P2X7R polymorphisms and MS disease severity, we performed an association study of five non-synonymous SNPs coding variants of the P2X7R gene: rs1718119 Ala348Thr, rs2230911 Thr357Ser, rs2230912 Gln460Arg, rs3751143 Glu496Ala, and rs28360457 Arg307Gln, modulating P2X7R expression in 128 MS patients . All patients were genotyped, and multiple sclerosis severity score (MSSS) was evaluated in every case; 189 healthy subjects were enrolled as well as controls. Results showed that P2X7R rs1718119(A) 348Thr and rs22390912(G) 464Arg, two SNPs of minor allele frequency (MAF) known to confer gain of function to the P2X7R protein, were associated with significantly higher MSSS in RRMS patients alone ). Interestingly, two whole haplotypes resulted in having significant association with MSSS in these same patients. Thus: (1) the P2X7R-4 “ACGAG” haplotype, characterized by the co-presence of the rs1718119-rs2230912 AG MAF alleles, was associated with higher MSSS (Beta: 1.11 p = 0.04), and (2) the P2X7R-1 “GCAAG” complementary haplotype, which contains the rs1718119 and rs2230912 GA wild-type alleles, was more frequently carried by patients with lower MSSS and less severe disease (Beta: −1.54 p < 0.001). Although being preliminary and needing confirmation in an ampler cohort, these results suggest that 348Thr and 464Arg variants have a role as modulators of disease severity in RRMS patients.Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS) that leads to progressive physical disability. Recent evidence has suggested that P2X7 receptor (P2X7R)-mediated purinergic signalling pathways play a role in MS-associated neuroinflammation, possibly contributing to disease pathogenesis. To evaluate possible associations between  Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS) that mainly affects young adults and leads to progressive physical disability . The aet+, NA+, and Ca+ through the cell membrane, and its activation can be driven by high concentrations of ATP and leads to the generation of large, cytolytic conductance pores [Activation of the purinergic receptor P2X7 (P2X7R) is believed to be deleterious in autoimmune diseases and was recently hypothesized to play a role in the pathogenesis of MS ,7. Thus,ce pores ,14. Notace pores ,16.P2X7R gene, were suggested to modulate P2X7R expression and the subsequent secretion of IL1β [A number of non-synonymous single nucleotide polymorphisms (SNPs) have been identified in the gene encoding the human P2X7R on chromosome 12q24.3. In particular, four SNPs (rs1718119(G>A) Ala348Thr, rs2230911(C>G) Thr357Ser, rs2230912(A>G) Gln460Arg, and rs3751143(A>C) Glu496Ala), which form a haplotype block spanning exons 11 to 13 of the  of IL1β .P2X7R gene [Recent results showed that glial cells expressing purinergic P2X7R are involved in the initial pathological mechanisms of disease, playing a role in experimental autoimmune encephalomyelitis (EAE) as well . Thus, eX7R gene ,23, resuX7R gene .P2X7R SNPs and haplotypes and clinical parameters of MS severity.In the attempt to shed further light on the role played by P2X7R in MS pathogenesis, we evaluated the possible correlations between p < 0.001) patients to reduce enrolling false negative individuals as HCs.Demographic and clinical data of the 128 MS patients (34 SPMS and 94 RRMS) and the 189 healthy controls (HCs) enrolled in the study are summarized in p < 0.001); expanded disability status scale (EDSS), as well as disease duration, was higher in SPMS than in RRMS (p < 0.01). HLA-DRB1*15 was significantly more expressed in overall MS patients (<0.001) compared to HCs, confirming the association between the disease and this allele.Not surprisingly, multiple sclerosis severity score (MSSS) was significantly higher in SPMS (7.0 ± 2.2) compared to RRMS (3.6 ± 2.7) patients (P2X7R SNPs: rs1718119(G>A) Ala348Thr, rs2230912(A>G) Gln464Arg, rs2230911(C>G) Thr357Ser, rs3751143(A>C) Glu496Ala, and rs28360457 (G>A) Arg307Gln. Genotype distribution showed that all the variants were in Hardy–Weinberg equilibrium in each group, and no statistical difference in distribution between groups was evidenced  Ala348Thr (p = 0.02) and higher MSSS values was evidenced in the overall group of MS patients. To summarize: (1) higher MSSS values were observed in patients carrying the minor allele frequency (MAF) rs1718119 (A):(AA+AG) compared to those carrying the major allele homozygous genotype (GG) (p = 0.005); and (2) higher MSSS scores were seen in patients carrying the MAF rs2230912(G):(GG+AG) genotype compared to those carrying the major allele homozygous genotype (AA) (p = 0.03) (Possible correlations between  = 0.03) .P2X7R genotypes were then analysed upon dividing MS patients into those with a diagnosis of either SPMS or RRMS. Results indicated the presence of a statistical association between rs1718119(A) and rs2230912(G) MAF and higher MSSS scores in RRMS  but not in SPMS patients.Correlations between MSSS scores and p < 0.001); similarly, rs2230912 (G)-carrying RRMS patients were characterized by higher MSSS scores compared to those carrying the (AA) genotype (p = 0.01). No associations between P2X7R variants and MSSS were observed in patients with a diagnosis of SPMS  than in those carrying the homozygous (GG) genotype   and rs2230912(G)  and MSSS scores in the overall group of MS patients and in those with a diagnosis of RRMS. Again, no association was observed in SPMS patients.A forward stepwise binary logistic regression analysis was finally performed to evaluate the possible association of MAF rs1718119(A) and rs2230912(G) (imputed as dependent variables) and MSSS after correction for factors known to influence disease severity, including age at onset, disease duration, 2 = 0.28 linkage disequilibrium was observed between rs1718119 (G>A) and rs2230912(A>G)  . Haploty912(A>G) .P2X7R-1 haplotype has a possible protective effect against MS severity. Thus, this haplotype was significantly associated with lower MSSS values both in the overall group of patients (Beta = −0.94 p = 0.01) and in RRMS patients (Beta: −1.54 p < 0.001). Notably, the P2X7R-4 “ACGAG” haplotype was instead weakly associated with higher MSSS in RRMS patients alone (Beta: 1.11 p = 0.04).Analysis of haplotype correlation with MSSS scores showed that the rs1718119(G>A), rs2230911(C>G), rs2230912(A>G), rs3751143(A>C), and rs28360457 (G>A) “GCAAG” called Once again, no significant correlation emerged between any haplotype and MSSS values observed in SPMS patients.P2X7R gene in relationship with MS development and disease severity. MS severity was assessed by MSSS, a disease-stratification method that combines disability scores (EDSS) and disease duration, and scores are calculated based on the distribution of EDSS within groups of MS patients of similar disease duration. Results showed that RRMS patients carrying the MAF of rs1718119(A) coding for a threonine in position 348 and the MAF of rs22390912 (G) coding for an arginine at position 464, two SNPs suggested to confer a gain of function to the P2X7R protein, had higher MSSS than those carrying the major alleles . Because patients with low MSSS tend to progress at slower rates than those with higher MSSS, these data indicate that increased P2X7R activity is associated to more severe disease in RRMS. Notably, the association of MAF of rs1718119(A) and rs22390912(G) with higher MSSS was confirmed by regression analysis that included age of disease onset, gender, and the major genetic risk factor for MS onset, HLA-DRB1*15, as covariates. No correlations were evidenced between MSSS and the other three P2X7R SNPs  we examined; these