12.0, 10.4, 4.8 Hz, 0.2H), 4.69 (dd, J = 10.4, 2.4 Hz, 0.2H), 4.49 (dd, J = 12.8, 2.4 Hz, 0.8H), 4.35 (dd, J = 12.8, 3.2 Hz, 0.2H), 4.14–4.09 (m, 3H), 4.05 (dd, J = 12.0, 5.6 Hz, 0.2H), 3.79 (s, 2.4H), 3.76 (s, 0.6H), 2.55 (dt, J = 12.8, 4.8 Hz, 1H), 2.14 (d, J = 3.2 Hz, 4H), 2.09 (d, J = 3.2 Hz, 1H), 2.06 (s, 2.2H), 2.05–2.02 (m, 6.4H), 1.89 (s, 3H), 1.26 (t, J = 7.2 Hz, 1H).13C NMR (200 MHz, CDCl3) δ 170.6, 170.4, 170.1, 161.5, 145.1, 107.5, 75.6, 70.6, 67.7, 61.9, 57.4, 52.6, 48.8, 23.4, 20.9, 20.8, 20.7. HRMS (ESI-Orbitrap) m / z calculated for C23H32NO16-[M+FA-H]-578.1727, found 578.1714. Methyl 7,8,9-tri-O-acetyl-2,3,4,5-tetradeoxy-2,3-didehydro-2,3-trideoxy-4′,5′-dihydro-2′- methyloxazolo[5,4-D]-D-glycero-D-talo-non-2-enonate (15) To the stirred solution of the protected methyl ester (14) (15.0 g, 28.1 mmol) in DCM (150 mL), BF3.Et2O (20.8 mL, 168.6 mmol) was added drop-wisely, and the mixture was stirred at room temperature under monitored with TLC for 14 hours. The reaction mixture was then diluted with ethyl acetate and washed gradually with saturated NaHCO3, water, and brine. The solvent was concentrated in vacuo and the residue was purified with a flash silica gel column chromatography (hexane:EtOAc = 1:4, by volume) to form the 4,5-oxazoline product 15 (10.5 g, 90% yield) as a white foam.1H NMR (800 MHz, CDCl3) δ 6.37 (dd, J = 4.0, 0.8 Hz, 1H), 5.67–5.60 (m, 1H), 5.49–5.40 (m, 1H), 4.81 (dd, J = 8.8, 4.0, 1H), 4.58 (dd, J =9531-111023-0312.0, 2.4 Hz, 1H), 4.21 (dd, J = 12.8, 6.4 Hz, 1H), 3.94 (t, J = 9.6 Hz, 1H), 3.80 (s, 3H), δ 3.42 (dd, J = 10.4, 2.4 Hz, 1H), 2.14 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 1.99 (s, 3H).13C NMR (200 MHz, CDCl3) δ 170.6, 169.8, 169.6, 167.2, 161.8, 147.1, 107.5, 76.7, 72.2, 70.2, 68.8, 62.0, 61.9, 52.5, 20.8, 20.8, 20.6, 14.1. Methyl 5-acetamido-7,8,9-tri-O-acetyl-4-azido-2,6-anhydro-3,4,5-trideoxy-D-glycero-D-galacto-non-2- enonate (16) The 4,5-oxazoline intermediate (15) (10.0 g, 24.2 mmol) was directly dissolved in dry tert-butanol (100 mL), TMSN3(6.4 mL, 48.4 mmol) was added, and the mixture was stirred at 80°C for 14 hours. After the reaction finished and cooled down, aqueous NaNO2 (1.0 g in 10 mL water) was added. HCl (1 M) was then added dropwise to the mixture until effervescence had ceased. The mixture was diluted with ethyl acetate and washed gradually with water, saturated NaHCO3, and brine. The solvent was concentrated by evaporation in vacuo, and the residue was purified by silica gel column chromatography (hexane:EtOAc = 2:4, by volume) to form the 4-azide derivative 16 (8.9 g, 81% yield) as a white solid.1H NMR (800 MHz, CDCl3) δ 5.98 (d, J = 2.4 Hz, 1H), 5.79 (d, J = 8.0 Hz, 1H), 5.45 (dd, J = 5.6, 2.4 Hz, 1H), 5.35–5.31 (m, 1H), 4.61 (dd, J = 12.8, 3.2 Hz, 1H), 4.52 (dd, J = 9.6, 2.4 Hz, 1H), 4.48 (dd, J = 8.8, 2.4 Hz, 1H), 4.19 (dd, J = 12.0, 6.4 Hz, 1H), 3.85–3.81 (m, 1H), 3.80 (s, 3H), 2.13 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H).13C NMR (200 MHz, CDCl3) δ 170.6, 170.4, 170.1, 161.5, 145.1, 107.5, 75.6, 70.6, 67.7, 61.9, 57.4, 52.6, 48.8, 23.4, 20.9, 20.8, 20.7. HRMS (ESI-Orbitrap) m / z calculated for C19H25N4O12- [M+FA-H]-501.1474, found 501.1463. Synthesis of 4N3Neu5Ac2en (5) The protected 4N3Neu5Ac2en methyl ester 16 (80 mg, 1 equiv.) was dissolved in MeOH and water (1:1) and treated with NaOH (pH>9), the reaction was monitored until completion by TLC (EtOAc:MeOH:H2O = 5:3:1 by volume) and confirmed with HRMS. The reaction mixture was concentrated, and the product 5 was purified by a Bio-gel P-2 column to form a light yellow solid (52.6 mg, 95% yield).1H NMR (800 MHz, D2O) δ 5.70 (d, J = 1.6 Hz, 1H), 4.32 (dd, J = 9.6, 2.4 Hz, 1H), 4.29 (d, J = 11.2 Hz, 1H), 4.23–4.19 (m, 1H), 3.94 (ddd, J = 8.8, 6.4, 2.4 Hz, 1H), 3.89 (dd, J = 12.0, 3.2 Hz, 1H), 3.66–3.62 (m, 2H), 2.07 (s, 3H).13C NMR (200 MHz, D2O) δ 174.5, 169.1, 149.3, 103.3, 75.2, 69.7, 68.0, 63.1, 59.2, 47.7, 22.1. HRMS (ESI-Orbitrap) m / z calculated for C11H15N4O7- [M-H]- 315.0946, found 315.0940. Synthesis of 4NH2Neu5Ac2en (3) The 4N3Neu5Ac2en (5) (30 mg, 1 equiv.) obtained above was dissolved in water and treated with 1M of trimethylphosphine (PMe3) in THF (0.2 mL, 2.0 equiv.). After 4 M NaOH solution was added to adjust the pH of the reaction to over 8.0. The reaction was stirred at room temperature for 3 hours and was monitored until completion by TLC (EtOAc:MeOH:H2O = 5:4:1 by volume) and confirmed with HRMS. The solvent was removed and purified using bio-gel P-2 column to form the product 3 as a white solid (23.69531-111023-03mg, 86% yield).1H NMR (800 MHz, D2O) δ 5.96 (d, J = 2.4 Hz, 1H), 4.43 (d, J = 10.4 Hz, 1H), 4.39 (t, J = 9.6 Hz, 1H), 4.26 (dd, J = 8.8, 2.4 Hz, 1H), 3.95 (ddd, J = 8.8, 5.6, 2.4 Hz, 1H), 3.88 (dd, J = 12.0, 3.2 Hz, 1H), 3.72 (d, J = 9.6, 1H), 3.67 (dd, J = 11.2, 5.6 Hz, 1H), 2.08 (s, 3H).13C NMR (200 MHz, D2O) δ 174.8, 165.3, 103.6, 103.5, 75.5, 69.7, 67.6, 62.9, 49.8, 45.6, 22.1. HRMS (ESI-Orbitrap) m / z calculated for C11H17N2O7- [M-H]- 289.1041, found 289.1034. Synthesis of 4NAcNeu5Ac2en (6) The conversion from 4-azido group to the 4-acetamido group was achieved by dissolving the protected 4N3Neu5Ac2en methyl ester 16 (40 mg, 1 equiv.) in pyridine (0.5 mL) with thioacetic acid (0.5 mL, 40 equiv.). Then the compound was de-protected using NaOH (reaction’s pH > 9) and a mixture solvent of MeOH and water (1:1). The reaction was monitored until completion by TLC (EtOAc:MeOH:H2O = 5:3:1 by volume) and confirmed with HRMS. The reaction mixture was concentrated upon completion and purified using a Bio-gel P-2 column to form compound 6 as a white syrup (27.3 mg, 94% yield).1H NMR (600 MHz, D2O) δ 5.56 (d, J = 1.8 Hz, 1H), 4.77 (d, J = 2.4 Hz, 1H), 4.34 (dd, J = 10.8, 1.2 Hz, 1H), 4.12 (t, J = 10.2 Hz, 1H), 3.97 (ddd, J = 9.0, 6.6, 2.4 Hz, 1H), 3.91 (dd, J = 12.0, 3.0 Hz, 1H), 3.68–3.64 (m, 2H), 2.01 (s, 3H), 2.00 (s, 3H), 1.92 (s, 2H).13C NMR (150 MHz, D2O) δ 174.4, 174.2, 169.5, 148.8, 105.5, 75.4, 69.8, 68.2, 63.1, 48.2, 47.9, 22.0, 21.9. HRMS (ESI-Orbitrap) m / z calculated for C13H19N2O8- [M-H]- 331.1147, found 331.1137. Methyl 5-acetamido-2,7,8,9-tetra-O-acetyl-4-amino-3,4,5-trideoxy-D-glycero-β-D-galacto-2- nonulopyranosonate (17) To the stirred solution of 4-azide derivative 16 (7.32 g, 16.1 mmol) in AcOH (36 mL) containing NaOAc (3.60 g), N-bromosuccinimide (3.40 g, 19.2 mmol) was added and the mixture was stirred at room temperature for 20 minutes. The reaction mixture was then diluted with DCM and washed gradually with brine, saturated NaHCO3, and brine. The solvent was concentrated in vacuo to give the crude product as a white solid, which contains two 3-Br isomers. The crude product was then directly dissolved in 1,6-dioxane (100 mL), Bu3SnH (18.1 mL, 67.2 mmol) was added and AIBN (279.2 mg, 1.7 mmol) in dioxane was added drop-wisely, and the mixture was heated at 80°C for 3 hours. The solvent was concentrated in vacuo and extracted with acetonitrile and hexane. The residue obtained was purified by silica gel column chromatography (EtOAc:MeOH = 2:1, by volume) to form the protected 4-amine derivative 17 (3.3 g, 42%) as a white solid.1H NMR (800 MHz, CDCl3) δ 5.96 (d, J = 9.6 Hz, 1H), 5.34 (dd, J = 4.8, 2.4 Hz, 1H), 5.00 (ddd, J = 7.2, 4.8, 2.4 Hz, 1H), 4.47 (dd, J = 12.8, 2.4 Hz, 1H), 4.08 (dd, J = 12.4, 6.9 Hz, 1H), 4.02 (dd, J = 10.4, 2.4 Hz, 1H), 3.73 (s, 3H), 3.63 (dd, J = 10.4, 20.8 Hz, 1H), 3.15–3.06 (m, 1H), 2.40 (dd, J = 13.6, 4.0 Hz, 1H), 2.07 (s, 3H), 2.07 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H), 1.93 (s, 3H), 1.72 (dd, J = 13.6, 12.0 Hz, 1H), 0.85 (t, J = 7.2 Hz, 1H).13C NMR (200 MHz, CDCl3) δ 171.2, 170.6, 170.4, 170.4, 168.4, 167.0, 97.6, 72.7,9531-111023-0371.7, 68.3, 62.2, 53.0, 52.6, 48.6, 39.2, 23.3, 20.9, 20.8, 20.8, 20.7. HRMS (ESI-Orbitrap) m / z calculated for C21H31N2O14- [M+FA-H]-535.1781, found 535.1769. 