Carica papaya L. is an important economic crop worldwide and is used as a model plant for sex-determination research. To study the different flower sex types, we screened sex-related genes using alternative splicing sequences (AS-seqs) from a transcriptome database of the three flower sex types, i.e., males, females, and hermaphrodites, established at 28 days before flowering using 15 bacterial artificial chromosomes (BACs) of C. papaya L. After screening, the cDNA regions of the three sex-related loci, including short vegetative phase-like (CpSVPL), the chromatin assembly factor 1 subunit A-like (CpCAF1AL), and the somatic embryogenesis receptor kinase (CpSERK), which contained eight sex-related single-nucleotide polymorphisms (SNPs) from the different sex types of C. papaya L., were genotyped using high-resolution melting (HRM). The three loci were examined regarding the profiles of the third whorl, as described below. CpSVPL, which had one SNP associated with the three sex genotypes, was highly expressed in the male and female sterile flowers  that lacked the fourth whorl structure. CpCAF1AL, which had three SNPs associated with the male genotype, was highly expressed in male and normal hermaphrodite flowers, and had no AS-seqs, whereas it exhibited low expression and an AS-seqs in intron 11 in abnormal hermaphrodite flowers. Conversely, carpellate flowers  showed low expression of CpSVPL and AS-seqs in introns 5, 6, and 7 of CpSERK, which contained four SNPs associated with the female genotype. Specifically, the CpSERK and CpCAF1AL loci exhibited no AS-seq expression in the third whorl of the male and normal hermaphrodite flowers, respectively, and variance in the AS-seq expression of all other types of flowers. Functional mapping of the third whorl of normal hermaphrodites indicated no AS-seq expression in CpSERK, low CpSVPL expression, and, for CpCAF1AL, high expression and no AS-seq expression on XYh-type chromosomes. Carica papaya L., 2n = 18) belongs to the family Caricaceae, which includes 35 species within six genera and is an extensively cultivated fruit in tropical and subtropical zones. It is a polygamous flowering plant with a small genome of 372 Mbp ), 1 μL of 10 mM dNTP, 1 μL of 10 mM forward primer, 1 μL of 10 mM reverse primer, 1 μL of Taq DNA polymerase (2 U/μL), 2 μL of DNA template, 5 μL of dimethyl sulfoxide (DMSO), and ddH2O up to a total volume of 50 μL. The PCR reaction mixture was pipetted into 0.2 mL microfuge tubes that were transferred to a PCR machine (BIOER-GenePro). The conditions of PCR–RFLP were as follows: 94°C for 3 min , followed by 35 cycles at 94°C for 40 s (for denaturation), 61°C for 40 s , and 72°C for 30 s (for elongation). The amplified PCR products were purified using a purification kit (Geneaid). We then examined the concentration of the PCR products. Four hundred nanograms of PCR product was reacted with restriction enzymes for the analyses. The PCR products of CpSVPL and CpSERK genes were digested with the Nde I and Spe I enzymes, respectively, using a suitable buffer at 37°C/8 h. Individual electrophoretic analyses of each state of the PCR product before and after the enzymatic reactions were performed, and UV images were acquired.The primers used for PCR–RFLP analysis were designed based on the sequences of the SNPs, and the product sizes were approximately 200 bp. Each tube comprised the following PCR constituents: 5 μL of 10× PC2 buffer . The PCR conditions were as follows: 95°C for 4 min, 35 cycles of 94°C for 40 s, 58°C for 40 s, 72°C for 20–120 s , a final extension at 72°C for 3 min, and a hold at 4°C. Five microliters of the RT–PCR product from each tube was used for individual electrophoretic analyses, and UV images were taken.We performed PCR assays to test the intron predictions for the 12 cDNA samples. The forward primer was designed for the sequences between two exons, and the reverse primer was designed for the sequences of the next exon. The PCR reaction mixture in each tube included the following constituents: 2.5 μL of 10× Taq buffer, 0.5 μL of 10 mM dNTP, 0.5 μL of 10 mM forward primer, 0.5 μL of 10 mM reverse primer, 0.5 μL of Taq DNA polymerase (2 U/μL), and 3 μL of cDNA template (80 ng/μL). The total volume of the reaction mixture was made up to 25 μL with ddH2O. The mixtures were transferred to 96-well plates, which were then sealed with a sealing membrane. The plates were transferred to a qPCR machine (Bio-Rad CFX Connect) that was operated using a qPCR software (Bio-Rad CFX Manager). The qRT–PCR conditions were as follows: 95°C for 10 min, followed by 39 cycles of 95°C for 15 s (for denaturation) and 60°C for 1 min . The conditions for the melting curve analysis were 95°C for 15 s, followed by 60°C for 30 s (for denaturation). The detection range was set from 65°C to 95°C, and the incremental speed was set to 0.5°C/10 s. Each tube of qRT–PCR product was subjected to individual electrophoretic analysis and UV images were taken.A qRT–PCR analysis was performed for each gene junction in the 12 cDNA samples  Tables. C. papaya L. plants .Regarding the results of the melting curves based on the SNP-HRM analysis, the primers  for the AL locus . The resanalysis . Regardi primers  Figs. NoCpSVPL gene in the fourth whorl of female flowers could be used to detect correctly the lengths of the fragments. Tests of introns 1 to 6 of the CpSVPL gene could be used to detect correctly the lengths of the fragments in the other sex types and abnormal hermaphrodite flowers, and there was no occurrence of AS. Therefore, we could distinguish female flowers from other-sex-type flowers based on this difference. Furthermore, the expression level of the CpSVPL gene was clearly elevated in the third whorl of both male and female sterile flowers. Thus, we could distinguish male and female sterile flowers from other-sex-type flowers based on this difference. There were occurrences of AS-seq polymorphism in introns 5, 6, 7, and 11 of the CpSERK and CpCAF1AL genes. In addition, polymorphism occurred in the fourth whorl of female and hermaphrodite flowers and the third whorl of abnormal hermaphrodite flowers. The expression level of the CpCAF1AL gene was clearly elevated in the third whorl of both male and normal hermaphrodite flowers. Therefore, we could distinguish between the third whorl of normal and abnormal hermaphrodite flowers by identifying the differences in AS-seq and in the expression level of the CpCAF1AL gene  with XYh-type SNPs; and no variance/AS-seq, low/no AS-seq, and low/AS-seq expression for carpellate flowers  with XYh-type SNPs  are extremely methylated and heterochromatized relative to their corresponding regions on the X chromosome [Papaya is a trioecious species with three sex types, i.e., female, male, and hermaphrodite. It is considered as an outstanding model system to study sex determination in plants. Because of recombination suppression, highly diverged sequences are present between the X and Y or Yelopment . MSY andromosome . These sromosome .C. papaya L. plants. We propose that the related genes in the fourth whorl of pistils from the male plants of C. papaya L. were inhibited. Our results indicate that the expression level of the CpSVPL gene was high in the third whorl of male flowers and in the third whorl of female sterile flowers; however, the expression level of the CpSVPL gene was relatively low in the third and fourth whorls of the flowers that contained the fourth whorl structure  [CpSVPL gene were comparable to those of earlier reports by Urasaki et al. (2012) and Ueno H et al. (2015) regarding sex determination in papaya. A genome comparison study of male and hermaphrodite differentiation showed that CpSVPL is a Y- and Yh-chromosome-specific gene, as this allele is not present in the X chromosome [h chromosomes carry different alleles of the SVPL gene; however, only the allele located in the Y chromosome encodes an intact protein with both the MADS-box and K-box domains. We propose that the gene expression level and mRNA sequence of the CpSVPL locus may differ substantially between the female and other sex types. These results suggest that amplification of the X chromosome occurred during its evolution in C. papaya L. [In the present study, in addition to the occurrence of abnormal hermaphrodite flowers, we also found the occurrence of hermaphrodite flowers that developed at the ends of the inflorescences of male tructure . Based o (>30°C) . The papet al. 20 regardinromosome . The Y aapaya L. . This amapaya L. . The NGSCpSVPL, CpSERK, and CpCAF1AL genes in C. papaya L. These SNPs include SNPs in intron 3  in