Liver sinusoidal endothelial cells (LSECs) are fenestrated endothelial cells (liver sieve) that undergo alteration during sepsis and H2S plays a role in this process. Substance P (SP) is encoded by the preprotachykinin A (PPTA) gene, and promotes inflammation in sepsis; however, its regulation by H2S is poorly understood. Furthermore, the interaction between H2S and SP in modulating LSEC fenestrations following sepsis remains unclear. This study aimed to investigate whether CSE/H2S regulates SP and the neurokinin-1 receptor (NK-1R) and modulates fenestrations in LSECs following caecal ligation and puncture (CLP)-induced sepsis. Here we report that the absence of either CSE or H2S protects against liver sieve defenestration and gaps formation in LSECs in sepsis by decreased SP-NK-1R signaling. Following sepsis, there is an increased expression of liver CSE and H2S synthesis, and plasma H2S levels, which were aligned with higher SP levels in the liver, lungs and plasma and NK-1R in the liver and lungs. The genetic deletion of CSE led to decreased sepsis-induced SP and NK-1R in the liver, lungs and plasma SP suggesting H2S synthesized through CSE regulates the SP-NK-1R pathway in sepsis. Further, mice deficient in the SP-encoding gene (PPTA) preserved sepsis-induced LSEC defenestration and gaps formation, as seen by maintenance of patent fenestrations and fewer gaps. In conclusion, CSE/H2S regulates SP-NK-1R and modulates LSEC fenestrations in sepsis.Cystathionine-γ-lyase (CSE) is a hydrogen sulfide (H H2S synthesized through cystathionine-γ-lyase (CSE), a predominant H2S-synthesizing enzyme in peripheral organs, promotes inflammation in various inflammatory conditions including sepsis [2S signaling promotes leukocyte infiltration and organ injury in sepsis [2S donors, such as sodium hydrosulfide (NaHS) and sodium sulfide (Na2S) promote liver and lung inflammation and systemic inflammatory response through ERK1/2-NF-κB and subsequent release of proinflammatory cytokines and chemokines [2S-mediated inflammatory response and organ injury in sepsis. These studies suggest that H2S has an important role in modulating the inflammatory process during sepsis.Hydrogen sulfide  is an 11 amino acid immunoregulatory neurokinin belonging to the tachykinin family and is encoded by the preprotachykinin A (PPTA) gene. Although SP is a peptide primarily of neuronal origin, its production from inflammatory cells such as macrophages, eosinophils and dendritic cells have been reported . The phy2S and SP in regulating inflammation of sepsis is poorly understood. For example, the H2S donor NaHS increased SP, which in turn promoted inflammation and lung injury through NK-1R activation following the induction of sepsis [2S-induced inflammation during sepsis. However, these studies were limited by the lack of use of specific pharmacological inhibitors  or activators  [2S and SP in sepsis.The interaction between Hf sepsis . The mecf sepsis ,14. Thisnd Na2S) ,14,15,162S is one of the key inflammatory mediators of LSEC damage during CLP-induced sepsis [2S contributes to LSEC damage and multiple organ damage in sepsis remains unknown.The liver has specialized endothelial cells called liver sinusoidal endothelial cells (LSECs). These are fenestrated in nature (liver sieve) with pore size in the range of 50–250 nm in diameter. The liver sieve acts as a permeable barrier between sinusoidal blood and hepatic parenchyma  and playd sepsis . However2S-synthesizing enzyme (CSE) and SP encoding gene (PPTA) were used to determine whether CSE/H2SregulatesSP-NK-1Rsignaling and SP modulates LSEC fenestration in sepsis.In the present study, mice deficient in the H2S-synthesizing activity and plasma H2S levels (* p < 0.05 vs. WT sham). Increased CSE/H2S signaling correlated with upregulated SP levels in the liver, lung, and plasma in sepsis (* p < 0.05 vs. WT sham). Mice deficient in the CSE gene had significantly lower levels of liver, lung and plasma SP compared to WT mice following sepsis(#p < 0.05 vs. WT sepsis), suggesting that CSE/H2S acts as an upstream regulator of SP . Decreased H2S levels following CSE gene deletion significantly decreased sepsis-induced NK-1R expression (#p < 0.05 vs. WT sepsis)  .2/mm2 vs. 0.06 ± 0.01 nm2/mm2; * p < 0.05 vs. WT sham). Mice, deficient in the PPTA gene had preserved LSEC morphology (fenestration frequency and porosity) and fewer gaps with sepsis, than WT with sepsis (gap area: 0.09 ± 0.01 nm2/mm2 vs. 0.12 ± 0.02 nm2/mm2)  .2S synthesized through CSE enzyme (using CSE deficient mice) regulates SP and NK-1R, suggesting that SP plays a key role in promoting H2S-mediated LSEC damage in sepsis.This is the first study demonstrating that H2S is one of the key mechanisms regulating SP and NK-1R in sepsis. The findings of the present study are consistent with earlier observations made using a pharmacological inhibitors of CSE [2S donors [2S on SP-mediated inflammation and organ injury in sepsis.The proinflammatory role of SP and NK-1R has already been reported in sepsis and endotoxaemia-associated organ injury ,22,23,24s of CSE ,14,16 anS donors ; these r2S levels, suggesting that H2S is an upstream regulator of SP [2S and SP, and suggests that both of these mediators serve as possible early stage diagnostic inflammatory markers in patients with sepsis.Previous clinical studies of patients with sepsis and septic shock have shown higher circulating levels of SP . To suppor of SP . The datPseudomonas aeruginosa) and endotoxaemia (induced by LPS) promote liver injury associated with LSEC defenestration [2S signaling protects against LSEC defenestration and gaps formation following CLP-induced sepsis [2S signaling occurs upstream to SP and regulates liver sieve fenestration.The present study showed that a sepsis-induced an increase in SP and promotes defenestration and gap formation in LSECs. Endotoxaemia and the sepsis-induced inflammatory response are known to regulate the structure of LSEC fenestrations, i.e., defenestration in LSECs ,26,27,28stration ,30. Furtstration ,31. A re2S-regulated SP is one of the key proinflammatory mediators in modulating the LSEC phenotype and therefore that pharmacological manipulation of the liver sieve through H2S/SP plays an important role in the management of sepsis-associated fibrosis and cirrhosis.Structural integrity of the fenestrated liver sinusoidal endothelium is believed to be essential for the normal homeostasis of fluids, particles and metabolites between hepatocytes and sinusoidal blood. Its alteration can have adverse effects on liver function. It is known that severe sepsis may lead to dysregulation of the LSEC . The res2S is one of the key mechanisms regulating SP in sepsis and the associated liver sieve phenotype, this is not a solitary mechanism. Mice deficient in the CSE gene have significantly reduced SP levels following sepsis; however, not to the extent of the control groups. This indicates there are other mechanisms through which SP is regulated in sepsis. During sepsis, an increased activation of immune cells such as macrophages plays an important role in liver sieve defenestration. Previous studies have suggested that an increased release of proinflammatory mediators, particularly TNF-α and biologically-active free radicals such as oxygen and nitrogen radicals lead to significant LSEC damage and that Kupffer cells, the resident macrophages in the liver, play a key role in this process [Although the present study shows that H process . Recent  process . Since S2S, is a key inflammatory mediator that promotes liver sieve disruption in sepsis.In conclusion, the current study shows that SP, regulated by Hn = 8 for each group); in the second experiment, WT (BALB/c) and PPTA KO mice were used (n = 4 for each group). All experiments were approved  by the University of Otago-Christchurch Animal Ethics Committee and performed according to the established guidelines.Both WT (C57BL/6J and BALAB/c) and KO (CSE KO on C57BL/6J background and PPTA KO on BALB/c background) mice were obtained from and maintained at Christchurch Animal Research Area (CARA). The CSE KO mice were generated as described previously . All mic2). The caecum was exposed by a small midline incision through the skin and peritoneum of the abdomen (under sterile conditions). With silkam 5.0 thread, the caecal appendage was ligated at the distal part of the caecum (8–10 mm from the tip) then perforated using a 22-gauge needle. A small amount of stool was squeezed through each hole and the bowel was repositioned. The abdomen was sutured using sterile permilene 5.0 thread. Sham mice went through the same protocol but with no bowel perforation. In the first experiment, mice were euthanized 8 h after surgery by IP injection of pentobarbital sodium (150 mg/kg) and organs and plasma were collected and stored at −80 °C for subsequent experiments. In the second experiment, 8 h after surgery, mice were anaesthetized and livers were perfusion fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer to measure LSEC fenestrations using SEM as described previously [CLP-induced sepsis was performed according to the previously described protocol with minor modifications ,27,36,37eviously .2S-synthesizing activity was measured as described previously [2S-synthesizing activity, 230 µL of homogenate was added to the reaction mixture containing L-cysteine , and pyridoxyal5′-phosphate . Samples were incubated for 30 min at 37 °C in a shaking water bath. Zinc acetate  was added to the tubes to trap evolved H2S. Next, a mixture of equal amounts of FeCl3 (30 µM) and N, N-dimethyl-p-phenylenediamine sulfate (20 µM)  was added. Samples were incubated in the dark for 20 min and the reaction was stopped by the addition of 10% (v/v) trichloroacetic acid (25 µL). Samples were centrifuged and the resultant supernatant was used to measure the H2S concentrations using a spectrophotometer at 670 nm. Results were calculated against a Na2S calibration curve and corrected by protein content. Results were expressed as nmol H2S formed/mg protein.Liver Heviously . Briefly2S levels were measured with a modified protocol based on methods described previously [w/v) zinc acetate (100 µL), a mixture of N, N-dimethyl-p-phenylenediamine sulfate (20 µM) in 7.2 M HCl and FeCl3 (30 µM) in 1.2 M HCl  and left to incubate at room temperature in the dark for 20 min. Following this, 10% (v/v) trichloroacetic acid (120 µL) was added to denature the protein and stop the reaction. Samples were centrifuged at 7700× g for 5 min and the absorbance of supernatant (150 µL) was measured with a 96-well microplate spectrophotometer at 670 nm. The H2S concentration was calculated against a calibration curve of Na2S. Results were expressed as µmole/mL.Plasma Heviously . Brieflyg, 20 min, 4 °C) and the supernatants were collected. These were adsorbed on C18 cartridge columns  as described [v/v) acetonitrile. The samples were freeze-dried and reconstituted in SP assay buffer. The levels of SP were determined using ELISA  according to the manufacturer’s instructions. The results obtained were then corrected for the protein content of tissue samples and expressed as nanograms per milligram of protein or nanograms per 100 µL of plasma.Liver and lung samples were homogenized in 1 mL of ice-cold sodium phosphate buffer for 20 s. The homogenates were centrifuged  and membranes blocked with 5% BSA for 1 h, followed by incubation with the primary antibody for CSE and NK-1R (1:1000) at 4 °C overnight, then by 2 h incubation with the secondary antibody  at room temperature before detection with a chemiluminescent substrate . Detection and quantification were performed on a chemi-doc system . Mouse anti-human CSE was purchased from Abnova  and mouse anti-mouse NK-1R antibody was purchased from Abcam . Rabbit anti-mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and goat anti-rabbit horseradish peroxidase (HRP)-conjugated antibodies were purchased from Santa Cruz Biotechnology .Tissue lysates (liver and lung) were prepared in ice-cold RIPA buffer supplemented with protease inhibitor cocktail by homogenization. The tissue homogenates were kept on ice for 30 min, followed by centrifugation at 10,000× To assess the liver sieve fenestrations using SEM, liver samples were perfusion fixed with phosphate buffered saline followed by paraformaldehyde. Perfusion fixed samples were cut into small blocks and processed  with hexamethyldisilazane (HMDS)) for SEM. The liver blocks were mounted on SEM slug mounts and a platinum coat was applied using a sputter coater. Using SEM , at 15 kV acceleration voltage, the blocks were examined. The fenestration diameter, frequency and total area were assessed using Image J (NIH) and porosity and number of gaps  were calculated [p < 0.05 was considered statistically significant.Data are expressed as mean ± standard error of mean. One-way ANOVA with post-hoc Tukey’s test was performed to compare multiple groups using GraphPad version 6.07.