4NH2Neu5Ac (7) To the stirred solution of protected 4-amine derivative 17 (3.0 g, 6.1 mmol) in MeOH (10 mL) and H2O (10 mL), aqueous NaOH (4 M) was added drop-wisely until the pH reached to 10. The reaction was neutralized after 30 minutes by aqueous HCl (1 M). The crude product was purified by a bio-gel P-2 column to form 1.3 g of compound 7 with 67% yield.1H NMR (800 MHz, D2O) δ 4.06 (d, J = 10.4 Hz, 1H), 3.98 (t, J = 10.4 Hz, 1H), 3.84 (dd, J = 12.0, 3.2 Hz, 1H), 3.76 (ddd, J = 9.6, 6.4, 3.2 Hz, 1H), 3.64–3.59 (m, 1H), 3.52 (d, J = 9.6 Hz, 1H), 3.43–3.39 (m, 1H), 2.19 (dd, J = 12.8, 4.0 Hz, 1H), 2.06 (s, 3H), 1.87 (t, J = 12.8 Hz, 1H).13C NMR (200 MHz, D2O) δ 176.48, 174.90, 95.50, 70.16, 70.01, 68.43, 63.21, 50.32, 48.50, 37.37, 22.08. HRMS (ESI-Orbitrap) m / z calculated for [M-H]-307.1147, found 307.1139. Synthesis of Neu5Acα2–3Gal / LacNAcβProNH-PEG4-Biotin (8c–d) and Neu5Acα2– 6Gal / LacNAcβProNH-PEG4-Biotin (8e–f) The acceptor (GalβProNH-PEG4-Biotin 18 or LacNAcβProNH-PEG4-Biotin 19) (20–30 mg), Neu5Ac (1.5 equiv.), and CTP (1.5 equiv.) were dissolved in water in a 50 mL centrifuge tube containing Tris-HCl buffer (100 mM, pH 8.5) and MgCl2 (20 mM). After adding NmCSS (0.5 mg), and PmST1_M144D (1 mg, for producing α2–3-linked sialosides) or Psp2,6ST_A366G (1 mg, for producing α2–6linked sialosides), water was added to bring the final concentration of acceptor to 10 mM. The reaction mixture was incubated at 30°C for 16 hours. The reaction progress was monitored using mass spectrometry. After 16 to 20 hours, the reaction mixture was incubated in a boiling water bath for 5 minutes to denature the enzymes, cooled down to room temperature, then centrifuged at 8000 rpm for 30 minutes at 4 °C. The supernatant was concentrated and purified by a preconditioned DSC-18 SPE cartridge (bed wt.10 g) eluting with MeOH in water (from 0 to 100%) to form the desired product. Neu5Acα2–3GalβProNH-PEG4-Biotin (8c) 39 mg, 91% yield.1H NMR (800 MHz, D2O) δ 4.60 (dd, J = 8.0, 4.8 Hz, 1H), 4.45 (d, J = 8.0 Hz, 1H), 4.42 (dd, J = 8.0, 4.0 Hz, 1H), 4.08 (dd, J = 9.6, 3.2 Hz, 1H), 3.98–3.52 (m, 30H), 3.38 (t, J = 5.4 Hz, 2H), 3.35–3.26 (m, 3H), 3.19 (d, J = 7.0 Hz, 1H), 2.99 (dd, J = 12.8, 4.8 Hz, 1H), 2.79–2.73 (m, 2H), 2.51 (t, J = 6.4 Hz, 2H), 2.27 (t, J = 7.2 Hz, 2H), 2.02 (s, 3H), 1.86–1.77 (m, 3H), 1.75–1.56 (m, 5H), 1.44–1.38 (m, 2H), 1.27 (d, J = 6.4 Hz, 1H).13C NMR (200 MHz, D2O) δ 176.91, 174.98, 173.83, 165.31, 102.51, 99.81, 75.82, 74.88, 72.82, 71.74, 69.62, 69.60, 69.58, 69.54, 69.48, 69.39, 69.15, 68.85, 68.33, 68.03, 67.64, 67.48, 66.78, 62.53, 62.05, 60.95, 60.22, 55.32, 51.65, 39.66, 39.62, 38.87, 36.42, 36.04, 35.43, 28.36, 27.84, 27.66, 25.10, 22.00, 21.98. HRMS (ESI-Orbitrap) m / z: [M-H]- calculated for C41H70N5O21S 1000.4289; found 1000.4308.9531-111023-03Neu5Acα2–3LacNAcβProNH-PEG4-Biotin (8d) 31 mg, 92% yield.1H NMR (800 MHz, D2O) δ 4.62–4.57 (m, 2H), 4.54 (d, J = 8.0 Hz, 1H), 4.49 (d, J = 8.0 Hz, 1H), 4.45–4.40 (m, 2H), 4.11–3.53 (m, 44H), 3.38 (t, J = 5.6 Hz, 2H), 3.36–3.14 (m, 7H), 3.01– 2.95 (m, 2H), 2.76–2.46 (m, 5H), 2.27 (t, J = 7.4 Hz, 2H), 2.03 (s, 3H), 2.02 (s, 3H), 1.83–1.52 (m, 6H), 1.41–1.38 (m, 3H).13C NMR (200 MHz, D2O) δ 183.77, 179.37, 176.89, 174.97, 174.44, 173.85, 165.37, 165.30, 102.54, 101.08, 99.78, 78.31, 75.44, 75.14, 74.75, 72.85, 72.37, 71.73, 69.62, 69.61, 69.60, 69.58, 69.55, 69.48, 69.39, 69.36, 69.27, 68.85, 68.32, 68.05, 67.92, 67.67, 67.44, 66.78, 62.54, 62.05, 61.95, 61.00, 60.22, 60.21, 60.17, 60.03, 55.33, 55.31, 55.29, 55.05, 51.65, 46.63, 39.67, 39.66, 39.59, 38.87, 37.59, 37.28, 36.21, 36.02, 35.43, 28.32, 28.22, 27.85, 27.82, 27.67, 27.65, 27.63, 25.62, 25.11, 25.09, 24.85, 22.13, 22.00. HRMS (ESI-Orbitrap) m / z: [M-H]- calculated for C49H83N6O26S 1203.5083; found 1203.5105. Neu5Acα2–6GalβProNH-PEG4-Biotin (8e) 38 mg, 89% yield.1H NMR (800 MHz, D2O) δ 4.60 (dd, J = 8.0, 4.0 Hz, 1H), 4.42 (dd, J = 8.0, 4.8 Hz, 1H), 4.36 (d, J = 8.0 Hz, 1H), 3.98–3.73 (m, 10H), 3.71–3.54 (m, 16H), 3.49 (dd, J = 10.4, 8.0 Hz, 1H), 3.41–3.23 (m, 6H), 2.98 (dd, J = 12.8, 4.8 Hz, 1H), 2.77 (d, J = 13.6 Hz, 1H), 2.72 (dd, J = 12.8, 4.8 Hz, 1H), 2.52 (t, J = 5.6 Hz, 2H), 2.27 (t, J = 7.2 Hz, 2H), 2.02 (s, 3H), 1.85–1.80 (m, 2H), 1.76–1.55 (m, 6H), 1.44–1.38 (m, 2H).13C NMR (200 MHz, D2O) δ 176.91, 175.03, 173.87, 173.37, 165.31, 102.81, 100.41, 73.41, 72.61, 72.54, 71.71, 70.66, 69.62, 69.60, 69.58, 69.55, 69.48, 69.39, 68.85, 68.56, 68.16, 67.73, 66.78, 63.35, 62.59, 62.05, 60.22, 55.31, 51.83, 40.20, 39.66, 38.87, 36.27, 36.06, 35.42, 29.55, 28.38, 27.84, 27.65, 25.10, 21.98. HRMS (ESI-Orbitrap) m / z: [M-H]- calculated for C41H70N5O21S 1000.4289; found 1000.4310. Neu5Acα2–6LacNAcβProNH-PEG4-Biotin (8f) 30 mg, 90% yield.1H NMR (800 MHz, D2O) δ 4.61–4.58 (m, 1H), 4.53 (d, J = 8.0 Hz, 1H), 4.45– 4.40 (m, 2H), 4.00–3.50 (m, 41H), 3.42–3.15 (m, 7H), 3.02–2.95 (m, 1H), 2.79–2.62 (m, 3H), 2.51 (t, J = 6.4 Hz, 2H), 2.27 (t, J = 7.2 Hz, 1H), 2.05 (s, 3H), 2.02 (s, 3H), 1.80–1.54 (m, 8H), 1.41 (q, J = 7.2 Hz, 2H).13C NMR (200 MHz, D2O) δ 183.75, 176.89, 174.88, 174.46, 173.84, 173.50, 165.30, 103.47, 100.88, 100.11, 80.77, 74.46, 73.66, 72.53, 72.45, 72.39, 71.67, 70.70, 69.62, 69.60, 69.58, 69.57, 69.55, 69.53, 69.48, 69.39, 68.85, 68.37, 68.33, 68.18, 67.67, 66.78, 63.30, 62.62, 62.05, 61.95, 60.36, 60.22, 60.21, 59.53, 55.33, 55.29, 54.83, 51.86, 46.64, 40.06, 39.67, 38.88, 37.27, 36.24, 36.02, 35.43, 28.35, 28.21, 27.85, 27.67, 27.63, 25.62, 25.11, 22.24, 22.00. HRMS (ESI-Orbitrap) m / z: [M-H]- calculated for C49H83N6O26S 1203.5083; found 1203.5107.9531-111023-03One-pot two-enzyme (OP2E) synthesis of 4NH2Neu5Ac-containing sialosides (9a–d) An acceptor (1 equiv., 10 mM), sialic acid 4NH2Neu5Ac (5) (21–87 mg, 1.5 equiv.), and CTP (56– 254 mg, 2.5 equiv.) were dissolved in water in a 50 mL centrifuge tube containing Tris-HCl buffer (100 mM, pH 8.5), MgCl2(20 mM), NmCSS (0.5 mg), and a sialyltransferase PmST1 (2 mg) or Psp2,6ST_A366G (3 mg). The reaction mixture was incubated at 30°C for 4 hours for α2–3-sialylation or 36 h for α2–6-sialylation. The reaction progress was monitored using TLC (EtOAc / MeOH / H2O = 3:5:1, by volume) and ultra-high-performance liquid chromatography (UHPLC). After the reaction reached completion, the reaction was cooked in a boiling water bath for 5 minutes. The resulting mixture was centrifuged, and the supernatant was concentrated by evaporation in vacuo. The final compounds (9a–9f) were purified using a reverse phase C18 column as white foams. 4NH2Neu5Acα2–3GalβpNP (9a) 108.0 mg, 92% yield.1H NMR (800 MHz, D2O) δ 8.27–8.25 (m, 2H), 7.37–7.12 (m, 2H), 5.30 (d, J = 8.0 Hz, 1H), 4.29 (dd, J = 9.6, 3.2 Hz, 1H), 4.17 (t, J = 10.4 Hz, 1H), 4.06 (d, J = 3.2 Hz, 1H), 3.96–3.89 (m, 3H), 3.87–3.81 (m, 2H), 3.77 (d, J = 6.4 Hz, 2H), 3.66–3.62 (m, 2H), 3.39 (dd, J = 12.0, 4.8 Hz, 1H), 2.86 (dd, J = 12.8, 4.4 Hz, 1H), 2.04 (s, 3H), 2.02 (t, J = 12.8 Hz, 1H).13C NMR (200 MHz, D2O) δ 175.0, 173.0, 161.7, 142.5, 126.1, 126.1, 116.4, 116.4, 99.7, 99.3, 75.4, 75.4, 73.1, 71.6, 68.8, 67.8, 67.1, 62.5, 60.6, 50.5, 47.8, 35.6, 22.1. HRMS (ESI-Orbitrap) m / z calculated for C23H32N3O15- [M-H]- 590.1839, found 590.1837. 4NH2Neu5Acα2–6GalβpNP (9b) 83.0 mg, 90% yield.1H NMR (800 MHz, D2O) δ 8.35–8.22 (m, 2H), 7.35–7.17 (m, 2H), 5.20 (d, J = 8.0 Hz, 1H), 4.04 (d, J = 4.0 Hz, 1H), 4.03 (d, J = 4.8 Hz, 1H), 3.98–3.96 (m, 1H), 3.88–3.83 (m, 3H), 3.82– 3.76 (m, 3H), 3.70 (dd, J = 10.4, 4.0 Hz, 1H), 3.61 (dd, J = 12.0, 6.4 Hz, 1H), 3.56 (dd, J = 8.8, 1.6 Hz, 1H), 3.06–2.97 (m, 1H), 2.72 (dd, J = 12.8, 4.0 Hz, 1H), 2.03 (s, 3H), 1.65 (t, J = 12.8 Hz, 1H).13C NMR (200 MHz, D2O) δ 175.1, 173.3, 161.8, 142.5, 126.1, 126.1, 116.4, 116.4, 100.1, 99.8, 74.0, 73.2, 72.3, 71.7, 70.2, 68.4, 68.1, 63.1, 62.6, 50.4, 49.8, 38.4, 22.0. HRMS (ESI-Orbitrap) m / z calculated for C23H32N3O15- [M-H]- 590.1839, found 590.1840. 4NH2Neu5Acα2–3GalβPorNH-PEG4-Biotin (9c) 40.1 mg, 95% yield.1H NMR (600 MHz, D2O) δ 4.62 (dd, J = 8.4, 5.4 Hz, 1H), 4.47 (d, J = 7.8 Hz, 1H), 4.44 (dd, J = 7.8, 4.2 Hz, 1H), 4.16–4.08 (m, 2H), 3.99–3.94 (m, 2H), 3.94–3.86 (m, 2H), 3.83–3.78 (m, 3H), 3.77–3.62 (m, 20H), 3.57 (dd, J = 9.6, 7.8 Hz, 1H), 3.41 (t, J = 5.4 Hz, 2H), 3.38–3.27 (m, 4H), 3.01 (dd, J = 12.6, 4.8 Hz, 1H), 2.84–2.77 (m, 2H), 2.54 (t, J = 6.0 Hz, 2H), 2.29 (t, J = 7.2 Hz, 2H), 2.06 (s, 3H), 1.97 (t, J = 12.6 Hz, 1H), 1.85 (p, J = 6.6 Hz, 2H), 1.78–1.72 (m, 1H), 1.70–1.57 (m, 3H), 1.46–1.41 (m, 2H).13C NMR (150 MHz, D2O) δ 176.9, 175.0, 173.9, 173.2, 165.3, 102.5, 99.3, 75.8, 74.9, 73.1, 71.6,9531-111023-0369.6, 69.6, 69.6, 69.6, 69.5, 69.4, 69.2, 68.9, 67.8, 67.6, 67.3, 66.8, 62.5, 62.1, 60.9, 60.2, 55.4, 50.4, 48.2, 39.7, 38.9, 36.4, 36.1, 35.9, 35.5, 28.4, 27.9, 27.7, 25.1, 22.1. HRMS (ESI-Orbitrap) m / z calculated for C41H71N6O20S- [M-H]- 999.4449, found 999.4447. 4NH2Neu5Acα2–3LacNAcβPorNH-PEG4-Biotin (9d) 30.3 mg, 92% yield.1H NMR (800 MHz, D2O) δ 4.61 (dd, J = 8.0, 4.8 Hz, 1H), 4.55 (d, J = 8.0 Hz, 1H), 4.50 (d, J = 8.0 Hz, 1H), 4.43 (dd, J = 8.0, 4.0 Hz, 1H), 4.11 (dd, J = 9.6, 3.2 Hz, 1H), 3.99 (dd, J = 12.0, 2.4 Hz, 1H), 3.95 (d, J = 3.2 Hz, 1H), 3.94–3.88 (m, 2H), 3.88–3.83 (m, 2H), 3.79–3.72 (m, 6H), 3.71– 3.66 (m, 16H), 3.66–3.63 (m, 1H), 3.63–3.61 (m, 3H), 3.60–3.55 (m, 3H), 3.39 (dd, J = 6.4, 4.8 Hz, 2H), 3.35–3.32 (m, 1H), 3.27 (dt, J = 13.6, 7.2 Hz, 1H), 3.18 (dt, J = 13.6, 7.2 Hz, 1H), 2.99 (dd, J = 12.8, 4.8 Hz, 1H), 2.90–2.81 (m, 1H), 2.78 (d, J = 12.8 Hz, 1H), 2.66 (dd, J = 12.8, 4.0 Hz, 1H), 2.52 (t, J = 6.4 Hz, 2H), 2.28 (t, J = 7.2 Hz, 2H), 2.06–2.01 (m, 6H), 1.80–1.75 (m, 2H), 1.75–1.55 (m, 5H), 1.46–1.38 (m, 2H).13C NMR (200 MHz, D2O) δ 176.9, 175.1, 174.4, 173.9, 165.3, 102.6, 101.1, 99.8, 78.3, 75.4, 75.2, 74.8, 73.8, 72.4, 71.7, 69.6, 69.6, 69.6, 69.5, 69.5, 69.4, 69.4, 69.4, 68.9, 68.1, 67.7, 67.4, 66.8, 62.5, 62.1, 61.0, 60.2, 60.0, 55.3, 55.0, 49.6, 39.7, 38.9, 36.2, 36.0, 35.4, 28.3, 27.8, 27.7, 25.1, 22.1, 22.0. HRMS (ESI- Orbitrap) m / z calculated for C49H84N7O25S- [M-H]- 1202.5243, found 1202.5223. General methods for converting 4NH2Neu5Ac-containing sialosides (9a–d) to 4-guanidino-Neu5Ac- containing sialosides (10a–d) The 4NH2Neu5Ac-containing sialoside (9a–d) (12 mg, 1 equiv.) was dissolved in 1 mL of anhydrous THF and 2 mL of MeOH.1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine (15–23 mg, 3 equiv.) was added to the mixture and triethylamine was added to adjust the pH to around 9. The reaction was stirred at 37°C for 24 hours and was monitored by TLC (iPrOH:H2O:NH4OH = 7:2:1, by volume). After the reaction was completed, the solvent was removed and 1.5 mL of trifluoroacetic acid was added. The reaction was stirred vigorously for 30–60 minutes at room temperature. After the reaction was completed, the solvent was removed by air dry and purified by a reverse phase C18 column. The sialosides were eluted at 20-60% acetonitrile in water, followed by lyophilization to obtain pure compounds (10a–d) as white foams. 