A novel whole blood assay to quantify the release of T cell associated cytokines in response to Bordetella pertussis antigens.

BACKGROUND: Bordetella pertussis continues to cause whooping cough globally even in countries with high immunisation coverage. Booster vaccinations with acellular pertussis vaccines are thus used in children, adolescents, and adults. T cell immunity is crucial for orchestrating the immune response after vaccination. However, T cell assays can be expensive and difficult to implement in large clinical trials. In this study, a whole blood (WB) stimulation assay was developed to identify secreted T cell associated cytokines in different age groups after acellular pertussis booster vaccination. MATERIAL AND METHODS: Longitudinal WB samples were collected from a small set of subjects (n = 38) aged 7-70 years participating in a larger ongoing clinical trial. For assay development, samples were diluted and incubated with purified inactivated pertussis toxin (PT), filamentous haemagglutinin (FHA), inactivated B. pertussis lysate, and complete medium (M) as stimulating conditions, with anti-CD28 and anti-CD49d as co-stimulants. Different timepoints around the vaccination (D0, D7, D14, D28), WB dilution factor (1:2, 1:4) and incubation time (24 h, 48 h, 72 h) were compared. Responses to 15 cytokines were tested with Luminex/multiplex immunoassay. RESULTS: The optimized assay consisted of WB incubation with M, PT, and FHA (including the two co-stimulants). After 48 h incubation, supernatants were collected for measurement of seven selected T cell associated cytokines (IL-2, IL-5, IL-10, IL-13, IL-17 A, IL-17F, and IFN-y) from samples before and 28 days after vaccination. PT stimulation showed a trend for upregulation of IL-2, IL-13, and IL-17 A/F for adult subjects, whereas the responses of all cytokines were downregulated for the paediatric subjects. Furthermore, PT and FHA-stimulated WB showed diverse cytokine producing profiles. CONCLUSIONS: The developed WB-based cytokine assay was shown to be less costly, easy to perform, and functional in differently aged individuals. Further, it requires only a small amount of fresh blood, which is beneficial especially for studies including infants. Our results support the use of this assay for other immunological studies in the future.

Trivalent oleanolic acid-glucose conjugates: Synthesis and efficacy against Influenza A virus.

Influenza A virus (IAV) leads to significant morbidity and mortality due to the seasonal epidemics and spread. We have demonstrated that oleanolic acid (OA) C28 glucose conjugates and OA trimers are capable of effectively blocking the recognition and interaction between the influenza virus and host cells. In this study, a series of OA-glucose trimers were designed and synthesized through the CuAAC reaction. All trimers underwent screening for anti-IAV activities in vitro. Among these, compounds 13a and 13b showed inhibitory activity against the influenza virus, with IC50 values of 0.68 µM and 0.47 µM, respectively, demonstrating greater potency than oseltamivir (IC50 = 1.36 µM). Results from the time-of-addition experiment and hemagglutination inhibition assay suggest that these OA-glucose trimers may disrupt the recognition between the HA protein of IAV and sialic acid receptors on host cells, thus blocking viral entry. Furthermore, it was found that compound 13b effectively inhibits IAV infection in BALB/c mice. This study has elucidated the structure-activity relationships of OA trimers against the influenza virus and highlighted the utility of multivalent OA conjugates for enhancing ligand-target interactions in anti-influenza virus drug design, laying a groundwork for future research into the antiviral applications of these natural products.

Dually-amplified electrochemical aptasensor based on the self-linking AuPt nanoflowers for ultrasensitive determination of hemagglutinin.