this study; in contrast, the occurrence of AS was not observed in the third whorl of the male flowers (XYm) and normal hermaphrodite flowers (XYh). The same specific bands were observed in both sex types (Drosophila melanogaster), a link between AS-seqs and sex type was identified [A. thaliana [AtSERK1 was noticed in closed flower buds before fertilization and in flowers with embryos at stages 1 through 7 after pollination [Gossypium hirsutum, GhSERK1 expression was associated with the development of anthers [GhSERK1 via RNAi studies revealed the presence of various levels of male sterility; higher levels of male sterility were observed in cases with lower expression levels of the GhSERK1 mRNA [Zea mays, ZmSERK1 was expressed in male and female reproductive organs, but showed higher expression in microspores [Glycine max by comparing the SNPs between wild and cultivated soybeans using genome-wide evolutionary analyses, which provided valuable tools for the identification of the functional divergence of this gene family [Occurrences of AS were discovered in the ex types . In a huentified . Differethaliana . A highelination . In Goss anthers . AnalysiRK1 mRNA . In Zea rospores . Recentle family .CAF1A gene may be related with X-chromosome inactivation and methylation of AS-seqs and DNA [C. papaya L. showed that CAF1 is one of the uniquely expressed candidate genes with sequence polymorphism located in the MSY or HSY that are responsible for male or hermaphrodite determination [A. thaliana, the CAF1 pathway inactivity interrupts and arrests the cell cycle during pollen development [CpCAF1AL gene in the third whorl.CAF1 plays a role in the assembly of newly synthesized DNA for the formation of mature nucleosomes. CAF1 dysfunction causes a reduction in the heterochromatic fraction. The  and DNA , 47. A gmination . In A. telopment . In accom, and Yh chromosomes. Hypothetically, at the CpCAF1AL locus, the pattern of AS-seq and low expression by the XX type may be replaced with the patterns of no AS-seq and high and stable expression by the XYm type and high and unstable expression by the XYh type, which may be related to the abnormal hermaphrodite flowers observed in the third whorl. Therefore, we suggest that the character of stable and normal hermaphrodite requires the CpCAF1AL locus with the XYm type  are difficult to study because of their intricate positioning, thus rendering the determination of males and hermaphrodites a difficult and complex process. Despite these issues, we performed a functional mapping of the three sex-related loci in different sex types and in the abnormal hermaphrodite flowers of C. papaya L. We also tried to perform functional mapping on the third whorl of the normal hermaphrodite flowers; however, there were no AS-seqs of CpSERK, the expression level of CpSVPL was low, and CpCAF1AL was expressed at high levels and lacked AS-seqs. The linkage maps of the stable normal hermaphrodite flowers, which were based on the sex-related SNP combinations, were also suggested Click here for additional data file.S1 Figh chromosome of BAC-49L11; (B) the X chromosome of BAC-50J21; (C) the Yh chromosome of BAC-50M09; (D) the Yh chromosome of BAC-53G04; (E) the Y chromosome of BAC-57M14; (F) the Yh chromosome of BAC-62H24; (G) the Yh chromosome of BAC-65D15; (H) the Yh chromosome of BAC-71E16; (I) the Yh chromosome of BAC-72J22; (J) the Yh chromosome of BAC-81O12; (K) the Yh chromosome of BAC-PH85B24; (L) the Yh chromosome of BAC-90D06; (M) the Y chromosome of BAC-PH94E22; (N) the Yh chromosome of BAC-PH95B12; (O) and the Yh chromosome of BAC-96A24. The 15 reference BACs were used to estimate the locations, and the functional proteins are represented on the left side and the right side for each individual linkage. Following each protein function, the “>” sign links with the ID number in the database. The reference BACs represent the three sex type linkage populations. F: females; M: males; H: hermaphrodites; underlined and bold information indicates that the same functional protein exists in the three sex types; bold information indicates the same functional protein exists in two sex types.(A) the Y(TIF)Click here for additional data file.S2 FigCpSVPL coding region; (B) Nucleotide sequence comparison of the CpSVPL cDNA of the three sex types.(A) AA-seq comparison of the (TIF)Click here for additional data file.S3 FigCpSERK coding region; (B) Nucleotide sequence comparison of the CpSERK cDNA of the three sex types.(A) AA-seq comparison of the (TIF)Click here for additional data file.S4 FigCpCAF1AL coding region; (B) Nucleotide sequence comparison of the CpCAF1AL cDNA of the three sex types.(A) AA-seq comparison of the (TIF)Click here for additional data file.S5 FigHRM curve assay results using the primers (A) CpSVPL_HRM_88; (B) CpSERK_HRM_30704; (C) CpSERK_HRM_34072; (D) CpSERK_HRM_34760; (E) CpSERK_HRM_34787; (F) CpCAF1AL_HRM_01; (G) CpCAF1AL_HRM_02; (H) CpCAF1AL_HRM_03; and (I) CpCAF1AL_HRM_04. F: females; M: males; H: hermaphrodites.(TIF)Click here for additional data file.S6 FigThe melting peak of HRM analysis of (A) CpSVPL_HRM_88; (B) CpSERK_HRM_30704; (C) CpSERK_HRM_34072; (D) CpSERK_HRM_34787; (E) CpSERK_HRM_34787; (F) CpCAF1AL_HRM_01; (G) CpCAF1AL_HRM_02; and (H) CpCAF1AL_HRM_04 for collective samples. Sex sample tests included F: females, 5 samples; M: males, 3 samples; H: hermaphrodites, 4 samples. Tests also included blind samples. f: female melting peak; m: male melting peak e; h: hermaphrodite melting peak; mh: not female melting peak; fh: not male melting peak.(TIF)Click here for additional data file.S7 FigThe analytical results of (A) CpSVPL_RFLP_Nde I; (B) CpSERK_RFLP_Spe I. M: marker (shown on the left side); H: hermaphrodites; FM: males of the Florida variety. The PCR products were digested using the restriction enzyme NdeI (shown behind the underline); the samples marked without an underline represent PCR products that were not subjected to restriction enzyme digestion; SF: females of Florida Variety; FM: males of the Florida variety; SH: hermaphrodites of Sunrise Solo. The PCR products were digested using the restriction enzyme Spe I (shown behind the underline).(TIF)Click here for additional data file.S8 FigCpSVPL; (B) CpSERK; (C) CpCAF1AL genes. ‘●’: start codon; ‘▲’: stop codon; black thin line: intron of gene; I#: number of introns; gray thick line: exon of gene; E#: number of exons; underline: primer name.(A) (TIF)Click here for additional data file.S9 Fig(A) Intron 1; (B) Intron 2; (C) Intron 3; (D) Intron 4; (E) Intron 5; (F) Intron 6 test. Arrows indicate the RT-PCR product sizes (in bp) from cDNA after normal splicing. M: marker; 1: 28 days before flowering female buds; 2: 28 days before flowering male buds; 3: 28 days before flowering normal hermaphrodite buds; 4: 28 female degradation hermaphrodite buds; 5: 28 days before flowering carpellody hermaphrodite buds; 6: female fourth whorl; 7: male third whorl; 8: normal hermaphrodite fourth whorl; 9: normal hermaphrodite third whorl; 10: female degradation hermaphrodite third whorl; 11: carpellody hermaphrodite fourth whorl; 12: carpellody hermaphrodite third whorl. B: blank.(TIF)Click here for additional data file.S10 Fig(A) Intron 1; (B) Intron 2; (C) Intron 3; (D) Intron 4; (E) Intron 5; (F) Intron 6; (G) Intron 7; (H) Intron 8; (I) Intron 9; (J) Intron 10 test. Arrows indicate the RT-PCR product sizes (in bp) from cDNA after normal splicing. M: marker; 1: 28 days before flowering female buds; 2: 28 days before flowering male buds; 3: 28 days before flowering normal hermaphrodites buds; 4: 28days before flowering female degradation hermaphrodite buds; 5: 28 days before flowering carpellody hermaphrodite buds; 6: female fourth whorl; 7: male third whorl; 8: normal hermaphrodite fourth whorl; 9: normal hermaphrodite third whorl; 10: female degradation hermaphrodite third whorl; 11: carpellody hermaphrodite fourth whorl; 12: carpellody hermaphrodite third whorl. B: blank.(TIF)Click here for additional data file.S11 Fig(A) Intron 1; (B) Intron 2; (C) Intron 3; (D) Intron 4; (E) Intron 5; (F) Intron 6; (G) Intron 7; (H) Intron 8; (I) Intron 9; (J) Intron 10; (K) Intron 11 test. Arrows indicate the RT-PCR product sizes (in bp) from cDNA after normal splicing. M: marker; 1: 28 days before flowering female buds; 2: 28 days before flowering male buds; 3: 28 days before flowering normal hermaphrodite buds; 4: 28 days before flowering female degradation hermaphrodite buds; 5: 28 days before flowering carpellody hermaphrodite buds; 6: female fourth whorl; 7: male third whorl; 8: normal hermaphrodite fourth whorl; 9: normal hermaphrodite third whorl; 10: female degradation hermaphrodite third whorl; 11: carpellody hermaphrodite fourth whorl; 12: carpellody hermaphrodite third whorl. B: blank.