4-Guanidino-Neu5Acα2–3GalβpNP (10a) 11.0 mg, 86% yield.1H NMR (600 MHz, D2O) δ 8.35–8.23 (m, 2H), 7.46–7.16 (m, 2H), 5.32 (d, J = 7.8 Hz, 1H), 4.30 (dd, J = 9.6, 3.0 Hz, 1H), 4.08–4.03 (m, 2H), 3.97–3.90 (m, 3H), 3.89–3.82 (m, 2H), 3.78 (d, J = 6.0 Hz, 2H), 3.70–3.61 (m, 3H), 2.76 (dd, J = 12.6, 4.2 Hz, 1H), 2.01 (s, 3H), 1.98 (dd, J = 13.2, 12.0 Hz, 1H).13C NMR (150 MHz, D2O) δ 174.6, 173.6, 161.7, 156.6, 142.6, 127.8, 126.1, 116.4, 115.9, 99.7, 99.4, 75.4, 75.4, 73.0, 71.7, 68.8, 68.0, 67.1, 62.5, 60.6, 51.7, 49.9, 37.0, 21.8. HRMS (ESI-Orbitrap) m / z calculated for C24H34N5O15- [M-H]- 632.2057, found 632.2059.9531-111023-034-Guanidino-Neu5Acα2–6GalβpNP (10b) 10.3 mg, 81% yield.1H NMR (600 MHz, D2O) δ 8.312–8.29 (m, 2H), 7.30–7.27 (m, 2H), 5.23 (d, J = 7.8 Hz, 1H), 4.06–4.03 (m, 2H), 4.02–3.99 (m, 1H), 3.92 (t, J = 9.6 Hz, 1H), 3.87 (m, 4H), 3.81 (dd, J = 10.2, 3.2 Hz, 1H), 3.72 (dd, J = 10.2, 3.6 Hz, 1H), 3.65–3.62 (m, 1H), 3.62–3.60 (m, 1H), 3.60–3.55 (m, 1H), 2.68 (dd, J = 13.2, 4.2 Hz, 1H), 1.99 (s, 3H), 1.78 (dd, J = 13.2, 12.0 Hz, 1H).13C NMR (150 MHz, D2O) δ 174.6, 173.3, 161.8, 156.5, 142.5, 126.1, 125.5, 116.4, 115.4, 100.0, 99.6, 74.1, 72.7, 72.3, 71.6, 70.3, 68.5, 68.1, 63.3, 62.5, 51.6, 50.0, 37.3, 21.8. HRMS (ESI-Orbitrap) m / z calculated for C24H34N5O15- [M-H]- 632.2057, found 632.2050. 4-Guanidino-Neu5Acα2–3Galβ PorNH-PEG4-Biotin (10c) 10.2 mg, 82% yield.1H NMR (800 MHz, D2O) δ 4.61 (dd, J = 8.0, 4.8 Hz, 1H), 4.46 (d, J = 8.0 Hz, 1H), 4.42 (dd, J = 8.0, 4.8 Hz, 1H), 4.12 (dd, J = 9.6, 3.2 Hz, 1H), 4.02 (t, J = 9.6 Hz, 1H), 3.98–3.93 (m, 2H), 3.91–3.89 (m, 1H), 3.87 (dd, J = 12.0, 2.4 Hz, 1H), 3.81 (d, J = 10.4 Hz, 1H), 3.78 (t, J = 6.4 Hz, 2H), 3.76–3.70 (m, 3H), 3.69 (s, 8H), 3.68–3.67 (m, 4H), 3.66–3.61 (m, 6H), 3.57–3.52 (m, 1H), 3.39 (t, J = 5.6 Hz, 2H), 3.35–3.32 (m, 1H), 3.32–3.27 (m, 2H), 2.99 (dd, J = 12.8, 4.8 Hz, 1H), 2.78 (d, J = 12.8.0 Hz, 1H), 2.70 (dd, J = 13.6, 4.8 Hz, 1H), 2.52 (t, J = 5.6 Hz, 2H), 2.27 (t, J = 7.2 Hz, 2H), 1.99 (s, 3H), 1.94 (t, J = 12.0 Hz, 1H), 1.84 (q, J = 7.2 Hz, 2H), 1.71–1.75 (m, 1H), 1.68–1.56 (m, 3H), 1.45–1.38 (m, 2H).13C NMR (200 MHz, D2O) δ 176.9, 174.6, 173.9, 173.8, 165.3, 156.6, 102.5, 75.7, 74.9, 72.9, 71.6, 69.6, 69.6, 69.6, 69.5, 69.5, 69.4, 69.2, 68.9, 68.0, 67.6, 67.3, 66.8, 62.5, 62.1, 60.9, 60.2, 55.3, 51.6, 49.9, 39.7, 38.9, 36.7, 36.4, 36.0, 35.4, 28.4, 27.8, 27.7, 25.1, 23.2, 21.8. HRMS (ESI-Orbitrap) m / z calculated for C42H73N8O20S- [M-H]- 1041.4667, found 1041.4652. 4-Guanidino-Neu5Acα2–3LacNAcβPorNH-PEG4-Biotin (10d) 7.3 mg, 88% yield.1H NMR (800 MHz, D2O) δ 4.61 (dd, J = 8.0, 4.8 Hz, 1H), 4.55 (d, J = 8.0 Hz, 1H), 4.51 (d, J = 8.0 Hz, 1H), 4.42 (dd, J = 8.0, 4.8 Hz, 1H), 4.14 (dd, J = 10.4, 3.2 Hz, 1H), 4.02 (t, J = 9.6 Hz, 1H), 3.99 (dd, J = 12.8, 2.4 Hz, 1H), 3.96 (d, J = 3.2 Hz, 1H), 3.93–3.89 (m, 2H), 3.87 (dd, J = 12.0, 2.4 Hz, 1H), 3.85–3.80 (m, 2H), 3.78 (t, J = 5.6 Hz, 2H), 3.75–3.71 (m, 4H), 3.71–3.70 (m, 3H), 3.69 (s, 8H), 3.67 (s, 3H), 3.65–3.61 (m, 6H), 3.59–3.55 (m, 2H), 3.41–3.37 (m, 2H), 3.36–3.32 (m, 1H), 3.27 (dt, J = 13.6, 6.4 Hz, 1H), 3.18 (dt, J = 13.6, 7.2 Hz, 1H), 2.99 (dd, J = 13.6, 5.6 Hz, 1H), 2.78 (d, J = 12.8 Hz, 1H), 2.69 (dd, J = 13.6, 4.8 Hz, 1H), 2.51 (t, J = 5.6 Hz, 2H), 2.27 (t, J = 7.2 Hz, 2H), 2.03 (s, 3H), 1.99 (s, 3H), 1.97–1.90 (m, 1H), 1.79–1.75 (m, 2H), 1.75–1.70 (m, 1H), 1.69–1.56 (m, 3H), 1.46–1.37 (m, 2H).13C NMR (200 MHz, D2O) δ 176.9, 174.6, 174.5, 173.9, 173.7, 165.3, 161.0, 156.6, 102.5, 101.1, 99.4, 78.3, 75.3, 75.1, 74.7, 72.9, 72.4, 71.6, 69.6, 69.6, 69.6, 69.5, 69.5, 69.4, 68.9, 68.0, 67.7, 67.2, 66.8, 62.5, 62.1, 61.0, 60.2, 55.3, 55.1, 51.6, 49.9, 39.7, 38.9, 36.2, 36.0, 35.4, 28.3, 27.8, 27.7, 25.1, 22.1, 21.8. HRMS (ESI- Orbitrap) m / z calculated for C50H86N9O25S- [M-H]- 1244.5461, found 1244.5435.9531-111023-03General methods for converting 4NH2Neu5Ac-containing sialosides (9a–d) to 4N3Neu5Ac-containing sialosides (11a–d) A 4NH2Neu5Ac-containing sialoside (9a–d) (12 mg, 1 equiv.) was dissolved in 0.5 mL H2O and 1 mL MeOH containing 15 mg of K2CO3and 10 mg of CuSO4∙5H2O, freshly prepared TfN3from NaN3(25 equiv.) was then added to the mixture and the reaction was stirring at room temperature for 16 hours. The reaction was monitored by TLC (EtOAc:MeOH:H2O = 5:3:1, by volume). After the reaction reached completion, the solvent was removed by rotavapor, and the final products were purified by a reverse phase C18 column. The sialosides were eluted at 20–60% acetonitrile in water, followed by evaporation in vacuo and lyophilization to produce pure compounds (11a–d) as white foams. 4N3Neu5Acα2–3GalβpNP (11a) 10.4 mg, 83% yield.1H NMR (600 MHz, D2O) δ 8.37–8.23 (m, 2H), 7.32–7.16 (m, 2H), 5.32 (d, J = 7.8 Hz, 1H), 4.27 (dd, J = 10.2, 3.0 Hz, 1H), 4.07 (d, J = 3.0 Hz, 1H), 4.02 (t, J = 10.2 Hz, 1H), 3.96–3.88 (m, 3H), 3.86 (dd, J = 12.0, 2.4 Hz, 1H), 3.81–3.76 (m, 3H), 3.68–3.59 (m, 3H), 2.82 (dd, J = 12.6, 4.2 Hz, 1H), 2.06 (s, 3H), 1.85 (t, J = 12.6 Hz, 1H);13C NMR (150 MHz, D2O) δ 174.8, 173.4, 161.7, 142.5, 127.0, 125.4, 116.4, 114.7, 99.7, 99.6, 75.5, 75.4, 73.2, 71.8, 68.8, 68.0, 67.2, 62.5, 60.7, 59.6, 49.8, 36.8, 22.0. HRMS (ESI-Orbitrap) m / z calculated for C23H30N5O15- [M-H]- 616.1744, found 616.1737. 4N3Neu5Acα2–6GalβpNP (11b) 10.9 mg, 87% yield.1H NMR (600 MHz, D2O) δ 8.38–8.23 (m, 2H), 7.31–7.24 (m, 2H), 5.21 (dd, J = 7.8, 1.2 Hz, 1H), 4.06–4.04 (m, 1H), 4.05–4.02 (m, 2H), 3.92–3.84 (m, 4H), 3.83–3.80 (m, 2H), 3.72–3.69 (m, 1H), 3.64–3.57 (m, 3H), 2.77 (dd, J = 12.6, 4.2 Hz, 1H), 2.04 (s, 3H), 1.67 (t, J = 12.6 Hz, 1H).13C NMR (150 MHz, D2O) δ 174.8, 173.2, 161.8, 142.5, 126.1, 125.6, 116.4, 113.3, 100.0, 99.8, 74.0, 72.9, 72.3, 71.7, 70.3, 68.4, 68.0, 63.0, 62.6, 59.4, 49.9, 37.2, 22.0. HRMS (ESI-Orbitrap) m / z calculated for C23H30N5O15- [M-H]- 616.1744, found 616.1756. 4N3Neu5Acα2–3GalβProNH-PEG4-Biotin (11c) 6.0 mg, 84% yield.1H NMR (800 MHz, D2O) δ 4.61 (dd, J = 8.0, 4.8 Hz, 1H), 4.45 (d, J = 8.0 Hz, 1H), 4.43 (dd, J = 8.0, 4.8 Hz, 1H), 4.09 (dd, J = 10.4, 3.2 Hz, 1H), 4.01–3.93 (m, 3H), 3.90–3.84 (m, 2H), 3.78 (t, J = 6.4 Hz, 2H), 3.76–3.66 (m, 18H), 3.62 (q, J = 5.6, 4.0 Hz, 4H), 3.55 (t, J = 8.8 Hz, 1H), 3.39 (t, J = 5.6 Hz, 2H), 3.35–-3.33 (m, 1H), 3.33–3.27 (m, 2H), 2.99 (dd, J = 12.8, 4.8 Hz, 1H), 2.78 (d, J = 12.8 Hz, 1H), 2.77–2.75 (m, 1H), 2.52 (t, J = 5.6 Hz, 2H), 2.28 (t, J = 7.2 Hz, 2H), 2.04 (s, 3H), 1.86–1.82 (m, 2H), 1.82–1.79 (m, 1H), 1.75–1.71 (m, 1H), 1.69–1.56 (m, 3H), 1.45–1.38 (m, 2H).13C NMR (200 MHz, D2O) δ 176.9, 174.8, 173.9, 173.4, 165.3, 102.5, 101.2, 75.8, 74.9, 73.2, 71.7, 69.6, 69.6, 69.6, 69.5, 69.5, 69.4, 69.1, 68.9, 67.9, 67.7, 67.5, 66.8, 62.5, 62.1, 60.9, 60.2, 59.6, 55.3, 49.8, 39.7, 38.9, 36.7, 36.4, 36.0, 35.4,9531-111023-0328.4, 27.8, 27.7, 25.1, 22.0. HRMS (ESI-Orbitrap) m / z calculated for C41H69N8O20S- [M-H]- 1025.4354, found 1025.4345. 4N3Neu5Acα2–3LacNAcβProNH-PEG4-Biotin (11d) 6.2 mg, 87% yield.1H NMR (800 MHz, D2O) δ 4.61 (dd, J = 8.0, 4.8 Hz, 1H), 4.54 (d, J = 7.2 Hz, 1H), 4.50 (d, J = 8.8 Hz, 1H), 4.43 (dd, J = 8.0, 4.8 Hz, 1H), 4.12 (dd, J = 10.4, 3.2 Hz, 1H), 4.01–3.96 (m, 2H), 3.96 (d, J = 3.2 Hz, 1H), 3.94–3.91 (m, 1H), 3.91–3.88 (m, 1H), 3.88–3.85 (dd, J = 11.9, 2.5 Hz, 1H), 3.84 (dd, J = 12.0, 4.8 Hz, 1H), 3.78 (t, J = 5.6 Hz, 2H), 3.75–3.69 (m, 7H), 3.69–3.66 (m, 12H), 3.66–3.61 (m, 5H), 3.61–3.55 (m, 3H), 3.39 (t, J = 4.8 Hz, 2H), 3.36–3.31 (m, 1H), 3.3–3.24 (m, 1H), 3.21–3.15 (m, 1H), 2.99 (dd, J = 12.8, 4.8 Hz, 1H), 2.78 (d, J = 12.8 Hz, 1H), 2.76 (dd, J = 12.8, 4.8 Hz, 1H), 2.52 (t, J = 5.6 Hz, 2H), 2.28 (t, J = 7.2 Hz, 2H), 2.09–1.09 (m, 6H), 1.83–1.69 (m, 4H), 1.70–1.55 (m, 3H), 1.46–1.38 (m, 2H).13C NMR (200 MHz, D2O) δ 176.9, 174.8, 174.4, 173.9, 173.5, 165.3, 102.5, 101.1, 99.6, 78.3, 75.4, 75.1, 74.7, 73.2, 72.4, 71.7, 69.6, 69.6, 69.6, 69.5, 69.5, 69.4, 69.3, 68.9, 67.9, 67.7, 67.4, 66.8, 62.5, 62.1, 61.0, 60.2, 60.0, 59.6, 55.3, 55.0, 49.8, 39.7, 38.9, 36.7, 36.2, 36.0, 29.6, 28.3, 27.8, 27.7, 25.1, 22.1, 22.0. HRMS (ESI-Orbitrap) m / z calculated for C49H82N9O25S- [M-H]- 1228.5148, found 1228.5128. General Methods for converting 4NH2Neu5Ac-containing sialosides (9a–d) to 4NAcNeu5Ac- containing sialosides (12a–d) The 4NH2Neu5Ac-containing sialoside (9a–d) (30 mg, 1 equiv.) was dissolved in 1 mL of methanol, acetic anhydride (10-50 µL, 10 equiv.) was added, 100 µL of triethylamine was then used to adjust the pH to around 9.0. The reaction was stirred at room temperature and was monitored by TLC (EtOAc:MeOH:H2O = 5:4:1, by volume). After the reaction was completed, the reaction mixture was dried, re-dissolved in 1 mL of H2O, the pH was adjusted to 11.0 and the reaction container was put on vacuum to remove the triethylamine, followed by purification via a reverse phase C18 column. The final products were eluted with 20–60% acetonitrile in water. After evaporation in vacuo and lyophilization, the pure 4-N-acetyl sialosides (12a–d) were obtained as white foams. 