SNPs were suggested to confer loss of receptor function. Finally, our results did not confirm the possible protective role of rs28360457 Arg307Gln against MS development [Herein, we performed an association study of five non-synonymous SNPs coding functional variants of the P2X7R polymorphisms was evidenced in patients with a diagnosis of SPMS. This discrepancy is likely the consequence of the fact that in this group of patients the disease severity, as evaluated by MSSS, was very high in all cases or, alternatively, could be due to the small number of SPMS patients analysed. It is nevertheless important to underline that, in comparison to RRMS, the extent of inflammation is lower in patients with SPMS, in whom degeneration prevails over inflammation [P2X7R SNPs, resulting in protein gain of function, are associated with higher MSSS in RRMS alone, a stage of the disease that is primarily driven by inflammation. Notably, an increased expression of P2X7R was observed in demyelinating plaques adjacent to blood vessels within activated microglial cells/macrophages of post mortem collected tissues from SPMS patients [In contrast with the results obtained in RRMS, no stratification of MSSS values in relationship with ammation . RRMS ispatients  and in cP2X7R SNPs we analysed were suggested to be in linkage disequilibrium [P2X7R-4 “ACGAG” haplotype, which sees the co-presence of rs1718119-rs2230912 AG MAF alleles, was associated with a higher MSSS, and (2) the P2X7R-1 “GCAAG” complementary haplotype, which contains the rs1718119 and rs2230912 GA wild-type alleles, was more frequently carried by patients with lower MSSS and a less severe course of the disease. Analyses of whole haplotypes are important because of the co-presence of polymorphisms responsible for either gain (rs1718119 Ala348Thr and rs2230912 Gln464Arg) or loss of function , which may counterbalance the effect of each other. It is noteworthy to underline that the P2X7R-4 “ACGAG” haplotype we observed to be associated with higher MSSS and more severe MS disease includes the haplotype previously shown to result in higher IL-1β production by monocytes, which could be completely suppressed by selective P2X7R antagonists [Since the ilibrium , haplotyP2X7R activation is suggested to be present in MS, and P2X7R-mediated purinergic signalling pathways are suspected to play a role in MS-associated neuroinflammation, possibly contributing to the pathogenesis of MS. This possibility was recently reinforced by results indicating that pharmacological blockade of P2X7R by brilliant blue G (BBG), its selective antagonist, delays the onset of the disease and alleviates clinical symptoms in EAE . These rP2X7R genotype and immune parameters in relationship with disease severity would be useful extensions of the current study. Finally, because RRMS can but does not always evolve into SPMS over time, it will be interesting to verify in longitudinal studies whether the presence of the gain-of-function P2X7R polymorphisms Arg464 and Thr348 SNPs will be able to predict which RRMS patients will develop SPMS.A limitation of this study is represented by the limited number of SPMS patients and by the lack of patients with a diagnosis of primary progressive disease (PPMS). To this end, a replication study focusing on a larger cohort of MS patients with different disease phenotypes is planned. Analyses of possible correlations between A total of 128 patients with a diagnosis of multiple sclerosis according to McDonald’s revised diagnostic criteria  were enrInformed consent was obtained from all the individuals before inclusion in the study. The study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the institutional review board of the Don Carlo Gnocchi ONLUS Foundation, Milan (protocol number: #11_27/06/2019).Whole blood was collected in 3 mL EDTA-containing vacutainer tubes ; genomic DNA was extracted from blood using standard phenol/chloroform procedures. Phenol chloroform extraction was performed by inducing cell lysis and DNA release with sodium dodecylsulfate (SDS) and proteinase K. Next, a phenol/chloroform/isoamyl alcohol mixture was added to the cell lysate to separate the proteins from the DNA . The amoP2X7R SNPs were evaluated by allelic discrimination real-time PCR using pre-designed TaqMan™ probes , in particular C_11704039_10 for rs1718119, C_15853705_20 for rs2230911, C_15853715_20 for rs2230912, C_27495274_10 for rs3751143, and C__59964938_10 for rs28360457. PCR consisted of a hot start at 95 °C for 10 min followed by 40 cycles of 94 °C for 15 s and 60 °C for 1 min. Fluorescence detection took place at 60 °C. Assays were performed in 10 μL reactions using 1 μL of DNA at 50 ng/μL, using TaqMan Genotyping Master Mix on 96-well plates using a CFX96 instrument . Control samples representing all possible genotypes and negative control were included in each reaction.HLA-DRB1*15 allele, either in heterozygous or homozygous form, was inferred by the genotyping of the tag SNP rs3135388 [The presence of the p values after Bonferroni’s correction (pc) for the degree of freedom (df) were calculated, and the significance threshold was set at p < 0.05. ANOVA comparisons were performed to correlate P2X7R SNP genotypes and MAF association with MSSS. Binary logistic forward stepwise regression analysis was performed to evaluate the association of MAF rs1718119(A):(AA+AG) and rs2230912(G):(GG+AG) (imputed as dependent variables) and MSSS values after correction for factors that may influence MS severity such as age at onset, disease duration, DRB1*15 positivity, and gender, which were calculated as covariates. The odds ratio (OR) and its 95% confidence interval (CI) were used to measure the MAF association with the clinical scores. Statistical analyses were performed using SPSS software .Chi-square analysis was applied to verify that populations were in Hardy–Weinberg equilibrium. Genotype and MAF comparisons between groups were performed by chi-square evaluation in 3X2 and 2X2 tables of contingency. Two-sided http://analysis.bio-x.cn, (accessed on 1 October 2022)) [http://pngu.mgh.harvard.edu/~purcell/plink/ (accessed on 1 October 2022)) software vs 2.020 [Haplotype analysis of distribution and MSSS association were performed by Shesis plus online software (r 2022)) ,33 and pvs 2.020 .P2X7R 348Thr and 464Arg variants have a role as modulators of disease severity in MS patients, which deserves to be further investigated.These results confirm that P2X7R plays a role in RRMS severity and suggest that P2X7R could be considered as a target to design personalised therapeutic and rehabilitative intervention protocols in MS. Specifically, evidence herein allows us to speculate that the