(TIF)Click here for additional data file.S12 Fig(A) Junction 1; (B) Junction 2; (C) Junction 3; (D) Junction 4; (E) Junction 5; (F) Junction 6. Arrows indicate the Q-PCR product sizes (in bp). M: marker; 1: 28 days before flowering female buds; 2: 28 days before flowering male buds; 3: 28 days before flowering normal hermaphrodite buds; 4: 28 days before flowering female degradation hermaphrodite buds; 5: 28 days before flowering carpellody hermaphrodite buds; 6: female fourth whorl; 7: male third whorl; 8: normal hermaphrodite fourth whorl; 9: normal hermaphrodite third whorl; 10: female degradation hermaphrodite third whorl; 11: carpellody hermaphrodite fourth whorl; 12: carpellody hermaphrodite third whorl.(TIF)Click here for additional data file.S13 Fig(A) Junction 1; (B) Junction 2; (C) Junction 3; (D) Junction 4; (E) Junction 5; (F) Junction 6; (G) Junction 7; (H) Junction 8; (I) Junction 9; (J) Junction 10. Arrows indicate the Q-PCR product sizes (in bp). M: marker; 1: 28 days before flowering female buds; 2: 28 days before flowering male buds; 3: 28 days before flowering normal hermaphrodite buds; 4: 28 days before flowering female degradation hermaphrodite buds; 5: 28 days before flowering carpellody hermaphrodite buds; 6: female fourth whorl; 7: male third whorl; 8: normal hermaphrodite fourth whorl; 9: normal hermaphrodite third whorl; 10: female degradation hermaphrodite third whorl; 11: carpellody hermaphrodite fourth whorl; 12: carpellody hermaphrodite third whorl.(TIF)Click here for additional data file.S14 Fig(A) Junction 1; (B) Junction 2; (C) Junction 3; (D) Junction 4; (E) Junction 5; (F) Junction 6; (G) Junction 7; (H) Junction 8; (I) Junction 9; (J) Junction 10; (K) Junction 11. Arrows indicate the Q-PCR product sizes (in bp). M: marker; 1: 28 days before flowering female buds; 2: 28 days before flowering male buds; 3: 28 days before flowering normal hermaphrodite buds; 4: 28 days before flowering female degradation hermaphrodite buds; 5: 28 days before flowering carpellody hermaphrodite buds; 6: female fourth whorl; 7: male third whorl; 8: normal hermaphrodite fourth whorl; 9: normal hermaphrodite third whorl; 10: female degradation hermaphrodite third whorl; 11: carpellody hermaphrodite fourth whorl; 12: carpellody hermaphrodite third whorl.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Tabletype_1: the genes are not expressed in one of the three sex types, and there are differences in their AA-seqs between those of the other two sex types; type_2: the genes are expressed in all three sex types, and there are differences in their AA-seqs; type_3: the genes are expressed in all three sex types, and there is no difference in AA-seqs between two sex types.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table28 days before flowering: different sex types at 28 days before flowering; 0 days before flowering: different sex types at 0 days before flowering; ND: not determined; -: no difference; x: no primer design; F: different female; M: different male; H: different hermaphrodite; HM3: different female degradation hermaphrodite; 4: fourth whorl of flower; 3: third whorl of flower.(DOCX)Click here for additional data file.S7 TableCpSVPL gene.Gray area: lowest expression junction of the (DOCX)Click here for additional data file.S8 TableCpSERK gene.Gray area: lowest expression junction of the (DOCX)Click here for additional data file.S9 TableCpCAF1AL gene.Gray area: lowest expression junction of the (DOCX)Click here for additional data file.


The emergence of H5, H7, and H9 avian influenza virus subtypes in humans reveals their pandemic potential. Although human-to-human transmission has been limited, the genetic reassortment of the avian and human/porcine influenza viruses or mutations in some of the genes resulting in virus replication in the upper respiratory tract of humans could generate novel pandemic influenza viruses. Current vaccines do not provide cross protection against antigenically distinct strains of the H5, H7, and H9 influenza viruses. Therefore, newer vaccine approaches are needed to overcome these potential threats. We developed an egg-independent, adenovirus vector-based, multi-epitope (ME) vaccine approach using the relatively conserved immunogenic domains of the H5N1 influenza virus . Our ME vaccine induced humoral and cell-mediated immune responses and caused a significant reduction in the viral loads in the lungs of vaccinated mice that were challenged with antigenically distinct H5, H7, or H9 avian influenza viruses. These results suggest that our ME vaccine approach provided broad protection against the avian influenza viruses. Further improvement of this vaccine will lead to a pre-pandemic vaccine that may lower morbidity, hinder transmission, and prevent mortality in a pandemic situation before a strain-matched vaccine becomes available. Human infections caused by H5, H7, and H9 avian influenza virus subtypes provide evidence of these strains’ pandemic potential , 2. The In 2013, a new H7N9 avian influenza virus strain emerged in China  and has For pandemic preparedness purposes, H5N1, H7N7, and H9N2 virus vaccines have been developed and clinically evaluated. However, the inactivated or subunit H5 vaccines were found to be low immunogenic, which required the administration of an adjuvant, higher HA doses, or the administration of multiple doses in preclinical and clinical trials –18. FurtAdenovirus (Ad) vector-based vaccines induce excellent humoral and cell-mediated immune (CMI) responses , 23 due The M2 ectodomain (M2e) –35, HA f293 (human embryonic kidney cells that express E1 gene products of human Ad-C5), 293Cre (293 cell line that expresses Cre recombinase), MDCK (Madin-Darby canine kidney), and BHH2C (bovine-human hybrid clone 2C)  cell linThe following low pathogenic influenza A viruses were used in this study: A/chukar/MN/14951-7/1998(H5N2), A/goose/Nebraska/17097/2011(H7N9), and A/Hong Kong/1073/1999(H9N2). These influenza viruses were grown in embryonated hen eggs and were titrated in eggs, MDCK, or both. These viruses will not cause significant morbidity or mortality in mice since they were not mouse-adapted. Therefore, the lung virus titers following challenge will be the best measurable parameter to evaluate the vaccine’s efficacy.Human Ad vectors were generated in 293Cre cells using Cre-recombinase site-specific recombination . The conFor the ME vaccine, the following M2e, HFD, TNP, and HαD domains of H5N1 (A/Vietnam/1203/2004) were separated by the AlaAlaAla, GlyGlyGly and GlyProGlyProGly linkers, respectively:M2e: MSLLTEVETPTRNEWECRCSDSSD;HFD: GLFGAIAGFIEGGW;TNP: TYQRTRALVR;HαD: RIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNA.The H5N1 ME (H5ME) gene cassette was synthesized commercially. The construct structures and their names are shown in 8 PFU of each of Ad vectors as follows: Ad-H5HA, Ad-H7HA, Ad-H9HA, Ad-H5ME, or empty vector Ad-ΔE1E3. The immunization was administered twice within a three-week interval. The animals were anesthetized with ketamine-xylazine (90 mg/kg ketamine and 10 mg/kg xylazine) via intraperitoneal injections three weeks after the second immunization. Blood samples were collected via retro-orbital puncture to evaluate the humoral immune responses, and the spleens were collected upon euthanasia to evaluate the cell-mediated immune responses. The mice under anesthesia were euthanized using cervical dislocation and the thorax was perforated to ensure death. These procedures were in accordance with the recommendations of the American Veterinary Medical Association’s Panel on Euthanasia.All animal experiments were conducted in a USDA-approved BSL-2+ facility at Purdue University with the approval and in accordance with the guidelines of the Institutional Biosafety Committee and Institutional Animal Care and Use Committee. Six-to-eight-week-old female BALB/c mice  were used to evaluate the breadth of immune responses and protection efficacy. Immunization and challenge experiments were conducted in two phases. During the first phase, five groups of mice  were immunized via intramuscular inoculation of 1050) of A/chukar/MN/14951-7/1998(H5N2), A/goose/Nebraska/17097/2011(H7N9) or A/Hong Kong/1073/1999(H9N2). Three days after the challenge, the animals were euthanized under anesthesia with ketamine-xylazine (90 mg/kg ketamine and 10 mg/kg xylazine) via intraperitoneal injections, and the lungs were collected and then