4NAcNeu5Acα2–3GalβpNP (12a) 31.5 mg, 98% yield.1H NMR (800 MHz, D2O) δ 8.28–8.26 (m, 2H), 7.27–7.25 (m, 2H), 5.31 (d, J = 8.0 Hz, 1H), 4.27 (dd, J = 9.6, 3.2 Hz, 1H), 4.06 (d, J = 3.2 Hz, 1H), 4.0–3.96 (m, 1H), 3.95–3.88 (m, 4H), 3.85 (dd, J = 12.0, 2.4 Hz, 1H), 3.82 (dd, J = 9.6, 1.6 Hz, 1H), 3.77 (s, 1H), 3.77 (s, 1H), 3.63–3.61 (m, 2H), 2.66 (dd, J = 12.8, 4.0 Hz, 1H), 1.97–1.96 (m, 6H), 1.86 (t, J = 12.8 Hz, 1H).13C NMR (200 MHz, D2O) δ 174.6, 173.7, 173.6, 161.7, 142.5, 126.1, 126.1, 116.4, 116.4, 99.7, 99.7, 75.4, 75.4, 73.6, 71.8, 68.8, 68.1, 67.1, 62.5, 60.7, 49.7, 48.5, 37.1, 21.9, 21.9. HRMS (ESI-Orbitrap) m / z calculated for C25H34N3O16- [M-H]- 632.1945, found 632.1947.9531-111023-034NAcNeu5Acα2–6GalβpNP (12b) 30.5 mg, 95% yield.1H NMR (800 MHz, D2O) δ 8.32–8.28 (m, 2H), 7.29–7.25 (m, 2H), 5.21 (d, J = 8.0 Hz, 1H), 4.03–4.02 (m, 2H), 4.0–3.97 (m, 1H), 3.95–3.92 (m, 1H), 3.88–3.80 (m, 6H), 3.68 (dd, J = 10.4, 3.2 Hz, 1H), 3.61 (dd, J = 12.8, 7.2 Hz, 1H), 3.57 (dd, J = 8.8, 1.6 Hz, 1H), 2.61 (dd, J = 12.8, 4.0 Hz, 1H), 1.95 (s, 3H), 1.94 (s, 3H), 1.66 (t, J = 12.8 Hz, 1H).13C NMR (200 MHz, D2O) δ 174.6, 173.7, 173.3, 161.8, 142.5, 126.1, 126.1, 116.4, 116.4, 100.2, 99.7, 74.0, 73.2, 72.3, 71.7, 70.3, 68.4, 68.2, 63.1, 62.6, 49.8, 48.3, 37.6, 21.9, 21.8. HRMS (ESI-Orbitrap) m / z calculated for C25H34N3O16- [M-H]- 632.1945, found 632.1948. 4NAcNeu5Acα2–3GalβProNH-PEG4-Biotin (12c) 5.2 mg, 99% yield.1H NMR (800 MHz, D2O) δ 4.61 (dd, J = 8.0, 5.6 Hz, 1H), 4.46 (d, J = 8.0 Hz, 1H), 4.42 (dd, J = 8.0, 4.8 Hz, 1H), 4.10 (dd, J = 9.6, 3.2 Hz, 1H), 3.97–3.94 (m, 3H), 3.913–3.90 (m, 1H), 3.90–3.84 (m, 2H), 3.79–3.77 (m, 3H), 3.75–3.70 (m, 3H), 3.69–3.66 (m, 13H), 3.65–3.61 (m, 4H), 3.54 (dd, J = 9.6, 8.0 Hz, 1H), 3.39 (t, J = 5.6 Hz, 2H), 3.36–3.32 (m, 1H), 3.32–3.27 (m, 2H), 2.99 (dd, J = 13.6, 5.6 Hz, 1H), 2.78 (d, J = 12.8 Hz, 1H), 2.61 (dd, J = 12.8, 4.0 Hz, 1H), 2.52 (t, J = 6.4 Hz, 2H), 2.28 (t, J = 7.2 Hz, 2H), 1.96 (s, 6H), 1.86–1.80 (m, 3H), 1.76–1.70 (m, 1H), 1.69–1.56 (m, 3H), 1.45–1.38 (m, 2H).13C NMR (200 MHz, D2O) δ 176.9, 174.5, 173.9, 173.7, 173.6, 165.3, 102.5, 99.7, 75.8, 74.9, 73.5, 71.8, 69.6, 69.6, 69.6, 69.5, 69.5, 69.4, 69.2, 68.9, 68.0, 67.6, 67.4, 66.8, 62.5, 62.1, 61.0, 60.2, 55.3, 49.7, 48.5, 39.7, 38.9, 37.1, 36.4, 36.0, 35.4, 28.4, 27.8, 27.7, 25.1, 21.9, 21.9. HRMS (ESI-Orbitrap) m / z calculated for C43H73N6O21S- [M-H]- 1041.4555, found 1041.4552. 4NAcNeu5Acα2–3LacNAcβProNH-PEG4-Biotin (12d) 5.2 mg, 98% yield.1H NMR (800 MHz, D2O) δ 4.61 (dd, J = 8.0, 5.6 Hz, 1H), 4.55 (d, J = 7.2 Hz, 1H), 4.50 (d, J = 8.8 Hz, 1H), 4.42 (dd, J = 8.0, 4.8 Hz, 1H), 4.12 (dd, J = 9.6, 3.2 Hz, 1H), 3.99 (d, J = 12.0 Hz, 1H), 3.98–3.88 (m, 5H), 3.87 (dd, J = 12.0, 2.4 Hz, 1H), 3.84 (dd, J = 12.0, 4.8 Hz, 1H), 3.80–3.76 (m, 3H), 3.75–3.70 (m, 6H), 3.69 (s, 8H), 3.67 (s, 3H), 3.64–3.59 (m, 5H), 3.59–3.55 (m, 2H), 3.39 (t, J = 5.6 Hz, 2H), 3.33 (dt, J = 9.6, 4.8 Hz, 1H), 3.27 (dt, J = 13.6, 7.2 Hz, 1H), 3.18 (dt, J = 13.6, 6.4 Hz, 1H), 2.99 (dd, J = 12.8, 4.8 Hz, 1H), 2.78 (d, J = 12.8 Hz, 1H), 2.61 (dd, J = 12.8, 4.0 Hz, 1H), 2.51 (t, J = 6.4 Hz, 2H), 2.27 (t, J = 7.2 Hz, 2H), 2.03 (s, 3H), 1.96 (s, 6H), 1.92–1.89 (m, 1H), 1.82 (t, J = 12.0 Hz, 1H), 1.78– 1.75 (m, 2H), 1.75–1.70 (m, 1H), 1.69–1.56 (m, 3H), 1.47–1.38 (m, 2H).13C NMR (200 MHz, D2O) δ 176.9, 174.5, 174.5, 173.9, 173.7, 165.3, 161.6, 102.6, 101.1, 99.7, 78.3, 75.4, 75.1, 74.7, 73.6, 72.4, 71.7, 69.6, 69.6, 69.6, 69.5, 69.5, 69.4, 69.4, 68.9, 68.1, 67.7, 67.3, 66.8, 62.5, 62.1, 61.0, 60.2, 60.1, 55.3, 55.1, 48.5, 39.7, 38.9, 37.0, 36.2, 36.0, 35.4, 28.3, 27.8, 27.7, 25.1, 23.2, 22.1, 21.9, 21.9. HRMS (ESI- Orbitrap) m / z calculated for C51H86N7O26S- [M-H]- 1244.5349, found 1244.5329.9531-111023-03High-throughput substrate specificity assays The substrate specificity assays were carried out in duplicate in a 384-well plate with a final volume of 20 µL. Each sialoside (0.3 mM) was incubated with a sialidase and an excess amount of β-galactosidase at 37°C for 30 minutes. High-concentration sialidase assays were carried out using 4-fold (Vc Sialidase) or 10-fold (other sialidases) sialidases for 1 hour. Assays were stopped with 40 µL of 0.5 M CAPS buffer (pH 11.5), and A405 nmreadings were obtained by a microplate reader. For every sialoside tested, duplicate reactions without a sialidase were used as negative controls and for background readings. The sialidase amounts and buffers used were: hNEU2 (0.6 µg), MES buffer (100 mM, pH 5.0); BiNanH2 (0.1 µg), NaOAc buffer (100 mM, pH 5.0); A. ureafaciens sialidase (1.0 mU), NaOAc buffer (100 mM, pH 5.5); C. perfringens sialidase (0.6 mU), MES buffer (100 mM, pH 5.0); V. cholerae sialidase (2.8 mU), NaCl (150 mM), CaCl2(10 mM), NaOAc buffer (100 mM, pH 5.5); SpNanA (14 ng), NaOAc buffer (100 mM, pH 6.0); SpNanB (5 ng), NaOAc buffer (100 mM, pH 6.0); SpNanC (20 ng), MES buffer (100 mM, pH 6.5); PmST1 (0.5 µg), NaOAc buffer (100 mM, pH 5.5), CMP (0.4 mM);. Amounts 4-fold (Vc Sialidase) or 10- fold (other sialidases) higher were used for high-concentration sialidase assays. Buffer conditions for IAV and IBV NAs: NaCl (150 mM), CaCl2 (1 mM), MES buffer (25 mM, pH 6.0). Amounts for low concentration sialidase assays: N1-Vic19 (0.08 µg), N1-BR18 (0.06 µg), N2-Dar21 (0.06 µg), N2-Kan17 (0.10 µg), NB-Aus21 (0.02 µg), NB-Phu13 (0.015 µg). Amounts 10-fold higher were used for high- concentration sialidase assays. Inhibition assays Percentage inhibition assays were carried out in duplicate in a 96-well plate with a final volume of 80 µL. Each reaction contained Neu5Acα2–3GalβMU (8.0 µM), an excess amount of β-galactosidase (12 µg), with or without an inhibitor (8.0 µM). The assay conditions were: hNEU2 (2.8 µg), MES buffer (100 mM, pH 5.0); N1-BR18 (0.06 µg), NaCl (150 mM), CaCl2 (1 mM), MES buffer (25 mM, pH 6.0). The reactions were carried out for 30 minutes and quenched with 80 µL of N-cyclohexyl-3-aminopropane sulfonic acid buffer (0.5 M, pH 11.5). The fluorescence of free MU formed was determined using a microplate reader with excitation at 360 nm and emission at 460 nm. Inhibition assays for obtaining IC50 values were carried out in duplicate in a 96-well plate similarly as described above except that a series of different concentrations of inhibitors were used. IC50values were obtained by fitting the obtained fluorescence values to the inhibitor concentration-response plots using GraphPad Prism 9. Enzyme-linked immunosorbent assays (ELISA) The ELISAs were carried out in duplicate in a 384-well neutravidin-coated plate. The wells were washed with 3 × 80 µL of washing buffer (1 × PBS buffer with 0.05% tween-20) and then were incubated with biotinylated glycans (10 µM, 20 µL) at 4 °C for overnight. Then the wells were washed with 3 × 80 µL of washing buffer and blocked with 60 µL of blocking buffer (1 × PBS buffer with 0.1% BSA). The wells9531-111023-03were then washed with 3 × 80 µL of washing buffer, and NA (10 µg / mL, 20 µL) was added to each well. The samples were incubated at 4°C for 30 minutes. The wells were then washed with 3 × 80 µL of washing buffer, and HRP-conjugated anti-His or anti-Strep antibodies (0.1 µg / mL or with proper dilution using 1 × PBS buffer with 0.1% BSA, 20 µL) were added. The samples were incubated at 4°C for 30 minutes. After washing with 3 × 80 µL of the washing buffer, 20 µL of TMB substrate was added to each well and the plate was incubated at room temperature for 30 minutes. The reaction was then stopped with 20 µL of 2 M sulfuric acid and absorbance readings at 450 nm were measured by a plate reader. Bio-lay interferometry (BLI) assays The BLI assays were carried out at room temperature similar to a reported procedure (Xiong et al., Front. Bioeng. Biotechnol.10, 962292, 2022), with modifications. The solutions containing biotinylated sialosides (100 nM, 200 µL each) were loaded to the wells of a 96-well plate. The analyte solutions of NAs (100 µL per well containing 1 × Octet® kinetics buffer with or without 1 µM NA or its 3 × serial diluted solutions) were loaded to the wells in a 384-well titled-bottom plate to minimize the materials required. Streptavidin (SAX) biosensor tips were hydrated in the kinetics buffer in a reservoir for 10 minutes before being mounted onto a sensor rack which was then placed in the Octet 384RED (Fortébio) sensor tray. The SAX biosensor tips were dipped into the biotinylated sialoside solutions (100 nM, 200 µL each in the 96- well plate) for 150 or 200 seconds or until the streptavidin biosensors reached coating saturation. After a baseline step of incubating the biosensor tips with the kinetics buffer (100 µL per well in the 384-well titled- bottom plate) for 60 seconds, the