SLC2A3 is upregulated in various cancer types and promotes proliferation, invasion, and metabolism. However, its role in the prognosis and immune regulation of head and neck squamous cell carcinoma (HNSCC) is still obscure. This study is aimed at exploring the prognostic and immunotherapeutic potential of SLC2A3 in HNSCC. SLC2A3 expression was evaluated using R software, TIMER, CPTAC, and HPA databases. The association between SLC2A3 expression and clinicopathologic characteristics was assessed by R software. The effect of SLC2A3 on survival was analyzed by R software and Kaplan-Meier Plotter. Genomic alterations in SLC2A3 were investigated using the cBioPortal database. Coexpression of SLC2A3 was studied using LinkedOmics and STRING, and enrichment analyses were performed with R software. The relationship between SLC2A3 expression and immune infiltration was determined using TIMER and TISIDB databases. Immune checkpoints and ESTIMATE score were analyzed via the SangerBox database.  All data were downloaded from TCGA database and integrated via R software. SLC2A3 expression was upregulated in HNSCC tissues compared to normal tissues. It was significantly related to TNM stage, histological grade, and alcohol history. High SLC2A3 expression was associated with poor prognosis in HNSCC. Coexpression analysis indicated that SLC2A3 mostly participated in the HIF-1 signaling pathway and glycolysis. Furthermore, SLC2A3 expression strongly correlated with tumor-infiltrating lymphocytes in HNSCC. SLC2A3 could serve as a potential prognostic biomarker for tumor immune infiltration in HNSCC. Head and neck squamous cell carcinoma (HNSCC) is one of the top 10 cancer types worldwide and has a high mortality rate , 3. The Recently, many endeavors have been made to explore the biomarkers for more effective treatments for HNSCC –6. TheraSLC2A3, also annotated as GLUT3, is a member of the solute carrier 2A (SLC2A) family participating in glucose transport across the hydrophobic cell membrane in mammalian cells. Cancer cells mainly rely on glycolysis for nutrient intake and energy production [SLC2A3 to research cancer diagnosis and therapy [SLC2A3 plays a crucial role in perineural invasion via the regulation of epithelial-mesenchymal transition [SLC2A3 could predict poor prognosis in gastric cancer, associated with macrophage infiltration in the tumor microenvironment (TME) [SLC2A3 overexpression in triple-negative breast cancer could activate inflammatory M1 tumor-associated macrophages resulting in the aggressiveness of tumor cells [SLC2A3 in diverse cancer types, there is a pressing need to research the correlation between SLC2A3 and immune infiltration. However, the relationship between SLC2A3 and HNSCC remains unclear.oduction . There i therapy . In coloansition . SLC2A3 nt (TME) . Similaror cells . With thSLC2A3 gene and protein between tumor tissues and normal tissues based on The Cancer Genome Atlas (TCGA) and several online databases. The interaction networks of SLC2A3 and enrichment analyses were performed. Prognostic value and clinicopathologic features of SLC2A3 in HNSCC were evaluated. We conducted immune infiltration analyses to explore the potential immune mechanisms of SLC2A3 in HNSCC. Our study suggests that SLC2A3 could act as a valuable prognostic biomarker. The study also sheds light on the association of SLC2A3 with immune infiltration in HNSCC.In the present study, we identified the differential expression levels of SLC2A3 and related clinical information was extracted from TCGA database . The RNA-seq gene expression with the format of FPKM was transformed into TPM format, and log2 conversion was conducted for further analyses. Gene expression data of SLC2A3 in HNSCC were acquired from TCGA and involved 502 tumor tissue and 44 adjacent normal tissue data. According to the median expression level of SLC2A3, gene expression data were divided into two subgroups: the high expression group and the low expression group. The samples contained several clinical characteristics, including TNM stage, clinical stage, histologic grade, gender, age, smoke condition, alcohol history, radiation therapy, primary therapy outcome, and lymphovascular invasion  is a user-friendly online web resource for the systematic analysis of immune infiltrates in different cancers (https://cistrome.Shinyapps.io/timer/). We used the TIMER database to verify the differential expression of SLC2A3 in pan-cancer [The gene expression of n-cancer .http://ualcan.path.uab.edu). Clinical Proteomic Tumor Analysis Consortium (CPTAC) is focused on molecular mechanisms of cancer via the application of proteome and genome analysis or proteogenomics (https://proteomics.cancer.gov/programs/cptac) [https://proteinatlas.org/) [SLC2A3 from CPTAC, and HPA was performed to analyze the protein expression of SLC2A3 in normal tissue and tumor tissue of HNSCC.UALCAN is a comprehensive web resource referring to publicly available cancer omics data (s/cptac) . The Humas.org/) . In the SLC2A3 expression and clinical pathological characteristics including TNM stage, clinical stage, gender, age, histologic grade, smoker condition, and alcohol history, we applied the “ggplot2” package based on the TCGA database. The statistical significance was set at P < 0.05.To explore the association between SLC2A3 using the “pROC” and “ggplot2” packages. The overall survival (OS) and disease-specific survival (DSS) between the high expression group and the low expression group were evaluated using the “survival” package and visualized using the “survminer” package. P < 0.05 was regarded as statistically significant. The Kaplan-Meier Plotter collected discovery and validation of survival biomarkers to analyze the correlations between gene expression and survival in multiple tumor types  [SLC2A3 expression levels in HNSCC. The prognostic value was statistically significant when the P < 0.05.The ROC curve was drawn to examine the cutoff value of alysis/) . AccordiSLC2A3 in HNSCC, we applied the “RMS” package to construct a predictive model of nomogram including several clinical characteristics. Calibration and discrimination analysis is a very effective predictive model evaluation method. In this study, variables of radiation therapy, primary therapy outcome, lymphovascular invasion, and SLC2A3 expression were brought in to map the nomogram-predicted probabilities. The 45-degree line in calibration curves represented the best predictive value. The C-index and ROC curves were used to examine the predictive accuracies of the nomogram and individual prognostic factors. The statistical significance was set at P < 0.05.To predict 1-, 3-, and 5-year survival probability associated with http://cbioportal.org). Thus, cBioPortal was used to investigate the genomic alterations in HNSCC [cBio Cancer Genomics Portal  is an online website for analyzing multidimensional cancer genomics data (in HNSCC .http://www.linkedomics.org/login.php). Thus, we applied LinkedOmics to examine the coexpression of SLC2A3 with Pearson's correlation coefficient [http://string-db.org, version 11.5). In this study, STRING analysis was conducted to explore the coexpression genes associated with SLC2A3 [The LinkedOmics database provides a public platform to analyze cancer multiomics data from all 32 TCGA cancer types and 10 CTPCA cancer cohorts  and SLC2A3 expression or clinical outcome in HNSCC. Tumor Immune System Interaction Database (TISIDB) integrates diverse data types for exploring the interaction between tumors and the immune system (https://cis.hku.hk/TISIDB) [SLC2A3 expression and the abundance of 28 tumor-infiltrating lymphocytes (TILs) acquired by gene set variation analysis. The association between immune or molecular subtypes and SLC2A3 expression in HNSCC was also explored via TISIDB. The difference at a P < 0.05 was regarded as statistically significant.We applied TIMER to investigate the association between immune infiltrates  . In thishttp://sangerbox.com/Tool) was used to investigate the correlation between SLC2A3 expression and immune checkpoint (ICP) genes, and ESTIMATE  score based on TCGA and GTEx databases. 