homogenized in 1 ml of sterile phosphate-buffered saline (PBS). A ten-fold serial dilution of the lung homogenates was used to infect either MDCK cells in 96-well plates  or specific-pathogen free (SPF) embryonated hen eggs . Following 72 h incubation at 37°C, the infected-cell supernatants or allantoic fluid samples were tested by hemagglutination assay (HA) using 0.5% turkey red blood cells (TRBC) to determine a 50% tissue culture infectious dose (TCID50) [for H5N2 and H9N2 virus-challenged groups] or 50% egg infectious dose (EID50) [for H7N9 virus-challenged groups] using the Reed-Muench method  and incubated at 37°C for 20 h. Thereafter the samples were inactivated by incubation at 56°C for 30 min. Two-fold dilutions of RDE-treated sera were conducted in 96-well plates first with a 1:10 dilution and followed by four hemagglutination units (HAU) of each virus diluted in PBS. Following incubation at room temperature for 1 h, 0.5% TRBC was added and the plates were incubated at room temperature for 20 min to allow hemagglutination to occur. The hemagglutination inhibition (HI) antibody titer was determined as the reciprocal of the highest dilution that completely prevented TRBC from agglutination, and the data are presented as the geometric mean HI titers.50 or 100 EID50 of an influenza virus, and the mixture was incubated for 1 h at 37°C. The mixture was then added to MDCK cells in 96-well plates or injected into the allantoic fluid of embryonated hen eggs and incubated for 72 h at 37°C. Thereafter, the HA activities of the cell supernatants or allantoic fluid samples were detected by HA assay with 0.5% TRBC. The virus neutralization (VN) titer was determined as the reciprocal of the highest dilution that completely neutralized the virus, and the data are presented as the geometric mean VN titers.The assays were performed as previously described . Two-folELISA was performed as described earlier , 55. TheThe assay was performed as previously described . The spl6 splenocytes (in RPMI-1640 medium with supplements) were incubated with either HA518 (IYSTVASSL) or NP147 (TYQRTRALV) peptides (H-2Kd-restricted cytotoxic T lymphocyte (CTL) epitopes for HA and NP, respectively) for 60 h at 37°C in a CO2 incubator. As a positive control, the splenocytes were cultured in the presence of phorbol myristate acetate/ionomycin . The cells were removed by washing, and a biotinylated rat anti-mouse antibody specific for INFγ  was added. The plates were incubated overnight at 4°C. Following the addition of streptavidin-AP  and washing, a chromogen mixture of BCIP/NBT  was added for color development. The reaction was stopped by rinsing the plates with deionized water. The ELISpot plates were read using a Bioreader 5000  to count the number of spot-forming units (SFU).PVDF-backed 96-well microplates  were coated overnight at 4°C with a monoclonal antibody for mouse interferon-gamma (INFγ) . 10p<0.05.For statistical significance determination, one- and two-way ANOVA tests were conducted with Dunnett's post-hoc analysis at a significance level of A Cre-recombinase site-specific recombination system was used to insert a coding sequence of the ME containing M2e, HFD, TNP, and HαD of H5N1 (H5ME) into the E1 region of Ad-C5 to generate Ad-H5ME . The recThe mice were inoculated i.m. with various Ad vectors, as described in the Material and Methods section. Three weeks following the booster inoculation, serum samples were collected and assayed for M2e-, HαD-, or HFD-specific IgG by ELISA . In thisMice vaccinated with Ad vector expressing H5ME (Ad-H5ME) showed high levels of M2e-specific IgG . For HαDThe serum samples from immunized mice were also analyzed for HI titers to determine the presence of HA-specific antibodies against heterologous H5 (H5N2), H7 (H7N9), and H9 (H9N2) avian influenza viruses, as we have previously elucidated that Ad vectors elicit enhanced HI titers against homologous avian influenza viruses . HI titeThe serum samples from various inoculated groups were also analyzed for neutralizing antibodies against the H5N2, H7N9, and H9N2 influenza viruses. Since the influenza viruses used for microneutralization assays have antigenically distinct HAs, lower levels of virus neutralizing antibody titers were anticipated depending on the differences between the H5, H7, and H9 HA sequences in the Ad vectors, and the HA sequences of the H5N2, H7N9, and H9N2 influenza viruses used for microneutralization assays. Mice vaccinated with Ad vectors containing the HA construct, Ad-H5HA, Ad-H7HA, or Ad-H9HA developed 246, 780, and 31 geometric mean VN titers against H5N2, H7N9, and H9N2 influenza viruses, respectively . Noticeain vitro stimulation with HA518 or NP147 peptide, respectively, using an IFNγ-specific ELISpot assay. Due to the fact that HA518 epitope, a CD8 T-cell epitope, is conserved in H5HA and H9HA, but not in H7HA, the animal groups that received Ad-H5HA or Ad-H9HA showed higher numbers of INFγ-secreting HA518-specific CD8 T cells compared to the empty vector (Ad-ΔE1E3) control group  was recognized by the host immune system as evident with significant levels of M2e-, HFD-, and HαD-specific antibody responses as well as the T-cell response to the TNP epitope. As expected, Ad-H5ME did not induce H5N2-specific HI or VN antibodies in immunized animals. However, following challenges with H5N2, H7N9, or H9N2, there was approximately 1.5, 1.2, and 1.8 log reductions in lung virus titers, respectively, which is indicative of cross-protection. The levels of cross-reactive humoral immune responses could vary significantly depending on the HA subtype antigenic group (H5 and H9 in Group 1 and H7 in Group 2) . The lowThe N terminal fusion domain of HA2 (HFD) is considered to be conserved among different influenza subtypes . HFD wasIn the present study, the immunization of mice with Ad-based ME vaccine failed to induce strong virus-neutralizing antibody responses. The Ad-H5ME vaccine failed to induce sterilizing immunity against high dose virus challenges, but our vaccine significantly reduced the virus replication in the lungs of mice challenged with antigenically distinct H5N2, H7N9, and H9N2 influenza viruses. The presence of heterosubtypic protection in the absence of strong neutralizing antibodies indicates the role of other players in providing cross-protection, including CMI responses and non-neutralizing humoral responses against M2e, HFD, and HαD. These findings are consistent with our previous results where the vaccination of mice with Ad-based vaccines expressing H1 HA induced high levels of broadly reactive, stalk-specific antibodies and reduced the lung viral titers of H5N1, H7N2, and H9N2 after challenges . SimilarIt is known that HA518-specific CTL are induced in BALB/c mice following influenza virus infection, but these responses are less pronounced than NP147-specific CTL responses . The rol8 PFU of an Ad vector expressing only the TNP resulted in approximately 0.4 log decreases in virus lung titers following challenge with an influenza virus compared to the animal group inoculated with the empty Ad vector [Ad-ΔE1E3]. These results suggest that in our ME vaccine study, humoral immune responses against M2e, HFD, and HαD domains contributed to approximately 1.1, 0.8, and 1.4 log reductions in lung virus titers against the H5, H7, and H9 influenza viruses, respectively. Additional studies will be required to evaluate the roles of individual epitopes in protection against the H5, H7, and H9 influenza viruses.NP is relatively conserved and represents a target for CTL-mediated immune responses critical to virus clearance and recovery after infection . Hence, Pre-existing vector immunity is considered to impact vector uptake and to reduce the level and duration of transgene expression. We previously demonstrated that moderate levels of pre-existing vector immunity  did not significantly hamper the protective efficacy of an Ad vector-based influenza vaccine in a mouse model . WhereasFuture developments toward a universal influenza vaccine may include relatively conserved B cell domains comprised of HA2 and M2e, and conserved T cell domains of NP. Further studies will be required to evaluate the best strategy to allow the improved presentation and recognition of the epitopes on HA2 and M2e for the development of strong, cross-protective humoral immune responses. Additional experiments are necessary to determine an approach for the vaccine formulation containing NP, HA2, and M2e, which would result in balanced humoral and CMI responses as well as enhanced cross-protection against different influenza subtypes.