Sphingosine kinase phosphorylates sphingosine to generate sphingosine 1 phosphate (S1P) following stimulation of the five plasma membrane G-protein-coupled receptors. The objective of this study is to clarify the role of S1P and its receptors (S1PRs), especially S1PR3 in airway epithelial cells.CCL20 in the airway inflammation, BALB/c mice were immunized with ovalbumin (OVA) and subsequently challenged with an OVA-containing aerosol to induce asthma with or without intraperitoneal administration of anti-CCL20. Finally, the anti-inflammatory effect of VPC 23019, S1PR1/3 antagonist, in the OVA-induced asthma was examined.The effects of S1P on asthma-related genes expression were examined with the human bronchial epithelial cells BEAS-2B and Calu-3 using a transcriptome analysis and siRNA of S1PRs. To clarify the role of ADRB2, PTGER4, and CCL20, in the bronchial epithelial cells. The knock-down of SIPR3 suppressed the expression of S1P-inducing CCL20. Anti-CCL20 antibody significantly attenuated the eosinophil numbers in the bronchoalveolar lavage fluid (P<0.01). Upon OVA challenge, VPC23019 exhibited substantially attenuated eosinophilic inflammation.S1P induced the expression of some asthma-related genes, such as S1P/S1PR3 pathways have a role in release of proinflammatory cytokines from bronchial epithelial cells. Our results suggest that S1P/S1PR3 may be a possible candidate for the treatment of bronchial asthma. Sphingosine 1 phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biological responses such as proliferation, migration, cytoskeletal organization, adherent junction assembly and morphogenesis .in vivo by sphingosine kinases (SPHK) 2 to biologically active FTY720-phosphate i response which is induced by protease activated receptor 2 agonist peptide and Alternaria extract may be the cause of difference of cellular response [The current study also showed that S1P induced siveness , 25. Thel asthma . It is il asthma , 29, andl asthma . In addil asthma . In contsymptoms . The prosymptoms . Thereforesponse .CCL20. A range of chemokines, including CCL20, CCL19, and CCL27, are primarily released by the bronchial ECs in response to stimulants, such as microbes or irritants [H2 cell cytokine release, inflammation, airway hyper-reactivity and the expression of the transcriptional activator STAT6 [In the current study, we focused on rritants . These crritants . Tumor nor STAT6 . The curIn conclusion, S1P/S1PR3 pathway has a role in release of CCL20, which has a role in experimental asthma mouse model, from bronchial ECs. Our results suggest that S1P/S1PR3 axis may be a possible candidate for the treatment of bronchial asthma.S1 Fig(XLSX)Click here for additional data file.


Mycobacterium smegmatis mc2155, 12 siphoviruses were recovered from long-term archival stocks stored in Japan. Their genome sequences were 46.0 to 61.3 kbp with 63 to 68% G+C contents, which allowed them to be categorized within cluster W and subclusters A1, A2, B3, A7, I1, and K4.Using  Mycobacterium spp., have been studied since their first isolation in the 1940s  (Phage genomic DNA samples were prepared as described previously . A singlgap.org) . These gMycobacterium virus D29 (GenBank accession number AF022214), the recovered phage D29 was considered to be the clonal strain of Mycobacterium virus D29 isolated in the 1950s (The Actinobacteriophage Database (PhagesDB) serves as a genome sequence repository for mycobacteriophages , 7, whicL5virus. Phage HC of subcluster I1 was classified into the genus Brujitavirus under subfamily Chebruvirinae. Phages Y2 and Y10 of cluster K were classified into the genus Tm4virus. Phages D12 and PR of cluster W could not be classified.The International Committee on Taxonomy of Viruses (ICTV) classification allowed the taxonomic classification of the recovered phages using these data . Phages GenBank accession numbers are shown in


Hyperactivation of Wnt/β-catenin signaling pathway is a critical step in colorectal tumorigenesis. In this study, we identified that V-set and transmembrane domain containing 2A (VSTM2A) was a top-downregulated secreted protein that negatively regulated Wnt singling pathways in colorectal cancer (CRC). We investigated the functional mechanisms and clinical implication of VSTM2A in CRC.Methods: Function of VSTM2A was investigated in vitro and in vivo. VSTM2A binding partner was identified by mass spectrometry, immunoprecipitation and Western blot. The clinical impact of VSTM2A was assessed in 355 CRC patients and TCGA cohort.Results: VSTM2A protein was prominently silenced in CRC tumor tissues and cell lines mediated by its promoter hypermethylation. VSTM2A DNA promoter hypermethylation and VSTM2A protein downregulation was associated with poor survival of CRC patients. Ectopic expression of VSTM2A inhibited colon cancer cell lines and organoid growth, induced CRC cells apoptosis, inhibited cell migration and invasion, and suppressed growth of xenograft tumors in nude mice. VSTM2A was released from CRC cells through a canonical secretion pathway. Secreted VSTM2A significantly suppressed Wnt signaling pathway in colon cancer cells. Wnt signaling co-receptor LDL receptor related protein 6 (LRP6) was identified as a cell membrane binding partner of VSTM2A. Using deletion/mutation and immunoprecipitation, we demonstrated that VSTM2A bound to LRP6 E1-4 domain with its IgV domain. VSTM2A suppressed LRP6 phosphorylation in a time and dose dependent manner, and induced LRP6 endocytosis and lysosome-mediated degradation, which collectively contributing to the inactivation of Wnt signaling.Conclusions: VSTM2A is a novel antagonist of canonical Wnt signaling by directly binding to LRP6 and induces LRP6 endocytosis and degradation. VSTM2A is a potential prognostic biomarker for the outcome of CRC patients. Colorectal cancer (CRC) is the third most common cancer and ranks the second in terms of cancer mortality worldwide based on the newly released Global cancer statistics 2018 VSTM2A gene spans 27 kb of genomic sequence on chromosome 7 and consists of five exons. VSTM2A protein composes of 240 amino acid (aa), containing a potential 25-aa N-terminal signal peptide, two putative glycosylation sites, and a single immunoglobulin V-set domain. Previous study suggested a role of VSTM2A in the regulation of the preadipocyte cell differentiation We analyzed 4642 predicted secreted proteins from the The Cancer Genome Atlas (TCGA) dataset for colon & rectum adenocarcinoma (COADREAD) gene expression by RNA sequencing and identified VSTM2A was one of the top-downregulated secreted proteins in CRC, but is readily expressed in normal colon tissue. The Three cohorts of 355 CRC patients were included in this study. Forty-six paired primary CRC and adjacent non-tumor fresh specimens and 158 CRC paraffin blocks were obtained during operation of CRC patients diagnosed in the Prince of Wales Hospital, the Chinese University of Hong Kong from 2002 to 2017. A cohort of 151 primary colorectal tumors and adjacent normal tissue RNA were obtained from Zhejiang University, China. Colorectal cancer patients with age >18 were enrolled in this study. Pregnant or nursing patients were excluded. Written informed consents were obtained from subjects or their authorized representatives. The study protocol was approved by the Ethics Committee of the Chinese University of Hong Kong and Ethics Committee of Zhejiang University. This study was carried out in accordance with the Declaration of Helsinki of the World Medical Association.The human CRC cell lines , L cell and L Wnt-3A cell were purchased from the American Type Culture Collection . 293T cell line was purchased from Invitrogen . Cell lines were maintained according to the protocols from American Type Culture Collection. All cell lines were obtained between 2013 and 2015. Routine Mycoplasma testing was performed by PCR. Cells were grown for no more than 25 passages in total for any experiment. CRC patient-derived organoid model were kindly provided by Prof. Catherine O'Brien, Department of Surgery at University Health Network and cultured as described previously http://tcga-data.nci.nih.gov/tcga/).A list of extracellular genes of 4,642 unique human proteins encoded by 3,641 unique genes were screened and evaluated in this study VSTM2A (NM_001301009.1), VSTM2A IgV domain (Δ144-240), and VSTM2A Δ N (Δ1-25) were amplified and cloned into the pcDNA3.1 expression vector. Human LRP6 deletions  are kind gifts of Prof. Christof Niehrs. Human full length LRP6 and deletion (ΔE1-2 and ΔC) were generated by PCR and cloned into pcDNA3.1.Sequence corresponding to the open reading frame (ORF) of human Cells were seeded for MTT and MTS assay to measure the cell viability. Cells were seeded on 6-well plates and stained with 0.5% crystal violet solution for 30 min. Colony with more than 50 cells per colony was counted. The experiment was conducted in three independent triplicates.Transfected cells were stained with Annexin V- Allophycocyanin (APC) (BD Biosciences) and 7-aminoactinomycin (7-AAD). The cells were sorted by FACS Calibur system.6 cells in 0.1 ml phosphate-buffered saline) were injected subcutaneously into the right dorsal flank of four 4-week-old male Balb/c nude mice, separately (5/group). Tumor diameter was measured every 3 days. Tumor volume (mm3) was estimated by measuring the longest and shortest diameter of the tumor and calculating as follows: volume = (shortest diameter)2 × (longest diameter) × 0.5. After 15 days, the mice were sacrificed, and the tumors were weighed and fixed in formalin for histological analysis. All experimental procedures were approved by the Animal Ethics Committee of the Chinese University of Hong Kong.Stable VSTM2A expressing or control HCT116 cells . Quantitative real-time PCR was performed by SYBR Green PCR Master Mix (Thermo Fisher Scientific) on 7500HT Fast Real-Time PCR System (Thermo Fisher Scientific). Primer sequences are listed in Table S2.Proteins were separated on 10-12% SDS-polyacrylamide gel electrophoresis and transferred onto Polyvinylidene difluoride membranes . Blots were immunostained with primary antibody and secondary antibody. Independent experiments were performed at least twice. The antibodies used and their dilutions are listed in Table S2. The results were scored independently by two pathologists.Tissue microarrays of CRC tissues were stained with VSTM2A antibody. IHC was performed using the EnVision system . The antibodies used and their dilutions are listed in oC, cells were chilled on ice followed by treatment of His-tag VSTM2A conditioned medium for 1 hour at 4oC. The cells were then incubated with 1.6 mM cell impermeable crosslinker 3,3'-dithiobis for 2 h on ice. Whole-cell lysates were incubated with 400 μl Ni-NTA agarose for 12 h. Potential binding proteins were eluted with 1.5 ml RIPA buffer containing 250 mM imidazole and precipitated with 10% trichloroacetic acid. Sample was loaded to 10% SDS-PAGE for separation followed by Coomassie Blue staining. The gel was excised into 11 segments and submitted to Shotgun proteomics analyses using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher) coupled with an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher) operating in the data-dependent acquisition (DDA) mode. The resulting spectra from each fraction were searched separately against Homo_sapiens_uniprot (169389 sequences) database by the search engines: Proteome Discoverer 2.2 .RKO cells were seeded on 10 X 100 mm Polyethylenimine  coated dishes. After 4 hours incubation with DMEM serum-free medium at 37The Dissociation Constant between VSTM2A and LRP6 was determined by Titration ELISA Wnt3a-conditioned medium was harvested from Wnt3a expressing L-cells according to the manufacturer's instructions (ATCC). VSTM2A-conditioned medium and control medium were harvested from 293T cells transiently transfect with VSTM2A expression or empty control constructs. Briefly, 293T cells were seeded in 10 cm dishes and transfected with 16 ug of p3.1-VSTM2A-His/myc or p3.1 empty vector using Lipofectamine 2000 as directed by the manufacturer (Thermo Fisher Scientific). Conditioned medium was harvested 48 h after transfection. Recombinant human VSTM2A His-tag protein (10037-VT), LRP6 (1505-LR-025) and Wnt3a (5036-WN-010) were purchased from R&D.Cells were labeled with EZ-Link Sulfo-NHS-SS-Biotin  prior to conditioned medium treatment to allow internalization of cell surface proteins performed as described previously Cells in a 24-well plate were co-transfected with luciferase reporter plasmid, and pRL-CMV control vector by lipofectamin 2000. Plasmids were co-transfected as indicated per well. After 24 h of transfection, luciferase activities were analyzed by Dual-Luciferase Reporter