BACKGROUND: Influenza virus can spread from person to person and cause epidemics. Therefore, rapid and sensitive diagnosis of virus is essential in controlling influenza outbreaks. Conventional virus diagnostic techniques are time-consuming, labor-intensive and requires large instruments. In this work, a sandwich electrochemical assay by a pair of aptamers was developed for ultrasensitive determination of hemagglutinin (HA) protein, which is one of the two surface glycoproteins of influenza A (H1N1) virus, using dual signal amplification techniques. RESULTS: HA was captured and magnetically separated by Fe3O4@SiO2-NH2@Au attached to aptamer 1 (Apt1), creating a sandwich structure with AuPt nanoflowers (AuPtNFs) connected to aptamer 2 (Apt2). Herein, AuPtNFs could catalyze H2O2/hydroquinone (HQ) to generate 1,4-benzoquinone (BQ), and achieved amplification of electrochemical signal detection through differential pulse voltammetry (DPV). The constructed aptasensor expressed a wide linear range (10 pg/mL-100 ng/mL) with limit of detection (LOD) of 2.4 pg/mL. Moreover, a novel strategy for dual signal amplification was developed to further enhance sensitivity. The innovative electrochemical aptasensor could achieve secondary amplification of the detection signal with LOD of 0.3 pg/mL and linear concentration range from 0.5 pg/mL to 100 ng/mL. The secondary amplification could be achieved only through the self-linking process, which allowed for the retention of numerous AuPtNFs by simple complementary base pairing to connect more AuPtNFs onto the above-mentioned sandwich structure. SIGNIFICANCE: Overall, the constructed aptasensor exhibited favorable sensitivity and accuracy, indicating the potential expanded application for the clinical detection of numerous viruses.

Effects of Alkaline Solutions on the Structure and Function of Influenza A Virus.

Influenza A virus (IAV) infection contributes to high annual morbidity and mortality, thus necessitating measures aimed at protecting against the disease. Alcohol-based disinfectants are commonly used to inactivate IAV, but they have several undesirable properties. In search of other means which would inactivate IAV, we focused on the effect of alkaline solutions on IAV. We found the viral infectivity remarkably decreased with treatment of an alkaline solution at pH 12.0 for 1 min, where destruction of the viral spikes was observed using an electron microscope. A more detailed examination revealed that the infectivity of IAV was remarkedly reduced by brief treatment with the alkaline solution at pH 11.75 or above, most likely due to the degradation of viral hemagglutinin protein. These results show that at a high pH, the haemagglutinin protein is degraded, resulting in very rapid inactivation of IAV.

Immunity and Protective Efficacy of a Plant-Based Tobacco Mosaic Virus-like Nanoparticle Vaccine against Influenza a Virus in Mice.

BACKGROUND: The rapid production of influenza vaccines is crucial to meet increasing pandemic response demands. Here, we developed plant-made vaccines comprising centralized consensus influenza hemagglutinin (HA-con) proteins (H1 and H3 subtypes) conjugated to a modified plant virus, tobacco mosaic virus (TMV) nanoparticle (TMV-HA-con). METHODS: We compared immune responses and protective efficacy against historical H1 or H3 influenza A virus infections among TMV-HA-con, HA-con protein combined with AddaVax™ adjuvant, and whole-inactivated virus vaccine (Fluzone®). RESULTS: Immunogenicity studies demonstrated robust IgG, IgM, and IgA responses in the TMV-HA-con and HA-con protein vaccinated groups, with relatively low induction of interferon (IFN)-γ+ T-cell responses across all vaccinated groups. The TMV-HA-con and HA-con protein groups displayed partial protection (100% and 80% survival) with minimal weight loss following challenge with two H1N1 strains. The HA-con protein group exhibited 80% and 100% survival against two H3 strains, whereas the TMV-HA-con groups showed reduced protection (20% survival). The Fluzone® group conferred 20-100% survival against two H1N1 strains and one H3N1 strain, but did not protect against H3N2 infection. CONCLUSIONS: Our findings indicate that TMV-HA and HA-con protein vaccines with adjuvant induce protective immune responses against influenza A virus infections. Furthermore, our results underscore the potential of plant-based production using TMV-like nanoparticles for developing influenza A virus candidate vaccines.

Antiviral Susceptibility of Swine-Origin Influenza A Viruses Isolated from Humans, United States.