ARHGAP22 rs4838605 is associated with T2D risk. Upon analyzing the subjects’ clinical characteristics, we found that for rs2811893, the TT genotype correlated with high creatinine levels, while the AA genotype of rs17045754 and the TT genotype of rs4838605 correlated with elevated triglyceride levels. In addition, the AA genotype of rs17376456 and the TT genotype of rs6214 (p = 0.006) correlated with elevated hemoglobin A1c levels. Lastly, those carrying the TT genotype of rs7772697 and the CA genotype of rs3918227 exhibited higher mean body mass index and Cystatin C than controls. Our results showing that the ARHGAP22 gene is associated with an increased risk of T2D, and that seven SNPs in MYSM1, PLXDC2, ARHGAP22 and HS6ST3 promote T2D progression and could help predict the clinical course of T2D in patients at risk.We investigated the correlation between type 2 diabetes (T2D)-related genes and the clinical characteristics of T2D in the Chinese Han population. Our study included 319 patients and 387 controls. Age, gender, clinical features, medications intake and biochemical blood profiles were analyzed. Genotyping was performed on a total of 18 single nucleotide polymorphisms previously reported to be associated with T2D. Our analyses revealed that the CT genotype of  Alterhttp://diabetes.diabetesjournals.org). Genetic susceptibility to T2D risk is multifactorial. In the past few years, many additional T2D risk loci have been identified including TCF7L2, FTO, HHEX, SLC30A8, HMG20A, IGF2BP2, MYSM1, TNFSF4, MYT1L, KIAA0825, VEGFA, PLXDC2, CREB5, NOS3, ARHGAP22, IGF1 [Previous studies have shown that T2D risk is largely determined by lifestyle habits including active smoking , quality22, IGF1 –21. HoweEarly T2D diagnosis can reduce long-term complications, such as diabetic retinopathy, kidney failure, cardiovascular disease and limb amputation –25. Unfop = 0.021), averaging 58.89 ± 12.01 and 57.05 ± 8.25 years, respectively.The demographic characteristics of the participants are presented in Table p > 0.05. However, we identified no significant association between SNPs and T2D risk under the allelic model , and Hardy–Weinberg Equilibrium (HWE) test, appears in Table MYSM1, KIAA0825, NOS3, ARHGAP2, and IGF1 were listed in Table p = 0.006), compared with the TC+CC genotype. The AA genotype of rs17045754 and the TT genotype of rs4838605 exhibited higher levels of triglycerides (TG) . Moreover, The AA genotype of rs17376456 and the TT genotype of rs6214 are associated with high levels of hemoglobin A1C (HbA1C) . We also found that the TT genotype of rs7772697 and the CA genotype of rs3918227 had a higher mean BMI  and Cystatin C (Cys-c) , respectively , fasting glucose, complications, biochemical criterion, drinking and smoking status, and hypoglycemic drug consumption. We investigated the relationship between candidate gene polymorphisms and the various clinical features listed in patients with T2D. Results for MYSM1 (rs2811893), LEKR1-CCNL1 (rs13064954), KIAA0825 (rs17376456), UST-TAB2 (rs7772697), NOS3 (rs3918227), ARHGAP22 (rs4838605), and IGF1 (rs6214) were shown in Table p = 0.003). Furthermore, we assumed that the minor allele of each SNP was a risk allele compared to the wild type allele. Four genetic models including log-additive, over-dominant, dominant and recessive were applied to analyze the associations between the SNPs and T2D risk using a logistic regression test in Table p = 0.018) . In addition, we found significant correlations between SNPs and clinical features including (1) rs2811893 (MYSM1) T to C change and Cr levels; (2) A allele in rs13064954 and C allele in rs4838605 (ARHGAP22) polymorphism and TG levels; (3) T allele in rs6214 (IGF1) and G allele in rs17376456 (KIAA0825) polymorphism and HbA1c levels; (4) C allele in rs7772697 (unknown) polymorphism and BMI; and (5) A allele in rs3918227 (NOS3) polymorphism and Cys-c levels.There are several biochemical markers for T2D including Cr, TG, HbA1C and Cys-c levels. An interplay between genetic and environmental factors seems to determine T2D risk. In this study, we selected previously published genetic loci associated with diabetic retinopathy to investigate their relationship with T2D risk and clinical characteristics of T2D in the Chinese Han population. Our results suggest that ARHGAP22) gene is associated with increased T2D risk (p = 0.018), consistent with previous studies. McAuley et al. found that diabetic retinopathy patients with rs4838605 polymorphism have a 1.58- fold increase in T2D risk under the co-dominant model [et al. provided the first evidence that ARHGAP22 polymorphisms were involved in endothelial cell angiogenesis [ARHGAP22 encodes for a GTPase protein in the Rho family, which is involved in the signal transduction pathway that regulates endothelial cell capillary tube formation during angiogenesis [ARHGAP22 gene has been implicated in a novel insulin-regulated pathway [ARHGAP22 was the only gene associated with T2D risk, which suggests that ARHGAP22 might function as an insulin regulator.We investigated the association between 18 SNPs and T2D risk. We found that the rs4838605 , our results indicated that seven SNPs in Our sample consisted of 706 Chinese Han individuals: 319 patients with T2D and 387 controls with normal glucose tolerance (NGT). These participants were recruited between August 2010 and December 2014 from clinics at the department of Endocrinology, Xi’an NO.1 Hospital, Xi’an, China. The key inclusion criteria of case subjects by the World Health Organization in 2010  were: (1MYSM1 (rs2811893), KIAA0825 (rs17376456), PLXDC2 (rs1571942), rs12219125, and ARHGAP22 (rs4838605) were selected from a GWAS concerning T2D in the Taiwanese population [http://www.ncbi.nlm.nih.gov/projects/SNP). The MAF of all SNPs was > 5% in the HapMap of the Chinese Han Beijing (CHB) population. The SNPs were genotyped using Sequenom MassARRAY RS1000  containing: PCR amplification assay, designed primers and probes, purification by Shrimp Alkaline Phosphatase (SAP), followed by addition of primers and extension of the special basic group, followed by stimulation cocrystallization combining sample analyte and chip substrates. Primers were designed for each SNP using Primer3 (http://frodo.wi.mit.edu).We genotyped a total of 18 SNPs previously reported to be associated with T2D. AA0825 rs376456, PDC2 rs15742, rs122t-test  for continuous variables and an additional Chi-square test for categorical variables. All populations also underwent testing for Hardy-Weinberg equilibrium (HWE). We used the PLINK software package (version 1.07) to assess genetic data. A multivariate logistic regression model, adjusting for age and gender, was used to estimate risk associations between genotypes and T2D by computing the odds ratio (OR) and 95% confidence intervals (95% CIs). To investigate potential models of inheritance, the log-additive, over-dominant, dominant and recessive genotypic models were applied.All demographic parameters are presented as mean ± standard deviation (mean ± SD), unless otherwise specified. Comparisons of participants’ characteristics were performed using an independent sample


S100 family contains at least 20 members, most of which are frequently dysregulated in human malignancies including breast cancer. However, the prognostic roles of each individual S100, especially the mRNA level, in breast cancer patients remain elusive. In the current study, we used “The Kaplan-Meier plotter” (KM plotter) database to investigate the prognostic values of S100 mRNA expression in breast cancer. Our results indicated that high mRNA expression of S100A8, S100A9, S100A11 and S100P were found to be significantly correlated to worse outcome, while S100A1 and S100A6 were associated with better prognosis in all breast cancer patients. We further assessed the prognostic value of S100 in different intrinsic subtypes and clinicopathological features of breast cancer. The associated results will elucidate the role of S100 in breast cancer and may further lead the research to explore the S100-targeting reagents for treating breast cancer patients.S100 family genes encode low molecular weight, acidic-Ca Breast cancer is a lethal disease that leads to 15% of cancer deaths in females worldwide in 20152+ binding proteins involving in a wide spectrum of biological processes, of which the first member was discovered in 1965571011121314151617181920et al. studied 16 members of S100 gene expression  in breast cancer, only S100A11 and S100A14 were related to poor outcome23S100 family members are small, acidic-Cahttp://www.ncbi.nlm.nih.gov/geo/). Currently, in that database, 3557 patients have relapsed free survival (RFS) data, 1610 have distant metastasis free survival (DMFS) data and 1117 have overall survival (OS) data2627KM plotter database was generated using gene expression data and survival information downloaded from GEO respectively and analyzed with setting different clinical parameters. Then, Kaplan-Meier survival plots with the number-at-risk indicated below, hazard ratio (HR), 95% confidence intervals (CI) and log rank P were obtained on the webpage. P value of <0.05 was considered to be statistically significant.The correlation of individual S100 family members mRNA expression to OS was analyzed on an online database, which was established using gene expression data and survival information of breast cancer patients downloaded from Gene Expression Omnibus (GEO)www.kmplot.com. Among all of them, 6 members were significantly associated with prognosis for all breast cancer patients . High mRNA expression of S100A8, S100A9, S100A11 and S100P were significantly associated with worse OS . However, S100A4 was not correlated with OS (p = 0.5414). The mRNA expression levels of the other S100 family members were not correlated with OS (p = 0.0578) was modestly associated with poor survival.We respectively examined the prognostic values of the mRNA expression of twenty S100 family members in breast cancer patients in patients . The sur with OS , althougp = 0.0007) and S100A9 (p = 0.0057), high mRNA expression of those S100A members