60 ICP genes were analyzed in this study. ESTIMATE is an algorithm to predict levels of infiltrating stroma and immune cells by calculating stroma and immune scores, further deducing tumor purity [P < 0.05 were regarded to be statistically significant.SangerBox website . Mann–Whitney SLC2A3 in 33 different types of cancers showed that SLC2A3 was upregulated in six cancer types, including cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), head and neck carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), and stomach adenocarcinoma (STAD) compared to normal tissues , breast invasive carcinoma (BRCA), cervical and endocervical cancer (CESC), glioblastoma multiforme (GBM), kidney chromophobe (KICH), kidney renal papillary carcinoma (KIRP), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) and uterine corpus endometrial carcinoma (UCEC). As displayed in SLC2A3 expression level was significantly higher in CHOL, COAD, ESCA, HNSC, KIRC, and STAD than in normal tissue. However, the SLC2A3 expression level was lower in BCLA, BRCA, KICH, KIRP, liver hepatocellular carcinoma (LIHC), LUAD, LUSC, and UCEC.Analysis of the mRNA expression of  tissues . HoweverSLC2A3 in 502 HNSCC tissues were significantly higher than those in 44 normal tissues . To explore the protein expression of SLC2A3, we performed analysis from HPA and CPTAC with UALCAN. The results revealed that the protein of SLC2A3 was overexpressed in HNSCC compared to normal tissues , N stage (P = 0.004), M stage (P = 0.012), histological grade (P = 0.002), and alcohol history (P = 0.015). However, no significant correlations were detected between SLC2A3 expression and other clinicopathologic variables, such as clinical stage (P = 0.071), gender (P = 0.501), age (P = 0.076), and smoker condition (P = 0.223) (The Wilcoxon rank sum test showed that the expression level of = 0.223) .SLC2A3 expression in HNSCC. Firstly, the ROC curve showed promising predictive power with an AUG value of 0.727 , and the time-dependent ROC curve to 1-, 3-, and 5-year OS showed the AUG value of 0.548, 0.578, and 0.535, respectively , CD8+ T cell , CD4+ T cell , macrophage , neutrophil , and dendritic cell . The cumulative survival was significantly associated with the abundance of B cells according to SLC2A3 and 28 TIL types across human cancers were explored in the TISIDB database  cells, type 1 helper (Th1) cells, regulatory T (Treg) cells, memory B (Mem B) cells, natural killer (NK) cells, natural killer T (NKT) cells, plasmacytoid dendritic cells (pDCs), macrophages, eosinophils, and mast cells (mast)  . Additio Figures . Immune SLC2A3 expression in HNSCC, in which 21 genes were inhibitors and 32 were stimulators (SLC2A3 expression and three kinds of ESTIMATE score (P = 3.1e − 47), immune score (P = 2.8e − 14), and ESTIMATE score (P = 3.1e − 34) in HNSCC.We found that 53 of 60 ICP genes significantly correlated to mulators . The assTE score . The resSLC2A3 was mainly expressed in the central nervous system, which relies on steady support for ATP generation [SLC2A3 was found in several cancers such as colorectal cancer, gastric cancer and breast cancer [SLC2A3-knockdown cell line accounted for the less effective demethylation and vitamin C in the AML and lymphoma cell lines suggesting SLC2A3 could act as a predictive biomarker in vitamin C treatment in AML [SLC2A3 played a vital oncogenic role in attenuating cell proliferation and glucose uptake via miR-106a [SLC2A3 was regarded as a cancer-promoting gene. The prognostic role of SLC2A3 in cancers has recently gained attention. High SLC2A3 expression resulted in shorter OS and disease-free survival in colorectal carcinoma (CRC) [SLC2A3 and prognosis in HNSCC based on transcriptome profiling using the TCGA database and several online websites. It was found that SLC2A3 is differentially expressed in many cancer types, which was consistent with the above results. Moreover, upregulation of SLC2A3 in HNSCC was demonstrated, and high expression of SLC2A3 was significantly related to TNM stage, histologic grade, and alcohol history, suggesting SLC2A3 functioned as an oncogene in HNSCC. A previous study revealed that siRNA-mediated silencing of SLC2A3 could suppress the growth and promote apoptosis in AML cells [SLC2A3 was associated with poor OS, DSS, and RFS compared to low expression. Similarly, the immunohistochemical analysis of SLC2A3 was conducted in OSCC tissues, and the results showed that positive expression of SLC2A3 was correlated to unfavorable prognosis outcomes [SLC2A3 could act as a biomarker for prognosis in HNSCC.The SLC2A family comprises 14 members from SLC2A1-SLC2A14, among which those genes exhibit a tissue-specific expression. For instance, SCL2A1 is widely expressed in most tissues, while SLC2A2 is predominantly expressed in liver tissues . SLC2A3 neration . Howevert cancer –12. In at in AML . In GBM,miR-106a . Thus, Ama (CRC) . We perfML cells . Furtheroutcomes . We concSLC2A3 could serve as an oncogene because of the synergistic action with upstream and downstream molecules. In GBM, knockdown of HDAC2 could induce GBM cell death by miR-3189-targeted SLC2A3 repression through reprogramming of glucose metabolism [SLC2A3/AMPK signaling pathway and epithelial-to-mesenchymal transition (EMT) [SLC2A3 was directly related to SOX9, TRIM66, and HMGA1, which could promote cancer cell proliferation, migration, and invasion [SLC2A3 in HNSCC. Interestingly, it was found that SLC2A3 was significantly correlated to glycolysis-related gene HK2 and hypoxia-related gene HIF1A, which indicated that the expression of SLC2A3 in HNSCC was regulated via glycolysis and HIF-1 signaling pathway according to enrichment analysis. In prostate cancer, SLC2A3 was a direct target of miR-29c to inhibit cell growth and glucose metabolism [SLC2A3 to suppress glucose consumption, lactate generation, cellular ATP levels, and glucose uptake in gastric cancer cells [SLC2A3 was significantly increased [SLC2A3 predicting unfavorable prognosis in HNSCC could associate with glucose metabolism and HIF-1 signaling pathway.Many studies have demonstrated that tabolism . YAP plaon (EMT) . Other sinvasion . Given ttabolism . Similarer cells . Furtherer cells . Howeverer cells . Hypoxiaer cells . In hyponcreased . CombiniSLC2A3 and immune infiltration in human cancers is not widely studied. A recent study observed that SLC2A3 was one of an-eight gene prognostic risk score model to predict the survival significantly related to immune cell infiltration [SLC2A3 strongly correlated to TILs in HNSCC by TIMER, such as B cell, CD8+ T cell, CD4+ T cell, macrophage, neutrophil, and dendritic cell. For example, macrophages have been proven to account for immune evasion and suppression, and CD8+ T cells could mediate the recognition of the antigens expressed by most tumor cells [SLC2A3 suggesting that SLC2A3 could serve as an immune-related prognostic biomarker. We also demonstrated a positive correlation between SLC2A3 expression and Tcm CD8, Th1, Treg, Mem B, NK, NKT, pDC, eosinophil, and mast cells. In prostate cancer, SLC2A3 was one of the high-risk genes, and the risk score was significantly related to NK cell infiltration [SLC2A3 which mediates glucose uptake [SLC2A3 was explored, and the results showed significantly differential expression of SLC2A3 in different immune and molecular subtypes. A previous study demonstrated that glucose metabolism could be applied in conjunction with several inflammatory diseases like rheumatoid arthritis, in which rheumatoid arthritis fibroblast-like synoviocytes were cultured with CD4 T cells or T cell conditioned medium. Then it was observed that the secretion of inflammatory genes including TNF-α, IFN-γ, IL-17A, IL-6, and IL-8 was augmented, and glucose transporters like GLUT1 and SLC2A3 were increased. [SLC2A3 could play an essential role in HNSCC immune microenvironment. Moreover, ICP genes are important immune regulators in maintaining self-tolerance and modulating the immune