Since 2013, a total of 167 human infections with swine-origin (variant) influenza A viruses of A(H1N1)v, A(H1N2)v, and A(H3N2)v subtypes have been reported in the United States. Analysis of 147 genome sequences revealed that nearly all had S31N substitution, an M2 channel blocker-resistance marker, whereas neuraminidase inhibitor-resistance markers were not found. Two viruses had a polymerase acidic substitution (I38M or E199G) associated with decreased susceptibility to baloxavir, an inhibitor of viral cap-dependent endonuclease (CEN). Using phenotypic assays, we established subtype-specific susceptibility baselines for neuraminidase and CEN inhibitors. When compared with either baseline or CEN-sequence-matched controls, only the I38M substitution decreased baloxavir susceptibility, by 27-fold. Human monoclonal antibodies FI6v3 and CR9114 targeting the hemagglutinin's stem showed variable (0.03 to >10 µg/mL) neutralizing activity toward variant viruses, even within the same clade. Methodology and interpretation of laboratory data described in this study provide information for risk assessment and decision-making on therapeutic control measures.

Collaborative study on the cross-reactivity of two influenza B viral components in single radial immunodiffusion assay using quadrivalent influenza vaccines in Japan from 2015/16 to 2021-22 influenza season.

A quadrivalent influenza vaccine (QIV) has been available in Japan since the 2015/2016 influenza season. Single radial immunodiffusion (SRID) assays are currently used worldwide to measure the hemagglutinin (HA) content of influenza vaccine components because they are simple, accurate, and the regulatory requirement, ensuring consistency in manufacture for the HA content. However, the cross-reactivity of antisera against the two lineages of the influenza B virus (IFVB) may cause inaccurate quantification of HA content in QIVs using the SRID assay. To examine cross-reactivity and develop an appropriate procedure for accurate measurement of vaccine potency, a collaborative study with four Japanese vaccine manufacturers was conducted to measure the HA contents of trivalent influenza vaccines (TIVs) and QIVs by SRID assay with a single and a mixture of reference antigens (refAgs) from each lineage of IFVB for seven influenza seasons from 2015/16 to 2021/22. The cross-reactivity of the two IFVB components in the SRID assay varied depending on the vaccine viruses. Our study demonstrated that it is useful to validate a suitable combination for each refAg and reference antiserum by selecting the combination showing similar HA contents between experimental TIV and QIV before lot release testing.

Influenza B Virus: Target and acting mechanism of antiviral drugs.

The influenza B virus is one of the causes of seasonal influenza, which has a long history of existence in various populations. Adolescents, children, pregnant women, the elderly, as well as patients with major diseases such as high blood pressure, diabetes, and cancer, and those with low immunity are more susceptible to infection by the influenza virus. During the influenza seasons, the influenza B virus can cause significant harm and economic burden. At present, neuraminidase inhibitors, hemagglutinin inhibitors and RNA polymerase inhibitors are the main antiviral drugs that are used in the clinical treatment of influenza B. Due to the repeated use of antiviral drugs in recent years, the emergence of resistant strains of the influenza virus exacerbated. By combining anti-viral drugs with different mechanisms of action or using a combination of traditional Chinese medicine and chemical drugs, the problem of reduced drug sensitivity can be improved. This article introduces the drug targets of the influenza B virus and the mechanism of virus resistance. It also emphasizes the clinically used antiviral drugs and their mechanisms of action, thereby providing a reference basis for the development of new anti-influenza drugs.

Hemagglutinin and neuraminidase of a non-pathogenic H7N7 avian influenza virus coevolved during the acquisition of intranasal pathogenicity in chickens.