were correlated to lower OS in luminal A type breast cancer patients. For S100A1 (p = 0.0123), S100A2 (p = 0.0297) and S100A6 (p = 0.0288), their mRNA expression levels were associated with longer OS in luminal A type cancers. S100A5 (p = 0.0549) was only modestly associated with better OS but without statistical difference. The rest of S100 members were not related with prognosis in luminal A breast cancer  and S100P (p = 0.014) was correlated to worse OS, however, S100B (p = 0.0042) was associated with better prognosis. The rest members of S100 were not correlated to prognosis in luminal B breast cancer  was modestly associated with OS (p = 0.0718).In HER2-overexpressing breast cancer patients, none of high mRNA expression levels of S100 family members were correlated with OS . The expp = 0.0061), S100P (p = 0.0139) and S100Z (p = 0.0491) were correlated to worse OS. However, S100A14 (p = 0.0169) was associated with better prognosis. We have observed the survival curves of the rest members of S100 in basal-like breast cancer were not associated with prognosis , S100A8 , S100A9  and S100A12  were associated with worse OS in grade II breast cancer. S100P high mRNA expression was associated with worse OS in grade I breast cancer patients . None of the S100 mRNA expression was found to be correlated to OS in grade III patients. As from p = 0.0031), S100A9  and S10010  were associated with worse survival in lymph node negative breast cancer patients. S100A13  was associated with better prognosis in lymph node negative breast cancer. p = 0.0055) and S100P  were correlated to worse OS in wild-p53-type breast cancer. However, S100A4 mRNA elevated expression was associated with better OS in mutant-p53-type breast cancer patients.Furthermore, we assessed the correlation of the prognostic values of S100 with other clinicopathological features, such as pathological grades, lymph node status and TP53 status. As we can see from In our study, S100A1 and S100A6 were significantly associated with better prognosis, while S100A8, S100A9, S100A11 and S100P were found to be correlated to worse outcome. Dysregulated S100 expression is a common feature in several human cancers. The alterative expression levels of S100 is correlated with progressive disease, but the mechanisms of how individual S100 family members contribute to disease aggression are largely unknownp = 0.059).S100A7 protein overexpression is associated with high grade and is an independent prognostic indicator in ER-negative invasive ductal carcinomas33S100A8 and S100A9 are originally identified in myeloid cells and naturally form a stable heterocomplex state, participating in myeloid cell differentiationS100A1 is abundantly expressed in cardiomyocytes, skeletal muscle fibers and neuronal populations and functions as regulation of energy metabolismS100A6 was preferentially expressed in proliferating but not quiescent fibroblasts cells42434445432+-induced growth arrest in human keratinocytes474950S100A11 is considered as a candidate tumor suppressor gene which regulates pathways for Caet al. reported the expression of short form of S100P indicated a worse survival in positive lymph node breast cancer patientsS100P was first identified in human placenta215455212+-dependent manner and consequently increase cell growthA crosstalk between S100 and estrogen may occur in breast cancer. S100A7 either inhibits or enhances the NF-κB–miR-29b–p53 pathway depending on the ER statusP53 protein is widely accepted as a tumor suppressor which is capable of inducing cell cycle arrest, senescence and apoptosis. Mutant p53, mostly missense mutations in exons 4–9, possesses a gain-of-function involving in tumorigenesis, invasion and metastasis67686970Our results indicated that S100A9, S100A11 and S100P were associated with worse outcome in all breast cancer patients according to the Kaplan-Meier survival curves and the log-rank P value based on the database. However, the survival curves of high and low mRNA expression of S100A9, S100A11 and S100P showed an intersection at the time of 200 months, which might indicated some confounding factors existing when doing these analysis. Multivariate analysis by COX regression which can eliminate the confounding factors couldn’t be achieved in this database. Thus the conclusion that S100A9, S100A11 and S100P correlated to worse outcome in breast cancer patients seems plausible, and it’s required to further study the precise prognostic significance of them in breast cancer.In summary, we assessed the prognostic values of 20 members of S100 mRNA expression in breast cancer patients by KM plotter database. Among them, 6 members were significantly associated with prognosis in breast cancer patients. Further assessment of prognostic values of S100 in breast cancer with different clinical features suggested that different S100 members may interact with different signaling pathways and exerted different functions in breast cancer development.Our study provides new insights regarding the contribution of S100 members to breast cancer progression and may be of help for the further discovering of S100-target inhibitors for treating breast cancer.How to cite this article: Zhang, S. et al. Distinct prognostic values of S100 mRNA expression in breast cancer. Sci. Rep.7, 39786; doi: 10.1038/srep39786 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The majority of viruses were effectively neutralized by 81.39a at a 50% effective concentration (EC50) of <0.01 to 4.9 μg/ml. Among group 2 HA viruses tested, a single A(H7N9) virus was not neutralized at 50 μg/ml; it contained HA2-Asp19Gly, an amino acid position previously associated with resistance to neutralization by the group 2 HA-neutralizing MAb CR8020. Notably, among group 1 HA viruses, H11-H13 and H16 subtypes were not neutralized at 50 μg/ml; they shared the substitution HA2-Asp19Asn/Ala. Conversely, H9 viruses harboring HA2-Asp19Ala were fully susceptible to neutralization. Therefore, amino acid variance at HA2-Asp19 has subtype-specific adverse effects on in vitro neutralization. Mice given a single injection (15 or 45 mg/kg of body weight) at 24 or 48 h after infection with recently emerged A(H5N2), A(H5N8), A(H6N1), or A(H7N9) viruses were protected from mortality and showed drastically reduced lung viral titers. Furthermore, 81.39a protected mice infected with A(H7N9) harboring HA2-Asp19Gly, although the antiviral effect was lessened. A(H1N1)pdm09-infected ferrets receiving a single dose (25 mg/kg) had reduced viral titers and showed less lung tissue injury, despite 24- to 72-h-delayed treatment. Taken together, this study provides experimental evidence for the therapeutic potential of 81.39a against diverse influenza A viruses.The pandemic threat posed by emerging zoonotic influenza A viruses necessitates development of antiviral agents effective against various antigenic subtypes. Human monoclonal antibody (hMAb) targeting the hemagglutinin (HA) stalk offers a promising approach to control influenza virus infections. Here, we investigated the ability of the hMAb 81.39a to inhibit IMPORTANCE Zoonotic influenza viruses, such as A(H5N1) and A(H7N9) subtypes, have caused severe disease and deaths in humans, raising public health concerns. Development of novel anti-influenza therapeutics with a broad spectrum of activity against various subtypes is necessary to mitigate disease severity. Here, we demonstrate that the hemagglutinin (HA) stalk-targeting human monoclonal antibody 81.39a effectively neutralized the majority of influenza A viruses tested, representing 16 HA subtypes. Furthermore, delayed treatment with 81.39a significantly suppressed virus replication in the lungs, prevented dramatic body weight loss, and increased survival rates of mice infected with A(H5Nx), A(H6N1), or A(H7N9) viruses. When tested in ferrets, delayed 81.39a treatment reduced viral titers, particularly in the lower respiratory tract, and substantially alleviated disease symptoms associated with severe A(H1N1)pdm09 influenza. Collectively, our data demonstrated the effectiveness of 81.39a against both seasonal and emerging influenza A viruses. Influenza A viruses of 16 hemagglutinin (HA) and nine neuraminidase (NA) antigenic subtypes have been detected in a vast natural reservoir of aquatic birds. Eight highly diverse HA subtypes are known to have caused infections in humans ; some of these subtypes  have caused pandemics and recurrent epidemics with various levels of severity. Interspecies transmission and zoonosis are central to the emergence of new viruses in humans. For example, a swine-origin virus contributed to the genesis of the 2009 H1N1 pandemic [A(H1N1)pdm09] and continues to circulate among humans . These two classes of influenza drugs are represented by small molecules that target highly conserved regions of two viral surface proteins, a transmembrane domain of M2 and a catalytic site of NA. The NAIs oseltamivir  and zanamivir  were the first two FDA-approved antivirals for treating influenza infections, with oseltamivir being the most widely prescribed. Intravenous peramivir has also recently been FDA approved, while intravenous zanamivir is undergoing clinical evaluation but has been available for compassionate use in patients with serious influenza-related illness . The intResistance to M2 blockers is widely present among seasonal influenza A viruses, and the U.S. Centers for Disease Control and Prevention (CDC) has not recommended their use since the 2005-2006 influenza season . Moreove0 molecules, which are cleaved by host proteases into HA1 and HA2 subunits  represent only a small fraction of the antibodies raised  and ferret  models, studies with additional challenge viruses are warranted.Further advancement has been achieved with discoveries of fully human MAbs that neutralize viruses of both HA groups. These MAbs are 39.29 and 81.39 ; FI6v3 ; CR9114 in vitro. The structural analysis showed that 39.29 and 81.39 MAbs utilized the same germ lines, with each containing 12 to 15 variable heavy-chain and 7 to 9 variable light-chain mutations  (group 1) and A(H3N2) (group 2) influenza