Assay System (Promega) and normalized to the control Renilla. The key cancer related signaling pathways including Wnt/β-catenin (TOPflash/FOPflash), nuclear factor-кB (NF-кB), MAPK (SRE), TGF-β (CAGA), JNK (AP-1), STAT3 (APRE), PI3K/AKT (FOXO), BMP (GCCG), and p53 (p53RE) were investigated.Cells were then treated with 2 μM demethylation reagent 5-aza-2'-deoxycytidine  for 5 days. The 5-Aza was replenished every day. Some cell lines were further treated with 300 nM of histone deacetylase inhibitor trichostatin A  for additional 24 h.C/ (HC + HT) × 100%, . Primer sequences for BGS are listed in Table Two μL bisulfite-treated DNA was amplified by primers of BGS and sequenced. Sequencing analysis was performed by SeqScape software (Thermo Fisher Scientific). Methylation percentage of each CpG site was calculated according this formula: Methylation% = Ht-test was used to compare the difference between two groups. The χ2 test was used to compare the clinicopathologic characteristics of CRC patients and VSTM2A IHC scores. Univariate and multivariate Cox regression analysis were performed to assess the prognostic value of VSTM2A. Overall survival in relation to VSTM2A methylation status and IHC scores were evaluated by Kaplan-Meier survival curve and log-rank test. Differences with P value < 0.05 were considered to be statistically significant.Values were expressed as mean ± standard deviation (SD). The Student's To identify the dysregulated secreted proteins, we adopted the list of candidate extracellular genes compiled by Gonzalez and colleagues, containing 4,642 unique human proteins encoded by 3,641 unique genes P < 0.05, Figure P < 0.01).We screened the VSTM2A expression status in normal human tissues and found that VSTM2A was predominantly expressed in gastrointestinal tract including colon Figure . CompareVSTM2A has four transcripts that can generate protein products (http://www.ensembl.org). The four protein isoforms share an identical immunoglobulin V-set (IgV) domain but different protein sequences at the C terminal. Among these isoforms, VSTM2A isoform 4 (NM_001301009) showed the highest expression level in normal mucosal specimens, but reduced significantly in CRC . VSTM2A markedly reduced cell viability , RKO (P < 0.01) and DLD1 (P < 0.01) by flow cytometry . Inhibition of the cell secretion by Brefeldin A (BFA), which prevents secreted protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, inhibited VSTM2A supernatant expression and induced cytosolic accumulation, suggesting a secreted property of VSTM2A , suggestive of a secreted nature  using titration ELISA. The Kd of recombinant human LRP-6 Fc chimera protein and recombinant human VSTM2A His-tag protein is 9.19 nM  domain VSTM2A protein contains a potential 25-aa N-terminal signal peptide and a single IgV domain spanning from aa 33-143  mRNA expression Figure K. OverexP = 0.04) and disease free survival  by multivariate Cox regression analysis . After adjustment for potential confounding factors, lower VSTM2A methylation was found to be an independent risk factor for overall survival in CRC patients by multivariate Cox regression analysis (RR 1.67 (95% CI 1.01 to 2.73), P = 0.044; Table We investigated the mechanism of VSTM2A downregulation in CRC. We found full or partial methylation in all seven CRC cell lines ; whilst no or low methylation was detected in the four normal colon tissues Figure . Demethyin vitro and in vivo functional experiments, VSTM2A possesses a tumor-suppressive function through inhibiting CRC cell proliferation, migration/invasion, and organoid growth, induced apoptosis in vitro and suppressed xenograft tumor growth in nude mice.Secreted proteins not only participate in various physiological processes but also play crucial roles in carcinogenesis Drosophila or C. elegans. However, no sequence similarity was found between DKKs and VSTM2A, suggesting a novel suppressor VSTM2A for LRP6. We measured that Kd of human LRP-6 Fc and VSTM2A His-tag protein is 9.19 nM by titration ELISA, which is lower than that of DKK1  Kd by titration ELISA requires pure preparations of both receptor and ligand. Considering the 80% purity of commercial available LRP-6 Fc, the Kd of LRP-6 Fc and VSTM2A should be further investigated by a cost-intensive SPR machine. Moreover, we observed that overexpression of VSTM2A showed double bands in western blot analysis as showed in Figure We revealed that VSTM2A attached to plasma membrane and interacted with plasma membrane protein LRP6 extracellular domain E1-E4 by LC MS/MS analysis and immunoprecipitation. Unlike the groups of Wnt antagonist of the SFRPs which suppressed the Wnt signaling through indirectly sequestering Wnt proteins, VSTM2A directly binds to the Wnt receptor LRP6, which is functionally more similar to the Wnt inhibitor DKKs family LRP6 is a critical co-receptor of Wnt ligands.Ligand binding with receptor often causes the receptor endocytosis In summary, we revealed that secreted protein VSTM2A is a critical tumor suppressor in colorectal carcinogenesis and a novel antagonist of Wnt signaling receptor LRP6. VSTM2A suppresses Wnt/β-catenin signaling by directly inhibiting LRP6 activity and inducing LRP6 endocytosis and lysosome mediated degradation. VSTM2A may serve as a new prognostic marker for CRC patients.


Identification of HIV-1-specific monoclonal antibodies (mAbs) with potent ADCC activity may help develop an effective HIV-1 vaccine. In present study, we isolated such human mAb, designated E10, from an HIV-1-infected patient sample by single B cell sorting and single cell PCR. E10 bound to gp140 trimer and linear peptides derived from gp41 membrane proximal external region (MPER). E10 epitope (QEKNEQELLEL) overlapped with mAb 2F5 epitope. However, E10 differentiated from 2F5 in neutralization breadth and potency, as well as ADCC activity. E10 showed low neutralization activity and narrow spectrum of neutralization compared to 2F5, but it mediated higher ADCC activity than 2F5 at low antibody concentration. Fine mapping of E10 epitope may potentiate MPER-based subunit vaccine development.Antibody-Dependent Cellular Cytotoxicity (ADCC) is a major mechanism of protection against viral infections  Antibody-Dependent Cellular Cytotoxicity (ADCC) has been repeatedly reported to be able to provide protection against HIV/AIDS. Antibodies that mediate ADCC activity can decrease viral load , 2, loweAccording to our and others' research, broadly neutralizing HIV-1 mAbs may not confer potent ADCC activity. Instead, non-neutralizing mAbs may mediate high ADCC activity, suggesting that neutralizing epitopes may be different from ADCC epitopes. More than a dozen of HIV-1 broadly neutralizing mAbs have been characterized for epitopes identified .AntibodySingle B cell sorting and antibody gene amplification platform have been a conventional tool for antibody identification , 31, 32.Forty eight HIV envelope specific single memory B cells  were sorted from PBMC of HIV patient Figure . One of Despite high affinity of E10 for gp41, it exhibited low neutralization potency compared to b12 and 2F5. We tested E10 for neutralization activity against GXC44(C), JRFL (B), PCNE3 (01_AE), Du172.17[C (ref.)], and 93MW959 (C (ref.)) by HIV-1 pseudo virus neutralization assay. Mab E10 can neutralize GXC44(C) and JRFL (B), but with high IC50s  Figure . It did With TF228 as target cell and pooled healthy donor PBMC as effector cell, IgG1 E10mediated ADCC activity was compared with IgG1 2F5 and IgG1 MZ76 .Unlike the proverbial neutralizing and ADCC antibody IgG 2F5, Mab E10 mediated higher ADCC activity at low antibody concentrations (below 0.1 μg/ml). Interestingly, ADCC activity of E10 did not increase significantly when antibody concentration exceeded 0.1 μg/ml, suggesting steric hindrance to E10 binding Figure . The conThe affinity ELISA results has confirmed that Mab E10 is gp41 specific. To localize E10 epitope, we constructed a yeast antigen library which displays HIV-1 envelope fragments. The yeast clones that specifically bind to Mab E10 were sorted and re-cultured. After yeast plasmid extraction and sequencing, we got a highly enriched sequence that encodes peptide F7 . To confirm the binding of E10 to F7 peptide, E10 were constructed to yeast vector PYD7 as single chain form. It can display on the surface of EBY100 recombinant yeast after galactose induction. F7 peptide were expressed as human Fc fusion protein and purified by protein G agarose. The binding of E10 toF7-Fc protein was confirmed by F7 ELISA Figure ; the binQEKNEQELLELDKWA)  and VL (IGKV3-20), the amino acid similarities are only 66.14% (VH) and 78.18% (VL), excluding the possibility of E10 as a variant of 4E10 Figure . To inve) Figure , suggest2F5 has been shown to be reactive with phospholipids, including CL, Ro (SSA), centromere B, and histones, as measured by an AtheNA antinuclear autoantibody assay (ANA) . And mAbTZM-bl cell line, TF228 cell line and consensus clade B 15-mer peptides (Cat No. 9480 Lot No. 8 098265) were obtained from the NIH ARRP(AIDS Research and Reference Program). HIV patient PBMC and HIV-1 Env plasmids was kindly provided by Linqi Zhang (Tsinghua University) . AntibodPBMC was purified by ficoll gradient from HIV infected patient, incubated with 1 μg/mlbiotinylated SF162gp140 trimer at 4°C for 30 min. The PBMC was then washed by PBS and stained with second antibodies:Pacific Blue™ anti-human CD19 , PE/Cy7 anti-human CD3, APC/Cy7 anti-human CD14 , APC anti-human IgG, 7-AAD(7-Aminoactinomycin D) , FITC anti-human CD27, PerCP/Cy5.5 anti-human IgM , and R-Phycoerythrin Streptavidin . SF162gp140-trimerbinding memory B cells  were sorted in 96 well PCR plate by FACS AriaIII(BD). The sorted cells were then lysedwith RNA directly reverse transcribed into cDNA according SuperScript™ III CellsDirectcDNA Synthesis System  manual. Single cell derived cDNA was used for antibody variable gene amplification.Antibody variable fragments were amplified using primers and strategies that described by Tiller . Paired For detection of affinity to envelope proteins, Bal-gp120 and bal-gp41-Fc were coated separately on ELISA plate at 1 μg/ml in phosphate buffer (PH9.6), after PBST wash and skimmed milk blocking, IgGE10 and control antibodieswas added to wells for incubation. Antibodies were three-fold serially diluted starting from 30 μg/ml. Seven dilutions and 3 duplicates were set for each antibody. Anti-human fab-HRP  was added as second antibody.EC50 (50% maximum binding) were determined using GraphPad Prism software. For affinity detection against SF162gp140 trimer, RSC3 and deltaRSC3, E10 was diluted staring from 90μg/ml with 8 dilutions. For ELISA based epitope mapping, HIV gp41 peptides  and pictures were generated by Flow Jo software.MabE10 variable fragments were constructed into SCFV format and co-transformed with linear PYD7 plasmid , 40 for A standardized TZM-bl cell line-based Env-pseudotyped neutralization assay was used as previously described , 42.4 labeled target cells were dispensed in 50 μl of RPMI 1640 medium in round-bottomed 96-well plate. Fifty microliters of diluted antibodies in triplicate were added to the wells and the plate incubated at 37°C for 15 min in CO2 incubator. Fifty microliters of unlabeled pooled PBMCs at a concentration of 2 × 107 cells/ml were added to each well and the plate incubated at 37°C for 4 h in CO2 incubator. One micro liter of 7-AAD solution was added to the wells and the plate incubated at 4°C in dark for 15 min. Cell mixtures were analyzed by flow cytometry. Percent ADCC was calculated as [ / (% maximum lysis – % spontaneous lysis)] × 100, in which “% spontaneous lysis” referred to percent lysis of target cells incubated with effector cells in the absence of IgGs, and “% maximum lysis” referred to percent lysis of target cells following heat shock by incubating the cells at 85°C for 30 min.TF228 was maintained in RPMI1640 containing the same supplements. Heparinized whole blood samples were used to isolate human PBMCs by Ficoll density gradient separation. A flow cytometry-based ADCC assay was set up using TF228 cells as target cells and healthy human PBMCs as effector cells at an T/E ratio of 1/50 , 43. In in vivo.In present study, we identified a new ADCC antibody that target the MPER region of gp41, adjacent to the epitope of 2F5, 4E10, and 10E8. The study emphasized the MPER region as a dominant ADCC epitope on HIV envelope. HIV MPER region is rich in alpha helix and antibodies  that target this region most probably recognize linear epitopes. Unlike the A32-like epitopes which is conformational and hard to mimic, the comparatively simpler structure of MPER may adapt itself in subunit vaccine design. In fact, a number of MPER based subunit vaccine design have