response by both stimulatory and inhibitory pathways [SLC2A3, a recent study observed that SLC2A3 knockdown significantly inhibited the expression of PD-L1 indicating SLC2A3 could participate in the immune response by regulating the PD-L1 immune checkpoint in CRC [SLC2A3 could enhance CD28 costimulation reprogrammed renal cell carcinoma CD8+ TIL metabolism, which is antagonized by the inhibitory and checkpoint immunotherapy receptors CTLA4 and PD-1 [SLC2A3 and ICP genes which provided a novel insight for targeted immunotherapy in HNSCC. In addition, we found that SLC2A3 was positively related to stromal, immune, and ESTIMATE score associated with tumor invasion.It has long been acknowledged that the comprehensive immunobiology of the TME is modulated by several factors, including TILs, location of the tumor, and tumor stromal and vasculature –44. The ltration . In thisor cells . Interesltration . While fe uptake . Then, tcreased. . Accordipathways . T cell-pathways . When itt in CRC . Similarand PD-1 . We founin vitro/in vivo studies to verify these results. Second, an in-depth analysis of the molecular mechanism of SLC2A3 in HNSCC is required. Moreover, further research is deserved for the lack of direct evidence of the association between SLC2A3 and immune regulation in HNSCC.However, we used diverse online databases and R software to cross-validate the results systematically in our study, but several issues remain to be addressed. First, it would be of value to perform SLC2A3 expression and HNSCC. We uncovered a role for SLC2A3 as a potential prognostic marker and immune therapeutic target in HNSCC via bioinformatics methods for the first time, and it suggested avenues for the precise treatment of patients with HNSCC.In summary, our findings indicated an intimate connection between


The results showed AddaVax-adjuvanted H5N8 inactivated vaccine could stimulate the mice to produce a stronger protective immune response with higher titers of IgG antibodies, hemagglutination inhibition (HI), neuraminidase inhibition (NI) and microneutralization (MN) antibodies than vaccine formulations with Al(OH)3 adjuvant or without adjuvant, and achieve a dose-sparing effect. Moreover, the AddaVax-adjuvanted formulation also exhibited potent cross-reactive response in HI antibodies against different clades of H5 viruses. A significant correlation and a curve fitting among HI, NI and MN were found by the correlation analysis to predict the protective effect of the vaccine. With these findings, our study demonstrates that AddaVax adjuvant can enhance the immunogenicity of H5N8 inactivated vaccine remarkably, and proposes an effective strategy for dealing with a potential H5N8 virus pandemic.Since some cases of human infections with H5N8 avian influenza virus have been reported and caused great concern in recent years, it is important to develop an effective vaccine for human use to prevent a potential H5N8 pandemic. In the present study, a vaccine candidate virus based on newly human-infected A/Astrakhan/3212/2020 H5N8 virus was constructed by reverse genetics (RG) technology. The immunogenicity of H5N8 whole virion inactivated vaccine was evaluated by various doses of vaccine antigen formulated with squalene-based adjuvant (AddaVax), aluminum hydroxide (Al(OH) Highly pathogenic avian influenza (HPAI) viruses of H5Nx originated from A/goose/Guangdong/1/1996-like avian influenza virus in 1996. The HPAI A/H5 avian influenza virus lineage has diversified into ten clades (0–9) and multiple subclades. In particular, clade 2.3.4.4 has further evolved into eight subclades (a–h) since 2019 [Most of the classified H5Nx clades and subclades can cause human infection, especially the H5N1 and H5N6 subtypes, which have been reported to infect humans . Since 2The whole-virion inactivated influenza vaccine is the main strategy for an avian influenza vaccine design for human use, while the development of a vaccine candidate virus is essential for pandemic preparedness. It has been found that the HA of various avian influenza viruses contains multi-basic cleavage sites that are lethal to chickens, such as arginine (R) and lysine (K) sites in H5Nx and H7Nx . After d3, AS03 and MF59, etc.) can improve, regulate and expand the immune response, thereby increasing the efficacy of vaccines [During the preparation of a pre-pandemic influenza vaccine, improving the immunogenicity of the vaccine and saving the dose of antigens need to be considered. Adjuvants (such as Al(OH)vaccines ,12,13. Fvaccines . AddaVaxvaccines .In this study, a vaccine candidate virus based on newly human-infected A/Astrakhan/3212/2020 H5N8 strain was constructed by RG. Meanwhile, we aimed to determine the immunogenicity of H5N8 inactivated vaccine formulated with different adjuvants, by measuring the HI, MN and NI antibodies and also its cross-reacting activity against different clades of H5 viruses. The results demonstrated that addition of the AddaVax adjuvant has a strong immunopotentiating- and therefore dose-sparing effect. ® CRL-3216™) and canine kidney cells  were maintained in DMEM medium  containing 10% fetal bovine serum  in incubator at 37 °C with 5% CO2.Human embryonic kidney 293T cell line (ATCCVaccine candidate virus A/Vietnam/1194/2004 NIBRG-14(H5N1) ) and A/Hubei/29578/2016(H5N6)-RG  are preserved by Shanghai Institute of Biological Products .Six internal genes  were derived from X-157 , a high-g for 30 min. Then, the virus solution was inactivated with 0.01% formaldehyde and concentrated five times via cross-flow ultrafiltration with a 500 kDa membrane cassette. The inactivated virus was further purified with Capto Core 700 resin  in AKTA Purifier 100 system. Finally, the purified bulk was filtered with a 0.22 μm Sartobran P filter and stored at 4 °C. Vaccine candidate viruses were inoculated into embryonated chicken eggs (10–11 days old) and incubated at 34 °C for 60 h. After incubation, the allantoic fluid was harvested and centrifugated at 1800× A/Vietnam/1194/2004 NIBRG-14(H5N1) virus obtained from NIBSC was also inoculated into embryonated chicken eggs, and then the allantoic fluid was harvested and centrifugated. The following procedures of the H5N1 vaccine preparation were the same as the H5N8 vaccine.For quantitation of haemagglutinin in the vaccine candidate virus, the virus concentrates of H5N8 and H5N1 viruses were deglycosylated using PNGase F and analyzed by SDS-PAGE. The amount of HA as a percentage of total protein was then calculated by dividing the ‘total HA’ by the ‘total protein’ . The totThe mice study protocol (NO. 2021005) was approved by the laboratory animal management committee and the laboratory animal ethics and welfare protection group of the Shanghai Institute of Biological Products.n = 5 per group) were intramuscularly vaccinated twice at day 0 and day 21 with an H5N8 inactivated vaccine (50 μL/dose) at doses of 0.015 μg, 0.15 μg or 1.5 μg HA antigen containing 0.5 mg/mL of Al(OH)3  or 25 μL of AddaVax ™  adjuvant or non-adjuvant; control mice were mock-vaccinated . Al(OH)3 and AddaVax were used according to the manufacturer’s instructions (adjuvant: antigen = 1:1). In addition, BALB/c mice (n = 5 per group) were also intramuscularly vaccinated twice at day 0 and day 21 with 0.15 μg HA antigen of A/Vietnam/1194/2004 NIBRG-14(H5N1) inactivated vaccine alone (50 μL/dose) or in combination with 25 μL of Al(OH)3 adjuvant. Serum samples were collected on day 21 and day 42 and stored at −20 °C for serological analysis.Six-week-old BALB/c mice  were coated with 100 μL/well of H5N8 inactivated vaccine at 4 °C overnight, and blocked