viruses utations . The theutations . Howeverin vitro. Furthermore, we demonstrated that a single administration of 81.39a protected mice and ferrets against lethal challenge with A(H1N1)pdm09, HPAI A(H5N2), HPAI A(H5N8), A(H6N1), and A(H7N9) influenza viruses. These studies provide further insight into the breadth of the antiviral activity of 81.39a and expand our understanding of this stalk-targeting MAb as a promising therapeutic option against seasonal and emerging influenza viruses.Here, we show that human IgG1 MAb 81.39a, a derivative of the parental 81.39, exerts a broadly neutralizing activity against a large panel of influenza A viruses covering 16 HA subtypes, including the emerging clade 2.3.4.4 HPAI H5Nx, HPAI A(H7N8), and A(H7N9) viruses. Here, we observed differences in 81.39a neutralization profiles and provide the molecular basis of 81.39a activity Guide for Animal Care and Use of Laboratory Animals , and the  Animals . Animal 2 sequence analyses were downloaded from the public database Global Initiative on Sharing All Influenza Data (GISAID).Seasonal and zoonotic influenza viruses used in this study were submitted to the WHO Collaborating Center for Surveillance, Epidemiology, and Control of Influenza at the Centers for Disease Control and Prevention (CDC) for virological surveillance, which includes antigenic characterization and antiviral susceptibility testing. Non-U.S. viruses were kindly shared with the CDC by the responsible authorities in designated countries. Two A(H7N9) clones of A/Taiwan/1/2013 virus used in this study were isolated using biological cloning (plaque purification) procedures. All viruses were propagated in Madin-Darby canine kidney (MDCK) cells  and underwent a limited number of passages in cultured cells to maintain their original properties. At least viral HA and NA gene sequences were confirmed by Sanger sequencing generated using an ABI Prism 3730 genetic analyzer  and assembled using Sequencher 5.0 . The amino acid substitution(s) was corroborated using the pyrosequencing method as previously described , 35. Proin vivo studies.The broadly neutralizing human 81.39a (IgG1) MAb for influenza A viruses was generated, purified, and produced in large quantity as previously described . Anti-inThe neutralization properties of 81.39a against 16 virus subtypes were examined using the focus reduction assay. Briefly, confluent MDCK-SIAT1 cell monolayers in 24-well plates were incubated with a mixture of 5-fold serial dilutions of MAb at 0.08 to 500 μg/ml and pretitrated virus (30 to 60 foci/well), which was allowed to react for 1 h at room temperature, for 1 h at 37°C. After removal of virus-MAb mixture, overlay  containing MAb was added to the cells, and they were further incubated for 16 to 20 h at 37°C. Overlay then was removed, and cells were washed and fixed. Visualization of infected cells was done by immunostaining with mouse anti-viral nucleoprotein (NP) antibody blend , followed by incubation with anti-mouse IgG (Fab specific)-fluorescein isothiocyanate (FITC) antibody . The number of foci (a focus is a cluster of five or more infected cells) was counted under a fluorescence microscope.50) assay.A virus yield reduction assay was applied to assess the inhibitory effect of 81.39a on virus replication in MDCK-SIAT1 cells. After 1 h of inoculation at 37°C as described above, the inoculum was replaced with fresh cell culture medium containing MAb, and cells were incubated for 24 h at 37°C. Thereafter, supernatants were collected and used for virus titration using the 50% tissue culture infectious dose (TCID5.6 50% egg infectious doses (EID50)  of H5N2 virus or 106.6 EID50 (∼1.6 MLD50) of A(H5N8) virus. Mice were then treated with either 81.39a (45 mg/kg of body weight) or control IgG  at 24 days postinoculation (dpi) via intravenous (tail vein) injection (50 μl/mouse) under anesthesia. Lungs (n = 12/treatment group) were collected at days 3 and 6 (6 mice per test day) as previously described  were housed and monitored for at least 3 days for acclimation prior to the start of the study. Groups of 22 mice were inoculated with 10escribed .5.1 TCID50 of A(H6N1) or 105.2 TCID50 of A(H7N9) virus; both inoculation doses corresponded to ∼5.0 MLD50. The experiment with A(H7N9) virus was done in duplicate, using 10 mice per group for the second round. 81.39a (15 or 45 mg/kg) or control IgG (45 mg/kg) was given once at 48 h postinoculation (hpi). Mice infected with A(H6N1) virus were treated only with the higher MAb concentration. Lungs (n = 6/treatment group) were collected at days 5 and 7 pi; for H7N9-infected mice, lungs were collected only from the first experiment. Viral titers in lung homogenates were determined using the TCID50 assay. Mice were observed daily for clinical signs and survival throughout the study.In the second set of experiments, groups of 14 mice were inoculated with 10Mustela putorius furo) aged 3 to 5 months , and serologically negative by hemagglutination inhibition (HI) assay for currently circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, were used in this study. Ferrets were housed in customized, individual cages  and monitored for at least 3 days for acclimation and to establish baseline body temperatures prior to the start of the study. Clinical signs of illness and body weight were recorded daily. The following activity status scores were used: 0, alert and playful; 1, alert and playful only when stimulated; 2, alert but not playful when stimulated; 3, neither alert nor playful when stimulated. Nasal and ocular discharge, sneezing, coughing, dyspnea (shortness of breath), and diarrhea were scored 0 if not present or 1 if present. The temperature was measured twice daily by subcutaneous implantable temperature transponders .Groups of four male ferrets  . The A(H1N1)pdm09 virus was used in this study. The infected ferrets were then divided into four groups : control IgG  given at 1 dpi and 81.39a (25 mg/kg) given at 24, 48, and 72 hpi. The antibodies  were administered once via intravenous injection through the cranial vena cava.Intranasal inoculation was performed under anesthesia, induced by intramuscular administration of a ketamine-xylazine-atropine mixture , using 1050 assay. Under anesthesia, all animals from each group were thoroughly exsanguinated prior to euthanization at 5 dpi, and viral titers were determined in nasal turbinates, trachea, and lung bronchoalveolar lavage fluid (BALF). The BALF was centrifuged at 1,000 × g for 10 min, and the inflammatory cell count and protein concentration in cell-free solution were determined. Harvested whole lungs were fixed in 10% neutral buffered formalin and used for histopathology and immunohistochemistry.Nasal washes were collected daily (under anesthesia) by flushing nostrils with 1 ml of sterile PBS and further processed for determination of infectious viral titers using TCIDHistopathology and detection of viral NP in the lung (immunohistochemistry) were performed at the Yerkes National Primate Research Center  according to procedures published previously . BrieflyP < 0.05). The unpaired two-tailed Student's t test was applied to evaluate the statistical significance in viral titers and other analyzed parameters of control IgG- and 81.39a-treated animals. The survival fractions were estimated by the Kaplan-Meier method and compared between treatment groups within the same virus strain using the log-rank (Mantel-Cox) test.Statistics were performed using the GraphPad Prism v.5.0. software, and the statistically significant difference was set at an alpha of 0.05  and H4 (<0.01 μg/ml) subtypes were the most sensitive to neutralization in HA groups 1 and 2, respectively. Notably, a total of 6 viruses representing five HA subtypes were not neutralized at 50 μg/ml, the highest concentration of 81.39a tested   . Specifi group 1 . Two H12oup 1 HA . An A(H7Asp19Gly .2 using a focus-forming reduction assay (2-Asp19 (wild type), clone S2 possessed HA2-Asp19Gly. In the first set of experiments, the maximum MAb concentration tested was 50 μg/ml. As shown in 2-Asp19 clone virus was inhibited by 3.55 μg/ml of 81.39a . The 50 μg/ml concentration of MAb failed to reach the 50%  infectivity inhibition for the HA2-Asp19Gly clone compared to that of the control . In the second set of experiments the maximal concentration of 81.39a was increased to 300 μg/ml. We found that the number of foci produced by the HA2-Asp19Gly clone in cells treated with 300 μg/ml of 81.39a was much lower. Approximately 48% inhibition  was observed. The results confirmed that the HA2-Asp19Gly substitution reduced the ability of the MAb to neutralize virus infectivity in vitro.We next compared the 81.39a neutralization potency against two clones of A/Taiwan/01/2013 [A(H7N9)] influenza virus that differed by a single amino acid substitution in HAon assay . While c2 protein, we examined HA sequences deposited in GISAID as of 12 November 2015. The majority (∼99%) of H7N9 viruses recovered from humans (n = 231) maintained HA2-Asp19; only two viruses, A/Jiangsu/02/2013 and A/Wuxi/1/2013, contained Asp19Asn, and a single virus, A/Shenzhen/SP39/2014, had Asp19Gly. Conversely, all HA sequences of H11 (n = 619), H13 (n = 176), and H16 (n = 58) viruses possessed Asn, while all H12 subtype sequences (n = 150) possessed Ala at this position. Peculiarly, while the H12 subtype belongs to the H9 cluster of group 1 HA, the presence of Ala19 in the A(H9N2) viruses tested had no effect on virus neutralization  viruses as a representative of group 1 HA viruses. Mice were inoculated with either an A(H5N2) (A/northern pintail/Washington/40964/2014) or A(H5N8)  influenza virus and given a single intravenous dose of 81.39a (45 mg/kg) at 24 hpi. 81.39a administration protected 100% of animals from death (10 (10 at day 3 (P < 0.0001 for both viruses) and virus was not detected (>5 log10 reduction) at day 6 (P < 0.0001 for both viruses), indicating that 81.39a controlled virus spread in the lower respiratory tract of mice. Further, 81.39a treatment prevented body weight loss ; no virus was detected at 7 dpi  (The 81.39a efficacy against another group 