already been proved successful at inducing neutralizing and ADCC antibody , 44, 45.According to previous reports, most MPER antibodies were limited in potency, and abilities to access MPER on envelope of primary viral isolates. In addition, lipid binding and autoreactivity were also considered to be the characteristics of MPER antibodies. E10 binds to autoantigen, but showed weaker binding activity in comparison with 2F5. Further improvement on accessibility and reduced autoreactivity may help E10 be used in combination with potent neutralizing antibodies in passive treatment. The potent ADCC activity of E10, and the potential roles in combination usage with other potent neutralizing antibodies in passive treatment, emphasizes the contribution of the effector functions of antibodies in prevention against HIV-1 infection.IgG E10 use a famous V gene segment IGHV1-69 allelic variants. Antibodies derived from this germline V gene tend to have a hydrophobic CDR2 and bind to hydrophobic alpha helix rich portions such as influenza hemagglutinin stem [e.g., monoclonal antibody CR6261 , F10 4747, CR911ZY, XL, MZ, ZS, and WY conceived and designed the experiment. XL, ZY, WT, ZS, and JL performed the experiments. ZY, MZ, WY, and XL wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli cell-free protein synthesis (CFPS) for mAb expression. In the CFPS step, we employed our original techniques: (1) ‘Zipbody’ as a modified Fab (fragment of antigen binding) format, in which the active Fab formation is facilitated by adhesive leucine zipper peptides fused at the C-termini of the light and heavy chains; and (2) an N-terminal SKIK peptide tag that can markedly increase protein production. By the Ecobody technology, we demonstrated rapid screening of antigen specific mAbs from immunized rabbits and Epstein-Barr Virus infected human B cells. We further obtained rabbit mAbs in E. coli expression system yielding to 8.5 mg of purified proteins from 1 L bacterial culture.Single B cell sampling following to direct gene amplification and transient expression in animal cells has been recognized as powerful monoclonal antibodies (mAbs) screening strategies. Here we report Ecobody technology which allows mAbs screening from single B cells in two days This technology uses  Monoclonal antibodies (mAbs) have become essential agents for research, diagnostic and therapeutic areas.Single B cell screening technologies, which can rapidly generate mAbs from sampled single B cells from immunized animals, have been proven to be powerful techniques to obtain the natural antibody repertoire ,2,3,4,5.Escherichia coli extract-based CFPS systems to produce fragments of antigen binding (Fab) derived from single B cells [E. coli cell-extract with PCR-amplified DNA templates, amino acids, nucleotides, T7 RNA polymerase and an energy source. However, practically available mAb screening was challenging due to the following technical problems. Firstly, active Fabs were sometimes not formed in the CFPS because of incorrect folding and assembling of heavy chain (Hc) and light chains (Lc). In particular, active Fabs were not produced at all in the case of rabbit mAb clones, probably because of the presence of too many Cys residues involved in disulfide bond formation [Escherichia coli CFPS and living cell expression systems [Taking advantage of CFPS systems, we developed a rapid mAb screening system named “Single-Cell Reverse Transcription-PCR linked in vitro Expression (SICREX)”, which utilizes  B cells ,10,11. Uormation . Therefoormation ,14. To o systems . Further systems . This seE. coli cytoplasmic expression system followed by refolding of inclusion bodies.Together with Zipbody and the SKIK peptide tag technologies, for improvement of Fab formation and protein production in CFPS, we have developed an improved SICREX system renewed as ‘Ecobody technology’ . Here, wEcobody technology will be beneficial to the field of mAb research and development as a high-throughput and low-cost mAb screening method.E. coli based cell-free protein synthesis (1.5 h). (vii) mAbs evaluation by ELISA (3 h).The scheme of Ecobody technology is illustrated in Vibrio parahaemolyticus NBRC 12711, E. coli O26 GTC14538 (verotoxin-1 producing strain), and non-toxic verotoxin 2 (VT2) were used as antigens for immunization of rabbits. Bacteria were obtained from the Biological Resource Center at the National Institute of Technology and Evaluation  and the National BioResource Project GTC Collection , cultured in Luria-Bertani (LB) medium at 37 °C overnight, and inactivated by incubating at 80 °C for 30 min in phosphate-buffered saline (PBS) containing 0.25% formalin. They were stored at −20 °C. Nontoxic VT2 (E167Q mutant) expression vector was prepared by PCR from VT2 producing E. coli O157 GTC 14535 strain with the primers AAGAAGGAGATATACATATGAAGTGTATATTATTTAAATGGGTACTG (forward) and TGGTGGTGGTGGTGCTCGAGGTCATTATTAAACTGCACTTCAGCAAAT (reverse) using KOD FX polymerase . After NdeI and XhoI treated PCR product and pET22b vector were ligated, inverse PCR introducing E167Q mutation into VT2a subunit was performed with primers ACAGCACAAGCCTTACGCTTCAG and TAAGGCTTGTGCTGTGACAGTG using KOD Plus polymerase (Toyobo). The His-tagged nontoxic VT2 was expressed in BL21 (DE3) pLysS and purified using Ni-Sepharose 6 Fast Flow resin  and HT-Hydroxy apatite  as described by He et al. [8 dead bacterial cells or 0.2 mg/mL of nontoxic VT2 supplemented with 2.5 mL of complete Freund’s adjuvant by hypodermic injection. The second and third boosters were given after intervals of 2 weeks and 10 days, respectively. A few mL of blood samples was collected 2 days after the final booster. This study was approved by the Committee of Animal Experiments of the Graduate School of Bioagricultural Sciences, Nagoya University (permit number 2015022611) and performed according to the Regulations on Animal Experiments in Nagoya University.Three types of antigens, bacteria e et al. . The two2. The proliferated cells (106 cells/tube) were stored in 1 mL of the same medium supplemented with 10% dimethyl sulfoxide in liquid nitrogen.The EBV infected cell line HEV0011, provided by the RIKEN BRC  through the National Bio-Resource Project of the MEXT/AMED , was onc6 cells/mL-PBS were stained with 1 µM ER-Tracker™ Green  at room temperature for 5 min then centrifuged at 1000× g for 2 min as described by Kurosawa et al. [E. coli DH5alpha-coated beads. After that, the remaining B cells were separated into each well (1 cell/10 µL-PBS) of 0.2 mm glass-bottomed 384-well imaging plates  and the bead-B cell complexes in each well were confirmed under a phase-contrast inverted microscope . Bürker-Türk plates were used for cell counting.After a few mL of blood samples were collected from the immunized rabbits, lymphocytes were separated by a previously reported protocol using density-gradient centrifugation . To selea et al. . In brieTM 647 Labeling Kit-NH2 . After replacing the solution with new PBS, the cells with double positive signals were separated into a 96 well PCR plate containing 10 µL of RT reaction mixture/well by the single cell sorting mode of cell sorter SH800 .The lymphocytes fraction separated by the same method as above was stained with ER-tracker and HiLyte Fluor 647 labeled nontoxic VT2 which was prepared by HiLyte Fluorg for 3 min) to replace the solution with 1 mL new PBS. The cells with higher HiLyte Fluor 647 fluorescence were collected by FACS . Next, the sorted cells (103 cells/100 µL PBS) were mixed with 5 µL of magnetic beads coated with E. coli DH5alpha (6 × 105 beads/µL PBS) and incubated at room temperature for 10 min. After removing beads fraction with a magnet stand, cells in the supernatant were mixed with 5 µL of magnetic beads coated with influenza vaccine  pdm09, A/Hong Kong/4801/2014 (H3N2), B/Phuket/3073/2013 (Yamagata strain), and B/Texas/2/2013 (Victoria strain)) (6 × 105 beads/µL PBS) and incubated as well. The cells bound to the beads were collected, diluted to 1 cell/10 µL PBS, and separated into a 384 well glass plate to be 1 cell/well.The stored HEV0011 cells were sub-cultured in a T25 culture flask overnight. Then, the cells were incubated with 11 µL/mL of HiLyte Fluor 647 labeled anti-Human IgG (Fc specific) antibody produced in goat  in PBS at 20 °C for 20 min. The cells were centrifuged  with oxidized glutathione (GSSG) and DsbC were used as recommended by the manufacturer. Proteins were expressed on a 10-µL scale containing 0.5 µL PCR product or 20 ng purified DNA fragments (each of Hc and Lc) as the template, at 37 °C for 90 min. FluoroTect™ GreenLys in vitro Translation Labeling System  was included to confirm protein synthesis in subsequent SDS-PAGE analysis.To express SKIK-fused Zipbody, the DNA templates of Hc and Lc were amplified from the above assembled vectors with primers annealed to upstream of the T7 promoter and downstream of the T7 terminator. E. coli were analyzed by ELISA using our standard protocol described previously. In brief, 50 µL of dead bacterial cells (OD600 nm = 0.1) or 10 µg/mL protein antigens (nontoxic VT2 or influenza vaccine), 0.4% Bovine Serum Albumin (BSA) in PBS as negative control were coated on the MaxiSorp plate (Thermo) overnight, and we followed the standard ELISA protocol using Zipbody as the primary antibody, and anti-rabbit Ig(G + M)-HRP conjugate , anti-His tag-HRP conjugate as the secondary antibody, or anti human IgG(H + L) polyclonal antibody-HRP conjugate produced in goat . Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo) was used for dilution of the secondary antibody.Unless otherwise stated, mAbs produced in CFPS or E. coli Shuffle T7 Express  as the host, grown in 50 mL LB medium supplemented with 100 µg/mL ampicillin with 1 mM IPTG induction at 16 °C for 24 h, as described in our previous study [E. coli BL21star (DE3) hovering plasmid pSJS1240 in the same medium with 100 µg/mL spectinomycin at 37 °C for 4.5 h. The amount of inclusion bodies of Hc and Lc was equalized and refolded together in the single dialysis tube. The KD values were measured by Bio-layer interferometry method.To produce the Zipbody with an SKIK tag, Hc and Lc were coexpressed in us study . The incus study . In the V. parahaemolyticus, E. coli O26, and nontoxic VT2. The details are described in the following sections.We followed the workflow of Ecobody technology shown in 6 lymphocytes were collected from a few milliliters of blood of a rabbit immunized with dead V. parahaemolyticus. The cells, about 30% (2.0 × 104 cells), showing higher fluorescence stained with ER tracker were collected by FACS. Subsequently, approximately 500 cells bound to magnetic beads coated with dead V. parahaemolyticus were separated by magnet, of which 19 were used for single-cell RT-PCR. From them, seven pairs of Lc and Hc genes were obtained . They were expressed in CFPS and evaluated by ELISA. All the clones, expressed as the Zipbody with the N-terminal SKIK tag, had higher binding activity toward V. parahaemolyticus than toward the other tested antigens  were obtained (data not shown). As the DNA of 3 M and 5 G were not amplified by PCR following Gibson assembly, in total, eight clones were expressed in CFPS and evaluated by ELISA, and each showed a higher signal with E. coli O26 than with BSA (negative control) (The mAb screening process for anti-control) b.5 cells were used and 165 cells (0.26%) were selected as ER tracker positive and antigen specific ones. From these, 47 cells were sorted into PCR tubes with single cell sorting mode. Of which, 8 pairs of Hc and Lc genes  were finally obtained and subjected to CFPS and ELISA. All clones showed high ELISA signals toward antigen  were sorted by FACS. After removing cells nonspecifically bound to croscope  and subjE. coli Shuffle T7 Express. Although most of the target proteins were confirmed in the insoluble fraction, they were also in the soluble fraction (data not shown). Therefore, we tried His tag purification from the soluble proteins and ELISA was conducted against individual influenza antigen, not vaccine mixture. As a result, this clone had reactivity to all  and human mAb (No. 14 for influenza virus vaccine) with high ELISA signals were expressed in E. coli. Consistent with our previous study about the SKIK peptide tag [E. coli expression has the potential to provide low cost mAb reagents applicable for diagnostics or research uses.Two rabbit mAb clones (22 G for tide tag , most oftide tag . InteresIn conclusion, we adopted two techniques, Zipbody and the N-terminal SKIK peptide tag, and succeeded in developing a mAb screening system, Ecobody technology, allowing rapid generation of mAbs derived from single B cells.Ecobody technology will be beneficial for rapid rabbit and human mAb screening.