Polybasic amino acid residues at the hemagglutinin (HA) cleavage site are insufficient to induce the highly pathogenic phenotype of avian influenza viruses in chickens. In our previous study, an H7N7 avian influenza virus named "Vac2sub-P0", which is nonpathogenic despite carrying polybasic amino acids at the HA cleavage site, was passaged in chick air sacs, and a virus with high intravenous pathogenicity, Vac2sub-P3, was obtained. Intranasal infection with Vac2sub-P3 resulted in limited lethality in chickens; therefore, in this study, this virus was further passaged in chicken lungs, and the resultant virus, Vac2sub-P3L4, acquired high intranasal pathogenicity. Experimental infection of chickens with recombinant viruses demonstrated that mutations in HA and neuraminidase (NA) found in consecutive passages were responsible for the increased pathogenicity. The HA and NA functions of Vac2sub-P3L4 were compared with those of the parental virus in vitro; the virus growth at 40 °C was faster, the binding affinity to a sialic acid receptor was lower, and the rate of release by NA from the cell surface was lower, suggesting that these changes enabled the virus to replicate efficiently in chickens with high intranasal pathogenicity. This study demonstrates that viruses that are highly pathogenic when administered intranasally require additional adaptations for increased pathogenicity to be highly lethal to intranasally infected chickens.

Rapid synthesis and screening of natively paired antibodies against influenza hemagglutinin stem via oPool+ display.

Antibody discovery is crucial for developing therapeutics and vaccines as well as understanding adaptive immunity. However, the lack of approaches to synthesize antibodies with defined sequences in a high-throughput manner represents a major bottleneck in antibody discovery. Here, we presented oPool+ display, which combines oligo pool synthesis and mRNA display to construct and characterize many natively paired antibodies in parallel. As a proof-of-concept, we applied oPool+ display to rapidly screen the binding activity of >300 natively paired influenza hemagglutinin (HA) antibodies against the conserved HA stem domain. Structural analysis of 16.ND.92, one of the identified HA stem antibodies, revealed a unique binding mode distinct from other known broadly neutralizing HA stem antibodies with convergent sequence features. Yet, despite such differences, 16.ND.92 remained broadly reactive and conferred in vivo protection. Overall, this study not only established an experimental platform that can be applied in both research and therapeutics to accelerate antibody discovery, but also provides molecular insights into antibody responses to the influenza HA stem, which is a major target for universal influenza vaccine development.

Mineralo-organic particles inhibit influenza A virus infection by targeting viral hemagglutinin activity.

Aim: Mineralo-organic particles, naturally present in human body fluids, participate in ectopic calcification and inflammatory diseases. These particles coexist with influenza A virus (IAV) in the same microenvironment during viral infection. Our objective was to investigate the functional consequences of the potential interactions between these particles and the virions.Materials & methods: We used in vitro models, including electron microscopy, fluorescence microscopy, hemagglutination assay and viral infection assays to examine the interactions.Results: Mineralo-organic particles bind to IAV virions through interactions involving particle-bound fetuin-A and mineral content, effectively engaging viral hemagglutinin. These interactions result in hindered viral infection.Conclusion: These findings uncover the novel interactions between mineralo-organic particles and IAV, highlighting the impact of virus microenvironment complexity.

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Avian influenza A H5N1 hemagglutinin protein models have distinct structural patterns re-occurring across the 1959-2023 strains.

Influenza A H5N1 hemagglutinin (HA) plays a crucial role in viral pathogenesis and changes in the HA receptor binding domain (RBD) have been attributed to alterations in viral pathogenesis. Mutations often occur within the HA which in-turn results in HA structural changes that consequently contribute to protein evolution. However, the possible occurrence of mutations that results to reversion of the HA protein (going back to an ancestral protein conformation) which in-turn creates distinct HA structural patterns across the 1959-2023 H5N1 viral evolution has never been investigated. Here, we generated and verified the quality of the HA models, identified similar HA structural patterns, and elucidated the possible variations in HA RBD structural dynamics. Our results show that there are 7 distinct structural patterns occurring among the 1959-2023 H5N1 HA models which suggests that reversion of the HA protein putatively occurs during viral evolution. Similarly, we found that the HA RBD structural dynamics vary among the 7 distinct structural patterns possibly affecting viral pathogenesis.