functionalized biosensor tips were dipped in the analyte solutions (100 µL per well in a titled bottom 384-well plate) containing various concentrations of NA (1 µM and 3 × serial diluted solutions) for 400 seconds. The sensor tips were then switched to be dipped in the kinetics buffer (100 µL per well in the titled bottom 384-well plate) for 600 or 900 seconds to allow dissociation. The sensorgrams obtained were fitted to a 1:1 binding model using Octet Analysis software. The equilibrium responses at the end of the association phase (400 seconds) were used for steady state analysis by fitting to a “one site-specific binding” model on GraphPad Prism 9 to obtain the KDvalues H1N1 virus detection by ELISA H1N1 / BR18 virus was grown in 10-day old specific pathogen free embryonated eggs for 3 days at 33℃ and the allantoic fluid was harvested and clarified by sedimentation (4,000 × g for 5 minutes). ELISA assays were carried out in duplicate using 96-well 2HB plates pre-coated with 25 µg / well streptavidin and blocked at 37°C for 1 hour with 200 µL of 1% BSA in PBS pH 7.4. Wells were washed with 3 × 200 µL of PBS pH 7.4 containing 0.1% Tween 20 (PBST) and incubated with 100 µL of the biotinylated glycans diluted to the indicated concentrations in PBST containing 0.1% BSA at 37 °C for 1 hour. Wells were washed with 3 × 200 µL PBST and 100 µL of H1N1 / BR18 virus in allantoic fluid diluted 1:4 in PBST was added to the wells at 37°C for 1 hour. Wells were washed 6 × 200 µL with PBST and incubated with 100 µL9531-111023-03of a rabbit polyclonal antibody against H1 (1 µg / mL) at 37°C for 1 hour. Wells were washed with 3 × 200 µL PBST and incubated with a HRP-conjugated goat-anti-rabbit secondary at a 1:20000 dilution at 37°C for 1 hour. Wells were washed again with 3 × 200 µL PBST and developed using 100 µL of OPD substrate at 37°C for 10 minutes and stopped by adding 50 µL / well of 2 N sulfuric acid and absorbance readings at 490 nm were measured with a plate reader. H1N1 virus isolation with streptavidin beads Dynabeads MyOne streptavidin T1 (10 mg) were washed with 2 × 10 mL virus buffer (25 mM MES pH 7, 150 mM NaCl and 1 mM CaCl2), resuspended with 1 mL of virus buffer containing 20 µM of the biotinylated glycan and incubated at 37°C for 1 hour. Beads were washed with 2 × 10 mL virus buffer and resuspended in 1 mL virus buffer. Clarified H1N1 / BR18 virus in allantoic fluid (~9 mL) was added to the beads and incubated at 37°C for 1 hour. Beads were sedimented (4,000 × g for 5 minutes) and the supernatant containing the unbound virus was retained. Beads were washed with 2 × 10 mL virus buffer and bound virus was eluted by incubating the beads with 200 µL virus buffer containing 50 mM Neu5Ac2en at 4°C for overnight. A magnet was then used to pulldown the beads and the supernatant was removed. HA titer determination, SDS-PAGE and DLS analysis HA titers were determined using 0.5% turkey red blood cells (TRBCs). Briefly, 50 µL samples were diluted two-fold in 96-well round bottom plates using PBS. Following the dilution, 50 µL of 0.5% TRBCs were added to each well, the plate was incubated at room temperature for 30 minutes, and HA titers corresponded to the last well where agglutination was observed were recorded. Equal samples volumes were mixed with sample buffer with or without 0.1 M dithiotreitol (DTT), heated at 50°C for 10 minutes and resolved using 4–12% SDS-PAGE gels. Gels were stained with SimpleBlue and imaged using an Azure C600 imaging system. Dynamic light scattering measurements were performed on the isolated virus diluted 1:10 in 0.2 µm filtered virus buffer (25 mM MES pH 7, 150 mM NaCl and 1 mM CaCl2) using a DynaPro NanoStar II. Data from three independent samples were collected using 10 × 5 sec intervals and each reading set was analyzed with the regularization setting to obtain the particle diameter and polydispersity measurements. Example 13: Chemoenzymatic Synthesis of Sialosides Containing 4-N-Substituted Neu5Ac as IAV and IBV Selective Substrates Since the report on the rational design of inhibitors selective against influenza NAs and effective in protecting cultured cells and model animals from viral challenges (von Itzstein et al., Nature 363 (6428), 418-423, 1993), the 4-N-substituted derivatives (Holzer et al., Glycoconj J 10 (1), 40-44, 1993) of sialidase transition analog 2,3-dehydro-3-deoxy-N-acetylneuraminic acid (Neu5Ac2en or DANA, 2) (Chong et al., Eur J Biochem 207 (1), 335-343, 1992) (FIG.23) have received much attention. Those with a C4-amino9531-111023-03(4NH2Neu5Ac2en, 3) or a C4-guanidino (4-guanidino-Neu5Ac2en or Zanamivir, 4) substitution inspired the development of the approved antiviral drugs Oseltamivir (trade name Tamiflu) (Li et al., Antimicrob Agents Chemother 42 (3), 647-653, 1998), peramivir, and Zanamivir (trade name Relenza) (von Itzstein et al., Nature 363 (6428), 418-423, 1993; von Itzstein et al., Nat Rev Drug Discov 6 (12), 967-974, 2007). The synthetic routes for 4NH2Neu5Ac2en (3) (Schreiner et al., Liebigs Annalen der Chemie 1991 (2), 129-134, 1991) were improved (on Itzstein et al., Carbohydr Res 1994, 259 (2), 301-305, 1994; Chandler et al., J Chem Soc Perkin 1 (9), 1173-1180, 1995), and additional derivatives including those with a C4-azido (4N3Neu5Ac2en, 5) or a C4-N-acetyl (4NAcNeu5Ac2en, 6) substitution (Holzer et al., Glycoconj J 10 (1), 40-44, 1993; Shidmoossavee et al., J Am Chem Soc 135 (36), 13254-13257, 2013) were produced and investigated as sialidase inhibitors. Sialosides containing 4-N-substituted Neu5Ac and a simple aglycone, such as methyl α-sialosides SiaαMe (Ciccotosto et al., Tetrahedron Lett 36 (30), 5405-5408, 1995) and para-nitrophenyl α-sialosides (SiaαpNPs) (Shidmoossavee et al., J Am Chem Soc 135 (36), 13254-13257, 2013) have been chemically synthesized. The latter compounds were used as potential substrates for sialidases from an H3N2 IAV (N2) and from the bacterium Micromonospora viridifaciencs (Shidmoossavee et al., J Am Chem Soc 135 (36), 13254-13257, 2013). α-Sialyl fluorides containing 3-fluoro and 4-amino- or 4-guanodino-Neu5Ac have also been synthesized and shown to be mechanism-based selective anti-influenza virus inhibitors (Kim et al., Science 340 (6128), 71-75, 2013). Due to the better resemblance to the native NA substrates, these inhibitors were believed to be less prone to suffering from the development of resistant strains compared to clinically used oseltamivir (Tai et al., Virus Res 210, 126-132, 2015). Chemically synthesized O-linked and S-linked α2–6-sialyl galactosides containing 4-N3, 4-NH2, or 4-acetamido-subsituted Neu5Ac (Sabesan, Bioorg Med Chem Lett 4 (20), 2457-2460, 1994) were shown to be resistant to hydrolysis by an NA from IAVs and 4- NH2-derivative of the Ο-linked sialoside was shown to be an inhibitor with a Ki of 150 µM and the corresponding S-linked sialoside had a Ki of 50 µM (Sabesan et al., Carbohydr Res 267 (2), 239-261, 1995). The binding preferences toward α2–6- or α2–3-linked sialic acid receptors of the hemagglutinins (HAs) of IAV isolates from human versus avian and equine species, respectively, are well documented (Connor et al., Virology 205 (1), 17-23, 1994; Rogers et al., Virology 173 (1), 317-322, 198). Synthetic α2– 3- and α2–6-linked sialyl galactoses containing a terminal 4,7-di-O-methyl Neu5Ac have been shown to be suitable for electrochemical detection of influenza NAs selectively (Cui et al., Chem Sci 8 (5), 3628-3634, 2017). Synthetic para-nitrophenylated sialyl galactosides (Siaα2–3 / 6GalβpNP) containing different sialic acid linkages and diverse sialic acid forms can be used for high-throughput substrate specificity studies of sialidases from various sources (Chokhawala et al., Chembiochem 8 (2), 194-201, 2007) and NAs of IAVs from different subtypes and groups (Li et al., Virology 415 (1), 12-19, 2011). Such probes containing a terminal sialic acid modified with 4-amino, 4-guanidino, and other 4-N-substitutions have the potential to differentiate influenza NAs and sialidases from other sources, similar to what was previously shown for9531-111023-03Siaα2–3 / 6GalβpNP compounds containing 7-N-acetyl derivative of Neu5Ac (Neu5Ac7NAcα2– 3 / 6GalβpNP) (Kooner et al., ACS Infect Dis 9 (1), 33-41, 2023). To assess these sialoside targets, 4-amino-N-acetylneuraminic acid (Li et al., J Am Chem Soc 144 (1), 424-435, 2022) (4NH2Neu5Ac, 7) (FIG.23) was chosen as a chemoenzymatic precursor or synthon to synthesize the 4-N- substituted derivatives of Neu5Acα2–3 / 6GalβpNP (Neu5AcαOR, 8a / b) including those with 4-amino- (9a / b), 4-guanidino- (10a / b), 4-azido- (11a / b), and 4-acetamido (12a / b)-derivatives using a one-pot two-enzyme (OP2E) chemoenzymatic synthesis strategy (Chen et al., Acc Chem Res 57 (2), 234- 246, 2024). Previously synthesized α2–3-sialyl GalβpNP containing naturally occurring 4-O-acetylated Neu5Ac (Neu4,5Ac2α2–3GalβpNP, 13a) (Yu et al., Org Biomol Chem 14 (36), 8586-8597, 2016) was included as a control for substrate-specificity studies, as it has been shown to be selectively cleaved by influenza NAs. 