Influenza epidemiological and virologic data from Georgia are limited. We aimed to present Influenza Like Illness (ILI) and Severe Acute Respiratory Infection (SARI) surveillance data and characterize influenza viruses circulating in the country over three influenza seasons.We analyzed sentinel site ILI and SARI data for the 2014–2017 seasons in Georgia. Patients’ samples were screened by real-time RT-PCR and influenza viruses isolated were characterized antigenically by haemagglutination inhibition assay and genetically by sequencing of HA and NA genes.32% (397/1248) of ILI and 29% (581/1997) of SARI patients tested were positive for influenza viruses. In 2014–2015 the median week of influenza detection was week 7/2015 with B/Yamagata lineage viruses dominating (79%); in 2015–2016—week 5/2016 was the median with A/H1N1pdm09 viruses prevailing (83%); and in 2016–2017 a bimodal distribution of influenza activity was observed—the first wave was caused by A/H3N2 (55%) with median week 51/2016 and the second by B/Victoria lineage viruses (45%) with median week 9/2017. For ILI, influenza virus detection was highest in children aged 5–14 years while for SARI patients most were aged >15 years and 27 (4.6%) of 581 SARI cases died during the three seasons. Persons aged 30–64 years had the highest risk of fatal outcome, notably those infected with A/H1N1pdm09 . A/H1N1pdm09 viruses analyzed by gene sequencing fell into genetic groups 6B and 6B.1; A/H3N2 viruses belonged to genetic subclades 3C.3b, 3C.3a, 3C.2a and 3C.2a1; B/Yamagata lineage viruses were of clade 3 and B/Victoria lineage viruses fell in clade1A.In Georgia influenza virus activity occurred mainly from December through March in all seasons, with varying peak weeks and predominating viruses. Around one third of ILI/ SARI cases were associated with influenza caused by antigenically and genetically distinct influenza viruses over the course of the three seasons. The monitoring of changes in seasonal influenza viruses plays a critical role in defining influenza vaccine composition [Influenza viruses affect people of all ages and cause mild to severe disease sometimes leading to fatal outcomes –4. Surveposition , 7. Moreposition –10 and n2 with a population of around 3.7 million people. Prior to 2006, a nationwide population-based surveillance system for influenza and upper respiratory tract infection provided influenza data based only on clinical diagnosis without laboratory confirmation. In 2007, in collaboration with US CDC and other international stakeholders, sentinel surveillance of ILI and SARI was initiated. The National Influenza Center (NIC) at the National Center for Disease Control and Public Health, Georgia (NCDC&PH) now screens specimens collected at ILI/SARI surveillance sites all year round to monitor activity of influenza viruses.Georgia is located in the Caucasus region and covers a territory of 69700 kmIn this study we describe epidemiological and virologic data for three consecutive influenza seasons obtained from ILI/SARI surveillance systems. The objectives were to: (1) define periods of influenza activity in Georgia; (2) assess the proportions of influenza infections among ILI and SARI cases; (3) determine most affected age groups; (4) describe epidemiological characteristics of influenza-associated fatal cases; and (5) determine antigenic and genetic profiles of influenza viruses circulating in the country.ILI surveillance was carried out in one outpatient clinic with a predetermined catchment of approximately 60,000 residents in the capital city Tbilisi (5% of 1.2 million population). The Children’s Central Hospital, the largest children’s clinic of the country also in Tbilisi, and four hospitals in Kutaisi (the biggest city in Western Georgia) were selected as SARI surveillance sites . MedicalThe case definitions were standardized for all clinics involved in influenza sentinel surveillance using the 2014 revision of WHO case definitions . ILI wasA systematic alternate day sampling method was used at the ILI and SARI surveillance sites . Both orViral RNA was extracted with QIAamp Viral RNA Mini Kits  according to manufacturer’s instructions. Real-time RT-PCR was performed for influenza virus detection, influenza A subtyping and influenza B lineage determination (the latter being implemented during the 2015–2016 season). Specimens were screened for influenza viruses using molecular kits, positive controls and protocols provided by the US CDC . LaboratReal Time RT-PCR positive specimens with Ct values <30 were inoculated onto Madin Darby Canine Kidney (MDCK) cells, incubated at 34°C and observed daily for 7 days for cytopathic effect. Haemagglutination assay was performed for confirmation of virus growth in cell culture using a 0.5% suspension of turkey red blood cells (RBCs). Preliminary antigenic characterization of influenza isolates was carried out by haemagglutination inhibition (HI) assay using sheep or goat antisera provided by the CDC, Atlanta in accordance with the WHO Manual . For det® terminator sequencing amplifications (Applied Biosystems) and the purified BigDye® terminator products were subsequently sequenced using an ABI 3130XL Analyser. DNA sequencing data were processed and assembled using Sequencher 5.0 software . Gene sequences of viruses from Georgia generated by Crick Worldwide Influenza Centre and CDC, and influenza viruses from various countries, were downloaded from the EpiFlu database of the Global Initiative on Sharing All Influenza Data (GISAID) (http://www.gisaid.org). Nucleotide and deduced amino acid sequences were aligned and analyzed using BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and maximum likelihood phylogenetic trees were estimated using RaxML v8.2X with a GTRGAMMA substitution model (https://sco.h-its.org/exelixis/software.html), followed by annotation with amino acid substitutions defining nodes and individual virus gene products using treesub (https://github.com/tamuri/treesub/blob/master/README.md). Phylogenies were based on full-length open-reading-frames with the signal peptide component being removed from HA genes and stop codons removed from all genes: H1-HA 1647, H3-HA 1650, N1-NA 1407, N2-NA 1407, B/Vic-HA 1710, B/Yam-HA 1707, B/Vic-Na 1398 and B/Yam-NA 1398 nucleotides, respectively. Trees were visualized using FigTree (http://tree.bio.ed.ac.uk/software/figtree/) and highlighted using Adobe Illustrator CC 2015.3 (http://www.adobe.com/uk/products/illustrator/features.html). Sequence accession numbers for all reference and test viruses are given in Sequence analysis of hemagglutinin (HA) and neuraminidase (NA) genes of influenza viruses was performed by NIC, Georgia; Crick Worldwide Influenza Centre, London, UK, and CDC, Atlanta, USA. A/H3N2 and A/H1N1pdm09 specific primers and the Sanger sequencing protocol were provided to NIC Georgia by Walter Reed Army Research Institute (WRAIR), USA. cDNAs were subjected to BigDyeWritten informed consent was not required for this study. Verbal consent from patients and guardians in the case of children was sufficient for sample and data collection as the study was carried out under Georgia’s routine public health surveillance practice.Fisher’s exact test was used for statistical analysis of data with chi squared estimation of significance. P values of ≤0.05 were considered as statistically significant. Data analysis was performed using Epi Info (version 7). Median, range, and interquartile range (IQR) were calculated to assess continuous variables. As Georgia is in the Northern Hemisphere, an influenza season is defined as the time period from week 40 of each year to week 20 of the following year, when the vast majority of influenza detections are made, and only data for these periods were analyzed. ILI consultation rates per 100 000 population and SARI vs total hospital admissions were used for evaluating season dynamics. The seasonal threshold was calculated using WHO guidelines and methodology [A total of 12,912 ILI and 10,218 SARI patients met the ILI/SARI case definitions at sentinel sites during three consecutive influenza seasons 2014–2017; of these, 1,248 (9.7%) ILI and 1,997 (19.5%) SARI patients were sampled and tested for influenza. Overall 30%  specimens were positive: 397 (32%) ILI and 581 (29%) SARI (including four specimens: three ILI and one SARI—positive for both influenza A and B viruses).Influenza detection median weeks and prevailing viruses varied between seasons: in 2014–2015 the median week of total influenza detections was week 7/2015 (IQR weeks 6–9) with influenza B viruses dominating (79%); in 2015–2016 the median week of total influenza detections was week 5/2016 (IQR weeks 3–7) with A/H1N1pdm09 viruses dominating (83%); and the 2016–2017 season showed a bimodal distribution of influenza detections—the first wave was caused by A/H3N2 (55%) with a median week of 51/2016 (IQR weeks 50–52) and the second by influenza B (45%) with a median week of detections of 9/2017 (IQR weeks 7–11) . Of the Of the 397 influenza virus-positive ILI samples over three seasons (2014–2017) 132 (33%) were A/H1N1pdm09, 121 (30%) were A/H3N2 and 147 (37%), influenza B (three were positive for both influenza A and B viruses). During influenza activity periods weekly outpatient visits for ILI rose in parallel with laboratory-confirmed influenza virus detection . Based oOverall influenza detection was highest 48% (133/276) among children aged 5–14 years  . ChildreOnly 18 of 1248 swabbed ILI participants had been vaccinated during the three seasons; of these, eight persons became ill with influenza A/H3N2 and two with A/H1N1pdm09.Out of 581 specimens testing positive for influenza viruses , 241 41 SARI admInfluenza virus detection rates among hospitalized patients were highest, almost equally, in the age groups 30–64 years  and 15–29 years  . PatientAmong 1,997 sampled SARI cases, only 25 were vaccinated against influenza; of these, three tested positive for A/H1N1pdm09 and three for A/H3N2.During the three influenza seasons, 27 (4.6%) patients out of 581 laboratory-confirmed influenza SARI cases died, all of whom were patients in Kutaisi SARI sentinel hospitals. Of the fatal cases 19 (70%) were male and 8 (30%) female. Influenza A/H1N1pdm09 was associated with 20 (74.1%), A/H3N2 five (18.5%) and influenza B two (7.4%) deaths . ThirteeEighteen (86%) of 21 viruses were successfully recovered by WHO CC London and studied by HI assay. All but two viruses were well recognized in HI assays, within 2-fold of the homologous titer, by antiserum raised against the vaccine virus, A/California/7/2009; A/Georgia/567/2015 and A/Georgia/791/2016 were recognized at titers 4- and 16-fold lower respectively. Both these viruses also showed reduced recognition, compared to the respective homologous titers, by antisera raised against several reference viruses. While A/Georgia/567/2015 carried an HA1 substitution N156S;  in a posSequencing of HA and NA genes of 19 A/H1N1pdm09 viruses was performed , S2 Fig.HA genes of nine viruses circulating early in the 2015–2016 season belonged to group 6B and encoded two additional amino acid substitutions K311T in HA1 and L37Q in HA2. The NA sequences of these viruses also fell in genetic group 6B and carried additional substitutions: T289I and I396M. The HA genes of six viruses from later in the 2015–2016 