PA-MSHA exerts potent activity against cetuximab-resistant colorectal cancer through the miR-7-5p/Akt3/Wnt-ß-catenin pathway.

Background: The prognosis and survival of individuals with cetuximab-resistant colorectal cancer (CRC) remain severely impacted by therapy for this disease. The study investigated the underlying mechanisms of Pseudomonas aeruginosa-mannose sensitive hemagglutinin (PA-MSHA), a type of therapeutic biological product approved in China, for cetuximab-resistant CRC. Methods: Cell proliferation, apoptosis, migration and invasion were detected by cell counting kit-8 (CCK-8) assay, flow cytometry, wound healing assay and transwell assay. Massively parallel sequencing of cetuximab-resistant CRC cells with PA-MSHA treatment was used to screen the differential expression profile of miRNAs. The directly target gene of miR-7-5p was revealed by dual luciferase assay. Apoptosis and invasion related proteins were detected by Western blot. Results: PA-MSHA could successfully stop the migrating and invading of cetuximab-resistant CRC cells while also inducing apoptosis. Tumor-bearing experiments in nude mice showed that PA-MSHA slowed tumor growth and lengthened mouse life. The sequencing data showed that miR-7-5p was considerably upregulated after PA-MSHA treatment. As anticipated, miR-7-5p overexpression improved PA-MSHA's anticancer properties both in vitro and in vivo. The target gene of miR-7-5p was confirmed to be Akt3 by dual luciferase assay, and Akt3 silencing undid the inhibition of PA-MSHA efficacy caused by miR-7-5p downregulation. Additionally, PA-MSHA therapy significantly reduced the activation of Wnt-ß-catenin pathway, and Akt3 expression was positively linked with several important Wnt-ß-catenin pathway genes, including Wnt and CTNNB1. Finally, we discovered that patients with CRC who had developed cetuximab resistance or disease progression had remarkably decreased serum miR-7-5p levels. Conclusions: PA-MSHA controlled the miR-7-5p/Akt3/Wnt-ß-catenin pathway to provide substantial efficacy against cetuximab-resistant CRC.

A Recombinant Mosaic HAs Influenza Vaccine Elicits Broad-Spectrum Immune Response and Protection of Influenza a Viruses.

The annual co-circulation of two influenza A subtypes, H1N1 and H3N2, viruses in humans poses significant public health threats worldwide. However, the continuous antigenic drift and shift of influenza viruses limited the effectiveness of current seasonal influenza vaccines, necessitating the development of new vaccines against both seasonal and pandemic viruses. One potential solution to this challenge is to improve inactivated vaccines by including multiple T-cell epitopes. In this study, we designed stabilized trimeric recombinant mosaic HA proteins named HAm, which contain the most potential HA T-cell epitopes of seasonal influenza A virus. We further evaluated the antigenicity, hemagglutinin activity, and structural integrity of HAm and compared its immunogenicity and efficacy to a commercial quadrivalent inactivated influenza vaccine (QIV) in mice. Our results demonstrated that the HAm vaccine was able to induce broadly cross-reactive antibodies and T-cell responses against homologous, heterologous, and heterosubtypic influenza-naive mice. Additionally, the HAm antigens outperformed QIV vaccine antigens by eliciting protective antibodies against panels of antigenically drifted influenza vaccine strains from 2009 to 2024 and protecting against ancestral viruses' lethal challenge. These results suggest that the HAm vaccine is a promising potential candidate for future universal seasonal and pandemic influenza vaccine development.

Recent Occurrence, Diversity, and Candidate Vaccine Virus Selection for Pandemic H5N1: Alert Is in the Air.