with 5% bovine serum albumin (BSA) in PBS for 1 h at 37 °C. Sera were tested at a starting dilution of 1:100 and applied to wells for 1 h at 37 °C, followed by incubation with rabbit anti-mouse IgG-peroxidase antibody for 1 h at 37 °C. The plates were developed using TMB, following the addition of 2 M Hreaction . The meaThe functional antibodies that inhibit hemagglutination was assayed by the hemagglutination inhibition (HI) assay . Serum w50 was added and incubated at 37 °C for 1 h, then the mixture samples were added into 2 × 104 MDCK cells each well and incubated for 72 h at 37 °C, 5% CO2. Freshly prepared 1% turkey erythrocytes were added, and were observed 1 h later. Neutralizing titers per milliliter of serum were calculated by the Reed and Muench method [The detection of influenza virus neutralizing antibody titers was performed according to the protocol recommended by the World Health Organization. Briefly, each serum treated with receptor-destroying enzyme (RDE) was diluted 2-fold with DMEM including 5 μg/mL TPCK-treated trypsin . An equal volume of virus at a concentration of 100 TCIDh method .The NI assay was performed according to the protocol. The median inhibitory concentration (IC50) was calculated as the reverse dilution of the serum, resulting in a 50% inhibition of NA activity ,23.p < 0.05.The data were counted and analyzed on GraphPad Prism 8.0 software. T-test was used for comparison between the two groups, and one-way ANOVA was used for comparison between multiple groups; 50/mL and an HA titer of 12 log2 HAU/50μL. The transmission electron microscope observation showed the H5N8 inactivated vaccine primary exhibited the small round (5–20 nm) structures. 3-adjuvanted or unadjuvanted H5N8 vaccines (p < 0.05)  < 0.05) , which sp < 0.05), and the HI titers of the other vaccine groups were all lower than or equal to 10 (p < 0.05), while the HI titers of the Al(OH)3-adjuvanted and unadjuvanted H5N8 vaccine were 256 and 128, respectively. More importantly, the group vaccinated with 0.15μg H5N8 vaccine combined with AddaVax adjuvant was found to have higher HI against H5N8 virus than the 1.5 μg unadjuvanted group. The data above indicate that the AddaVax adjuvant had immuno-enhancing effect in stimulating HI antibodies, as well as a better antigen-sparing effect. HI antibody is the gold standard for evaluating the immunogenicity of influenza vaccines. In our experiment, only 1.5 μg AddaVax-adjuvanted H5N8 vaccine could elicit HI titers of up to 80 after the first vaccination . The MN antibodies induced by the AddaVax-adjuvanted H5N8 vaccine were higher than those induced by Al(OH)3 or non-adjuvant groups . The dose-dependent effects of vaccination on enhancing NI titers were not observed in the groups vaccinated with Al(OH)3-adjuvanted vaccine doses reaching 0.15 μg and 1.5 μg en group .3 only induced a small amount of HI antibody titers against H5N6 virus, meanwhile failing to induce a cross reaction to H5N8 virus, as evidenced by the absence of HI titers I titers .Overall, AddaVax-based adjuvant H5N8 vaccines induced not only antibody responses against homologous viruses, but also cross-reactions against different clades of H5 viruses, and even achieved antigen-sparing effects.2 > 0.8), indicating that the three can be used as evaluation indices of immunogenicity. The increase in one antibody level was also related to corresponding increases in other antibody levels, which can be used as cross-reference indices for the evaluation of immunogenicity.The three humoral immune parameters of HI, NI and MN antibodies can be used to evaluate the immunogenicity and immune efficacy of vaccines. In order to study their correlation, the data were subjected to curve fitting test, and there was a strong correlation among HI, NI and MN . The curThe prevalence of the H5N8 virus has raised great concern since seven cases of human infection with H5N8 influenza viruses were reported in the Russian Federation in 2020. The most cost-effective way to prevent the spread of highly pathogenic avian influenza disease is to immunize humans through widespread vaccination. In this study, the HA gene of H5N8 virus was modified at the cleavage site, which can reduce the pathogenicity of avian influenza virus to avian embryos and poultry, and the vaccine candidate virus based on A/Astrakhan/3212/2020 H5N8 was constructed by RG. We examined an H5N8 inactivated vaccine, both with and without AddaVax adjuvant, at different dose levels, and found that the AddaVax adjuvant significantly enhanced HI, NI and MN antibody responses. Moreover, the AddaVax-adjuvanted H5N8 inactivated vaccine also elicited a certain degree of cross-reacting antibodies against different clades of H5 viruses. Our results illustrated that squalene-based adjuvant may confer a superior formulation for enhancing H5N8 vaccine efficacy, affording a dose-sparing effect.3 adjuvant has resulted in an improvement of the immune response in humans [3 adjuvant, the safety and efficacy of squalene-based immunogenic adjuvants such as MF59 have been well proved in many human clinical trials [3 [Most clinical studies have shown that avian influenza vaccines are less immunogenic than seasonal influenza vaccines, or induce weaker immune memory in humans, thus requiring adjuvantation or double-dose administration to improve vaccine efficacy ,28. To on humans . In addil trials . So far,l trials ,31,32. Sl trials . Furtherrials [3 . Similar3-adjuvanted H5N1 inactivated vaccine failed even to elicit NI and MN antibodies against H5N8 virus, suggesting that the H5N1 vaccine might not produce cross-neutralizing antibodies against the H5N8 virus.HI, NI and MN antibodies play an indispensable role in preventing viral infections . The H5N3-adjuvanted H5N1 inactivated vaccine. Since phenotypic diversity is one of the characteristics of H5 clade 2.3.4.4 viruses, genetic changes among different strains can induce antigenic alteration, and the antigenicity of viruses in different subclades varies widely, resulting in different HI activities among viruses [3 adjuvant or without adjuvant. Similarly, other studies have also illustrated that MN titers are much higher than HI titers in the clinical results of H5N1 vaccine combined with MF59 [Unexpectedly, the homologous HI antibodies stimulated by AddaVax-adjuvanted H5N8 vaccine were slightly lower than those stimulated by Al(OH) viruses . Previou viruses . Furtherith MF59 , and in ith MF59 , which sMoreover, NA is more conservative among circulating strains. NI corresponds with cross-protection and is highly correlative with protective immunity ,41. ClinMore importantly, our observations that the HI antibody titers induced by the 0.15 μg AddaVax-adjuvanted H5N8 vaccine were much higher than those of the 1.5 μg antigen vaccine alone, which suggested that the squalene-based adjuvant economized on the use of the immunogenic antigen of H5N8 vaccine at least 10-fold. Previous studies have also indicated that AddaVax formula, which provided sufficient seroprotective titers, could achieve better antigen-sparing effect and cross-reactivity to H7 subtype virus . TherefoIn conclusion, this study conducted a preliminary evaluation of the immunogenicity and efficacy of the H5N8 inactivated vaccine, explored an available adjuvant, and evaluated the cross-reactivity. The data reported here show that the vaccine can induce the humoral immune response with higher titers of HI, NI and MN antibodies, and can provide a potent cross-reaction with different clades of H5 viruses, which may serve as a reference in preparing for a potential H5N8 pandemic.