season fell into genetic subgroup 6B.1 characterized by encoding the substitutions S84N, S162N  and I216T in HA1 and clustered with reference virus A/Slovenia/2903/2015. NA genes of the same viruses also belonged to subgroup 6B.1, encoding the substitutions V13I, K264I, N270K and I314M in the NA glycoprotein.A/H3N2 viruses have undergone extensive evolution in recent years and a number of genetic clades/groups have been designated, indicated below in parentheses after virus names. Of 28 A/H3N2 viruses from the three seasons 22 (79%) were recovered by the WHO CCs.In the 2014–2015 season all four A/H3N2 viruses were cultured from clinical samples but only one virus, A/Georgia/532/2015, had HA activity. It was poorly recognized by antisera raised against the 2014–2015 season vaccine virus, egg-propagated A/Texas/50/2012 (3C.1), and antisera raised against both cell culture-propagated and egg-propagated cultivars of the 2015–2016 season A/Switzerland/9715293/2013 (3C.3a) vaccine virus. However, A/Georgia/532/2015 was recognized well by antisera raised against cell culture-propagated A/Victoria/361/2011(3C.1) and both cell culture-propagated and egg-propagated cultivars of A/Stockholm/6/2014 (3C.3a).Five of ten viruses from the 2015–2016 season were recovered, based on detection of sialidase activity, but only three had HA activity. Antiserum raised against the 2015–2016 season vaccine virus, egg-propagated A/Switzerland/9715293/2013 (3C.3a), recognized all three viruses poorly, but they were recognized by antiserum raised against the 2016–2017 season vaccine virus, egg-propagated A/Hong Kong/4801/2014 (3C.2a), at titers only 4-fold lower than the homologous titer of the antiserum.Twelve viruses were isolated from 14 samples of the 2016–2017 season, but 11 were unable to agglutinate RBCs and only one virus (A/Georgia/1819/2016) was analyzed by HI assay. It was recognized by an antiserum raised against the recommended 2017–2018 season vaccine virus, egg-propagated A/Hong Kong/4801/2014, at a titer within 4-fold of the homologous titer of the antiserum.In total, 27 A/H3N2 viruses were sequenced , S3 Fig.HA gene sequence analysis of 2015–2016 season viruses showed that four of the five fell in subclade 3C.2a1 and one in clade 3C.3a, the A/Switzerland/9715293/2013 clade. The HA gene of the 3C.3a virus encoded HA1 amino acid substitution Y94H, commonly seen in 3C.3a viruses collected during the 2015–2016 season. The NA gene of this virus clustered with other 3C.3a subclade viruses. 3C.2a1 viruses from Georgia had, in addition to the clade defining substitutions (N171K in HA1 and I77V and G155E in HA2), the HA1 amino acid substitution S46Y. The NA genes of these four viruses clustered with other 3C.2a1 subclade viruses and differed from the A/Switzerland/9715293/2013 vaccine virus by encoding the substitutions S245N (resulting in the gain of a new glycosylation motif), S247T (a substitution that maintained a glycosylation motif at residues 245–247), T267K, D339N, I380V, T392I and P468H.All 18 sequenced viruses of the 2016–2017 season from Georgia belonged to subclade 3C.2a1. Two viruses showed mixed sequences encoding the glycosylation motif at residues 158–160 in HA1 following culture; one of them, A/Georgia/1819/2016, was able to agglutinate RBCs. The HA genes of viruses from Georgia clustered in two subgroups: the first encoding an HA1 R142G substitution (12 viruses) and the second with HA1 amino acid changes K92R, N121K and H311Q (6 viruses). The NA genes of viruses from Georgia had the same clustering pattern as the HA genes and the NAs of all differed from that of the A/Hong Kong/4801/2014 vaccine virus by 7 amino acid substitutions: S245N (resulting in the gain of a new glycosylation site), S247T (maintaining the glycosylation motif), I231V, T267K, I380V, T392I and P468H. The NAs of 12 viruses also carried the amino acid substitutions L140I, V143M, S315R and D339N.B/Yamagata lineageEleven (92%) of twelve viruses of the 2014–2015 season were successfully recovered. In HI analyses all but two viruses showed reduced recognition by antiserum raised against the egg-propagated B/Massachusetts/02/2012 vaccine virus (clade 2) used in the 2014–2015 season; however, all test viruses were recognized very well by antisera raised against egg-propagated viruses from clade 3 .Sequence analysis of eight viruses showed that HA and NA genes belonged to clade 3, the B/Wisconsin/1/2010 –B/Phuket/3073/2013 clade  Figs. AlB/Victoria lineageSeven (54%) of 13 viruses from the 2015–2016 season were recovered and analyzed by HI. Two viruses reacted well with post-infection ferret antiserum raised against egg-propagated B/Brisbane/60/2008, the recommended influenza B component in northern hemisphere quadrivalent vaccines. The other five were recognized poorly by the same antiserum, but were recognized well by antisera raised against two cell culture-propagated viruses  genetically closely related to, and serving as surrogates for, cell-propagated B/Brisbane/60/2008.Eleven (92%) of 12 viruses from the 2016–2017 season were recovered. All but two were poorly recognized by antiserum raised against the vaccine virus egg-propagated B/Brisbane/60/2008 but recognized well by antisera raised against cell culture-propagated reference viruses  at titers generally similar to the respective homologous titers of the antisera, all of which were low.HA and NA genes of the 18 viruses fell into clade 1A, the B/Brisbane/60/2008 vaccine virus clade, but carried amino acid substitutions compared to B/Brisbane/60/2008: I117V and N129D in HA1 and I120V, K220N, S295R, N340D, E358K and D384G in the NA. Nine of 11 viruses from the 2016–2017 season carried additional substitutions of I45M, T72A, P336S in the NA  Figs.We present an analysis of data obtained from ILI and SARI sentinel surveillance sites and virus characterization to provide an overview of three consecutive influenza seasons in Georgia. The majority of laboratory-confirmed influenza detections were seen from December through to March, but seasonal peaks occurred in different weeks and coincided with those observed in the European region –16. InflDuring a season approximately one third of both ILI and SARI cases sampled by sentinel sites in Georgia were associated with influenza virus infection. Increased ILI consultation rates at outpatient clinics correlated with raised numbers of laboratory-confirmed influenza infections. The same trend was observed for SARI hospital admissions: the proportion of SARI cases among total hospitalizations doubled during peak weeks of influenza activity compared to weeks with low or no influenza detections. The number of SARI hospitalizations was lowest in 2014–2015, an influenza B season, and highest in 2015–2016 when A/H1N1pdm09 viruses predominated.Children under 5 years of age were influenza virus-positive less frequently compared to older patients, reflecting the reports of a recent Egyptian study . In our Among ILI patients those aged 5–14 years were most often infected with influenza A/H3N2 or B viruses, while A/H1N1pdm09 infection was most commonly observed in individuals aged 30–64 years; similar observations have been described in Bulgaria . The proOf influenza-confirmed SARI cases in Georgia approximately 5% died while the percentage of fatal outcomes among hospitalized influenza-confirmed cases has been reported to vary from 0 to 4% in other countries , 23–25. Antigenic and genetic characterization of influenza viruses from Georgia mostly showed similarities to strains circulating worldwide. Since their emergence A/H1N1pdm09 viruses have evolved forming eight genetic groups . Viruseshttp://www.who.int/influenza/vaccines/virus/recommendations/en/). In 2014–2015, viruses from Georgia fell in genetic clades 3C.2a and 3C.3b while the majority of A/H3N2 viruses detected in the northern hemisphere belonged to clades 3C.2a and 3C.3a and the vaccine was reported to have low effectiveness [Over the period of this study A/H3N2 viruses evolved rapidly genetically and showed some antigenic change, antigenic drift, such that three vaccine viruses were recommended: A/Texas/50/2012-like (3C.1) for 2014–2015, A/Switzerland/9715293/2013-like (3C.3a) for 2015–2016 and A/Hong Kong/4801/2014-like (3C.2a) for 2016–2017 seasons respectively , as approximately 70% of the influenza B/Yamagata lineage viruses circulating in the European region , and difInfluenza B/Victoria lineage viruses circulating in Georgia during 2015–2016 and 2016–2017 seasons belonged to clade 1A, the B/Brisbane/60/2008 clade. Trivalent influenza vaccines for 2015–2016 contained a virus of the B/Yamagata lineage (a B/Phuket/3073/2013-like virus) while the vast majority of influenza B viruses detected in the European region were B/Victoria lineage . GeneticOur study had several limitations that could influence the results of analyses performed. False negative laboratory results may have been obtained due to the late referral of patients to clinics. Individual questionnaires were filled out during sampling and subsequent data regarding course of disease were not collected so we were unable to assess disease severity characteristics . For the same reason, we did not evaluate chronic conditions among non-fatal ILI/SARI cases to determine risk factors associated with any severe courses of disease.Despite limitations, this study contributes to a much better understanding of the epidemiological and virologic characteristics of influenza in Georgia. Influenza virus activity in the country was mainly observed from December through March each season with varying peak weeks and predominant viruses. On average one third of patients with ILI/SARI screened in each season had influenza virus infection. Among ILI cases influenza detection was highest in patients aged 5–14 years while the highest proportions of influenza-confirmed SARI cases were among adults. Persons aged 30–64 years had a higher risk of a fatal outcome. The observed circulation of antigenically and genetically variable viruses in Georgia and selection of A/Georgia/532/2015 as a reference virus by WHO CC, London illustrates the need for routine surveillance to contribute to the work of the WHO Global Influenza Surveillance and Response System.S1 FigSentinel site cities are marked in dark green.(TIF)Click here for additional data file.S2 FigVirus annotation is as for (TIF)Click here for additional data file.S3 FigVirus annotation is as for (TIF)Click here for additional data file.S4 FigVaccine viruses are indicated in red, viruses from Georgia in the 2014–2015 season are shown in blue. Reference and vaccine viruses against which post-infection ferret antisera were raised for use in HI assays are in bold type. The scale bar represents nucleotide substitutions per site.(TIF)Click here for additional data file.S5 FigVirus annotation is as for (TIF)Click here for additional data file.S6 FigVaccine virus is indicated in red; 2015–2016 and 2016–2017 viruses from Georgia in pink and brown respectively. Reference and vaccine viruses against which post-infection ferret antisera were raised for use in HI assays are in bold type. The scale bar represents nucleotide substitutions per site.(TIF)Click here for additional data file.S7 FigVirus annotation is as for (TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(TIF)Click here for additional data file.