4NH2Neu5Ac (7) was synthesized from commercially available Neu5Ac (1) in seven steps via a fully protected 4-azido-Neu5Ac2en intermediate (16) (FIG.24) similar to that reported previously (Li et al., J Am Chem Soc 144 (1), 424-435, 2022). Briefly, the carboxyl group in Neu5Ac (1) was methylated and its hydroxyl groups were protected by acetylation to form per-O-acetylated methyl ester of Neu5Ac (14) in two steps in an overall 90% yield. Treatment of 14 with BF3.Et2O formed oxazoline 15 (Morais et al., Tetrahedron Lett 50 (14), 1642-1644, 2009; von Itzstein et al., Carbohydr Res 244 (1), 181-185, 1993), which reacted with trimethylsilyl azide (TMSN3) to produce 4-azido methyl ester 16 (von Itzstein et al., Carbohydr Res 244 (1), 181-185, 1993). Treatment of 16 with N-bromosuccinimide (NBS) followed by debromination and reduction using tri-n-butyltinhydride (Ciccotosto et al., Tetrahedron Lett 36 (30), 5405- 5408, 1995; Lu et al., Carbohydr Res 342 (12-13), 1636-1650, 2007) yielded 4-amino methyl ester 17 in 42% yield over two steps. Deprotection of 17 with sodium methoxide in methanol and water (Li et al., J Am Chem Soc 144 (1), 424-435, 2022) formed the target 4NH2Neu5Ac (7). The protected 4-azido methyl ester 16 was then used as an intermediate for synthesizing the 4-N- derivatives of the transition state analog-based inhibitor of NAs including 4N3Neu5Ac2en (5), 4NH2Neu5Ac2en (3), and 4NAcNeu5Ac2en (6). Briefly, deprotection of the fully protected 4N3Neu5Ac2en (16) in NaOH aqueous solution formed 4N3Neu5Ac2en (5) in 95% yield. Treating compound 5 with PMe3under a basic condition (Simonsen et al., Chembiochem 3 (12), 1223-1228, 2022), 4NH2Neu5Ac2en (3) was synthesized in 86% yield. On the other hand, the azido group in compound 16 was converted to 4-acetamido by incubation with thioacetic acid (AcSH) in pyridine and after deprotection with NaOH in MeOH (Simonsen et al., Chembiochem 3 (12), 1223-1228, 2022), 4NAcNeu5Ac2en (6) was obtained in 94% yield. With 4NH2Neu5Ac (7) in hand, para-nitrophenol (pNP)-tagged sialosides 4NH2Neu5Acα2– 3GalβpNP (9a) and 4NH2Neu5Acα2–6GalβpNP (9b) (FIG.25) were readily prepared in 92% and 82% yields, respectively, using a one-pot two-enzyme (OP2E) sialylation systems (Chen et al., Acc Chem Res 57 (2), 234-246, 2024) containing Neisseria meningitidis cytidine 5’-monophosphate (CMP)-sialic acid synthetase (NmCSS) (Yu et al., Bioorg Med Chem 12 (24), 6427-6435, 2004) and Pasteurella multocida9531-111023-03α2–3-sialyltransferase-1 (PmST1) (Yu et al., J Am Chem Soc 127 (50), 17618-17619, 2005) or Photobacterium sp. α2–6-sialyltransferase A366G mutant (Psp2,6ST_A366G) (Ding et al., Carbohydr Res 408, 127-133, 2015). Derivatizing the C4-NH2 group in these pNP-tagged sialosides 4NH2Neu5Acα2–3 / 6GalβpNP (9a / b) formed other C4-modified derivatives (10a / b–12a / b) (FIG.25). For example, 4-guanidino-Neu5Acα2– 3 / 6GalβpNP (10a / b) were obtained in 86% and 81% yields, respectively, by treating 9a / b with 1,3-di-Boc-2- (trifluoromethylsulfonyl)guanidine (Baker et al., J Org Chem 65 (26), 9054-9058, 2000) in tetrahydrofuran and methanol under basic reaction conditions followed by removing the Boc protecting group in trifluoroacetic acid. Treating 9a / b with freshly prepared triflate azide (TfN3) in the presence of catalytic CuSO4 and triethylamine (Lau et al., Chem Commun (Camb) 46 (33), 6066-6068, 2010) produced 4N3Neu5Acα2–3 / 6GalβpNP (11a / b) in 83% and 87% yields, respectively. On the other hand, treating 9a / b with acetic anhydride in the presence of triethylamine (Baker et al., J Org Chem 19 (11), 1786-1792, 1954) produced 4NAcNeu5Acα2–3 / 6GalβpNP (12a / b) at 98% and 95% yields, respectively. 4N3Neu5Acα2–3 / 6GalβpNP (11a / b), in addition to Neu4,5Ac2α2–3GalβpNP (13a), are selective substrates for influenza NAs. The obtained sialosides containing 4-N-substituted Neu5Ac (9a / b–12a / b) were used together with Neu5Acα2–3 / 6GalβpNP (8a / b) (Chokhawala et al., Chembiochem 8 (2), 194-201, 2007) and Neu4,5Ac2α2–3GalβpNP (13a) (Yu et al., Org Biomol Chem 14 (36), 8586-8597, 2016) as potential substrates for multiwell plate-based high-throughput substrate specificity studies of human, bacterial, and influenza NAs. These include NAs from two recent H1N1 IAV strains A / Victoria / 2570 / 2019 (N1-Vic19) and A / Brisbane / 02 / 2018 (N1-BR18), and two recent H3N2 IAV strains A / Darwin / 9 / 2021 (N2-Dar21) and A / Kansas / 14 / 2017 (N2-Kan17); and NAs from two IBV strains B / Austria / 1359417 / 2021 (NB-Aus21) and B / Phuket / 3073 / 2013 (NB-Phu13) (Mishra et al., Angew Chem Int Ed Engl 63 (29), e202403133, 2024). To compare relative substrate preferences, NA amounts in the low-enzyme-concentration assays were standardized so that Neu5Acα2–3GalβpNP cleavage during a 30-minute reaction at 37°C reached 40– 80%. Each sialoside, in a 384-well plate, was incubated with an NA in the presence of an excess amount of β-galactosidase which catalyzes the cleavage of the NA product GalβpNP to produce p-nitrophenol. Following the addition of N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer (pH 11.5) to adjust the pH of the reaction mixture to above 9.5, A405 nmvalues (to quantify the formation of p-nitrophenolate) were determined using a plate reader. As shown in FIGS.26A-26D, 4N3Neu5Acα2–3GalβpNP (11a) and Neu4,5Ac2α2–3GalβpNP (13a) were selectively cleaved off by all influenza NAs tested but not human NEU2 or bacterial sialidases. The selectivity for influenza NAs was observed under both assay conditions but was more pronounced with the high-enzyme-concentration assay conditions which used 10-fold higher enzyme concentrations (except for Vc sialidase used at a 4-fold higher concentration) and a reaction time of 1 hour. Unlike Neu4,5Ac2α2– 3GalβpNP (13a) which was a suitable substrate for influenza NAs but not human or bacterial sialidases (Yu9531-111023-03et al., Org Biomol Chem 14 (36), 8586-8597, 2016), its more stable 4-N-acetyl derivative 4NAcNeu5Acα2– 3GalβpNP (12a) was resistant to cleavage by all of the sialidases tested. Example 14: 4NH2Neu5Ac- and 4-Guanidino-Neu5Ac-Sialosides Are Selective Inhibitors Against Influenza NAs It is well-known that both 4NH2Neu5Ac2en (1) and 4-guanidino-Neu5Ac2en (or Zanamivir, 2) bind to influenza NAs much stronger than Neu5Ac2en (Holzer et al., Glycoconj J 10 (1), 40-44, 1993). The observation that both 4NH2Neu5Acα2–3 / 6GalβpNP (9a / b) and 4-guanidino-Neu5Acα2–3 / 6GalβpNP (10a / b) were very poor substrates for the influenza NAs tested under both assay conditions (FIG.26B) indicated that these sialosides could be effective inhibitors. To test this hypothesis, a fluorescence-based inhibition assay platform was developed. In this assay, a 4-methylumbelliferyl (MU)-tagged sialoside Neu5Acα2–3GalβMU (18, see ESI for synthesis) (8.0 µM) was used as a sialidase substrate in a 96-well plate. In the absence of an inhibitor, a sialidase can catalyze the cleavage of Neu5Ac to form GalβMU, which is then cleaved by the excess amount of β-galactosidase in the reaction mixture to release the fluorescent molecule MU that can be detected by a plate reader. Indeed, when Neu5Acα2–3GalβMU (8.0 µM) was used as the substrate and the same concentration of a pNP-tagged sialoside was used as an inhibitor, 4-guanidino-Neu5Acα2–3GalβpNP (10a) and 4- guanidino-Neu5Acα2–6GalβpNP (10b) showed 80–98% and 67–93%, inhibitory activities, respectively, against the influenza NAs tested (Table 6). Inhibitory activity of 4NH2Neu5Acα2–3GalβpNP (9a) was also significant against N1-Vic19 (48.3 ± 7.9%) and N1-BR18 (47.2 ± 3.3%), which aligned well with previously reported results (Sabesan et al., Carbohydr Res 267 (2), 239-261, 1995). In contrast, no significant inhibitory activity of the pNP-tagged sialosides against human cytosolic sialidase hNEU2 (Li et al., Mol Biosyst 7 (4), 1060-1072, 2011), bacterial Streptococcus pneumoniae sialidase SpNanA (Tasnima et al., Org Biomol Chem 15 (1), 160-167, 2016), and the sialidase activity of a multifunctional Pasteurella multocida α2–3- sialyltransferase 1 (PmST1) (Yu et al., J Am Chem Soc 127 (50), 17618-17619, 2005) in the presence of cytidine-5’-monophosphate (CMP) (Table 7, ESI) was observed under the same assay conditions. Compared to 4NH2Neu5Acα2–3GalβpNP (9a), the inhibitory activity of 4NH2Neu5Acα2–6GalβpNP (9b) was weaker against NB-Aus21 and NB-Phu13 and insignificant against the N1 or N2 NAs tested. Table 6. Percentage inhibition (%) of pNP-tagged sialosides containing a 4-N-modified Neu5Ac and Sia2en compounds against IAV NAs (N1-Vic19, N1-BR18, N2-Dar21, N2-Kan17) and IBV NAs (NB-Aus21, NB- Phu13). Percentage inhibitions higher than 47% are in bold. NA N1-Vic19 N1-BR18 N2-Dar21 N2-Kan17 NB-Aus21 NB-Phu13 9531-111023-03NA N1-Vic19 N1-BR18 N2-Dar21 N2-Kan17 NB-Aus21 NB-Phu13 Inhibitor 6 5 8 3 3 5 6 . Sia2en derivatives against PmST1, SpNanA, and hNEU2. Inhibition levels higher than 47% are in bold. NA PmST1 WT SpNanA hNEU2 Inhibitor Neu5Ac2en displayed general inhibitory activity against all sialidases tested except for the sialidase activity of PmST1 (Table 6 and Table 