The prevalence of the highly pathogenic avian influenza virus H5N1 in wild birds that migrate all over the world has resulted in the dissemination of this virus across Asia, Europe, Africa, North and South America, the Arctic continent, and Antarctica. So far, H5N1 clade 2.3.4.4.b has reached an almost global distribution, with the exception of Australia and New Zealand for autochthonous cases. H5N1 clade 2.3.4.4.b, derived from the broad-host-range A/Goose/Guangdong/1/96 (H5N1) lineage, has evolved, adapted, and spread to species other than birds, with potential mammal-to-mammal transmission. Many public health agencies consider H5N1 influenza a real pandemic threat. In this sense, we analyzed H5N1 hemagglutinin sequences from recent outbreaks in animals, clinical samples, antigenic prototypes of candidate vaccine viruses, and licensed human vaccines for H5N1 with the aim of shedding light on the development of an H5N1 vaccine suitable for a pandemic response, should one occur in the near future.

Recombinant Marek's disease virus type 1 provides full protection against H9N2 influenza A virus in chickens.

The H9N2 subtype of the avian influenza virus (AIV) poses a significant threat to the poultry industry and human health. Recombinant vaccines are the preferred method of controlling H9N2 AIV, and Marek's disease virus (MDV) is the ideal vector for recombinant vaccines. During this study, we constructed two recombinant MDV type 1 strains that carry the hemagglutinin (HA) gene of AIV to provide dual protection against both AIV and MDV. To assess the effects of different MDV insertion sites on the protective efficacy of H9N2 AIV, the HA gene of H9N2 AIV was inserted in UL41 and US2 of the MDV type 1 vector backbone to obtain recombinant viruses rMDV-UL41/HA and rMDV-US2/HA, respectively. An indirect immunofluorescence assay showed sustained expression of HA protein in both recombinant viruses. Additionally, the insertion of the HA gene in UL41 and US2 did not affect MDV replication in cell cultures. After immunization of specific pathogen-free chickens, although both the rMDV-UL41/HA and rMDV-US2/HA groups exhibited similar levels of hemagglutination inhibition antibody titers, only the rMDV-UL41/HA group provided complete protection against the H9N2 AIV challenge, and also offered complete protection against challenge with MDV. These results demonstrated that rMDV-UL41/HA could be used as a promising bivalent vaccine strain against both H9N2 avian influenza and Marek's disease in chickens.

Hemagglutinin from the Root Tuber of Dioscorea preussii Pax.

Objectives: In this study, Dioscorea preussii root tuber hemagglutinin was purified and its physicochemical properties were determined. The antioxidative and anti-hemolytic activities of the hemagglutinin were also investigated. Materials and Methods: The hemagglutinating assay was used to detect the presence of lectin in the phosphate-buffered saline extract of the D. preussii root tuber. The lectin was purified using ammonium sulfate fractionation and molecular sieve chromatography. The optimum pH and temperature were determined. In addition, antioxidant activity was assessed using 2,2 diphenylpicrylhydrazyl (DPPH) radical scavenging, metal chelating, ferric reducing antioxidant power (FRAP), and lipid peroxidation inhibition assays. Red blood cells subjected to oxidative damage caused by H2O2 were employed to evaluate their antihemolytic ability. Results: Starch inhibited hemagglutinin activity. Dioscorea preussii hemagglutinin (DPH) maintained full hemagglutinating activity from 30 °C to 60 °C and pH 5-13. Ethylene diamine tetraacetic acid did not affect the hemagglutinating activity of hemagglutinin. All denaturing agents (Guanidine-HCl, urea, and ß-mercaptoethanol) reduced the hemagglutinating activity of the hemagglutinin to different degrees. The hemagglutinin scavenged the DPPH radical and chelated iron metal with half maximum inhibitory concentration (IC50) of 0.727 ± 0.035 mg/mL and 0.583 ± 0.078 mg/mL, respectively, while the FRAP assay showed that it contained 76 mg of ascorbic acid equivalent per gram of the purified hemagglutinin. In the absence of hemolytic agents and at lower concentration tested, hemagglutinin showed positive membrane integrity protection. Conclusion: This study provides information on the antioxidant properties of D. preussii root tuber hemagglutinin as well as its cell membrane protective ability. The lectin is a starch-binding, which makes it a novel lectin.