A-adjuvanted A/turkey/Turkey/1/2005, A/Turkey), or heterologous  prime-boost vaccination regimens were analyzed by hemagglutination inhibition and microneutralization assays against 8 wild-type HPAI A(H5Nx) viruses from 6 genetic clades. Molecular, structural and antigenic features of the A(H5Nx) viruses that could influence the cross-clade antibody responses were also explored. Compared with homologous prime-boost vaccinations, priming with a clade 2.1.3.2 antigen (A/Indo) followed by one booster dose of a clade 2.2.1 antigen (A/Turkey) administered 18 months apart did not compromise the antibody responses to the booster vaccine (A/Turkey), it also broadened the cross-clade antibody responses to several antigenically drifted variants from 6 heterologous clades, including an antigenically distant A(H5N8) virus  that caused recent outbreaks in US poultry. The magnitude and breadth of the cross-clade antibody responses against emerging HPAI A(H5Nx) viruses are associated with genetic, structural and antigenic differences from the vaccine viruses and enhanced by the inclusion of an adjuvant. Heterologous prime-boost vaccination with AS03A adjuvanted vaccine offers a vaccination strategy to use existing stockpiled vaccines for pandemic preparedness against new emerging HPAI A(H5Nx) viruses.Highly pathogenic avian influenza (HPAI) A(H5Nx) viruses continue to pose a pandemic threat. US national vaccine stockpiles are a cornerstone of the influenza pandemic preparedness plans. However, continual genetic and antigenic divergence of A(H5Nx) viruses requires the development of effective vaccination strategies using stockpiled vaccines and adjuvants for pandemic preparedness. Human sera collected from healthy adults who received either homologous  vaccination regimen that induced a broader cross-clade response when compared with homologous vaccination. In the study, adults primed with a clade 2.1.3.2 antigen (A/Indo) followed by one booster dose of a clade 2.2.1 antigen (A/Turkey) presented with enhanced hemagglutinin inhibition and neutralizing antibody titers to eight A(H5Nx) viruses without limiting the antibody response to the A/Turkey booster vaccine. Given that no individual H5 clade has led to protection against all H5 viruses, heterologous vaccination strategies that provide cross-clade reactivity may lead to more effective protection against influenza virus infection. Since the identification of human infections with the highly pathogenic avian influenza (HPAI) A(H5N1) viruses over two decades ago, these viruses have become endemic in poultry and wild birds in many countries. They continue to cause sporadic human infections with high disease severity and high mortality . By December 2018, the World Health Organization (WHO) reported 860 cases of human infections with A(H5N1) viruses since 2003.9 In the United States, development of national vaccine stockpiles is a cornerstone of the pandemic preparedness plans.10 In addition, candidate vaccine viruses (CVVs) are developed to match emerging strains. However, rapid evolution of the A(H5Nx) viruses requires constant updating of such CVVs. In the face of a pandemic, the likelihood of antigenic mismatch between the pandemic strain and the existing stockpiled vaccine strains is fairly high. Even when the CVV to the pandemic strain becomes available, the existing vaccine manufacturing capacity may not be sufficient to provide enough quantities of matched vaccines rapidly to protect the entire population against the pandemic virus. Thus, improved influenza A(H5Nx) vaccination strategies utilizing existing stockpiled vaccines that can induce broader cross-reactive neutralizing antibody responses to emerging A(H5Nx) pandemic strains are highly desirable.Vaccination is a critical component of public health measures to protect the population against influenza infections. Previous studies have demonstrated that two doses of adjuvanted A(H5N1) vaccines administered as a prime-boost regimen are needed to elicit sufficient levels of antibody responses thought to confer protection.11 In the current study, we evaluated the magnitude and the breadth of the cross-clade antibody responses following either homologous or heterologous prime-boost vaccination in adults against wild type A(H5Nx) HPAI viruses from 6 genetic clades that caused either human diseases or animal outbreaks in the recent years. We chose antigens and adjuvants that are currently a part of the US National Pre-pandemic Influenza Vaccine Stockpile: homologous vaccination with 2 doses of a AS03A-adjuvanted clade 2.2.1 virus , versus heterologous vaccination primed with an AS03A-adjuvanted clade 2.1.3.2 virus  followed by a single booster dose with AS03A-adjuvanted A/Turkey, 18 months apart. We also explored the genetic, structural and antigenic characteristics of the A(H5Nx) viruses that may influence the levels of cross-clade antibody responses induced by the prime-boost vaccinations.Recently, we demonstrated that heterologous boost vaccination with MF59-adjuvanted clade 2.3.4 A(H5N1) virus following a non-adjuvanted clade 1 virus prime can elicit modest cross-clade neutralizing antibodies against newly emerged A(H5Nx) viruses from clade 2.3.4.4.12 had ≥128-fold two-way reduction by HI when compared to both vaccine viruses (Table HPAI AH5N1) and A(H5N8) viruses from genetic clades that caused human infections and animal outbreaks in recent years were selected to evaluate the breadth of the antibody responses following either homologous or heterologous prime-boost vaccination with A(H5N1) vaccines. A(H5Nx) viruses have diverged into multiple phylogenetic clades; 8 viruses from 6 genetic clades were included in the study  vaccines  influenza viruses before receiving A(H5N1) vaccination with baseline titers below the limit of detection to almost all 8 A(H5Nx) viruses . Following vaccination, participants in heterologous prime-boost groups (groups 2 & 3) that only received one dose of A/Turkey post A/Indo prime had higher HI and MN antibody titers to the vaccine virus A/Turkey than those in the homologous prime-boost group (group 1) that received 2 doses of A/Turkey Tables .Table 3Cp < 0.05, Fig. p > 0.05).Compared with the homologous prime-boost group (group 1), subjects in the heterologous prime-boost groups (groups 2 & 3) also mounted stronger and broader cross-clade HI and MN antibody responses to antigenically diverse variants. HI Table  and neutA-adjuvanted antigens are broadened against viruses from heterologous clades, with lower reductions in titers and higher GMT ratios when comparing responses to heterologous clade versus those to the vaccine viruses  achieved the best SPR rate, with lower limit of 95% CI of SPR ≥70% to 4 (by MN) and 3 (by HI) out of 8 viruses. The lowest antibody response was to the A(H5N8) virus from clade 2.3.4.4. It should be noted that subjects included in the current study were a subset that was randomly selected from strong responders to the vaccine components from the initial immunogenicity evaluation.13 As such, these SPR and SCR do not necessarily reflect the performance of the study groups from the original randomized trial. Regardless, these data still suggest that heterologous prime-boost strategy with AS03A-adjuvanted A(H5N1) A/Indo-A/Turkey vaccination, even administered at 18 months apart, may offer a broader range of cross-protection to drifted A(H5Nx) viruses.The US Food and Drug Administration (FDA) accelerated approval vaccine licensure criteria requires that based on HI titers, the lower limits of 95% CI for SPR are ≥70% and SCR are ≥40% for healthy adults.r) between HI and MN are r > 0.86 to 7 out of 8 viruses (p < 0.0001) and r > 0.58 for all 8 viruses (p < 0.0001)  wild-type viruses from 8 clades measured by HI correlated well with neutralizing antibody responses measured by MN to each of the 8 viruses. The spearman correlation co-efficient (p < 0.01) than the homologous group against A/Indo/12, a virus from clade 2.1.3.2. Sequences of A(H5Nx) viruses were structurally aligned, positions equivalent to A(H3N2) and A(H1N1) antigenic sites on the HAs were analyzed  and A/duck/Bangladesh/19097/2013 (2.3.2.1a) both have an additional glycosylation site at position 140 of the HA  virus (A/GF/14) has the lowest amino acid homology in antigenic sites compared to the vaccine viruses. It only has 64, 68% sequence identity compared to A/Turkey, and 61, 68% sequence identity compared to A/Indo in antigenic sites A and B, respectively  (Supplementary Tables 19 without compromising the antibody responses to the booster vaccine (A/Turkey).Identifying effective and efficient vaccination and stockpile strategies against a potential influenza pandemic is an essential component of the influenza pandemic preparedness. A prime-boost vaccination utilizing existing stockpiled vaccines in combination with newly developed vaccines containing a CVV antigenically well-matched with the pandemic virus may offer a strategy to broaden the protective efficacy of the vaccines and ease the surge in demand for vaccine doses in the event of a rapid onset of the pandemic. Pre-pandemic stockpiled vaccines can be used to prime the high-risk populations either at the early stage of a pandemic (pandemic scenario), or in advance (pre-pandemic scenario), followed by a boosting dose of vaccine that is well-matched with the pandemic strain when it becomes available. In addition, prior to the widespread availability of a well-matched pandemic vaccine, a prime-boost strategy solely based on the existing antigenically related stockpiled vaccines that offer partial protection against pandemic strains can also be of value. Here, we demonstrated that priming with a clade 2.1.3.2 antigen (A/Indo) followed by one booster dose of a clade 2.2.1 antigen (A/Turkey) broadened the cross-clade antibody responses to several antigenically drifted variants from 6 heterologous clades, including an antigenically distant A(H5N8) virus20 suggest that a heterologous prime-boost vaccination using A(H5N1) vaccine antigens with different antigenic properties may provide broader protection against novel strains by inducing more cross-reactive responses. In addition, the inclusion of oil-in-water emulsion-based adjuvants such as AS03A and MF59 may further broaden antibody responses.HPAI A(H5N1) viruses have exhibited increasing genetic and antigenic diversity since their re-emergence in 2003. No viruses from one single H5 clade can serve as a vaccine that provides sufficient protection against all H5 viruses from heterologous clades. To-date, it is still impossible to predict which H5 viruses may gain efficient and sustained human-to-human transmission and cause the next influenza pandemic. Results from the current study, together with others,21 Residues on the viral hemagglutinin protein that are around the receptor binding sites, key antigenic sites and those that alter glycosylation are more likely to have bigger impact on altering virus antigenicity.23 Furthermore, immune responses in humans following A(H5N1) vaccination can also be confounded by the use of potent oil-in-water adjuvants (for example stockpiled AS03A and MF59),26 multiple doses, and the complex immune exposure history to seasonal influenza viruses. Compared to antigenic characterization using post-infection ferret antisera, antibody responses in humans following A(H5N1) vaccination are broadened against viruses from heterologous clades, with less fold-reductions in titers and higher GMT ratios to antigenically drifted viruses versus those to the vaccine viruses, even among the current study participants that had no pre-vaccination titers to A(H5N1)  Fig. . Others A adjuvant elicited broad cross-clade antibody responses against multiple heterologous viruses. In subjects that were primed with an older antigen from clade 2.1.3.2 (A/Indo), a single booster dose with a clade 2.2.1 virus resulted in significantly higher antibody responses to a newer virus from the same priming clade (A/Indo/12) than those subjects that