Humoral immunity to influenza neuraminidase (NA) was evaluated among different groups of people including patients with acute influenza infection and healthy people in different age groups using an enzyme linked lectin assay (ELLA). The amino acid composition of NA of seasonal influenza viruses A/Victoria/361/2011(H3N2) and A/Hong Kong/4801/2014(H3N2) differed by 2%, while cross-reacting neuraminidase-inhibiting (NI) antibodies to them in the same serum samples were detected in 10% of cases. Middle-aged patients born from 1977 to 2000 had a high level of hemagglutination-inhibiting (HI) antibodies to A/Hong Kong/4801/2014(H3N2), but almost no NI antibodies, which may indicate that in the case of a change in the hemagglutinin (HA) subtype, this age group will be susceptible to influenza A/H3N2 viruses. Therefore, it could mean there is a need for priority vaccination of this age group with a vaccine against the appropriate strain. It was shown that after intranasal administration of live influenza vaccine (LAIV) for the 2017–2018 season, serum antibody response was not lower compared to that during natural infection. In older people, antibodies to archival A/H2N2 viruses were detected more often than to modern A/H3N2. Since the conversion of antibodies to HA and NA often did not coincide, antibodies to NA can serve as an additional criterion for assessing the immunogenicity of influenza vaccines. The influenza viruses cause annually about 3 to 5 million cases of severe illness, and about 290,000 to 650,000 influenza-related deaths . Due to HA is the dominant antigenic component of the influenza virus, and antibodies directed against HA serve as the main criterion for assessing the post-infection or post-vaccination humoral immune response to influenza. Anti-NA antibodies reduce the severity of the infection and may provide protection ,6. When Moreover, the detection of antibodies to NA may be used to provide data on population immunity in cases when new antigenic variants of influenza viruses emerge, since subjects often do not have antibodies to the modified HA. For example, there are no HI antibodies to viruses A/H5N1 in the population, but there are cross-reactive antibodies to NA . The evaA phylogenetic analysis of NA subtype N2 of human influenza vaccine strains isolated in different years, in particular strains A/17/Hong Kong/4801/2014 (H3N2) and A/Victoria/361/2011(H3N2), was carried out.The results of the phylogenetic analysis of NA amino-acid sequences of A/H2N2 and A/H3N2 influenza viruses are shown in The NA of the influenza virus A/Victoria/361/2011(H3N2) had a high degree of amino acid sequence homology (96–98%) with other A/H3N2 strains isolated from the human population from 2004 to 2018, as well as strain A/Hong Kong/4801/2014(H3N2). The most distant strains were isolated before 1968 (the degree of homology was 83–85%). The strains A/Victoria/361/2011(H3N2) and A/Hong Kong/4801/2014(H3N2) were 98% identical in amino acid composition. The strains differ by only 5 amino acid substitutions: E221D (Glu-Asp), V231I , K258E (Lys-Glu), T329N (Thr-Asn), I392T (Ile-Thr). Amino acid substitutions were located in the NA “head” region outside the active sites. Using the Chimera software , a spatip > 0.05), but the levels of NI antibodies were significantly higher to A/Victoria/361/2011(H3N2) than to A/Hong Kong/4801/2014(H3N2) virus  and A/Hong Kong/4801/2014(H3N2) influenza viruses in paired sera of patients hospitalized in the 2017–2018 flu season. In the 2017–2018 flu season, the mean levels of HI antibodies to both A/H3N2 viruses at the beginning of hospitalization were similar (2) virus A,B. The 2) virus B. This mNext, we assessed the formation of antibodies to HA and NA of A/Hong Kong/4801/2014(H3N2) virus in patients hospitalized with influenza in the 2018–2019 influenza season. Data in The levels of antibodies to surface antigens of the A/Hong Kong/4801/2014(H3N2) strain were assessed in the sera collected from healthy persons of different ages examined in 2017–2018 flu season. For this, we used HI test with the A/17/Hong Kong/2014/8296(H3N2) vaccine strain and ELLA with A/H6N2 virus. The highest average level of HI antibodies was found among younger patients and the lowest among middle-aged patients .The highest mean anti-NA antibody titers were found in the group of volunteers born between 1924 and 1956, while the lowest mean antibody titers were noted in the group of patients born between 1977 and 2000 .‘Herd’ immunity was assessed by the proportion of individuals with protective HI antibody titers ≥1:40 . For NI The lowest level of protective HI antibody titers to A/Hong Kong/4801/2014(H3N2) virus was in the group of middle-aged patients, while 10% of them had NI antibody titers ≥1:20.Antibodies to NA of A/Hong Kong/4801/2014(H3N2) influenza virus were also evaluated in individuals vaccinated with trivalent live influenza vaccine (LAIV) in the fall of 2017. LAIV included vaccine strains A/17/New York/15/5364(H1N1)pdm09, A/17/Hong Kong/2014/8296(H3N2), and B/60/Brisbane/08/83 (antigenic lineage B/Victoria). The distribution of serum antibody titers to NA and HA strain A/Hong Kong/4801/2014(H3N2) among 18 vaccinated patients is shown in In And finally, we wanted to compare the effect of age on the level of NI antibodies to N2 of the A/H2N2 and A/H3N2 viruses. To do this, we used archival samples of patients of different ages left over from routine laboratory tests conducted in 2013.p < 0.0001). The highest main titer of HI antibodies was detected against the 1966 strain A/California/1/1966(H2N2), while the level of HI antibodies to A/Victoria/361/2011(H3N2) was four times lower (Wilcoxon Matched Pairs test: p = 0.0002). HI antibodies to A/Leningrad/134/17/57(H2N2) were not detected. The levels of anti-N2 antibodies to the influenza viruses of 1957, 1966 and 2011 were also statistically significantly different  in the examined patients over 50 years of age (p = 0.013). The study included six sera from adults aged 21–38 years which did not have HI antibodies to A/H2N2 viruses. Mainly antibodies to the HA and NA of seasonal influenza virus A/Victoria/361/2011(H3N2) were detected among these patients. Certain levels of NI antibodies to A/Leningrad/134/17/1957(H2N2) may be attributed to the cross-reactivity of antibodies to N2 of A/H3N2 viruses with which these patients may have previously been in contact.The levels of serum HI antibodies to A/Leningrad/134/17/1957(H2N2), A/California/1/1966(H2N2) and A/Victoria/361/2011(H3N2), shown in s of age A. The mas = 0.525).The herd immunity to influenza viruses A/Leningrad/134/17/1957(H2N2), A/California/1/1966(H2N2) and A/Victoria/361/2011(H3N2) as assessed by antibody levels ≥1:40, are presented in The percentage of seropositive elderly individuals to the A/Victoria/361/2011(H3N2) strain according HI test was only 8.6%, although none of examined elderly individuals had a titer of NI antibodies to seasonal influenza virus A/Victoria/361/2011(H3N2) ≥1:40.Although antibodies to influenza NA are neutralizing, antibodies to the second antigenic component–NA-can also provide protection, especially against viruses with novel HA variants. The most clear and substantiated results indicating the protective effect of NA antibodies were obtained during the flu pandemic in 1968 on a group of people with obviously absent or low levels of antibodies to the influenza virus A/Hong Kong/1/68(H3N2). Among the observed group of 274 people aged 20 to 45 years, the frequency of infection with the A/H3N2 influenza in November 1968–January 1969, detected by HI seroconversions, as well as severity, were inversely related to the level of antibodies to N2 . It was Our study showed that minor changes in the structure of N2 can lead to changes in antigenic properties. We identified five amino acid substitutions located in the NA “head” differed between NA of viruses A/Hong Kong/4801/2014(H3N2) and A/Victoria/361/2011(H3N2). One such substitution at position 221 was described earlier. It was previously reported that an asparagine residue at position 221 changed to histidine when analyzing A/H2N2 and A/H3N2 influenza viruses isolated between 1957 and 1975 [The fact that groups of patients had a high level of antibodies to A/Hong Kong/4801/2014(H3N2) HA but almost no NI antibodies may indicate more frequent contacts with viruses of this subtype. The lack of formation of antibodies