1 HA virus (A/Taiwan/2/2013) of A(H6N1) subtype was also tested in mice. In this experiment, the same dose (45 mg/kg) of antibody as that used for H5-infected animals was administered at 48 hpi. As expected, control IgG-treated animals exhibited more severe signs of disease and succumbed to infection between 8 and 9 dpi . Convers 0.0001) . Altoget2-Asp19 (wild type) and HA2-Asp19Gly A(H7N9) (A/Taiwan/1/2013) influenza viruses in our experiments.Antiviral efficacy of 81.39a against group 2 influenza virus was evaluated in the A(H7N9) lethal model. Two doses, 15 and 45 mg/kg, were given to animals at 48 hpi. Viral titers in lungs were determined at 5 and 7 dpi, and morbidity and mortality were monitored for 14 days. To maximize the translational relevance, we used both HA10 (P = 0.004) and 3.1 log10 (P < 0.0001) for lower and higher doses, respectively (10) in control IgG-treated mice, while no virus was detected in animals treated with either dose of 81.39a  and 16 of 18  mice inoculated with the wild-type virus  mice and the lower dose protected 11 of 18   viruses caused ∼90% mortality in control IgG-treated animals  and O. Tpe virus . In mice= 0.013) . Therefo2-Asp19Gly A(H7N9) virus. The lower and higher doses reduced mean lung viral titers by 1.2 log10 (P = 0.002) and 1.5 log10 (P = 0.003) at day 5, respectively (10), while those that received 81.39a had significantly lower lung viral titers, 3.7 log10 with 15 mg/kg (P = 0.004) and 3.4 log10 with 45 mg/kg (P = 0.001). Overall, the antiviral effect of 81.39a was less pronounced in the HA2-Asp19Gly-infected group than in the mice inoculated with the wild-type A(H7N9) virus.The inhibitory effect of 81.39a on virus replication was also observed in lungs of mice inoculated with the HAectively . At 7 dp2-Asp19Gly virus.Other clinical indices mirrored the antiviral effect observed in the lungs of 81.39a-treated mice. Disease manifestations such as lethargy, ruffled fur, hunched posture, and dyspnea were apparent in control groups infected by either H7N9 virus but were rarely to occasionally present in the majority of 81.39a-treated animals. The body weight loss in 81.39a-treated animals was less pronounced and their recovery occurred much faster than that of the control groups  and N. Tin vitro antiviral activity is not always a predictive factor of efficacy in mammalian animal models.Overall, these data indicate that 81.39a was also effective in mice infected with a group 2 HA influenza virus, demonstrating that We next utilized a ferret model to assess the effect of 81.39a treatment on viral replication in the upper and lower respiratory tracts. A/California/12/2012, a representative of an A(H1N1)pdm09 virus, was used to analyze 81.39a effectiveness in ferrets. A single intravenous treatment was given to ferrets at 24, 48, or 72 hpi to assess the effect of delayed treatment. Either 81.39a or control IgG was delivered at a dose of 25 mg/kg. Nasal wash samples were collected from 1 through 5 dpi before animals were humanely sacrificed to harvest respiratory organs.2]) below 90%, which, if present, would indicate hypoxemia and/or respiratory failure.Infection of ferrets with the A(H1N1)pdm09 virus caused moderate to severe signs of disease . All ino10), which is not an unusual phenomenon when a high dose (106 TCID50) of virus is used for inoculation. The 24-h delayed treatment with 81.39a resulted in a significant viral titer reduction  detected at 3 dpi. When given at 48 hpi, 81.39a treatment significantly reduced nasal wash viral titer at 3  and 4  dpi. Notably, a statistically significant difference in viral titer reduction was also detected at 4 dpi, even when 81.39a treatment was delayed for 72 h .All inoculated animals shed virus throughout the 5-day experiment ; nasal w10) not only in the nasal turbinate but also in the trachea and, to some extent, in bronchoalveolar lavage fluid (BALF) of control IgG-treated ferrets  in the nasal turbinate (P = 0.01 for both time points) and BALF (P = 0.005 for both time points). The results suggest a superior antiviral activity of 81.39a in the lower respiratory tract compared to the upper respiratory tract.The A(H1N1)pdm09 influenza virus replicated in the lung tissues, consistent with previous reports , 41, 42. ferrets  to D at urbinate ; these curbinate  and C. VP = 0.004), 48 (P = 0.006), or 72 (P = 0.008) hpi  hpi . Althoug008) hpi , suggestTo further investigate the therapeutic effect of 81.39a in the lower respiratory tract of ferrets, we examined the pulmonary histopathology and the presence of viral NP in the lung sections of each lobe of virus-inoculated animals. Lung tissues of control IgG-treated ferrets showed severe multifocal alveolar, bronchial, perivascular, and interstitial infiltrates of inflammatory cells composed of neutrophils, macrophages, and a few lymphocytes intermixed with occasional glandular and lymphoid necrosis, fibrin deposition, and edema . CongestTo confirm that the observed lung damage was caused by virus replication and to examine the effect of 81.39a administration on the degree of virus replication in the lungs, we stained lung tissue samples for viral NP using immunohistochemistry. The lung sections derived from control animals showed frequent NP staining in the bronchiolar and peribronchial lymphoid and glandular tissues . Conversin vitro neutralization method, while previous studies on group 1- and 2-targeting MAbs only tested selected subtypes in the neutralization assay . This panel contained viruses collected from various geographic locations and included seasonal viruses, zoonotic viruses, HPAI A(H5N2) and A(H5N8) viruses, subtypes responsible for an unprecedented outbreak in poultry in the U.S., and virus variants resistant to one or both classes of FDA-approved anti-influenza drugs. The strength of this study is the direct testing of this diverse group of viruses using the same on assay –25. In an assay –. This ph50s ranged widely (≥500-fold), from <0.01 to 4.85 μg/ml, and there was no consistent difference between group 1 and group 2 viruses. The EC50s determined in our study were similar to those reported for viruses tested with the parental MAb 81.39   . Severall egress , 25, 43.l egress . Indeed, evident . Taken t evident .2-19 replaced with Asn, Ala, or Gly. All but one virus belong to H11, H12, H13, and H16 subtypes (group 1), which are not known to cause human infections. A single virus from group 2, a drug-resistant A(H7N9) clone, had HA2-Asp19Gly. It is noteworthy that substitutions at this position are rarely found among A(H7N9) virus recovered from humans. The HA2-Asp19Gly virus escaped neutralization by 81.39a at a concentration of 50 μg/ml. However, when the MAb concentration was increased to 300 μg/ml, some degree of inhibition of virus infectivity was observed, suggesting a therapeutic benefit of higher drug doses. It is worth noting that while group 1-targeting MAbs D8 and F10 effectively neutralized a pseudotyped virus of H11 subtype carrying Asp19Asn  and A(H7N7) viruses by CR8020  in the H3 subtype, while the binding affinity of the H13 (dK = 6.3 nM) and H16 (dK = 20 nM) subtypes carrying HA2-Asn19 was similar to that of the H3 subtype carrying HA2-Asp19 (dK = 33 nM) , similar to that of the H1 subtype. The H9 and H12 subtypes are phylogenetically closely related; H12 belongs to the H9 cluster of group 1 HA  viruses are known to infect humans  . Moreoveoup 1 HA , 40. In t humans , 46, the50s in the neutralization assay. Both H5Nx viruses used in this study have been shown to efficiently replicate in the lungs of mice without prior adaptation , A(H5N8), A(H6N1), and A(H7N9), which showed similar ECaptation . Regardll models , 28, 30.2-Asp19Gly despite its lack of neutralization in cell culture. Similarly, the MAbs VIS410  region of broadly neutralizing HA stalk-specific antibodies and Fc receptors (FcγRs) for IgG are required for protection against influenza virus in vivo (in vivo efficacy. One or more of these mechanisms could contribute to the therapeutic benefits observed for 81.39a against HA2-Asp19Gly-carrying A(H7N9) viruses in vivo: antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent respiratory burst (ADRB) activity, and complement-dependent cytotoxicity (CDC). A similar in vivo phenomenon was also observed with MAbs 1H5 and 1H10, despite the lack of remarkable in vitro neutralizing activity  virus carrying HAs VIS410  and 1H5 s VIS410 , as wells VIS410 , were al viruses . Therefo in vivo . In agre in vivo , strengtactivity .In the ferret model, the antiviral effect of delayed 81.39a treatment was analyzed both in the upper and lower respiratory tracts. While viral titers in nasal washes and nasal turbinate were moderately reduced, a stronger inhibition of viral replication was detected in the trachea and BALF after a single-dose administration of 81.39a, indicative of a higher level of 81.39a activity in the lower respiratory tract compared to the upper respiratory tract. This result was further supported by lung tissue analyses showing a strong reduction of viral NP expression upon 81.39a treatment. In addition, histopathological analyses demonstrate that lung tissues of an uninfected ferret treated with 81.39a displayed no significant lesion, similar to what is observed in lung tissues of an uninfected and untreated control animal (data not shown), indicating that treatment with 81.39a exerts no apparent toxicity.A previous study suggested that IgA is more efficiently transported to mucosal surfaces, while IgG is found predominantly in the lungs of influenza virus-infected animals , 50. TheTo the best of our knowledge, this is the first study in which all 16 HA subtypes of influenza A viruses present in aquatic birds and mammals were used to evaluate a group 1- and 2-targeting bnAb in the neutralization assay. Our data demonstrate that treatment administration of 81.39a is effective in protecting mammals from disease caused by emerging influenza viruses, including H5Nx viruses. Collectively, this study revealed experimental evidence of the therapeutic potential of 81.39a and provided a basis for future clinical trials.