were not primed with A/Indo, suggesting a strong recall response to those shared epitopes between the older and newer clade 2.1.3.2 viruses in the heterologous prime-boost groups. Moreover, the addition of glycans on critical antigenic sites of the viruses may shield epitopes and alter antigenicity of the viruses. We hypothesize that an additional glycosylation site at position 140 in the antigenic site A of the two viruses from clade 2.3.2.1c and 2.3.2.1a may contribute to the moderate level of cross-reactivity detected to these two viruses following vaccination with A/Indo and A/Turkey, which both lack glycosylation site at this position. Likewise, a shared glycosylation site at position 154 of the HA on antigenic site B between A/Vietnam and A/Indo may affect immune responses to A/Vietnam following A/Indo priming. Additional studies are warranted to further identify structural epitopes that may influence the breadth of the antibody responses from A(H5N1) vaccination.In the current study, an A/Indo-A/Turkey prime-boost with AS0311 Other studies also suggested that compared with non-adjuvanted antigen priming, priming with AS03A-adjuvanted vaccines can promote stronger memory responses upon boosting with heterologous strains.27 The differences in the cross-clade response against the antigenically distant A(H5N8) virus are likely due to the difference in antigen and adjuvant prime-boost combinations used in these studies. It is also to be noted that even in the heterologous prime-boost groups, licensure criteria was only achieved for some but not all of the antigenically drifted A(H5Nx) viruses.Surprisingly, despite the large antigenic, genetic and structural differences between the A(H5N8) virus and the two vaccine viruses, 63–74% of subjects in the heterologous groups still achieved post vaccination titers ≥40 and seroconversion by MN to A/GF/14, a recently emerged clade 2.3.4.4 virus that caused multiple outbreaks in domestic poultry and wild birds in the U.S. More interestingly, the antibody responses detected against A(H5N8) virus with A/Indo-A/Turkey prime-boost in the current study are superior than those detected from another heterologous prime-boost regimen we evaluated in a similar age cohort in adults. Subjects in the previous study that were primed with unadjuvanted A/VN/1194/2004 (clade 1) and boosted by a MF-59 adjuvanted clade 2.3.4.4. virus (A/Anhui/1/2005) administered 21 months later mounted little neutralizing antibody responses to the same A(H5N8) virus.29 In a naïve population, two doses of A(H5N1) vaccination are needed to induce sufficient antibody responses to provide protection. Here, we demonstrated that in a primed cohort, a single booster dose administered 18 months after the original priming offered rapid (10 days post-boost) and broad coverage to a panel of genetically and antigenically diverse A(H5Nx) viruses, suggesting a strong recall of immune memory and quick expansion of the long-lived memory B cells and plasma cells capable of differientating into plasmablasts and secreting HI and neutralizing antibodies, even after 18 months post-priming.29 The cross-clade heterologous antibody responses were detected by both HI and neutralizing assays with good correlation in titers. Moreover, there is no significant difference between the high and low HA dose heterologous prime-boost groups, consistent with previous studies that demonstrated the role of AS03A on antigen sparing.9 Pandemics can occur rapidly, even in the matter of weeks after emergence.30 Our study only evaluated prime-boost schedule that is 12–18 months. In a pandemic setting, the relevance of heterologous prime-boost to broaden protection when the booster dose is administered at a short interval after the prime  remains to be understood. Nevertheless, the long term booster schedule demonstrated in the current study offers flexibility in pre-pandemic planning. The quick rise and broad responses following a single booster dose of vaccine are encouraging. Surprisingly, even the homologous prime-boost group induced cross-reactive antibody responses to several A(H5Nx) drift variants, this could be especially valuable for countries that do not hold stockpiles of vaccines against multiple A(H5Nx) viruses.A vaccination regimen that can induce long term immunity and provide protection to potential emerging pandemic strains is highly desirable for pandemic preparedness.Our study has several limitations, first, we could not include all A(H5Nx) viruses that caused recent human cases and outbreaks, as A(H5Nx) viruses continue to evolve, further studies may be needed when new antigenic variants emerge; second, our analysis is only focused on the strong responders from the immunogenicity evaluation of the original study, those that initially did not respond to the vaccine viruses would likely have little or no cross-reactive antibody responses to A(H5Nx) antigenic variants; Third, the two dose of homologous vaccination were given at 6 months interval, whereas the two doses of heterologous vaccination were given at 18 months interval, this difference in vaccination intervals may impact the comparisons.In conclusion, this study demonstrated that heterologous prime-boost with adjuvanted stockpiled A/Indo and A/Turkey antigens can induce broad and robust cross-clade antibody responses to recent emerging strains. Our results also suggest that the levels of cross-clade antibody responses against novel viruses vary, depending on the antigenic distance from the vaccine viruses. The use of potent adjuvants likely broadens the responses. The molecular, structural and antigenic determinants of A(H5Nx) viruses that can drive the breadth of the vaccine responses following a prime-boost strategy, and the mechanism of boosting of immune memory in humans against novel A(H5Nx) viruses, must be better understood in order to design the most effective vaccination strategies utilizing stockpiled vaccines against novel viruses that could cause an influenza pandemic.13 The original trial was a randomized, observer-blind, placebo controlled study including 7 study arms.13 Sera from subjects in three study arms  with homologous prime-boost (group 1), and heterologous prime-boost (group 2 & 3), were included in the current study.A(H5N1) vaccine sera were provided by GSK Vaccines. Stored sera were collected from healthy adults (≥18 years) enrolled in a GSK’s A(H5N1) vaccine trial .ms Table  who receA adjuvanted A/Turkey inactivated split virus antigen at 3.75 µg HA per dose (standard dose with AS03A adjuvant), administered 12 months apart. Subjects from the heterologous prime-boost groups (groups 2 and 3) were primed with AS03A-adjuvanted A/Indo inactivated split virus antigen at day 0, and boosted with AS03A-adjuvanted inactivated split virus antigen A/Turkey 18 months later. Subjects from the two heterologous prime-boost groups (A/Indo-A/Turkey) differed by the vaccine antigen doses received: they either received 7.5 µg  or 3.75 µg HA antigen per dose .All subjects received 3 intramuscular injections (2 vaccines and 1 placebo) during the course of 18 months (Table 13 Thirty-five subjects were randomly selected from strong responders for each of the 3 study groups that received either homologous or heterologous prime-boost A(H5N1) vaccination.The subjects evaluated in the current study are a subset of strong responders that achieved seroconversion by HI from the original immunogenicity evaluation of the clinical trial, which represents 42.9% of the study participants in the homologous group that received 2 doses of A/Turkey (group 1), 87.5% of the participants from the heterologous group that received A/Indo-A/Turkey at 7.5ug HA per dose (group 2), and 84.1% of the participants from the heterologous prime-boost group that received A/Indo-A/Turkey at 3.75 µg HA per dose of vaccines (group 3).Paired sera collected at day 0 (pre-vaccination), and day 559 (approximately 10 days post- last dose) were analyzed. All subjects included in the current study gave consent for the testing of stored sera. Written informed consent were obtained from all participants. The use of sera was approved by the Centers for Disease Control and Prevention, National Centers for Immunization and Respiratory Diseases Human Research Determination Review.A total of 8 wild-type HPAI A(H5Nx) viruses from 6 genetic clades were used in the study, including the wild-type version of the vaccine strains and a panel of HPAI A(H5Nx) viruses from 6 genetic clades: A/Turkey (clade 2.2.1), A/Indo (clade 2.1.3.2), A/Vietnam/1194/2004 , A/Indonesia/NIHRD-12379/2012 , A/Egypt/N04915/2014 (clade 2.2.1), A/duck/Bangladesh/19097/2013 (clade 2.3.2.1a), A/duck/Vietnam/NCVD-1584/2012 (clade 2.3.2.1c), and A/gyrfalcon/Washington/410886/2014 . Viruses were propagated in the allantoic cavity of embryonated hen’s eggs at 37 °C for approximately 24 h. All research with A(H5N1) and A(H5N8) HPAI viruses was conducted in Biosafety level 3 Enhanced facilities.Sera were tested by hemagglutination inhibition (HI) and microneutralization (MN) assays. All assays were conducted in Biosafety level 3 Enhanced (BSL3E) laboratories following select agent requirements defined by US Department of Agriculture and the Select Agent Program.11 Human sera were heat inactivated, tested for non-specific agglutinins and adsorbed with packed horse erythrocytes as needed. All sera were then treated with receptor destroying enzyme (RDE) at 37 °C for 18–20 h, followed by heat inactivation prior to HI assays. Sera were two-fold serially diluted and incubated with 4 hemagglutination units (4HAU)/25 µl of virus for 30 min. Horse erythrocytes were added to the wells and incubated with virus and sera mixture at room temperature. Hemagglutination was read after 60 min. HI titer was defined as the reciprocals of the last dilutions of serum that completely inhibited hemagglutination. Antibody titers less than 10  were reported as 5 for calculation purposes.A modified HI assay using horse erythrocytes was performed to measure HI antibody responses.31 Human sera were heat inactivated at 56 °C for 30 min. Two-fold serial diluted sera were incubated with 100 tissue culture infectious dose 50 (TCID50) of influenza viruses and incubated at 37 °C 5% CO2 for 1 h. The virus-sera mixture was used to infect 1.5 × 104/well Madin-Darby Canine Kidney (MDCK) cells, and incubated for 18–20 h at 37 °C with 5% CO2. After cold acetone fixation, the presence of influenza viral protein was quantified by an ELISA using influenza A nucleoprotein (NP) monoclonal antibodies. MN titers were defined as the reciprocal of the highest dilutions of serum that gave 50% neutralization. Antibody titers less than 10  were reported as 5 for calculation purposes.MN assays were performed to measure neutralizing antibody responses.32 For structural-based sequence alignment, HA surface residues were determined based on A/Vietnam/1203/2004 (pdb: 2fk0),33 virus A(H5Nx) sequences were compared against A/Turkey. HA structural models were generated using PyMOL .A(H5Nx) models were generated by homology modeling.t-tests. Sample random selection and statistical analysis were performed using SAS® 9.3 and GraphPad Prism 5.Geometric mean titers (GMTs) of at least 2 replicates were reported as final titers for HI or MN. For each study group, GMT and their 95% confidence intervals (CI) were determined. “GMT ratio” is calculated as the GMT to the test virus divided by the GMT to the vaccine virus post vaccination for each study group. Seroprotection Rate (SPR) is the proportion of the subjects (%) that achieved a post-vaccination titer of ≥40. Seroconversion is defined as a 4-fold or greater rise from pre-vaccination to post-vaccination titer with post-vaccination titers of ≥40. Seroconversion rate (SCR) is the proportion of subjects (%) that achieved seroconversion per study group. Statistical comparison was analyzed using ANOVA and paired two-sided Further information on experimental design is available in the Supplementary MaterialReporting Summary