to N2 can be explained by the probable primary contact with the A/H1N1 viruses that caused the 1977 pandemic and continue to circulate until now. In the case of a change in the HA subtype, the group of those born in from 1977 to 2000 will be susceptible to influenza A/H3N2 viruses. Therefore, it could mean a need for priority vaccination of this age group with the appropriate vaccine strain.It was shown that the intensity of serum antibody response after intranasal administration of trivalent LAIV was not lower compared to natural infection with the influenza A virus. It should have been taken into account that during immunization with LAIV, three vaccine viruses are introduced at once, to which an immune response is formed. Influenza infection, on the other hand, is supposed to be caused most often by one virus, but given the phenomenon of antigenic ‘sin’, antibodies may also form against previous variants with which the body has previously contacted. It was previously reported that the immune response to NA and HA differs between infection and vaccination. Thus, it has been shown that more NA-reactive than HA-reactive B cells are activated during H3N2 infection . This isSeasonal strains of the A/H3N2 subtype, having significant antigenic differences in hemagglutinin with A/H2N2 strains that have left the circulation since 1968, represent the same NA subtype–N2. Influenza viruses of the A/H2N2 subtype are considered as potentially pandemic, since the majority of the population does not have immunity to them. This is also shown in our study in regard to HI antibodies. Thus, the presence of a group of people vaccinated with a seasonal trivalent live influenza vaccine (LAIV) of the composition A (H1N1) + A (H3N2) + B, or those who had a natural infection caused by A/H3N2 influenza, may be protected against the A/H2N2 pandemic strain at the initial stage until mass vaccination of the population is deployed. Previously, it was shown that the antibody titers against N2 and influenza B virus NA were moderately high compared to N1, which may be due to differences in stability between N1 and N2 . The facOur data on high levels of antibodies to the A/California/1/1966(H2N2) virus in the elderly may suggest that a significant part of older people was in direct contact with the A/H2N2 virus isolated in 1966, in addition to similar viruses. The formation of a high level of NI antibodies to the A/California/1/1966(H2N2) in a large number of volunteers may also point to repeated contacts with other A/H2N2 viruses during the 1957 to 1968 circulation period. The cross-reaction of anti-N2 antibodies acquired during this period can explain the high percentage of individuals with a detectable titer of antibodies to the NA strain A/Leningrad/134/17/57(H2N2) in the absence of HI antibodies.Data on the low antibody levels to the A/Victoria/361/2011(H3N2) in the elderly in 2013 may indicate a not very frequent contact with such viruses. A/H2N2 viruses isolated between 1957 and 1968 differ significantly from each other both antigenically and genetically . The mosThis study has potential limitations. We used ELLA to detect functional NA-inhibiting antibodies, while not taking into account non-NI antibodies that may potentially participate in Fc-mediated reactions.n = 86), as well as paired sera from 18 healthy adults vaccinated with seasonal live influenza vaccine (LAIV) in the fall of 2017. Written informed consent was obtained from each subject. Also, in this study we used 15 pairs of sera from patients hospitalized during 2017–2018 flu season with PCR confirmed type A influenza. Additionally, we used 41 archive sera from healthy adults collected in 2013. All sera were left after routine lab tests. The study was approved by the Local Ethics Committee at Institute of Experimental Medicine (protocol dated 25 April 2019). After obtaining the approval of the local ethical committee, the sera were transferred to the researchers, none of whom had access to the personal data of the patients.In this retrospective study we examined blood sera from patients at the “IEM” Medical Research Center collected in 2018–2019 (To determine neuraminidase inhibiting (NI) antibodies in human sera, we used influenza A/H7N2 viruses that contained HA from A/horse/Prague/1/57(H7N7) and NA from A/Leningrad/134/17/1957(H2N2), A/California/1/1966(H2N2) or A/Victoria/361/2011(H3N2). One more virus was A/H6N2, which contained HA from A/herring gull/Sarma/51c/2006 (H6N1) and NA from A/Hong Kong/2014/8296(H3N2). To determine serum anti-HA antibodies, strains A/Leningrad/134/17/1957(H2N2), A/17/California/66/395(H2N2), A/17/Victoria/2011/89 (H3N2) and A/17/Hong Kong/2014/8296 (H3N2) obtained from the collection of the Institute of Experimental Medicine were used. All viruses were propagated in developing chicken eggs at 33 °C.The levels of antibodies to NA of the A/H3N2 strains were evaluated using enzyme-linked lectin assay (ELLA) after determining the optimal conditions for the enzymatic reaction. For this, reassortant viruses A/H7N2 and A/H6N2 were purified by ultracentrifugation on a stepwise sucrose gradient. The working concentration of the virus was defined as 128 hemagglutinating units (HAU) in 50 μL. This dose provides a signal level when reading the results on a spectrophotometer in the range of optical density (OD) at 450 nm (OD450) from 0.45 to 0.70. The titer of serum NA inhibiting (NI) antibodies was calculated as the reciprocal of the dilution of the sample giving 50% inhibition of NA activity, i.e., a two-fold decrease in OD compared with the viral control when reading the results on the ELx800 spectrophotometer .Vibrio cholerae neuraminidase extract . The reaction results were taken into account after erythrocyte sedimentation in control wells without virus. Serum anti-hemagglutination antibody titer was defined as the highest dilution that resulted in the inhibition of erythrocyte agglutination.HI was performed according to the previously described method  in 96-weAlignment of amino acid sequences taken from the database of influenza viruses of the National Center for Biotechnology Information  was performed using the UGENE program from UniPro (version 1.19.0) . The desThe remote nearest neighbor method (neighbor joining) was used to reconstruct phylogenetic trees. The stability of the tree topology was assessed using bootstrap analysis based on the results of building and matching phylogenetic trees for 1000 new sets of amino acid sequences generated based on the original alignment.p < 0.05.The results were processed using the statistical package “Statistica” (version 6.0). The geometric mean titers (GMT), arithmetic mean (m) and standard deviation (σ) were used to describe the obtained data. For statistical analysis, antibody titers were expressed as log2 reciprocals of the final dilution. Checking the normality of the distribution was carried out using the Shapiro-Wilk test. If the assumptions about the normal distribution of the dependent variable within each group and the homogeneity of the variance were not met, Kruskal-Wallis rank analysis of variance was used. The comparison of dependent samples was carried out using the Wilcoxon test. To compare multiple independent groups, we used a Kruskal-Wallis analysis of variance (ANOVA) with subsequent multiple pairwise comparisons based on Kruskal-Wallis sums of ranks (Dunn test). For nominal data, Fisher’s exact two-tailed test was used. The presence of a statistical relationship between the variables was estimated using correlation analysis by the Spearman’s method. Criteria-tested null hypotheses were rejected at In this work, we analyzed the molecular and antigenic changes in NA subtype N2 with influenza infection and vaccination. The formation of NI antibodies in patients with acute influenza infection was studied. Using ELLA, antibodies to N2 were detected in patients of various ages. The lowest levels of antibodies to NA subtype N2 were noted in the group of volunteers born between 1977 and 2000, which may indicate the need for priority vaccination of this age group with the appropriate vaccine strain. In the group of patients vaccinated with LAIV, there was a significant increase in the average titers of NI antibodies to A/Hong Kong/4801/2014(H3N2). Thus, when evaluating the immunogenicity of an influenza vaccine, the increase in anti-NA antibodies should be taken into account.