7, ESI). Its inhibitory activity against N2s and hNEU2 was generally lower than other sialidases tested. In comparison, 4NH2Neu5Ac2en (3) and Zanamivir (4) were selective inhibitors against influenza NAs as demonstrated previously (von Itzstein et al., Nature 363 (6428), 418- 423, 1993). Selective inhibitory activity of 4N3Neu5Ac2en (5) was observed for the IBV NAs NB-Aus21 (78.8 ± 3.7%) and NB-Phu13 (75.6 ± 1.6%).9531-111023-03IC50values (µM) were further determined for the compounds with significant activities against IAV and IBV NAs (Table 6) in the inhibition assays. As shown in Table 8, Neu5Ac2en was a micromolar inhibitor whereas Zanamivir (4) was a nanomolar inhibitor against influenza NAs consistent with previous reports (Holzer et al., Glycoconj J 10 (1), 40-44, 1993; Kim et al., Science 340 (6128), 71-75, 2013; Ferraris et al., Antiviral Res 68 (1), 43-48, 2005; Nishikawa et al., PLoS One 7 (9), e45371, 2012; Wetherall et al., J Clin Microbiol 41 (2), 742-750, 2003). The inhibition efficiency of sialoside 4-guanidino-Neu5Acα2– 3GalβpNP (10a) was better than 4NH2Neu5Ac2en (3) against N1-Vic19 and N1-BR18 (about 4-fold better), as well as N2-Kan17 and N2-Dar21 (about 10-fold better), but it was still ~10-fold less efficient than Zanamivir (4) in inhibiting these IAV NAs. In addition, 4-guanidino-Neu5Acα2–6GalβpNP (10b) with an α2–6-sialyl linkage was generally a weaker NA inhibitor than its α2–3-sialyl linked counterpart 4- guanidino-Neu5Acα2–3GalβpNP (10a), agreeing well with the data shown in Table 6. Therefore, α2–3- linked 4-guanidino-Neu5Ac-sialosides can serve as a new class of sialidase inhibitors (Keil et al., J Med Chem 65 (20), 13574-13593, 2022) that are based on substrate-analogs. These inhibitors have the advantage of allowing easy immobilization or conjugation via a linker readily installed at their reducing end as demonstrated below. Table 8. IC50 values (µM) of 4-guanidino-sialosides (10a / b), Neu5Ac2en (2), its 4-N-derivatives (3–5). NA N1-Vic19 N1-BR18 N2-Dar21 N2-Kan17 NB-Aus21 NB-Phu13 Inhibitor 0 0 0 Example 15: Chemoenzymatic Synthesis of Biotinylated Sialosides as Affinity Ligands for Influenza NAs The strong inhibitory activity of 4-guanidino-Neu5Acα2–3GalβpNP (10a) against the NAs from IAVs (N1 and N2) indicated that this compound could potentially be used as a selective affinity ligand to purify NAs and IAVs for vaccine development. To test this hypothesis, biotinylated and polyethylene oligomer (PEG4)-linked galactosides GalβProNH-PEG4-Biotin (19) and LacNAcβProNH-PEG4-Biotin (20) were synthesized (ESI) and used as sialyltransferase acceptors and negative controls for binding studies. α2– 3-Linked sialosides 4NH2Neu5Acα2–3GalβpProNH-PEG4-Biotin (9c) and 4NH2Neu5Acα2– 3LacNAcβpProNH-PEG4-Biotin (9d) were chemoenzymatically synthesized from 4NH2Neu5Ac (7) and the corresponding biotinylated galactoside (19) and biotinylated LacNAc-glycoside (20), respectively, using the9531-111023-03one-pot two-enzyme (OP2E) sialylation systems similar to that described above and illustrated in FIG.25. They were chemically derivatized at the 4-amino group of the 4NH2Neu5Ac (7) to form sialosides containing 4-guanidino-Neu5Ac (10c and 10d), 4N3Neu5Ac (11c and 11d), and 4NAcNeu5Ac (12c and 12d), respectively. The corresponding Neu5Acα2–3Gal / LacNAcβProNH-PEG4-Biotin (8c / d) with an α2–3- sialyl linkage and Neu5Acα2–6Gal / LacNAcβProNH-PEG4-Biotin (8e / f) with an α2–6-sialyl linkage were synthesized from Neu5Ac using a similar OP2E sialylation strategy and used as controls. Enzyme-linked immunosorbent assays (ELISA) using neutravidin-coated 384-well plates showed (FIG.27) that both 4NH2Neu5Acα2–3Gal / LacNAcβProNH-PEG4-Biotin (9c / d) and 4-guanidino- Neu5Acα2–3Gal / LacNAcβProNH-PEG4-Biotin (10c / d) indeed were suitable ligands for immobilizing the NAs from both IAVs and IBVs as detected by a horseradish peroxidase (HRP)-conjugated anti- histidine / strep antibody followed by adding an HRP substrate 3,3’,5,5’-tetramethylbenzidine (TMB). In comparison, galactoside (19) and LacNAc-glycoside (20) and the corresponding Neu5Ac-sialosides (8c–8f), 4N3Neu5Ac-sialosides (11c / d), and 4NAcNeu5Ac-sialosides (12c / d) were not suitable affinity ligands. The lack of the binding can be due to the absence of or weak interactions between the glycosides and the NAs (e.g., galactoside 19, LacNAc-glycoside 20, and 4NAcNeu5Ac-sialosides 12c / d), or cleavage of the terminal sialic acids (e.g., Neu5Ac-sialosides 8c–f, 4N3Neu5Ac-sialosides 11c / d).4NH2Neu5Ac-sialosides (9c / d) showed weaker binding to N2-Dar21 and N2-Kan17, compared to other influenza NAs, suggesting NAs from these H3N2 IAVs generally have lower affinity for 4NH2Neu5Ac-sialosides (9c / d). Bio-layer interferometry (BLI) experiments were carried out to investigate the binding affinity between biotinylated sialosides immobilized on streptavidin-coated biosensors and influenza NAs as analytes. Real-time BLI assays for N2-Kan17 with sialyl LacNAc-glycosides and sialyl Gal-glycosides containing 4NH2- or 4-guanidino-derivative of Neu5Ac (FIG.34, ESI) showed that sialyl LacNAc- glycosides were better binders than their sialyl Gal-glycoside counterparts, aligning well with the ELISA results (FIG.27). Therefore, the α2–3-linked sialyl LacNAc-glycosides were chosen for more detailed BLI assays. As shown in FIGS.28A-28B, 4-guanidino- (10d) and 4NH2-(9d) derivatives are significantly stronger binders than other sialosides. Weak binding of N1-Vic19, N2-Dar21, and the IBV NAs to the 4N3- derivative (11d) and weak binding of N1-Vic19 and N2-Dar21 to the 4NAc-derviative (12d) were also observed. To obtain binding kinetic data of 4NH2- or 4-guanidino- Neu5Acα2–3LacNAcβProNH-PEG4- Biotin (9d or 10d), influenza NAs in serial dilutions were used as analytes in a steady-state analysis (FIGS. 29A-29B). The results showed that 4NH2-modified sialoside (9d) bound to the NAs but was partially cleaved off during the incubation. In comparison, the 4-guanidino-modified sialoside (10d) was resistant to the NA-catalyzed cleavage. By fitting equilibrium response curves during the association phase and the NA concentration to the dissociation rate constant equation (FIGS.35A-35B, ESI), the KDvalues (2.90–8.49 × 10-8M) were in the nanomolar range. The KD value (4.55 × 10-8M) obtained for the interaction of N1-BR18 to 4-guanidino-Neu5Acα2–3LacNAcβProNH-PEG4-Biotin (10d) was comparable to the IC50value,9531-111023-03(4.3±0.4) ×10-8M, determined by the inhibition studies for the counterpart 4-guanidino-Neu5Acα2– 3GalβpNP (10a) (Table 8). Example 16: Biotinylated 4-Guanidino-Sialosides as Affinity Ligands for Purifying Influenza viruses Together, the results above indicated that the immobilized 4-guanidino-Neu5Acα2– 3LacNAcβProNH-PEG4-Biotin (10d) and related 4-guanidino-Neu5Ac-sialosides could be used as affinity ligands to isolate influenza viruses for analysis and downstream applications. Supporting this concept, specific binding of the vaccine strain A / Brisbane / 02 / 2018 (H1N1) in egg allantoic fluid was observed by ELISA using streptavidin-coated 96-well plates (FIG.30A). In addition, 4-guanidino-Neu5Acα2– 3LacNAcβProNH-PEG4-Biotin (10d)-immobilized streptavidin beads were efficient in retaining H1N1 / BR18 virions (FIG.30B) which were readily eluted using 50 mM Neu5Ac2en. In comparison, streptavidin beads immobilized with the control glycan LacNAcβProNH-PEG4-Biotin (20) did not retain influenza virions. SDS-PAGE analysis (FIG.30C) indicated that the elution fraction contained the expected viral protein bands with almost no contaminating allantoic fluid proteins. Dynamic light scattering analysis showed that eluted virus was homogeneous with reasonable polydispersity (Pd < 30%) and a mean diameter of ~125 nm (FIG.30D), confirming that the approach had indeed isolated virus efficiently. Conclusions A series of α2–3- and α2–6-linked sialyl glycosides containing a 4-N-substituted sialic acid have been synthesized by highly efficient one-pot two-enzyme (OP2E) sialylation systems using chemically synthesized 4NH2Neu5Ac (7) as a chemoenzymatic synthon. Colorimetric high-throughput substrate specificity studies identified those with a terminal α2–3-linked 4N3Neu5Ac, together with a terminal α2–3- linked Neu4,5Ac2, as selective substrates for NAs from both IAVs and IBVs. They can serve as useful diagnostic tools for influenza virus infection. On the other hand, sialosides containing a terminal 4NAcNeu5Ac are resistant to NA cleavage. Significantly, sialosides containing a terminal 4-guanidino- Neu5Ac have been shown to be strong and effective inhibitors and affinity ligands selective for IAV and IBV NAs. The application of 4-guanidino-Neu5Ac-sialosides as affinity ligands have been demonstrated for purifying influenza virions from egg allantoic fluid. These probes will contribute to the continuous efforts in advancing knowledge in influenza virus infection and developing next-generation vaccines, diagnostics, and therapeutics against influenza virus infection. It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