Computational exploration and molecular dynamic simulation for the discovery of antiviral agents targeting Newcastle disease virus.

Newcastle disease virus (NDV) is a highly infectious viral disease that impacts birds globally, especially domestic poultry. NDV is a type of avian paramyxovirus which poses a major threat to the poultry industry due to its ability to inflict significant economic damage. The membrane protein, Hemagglutinin-Neuraminidase (HN) of NDV is an attractive therapeutic candidate. It contributes to pathogenicity through various functions, such as promoting fusion and preventing viral self-agglutination, which allows for viral spread. In this study, we used pharmacophore modeling to identify natural molecules that can inhibit the HN protein of NDV. Physicochemical characteristics and phylogenetic analysis were determined to elucidate structural information and phylogeny of target protein across different species as well as members of the virus family. For structural analysis, the missing residues of HN target protein were filled and the structure was evaluated by PROCHECK and VERIFY 3D. Moreover, shape and feature-based pharmacophore model was employed to screen natural compounds' library through numerous scoring schemes. Top 48 hits with 0.8860 pharmacophore fit score were subjected towards structure-based molecular docking. Top 9 compounds were observed witihin the range of -8.9 to -7.5 kcal/mol binding score. Five best-fitting compounds in complex with HN receptor were subjected to predict biological activity and further analysis. Top two hits were selected for MD simulations to validate binding modes and structural stability. Finally, upon scrutinization, A1 (ZINC05223166) emerges as potential HN inhibitor to treat NDV, necessitating further validation via clinical trials.

An explainable language model for antibody specificity prediction using curated influenza hemagglutinin antibodies.

Despite decades of antibody research, it remains challenging to predict the specificity of an antibody solely based on its sequence. Two major obstacles are the lack of appropriate models and the inaccessibility of datasets for model training. In this study, we curated >5,000 influenza hemagglutinin (HA) antibodies by mining research publications and patents, which revealed many distinct sequence features between antibodies to HA head and stem domains. We then leveraged this dataset to develop a lightweight memory B cell language model (mBLM) for sequence-based antibody specificity prediction. Model explainability analysis showed that mBLM could identify key sequence features of HA stem antibodies. Additionally, by applying mBLM to HA antibodies with unknown epitopes, we discovered and experimentally validated many HA stem antibodies. Overall, this study not only advances our molecular understanding of the antibody response to the influenza virus but also provides a valuable resource for applying deep learning to antibody research.

Variation of Site-Specific Glycosylation Profiles of Recombinant Influenza Glycoproteins.

This work presents a detailed determination of site-specific N-glycan distributions of the recombinant influenza glycoproteins hemagglutinin (HA) and neuraminidase. Variation in glycosylation among recombinant glycoproteins is not predictable and can depend on details of the biomanufacturing process as well as details of protein structure. In this study, recombinant influenza proteins were analyzed from eight strains of four different suppliers. These include five HA and three neuraminidase proteins, each produced from a HEK293 cell line. Digestion was conducted using a series of complex multienzymatic methods designed to isolate glycopeptides containing single N-glycosylated sites. Site-specific glycosylation profiles of intact glycopeptides were produced using a recently developed method and comparisons were made using spectral similarity scores. Variation in glycan abundances and distribution was most pronounced between different strains of virus (similarity score = 383 out of 999), whereas digestion replicates and injection replicates showed relatively little variation (similarity score = 957). Notably, glycan distributions for homologous regions of influenza glycoprotein variants showed low variability. Due to the multiple possible sources of variation and inherent analytical difficulties in site-specific glycan determinations, variations were individually examined for multiple factors, including differences in supplier, production batch, protease digestion, and replicate measurement. After comparing all glycosylation distributions, four distinguishable classes could be identified for the majority of sites. Finally, attempts to identify glycosylation distributions on adjacent potential N-glycosylated sites of one HA variant were made. Only the second site (NnST) was found to be occupied using two rarely used proteases in proteomics, subtilisin and esperase, both of which did selectively cleave these adjacent sites.