9531-111023-03CLAIMS 1. A method for detecting the presence or absence of influenza virus neuraminidase (NA) inhibitory (NAI) antibodies in a biological sample, comprising: (i) incubating the biological sample in a reaction vessel comprising recombinant NA protein or comprising influenza virus containing NA protein, under conditions sufficient for any NAI antibodies present in the biological sample to bind the recombinant NA protein or the NA protein of the influenza virus; (ii) adding a NA inhibitor (NAi) to the reaction vessel, wherein the NAi binds to the NA active site in the absence of NAI antibodies in the biological sample; (iii) adding a NA reporter substrate to the reaction vessel, wherein the NA reporter substrate comprises a reporter molecule covalently linked to sialic acid or a sialic acid derivative, and the reporter molecule is detectable upon cleavage of the sialic acid or the sialic acid derivative; and (iv) detecting the presence or absence of the detectable reporter molecule, wherein: an increase in the detectable reporter molecule compared to a control sample indicates the biological sample contains NAI antibodies; no change in the detectable reporter molecule compared to a control sample indicates the biological sample does not contain NAI antibodies; and a decrease in the detectable reporter molecule compared to a control sample indicates the biological sample contains NAI antibodies that bind to the NA active site.

2. The method of claim 1, wherein the biological sample comprises blood or serum.

3. The method of claim 1 or claim 2, wherein the method further comprises serially diluting the biological sample prior to adding the biological sample to the reaction vessel.

4. The method of claim 3, wherein at least two, at least three, at least four, or at least five dilutions of the biological sample are incubated in separate wells of the reaction vessel.

5. The method of any one of claims 1-4, further comprising diluting the recombinant NA protein or the influenza virus containing NA protein prior to adding to the reaction vessel.

6. The method of claim 5, wherein the NA protein or the influenza virus containing NA protein is diluted such that the resulting NA activity is about 0.25-0.5 pmol / second of substrate.9531-111023-037. The method of any one of claims 1-6, wherein the biological sample is incubated with the recombinant NA protein or the influenza virus containing NA protein in step (i) for approximately 60 minutes.

8. The method of any one of claims 1-7, wherein the NAi is incubated with the biological sample and the recombinant NA protein or the influenza virus containing NA protein in step (ii) for about 3 hours.

9. The method of any one of claims 1-8, wherein the presence or absence of the detectable reporter molecule is detected at multiple timepoints after addition of the NA reporter substrate.

10. The method of any one of claims 1-8, wherein the presence or absence of the detectable reporter molecule is detected at a single timepoint following addition of the NA reporter substrate.

11. The method of claim 10, further comprising adding a stop solution prior to detecting the presence or absence of the detectable reporter molecule.

12. The method of claim 11, wherein the stop solution comprises 0.133 mM glycine, 0.06 M NaCl, and 0.083N Na2CO3 at pH10.

7.

13. The method of any one of claims 1-12, wherein the detectable reporter molecule is a fluorescent molecule, a colorimetric molecule, or a chemiluminescent molecule.

14. The method of claim 13, wherein the fluorescent molecule is 4-methyl umbelliferone.

15. The method of any one of claims 1-14, wherein the NA reporter substate is 4- methylumbelliferyl-N-acetyl-α-D-neuraminic acid (MUNANA).

16. The method of any one of claims 1-15, wherein the NAi is a monoclonal antibody.

17. The method of any one of claims 1-15, wherein the NAi is a synthetic molecule.

18. The method of claim 17, wherein the synthetic molecule comprises one of the following molecules bound to streptavidin: 4-guanidino-Neu5Acα2–3Galβ1–4GlcNAc–βProNH-PEG4-Biotin; 4-guanidino-Neu5Acα2–3GalβProNH-PEG4-Biotin; 4-guanidino-Neu5Acα2–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβProNH-PEG4-Biotin;9531-111023-034-guanidino-Neu5Acα2–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcβProNH-PEG4-Biotin; 4-guanidino-Neu5Acα2–6Galβ1–4GlcNAcβProNH-PEG4-Biotin; 4-guanidino-Neu5Acα2–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβProNH- PEG4-Biotin; 4-guanidino-Neu5Acα2–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1– 4GlcNAcβProNH-PEG4-Biotin; or 4-guanidino-Neu5Acα2–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1– 4GlcNAcβ1–3Galβ1–4GlcNAcβProNH-PEG4-Biotin.

19. The method of any one of claims 1-18, wherein the reaction vessel further comprises detergent when influenza virus containing NA protein is used in the method.

20. The method of any one of claims 1-19, wherein the reaction vessel comprises a multi-well plate.

21. The method of any one of claims 1-20, further comprising calculating the NAI antibody titer in the biological sample.

Images