HLA class II  alleles could provide further clues to the identity of the infection, since HLA molecules exhibit allotype-restricted binding of infection-derived antigenic peptides. We clustered >30 HLA-DPB1 alleles into six predicted peptide-binding supertypes , based on amino acid di-morphisms at positions 11 (G/L), 69 (E/K), and 84 (G/D) of the DPβ1 domain. We found that the DPβ11-69-84 supertype GEG (DP2), was 70% more frequent in BCP ALL , and 98% more frequent in cases diagnosed between 3 and 6 years (P<10−4), but not <3 or >6 years, than in controls. Only one of 21 possible DPB1 supergenotypes, GEG/GKG (DP2/DP4) was significantly more frequent in BCP ALL (P=0.00004) than controls. These results suggest that susceptibility to BCP ALL is associated with the DP2 supertype, which is predicted to bind peptides with positively charged, nonpolar aromatic residues at the P4 position, and hydrophobic residues at the P1 and P6 positions. Studies of peptide binding by DP2 alleles could help to identify infection(s) carrying these peptides.Childhood B-cell precursor (BCP) ALL is thought to be caused by a delayed immune response to an unidentified postnatal infection. An association between BCP ALL and  However, the P4 peptide-binding pocket, at position β69, also makes an important contribution to antibody and peptide-binding . We compared their frequencies in childhood BCP ALL, non-BCP leukaemia and solid tumours recruited as part of the Peptide binding by HLA class II allotypes, including DP, is the outcome of interactions between the amino acid side chains of the peptide and four major peptide-binding pockets  or non-BCP acute leukaemia. The non-BCP leukaemias were the sum of Pro-B ALL , T-ALL , and AML (n=208). Diagnostic immunophenotyping was carried out according to the protocol for UK Medical Research Council leukaemia trials (n=409) were also recruited as part of the UKCCS (n=864) born in Manchester UK between 1991 and 1997 were used as controls  were validated by the UKCCS data centre at the Epidemiology and Genetics Unit, University of York.Childhood leukaemia cases were recruited between 1992 and 98 as part of the UK Childhood Cancer Study . Leukaema trials . Childhohe UKCCS . UmbilicHLA-DPB1 molecular typing was carried out as previously described in detail . Supertype allele and genotype frequencies were compared in cases and controls using global and univariate statistical analysis. As discussed previously  used for supertype clustering was included in the analysis. Global case–control supertype frequencies were compared using the CLUMP programme of χ2 statistic (T1) from a series of simulated case–control tables. In univariate analysis, cross-product odds ratios (ORs), and 95% confidence intervals were calculated from case–control supertype and genotype frequencies by the RERI program in the Linkage Utility Package, P-values for case–control supertype and genotype differences using Fisher's Exact test. Six supertypes require an uncorrected P-value <0.008, and 21 supergenotypes an uncorrected value <0.002 to achieve significance (P=0.05). No correction for the total number of classical DP alleles was applied. DPB1 and DQA1, or DQB1 alleles.HLA-DPB1 types for 982 cases of childhood leukaemia , not including childhood lymphoma (n=864) were used as controls. Male–female ratios were slightly higher in the leukaemia cases (1.22) than the solid tumours (1.14) and controls (1.01).The UKCCS is an epidemiological case–control study designed to test the role of environmental factors in the aetiology of childhood cancer and leukaemia . As partia cases . A mixedDPB1 alleles in the cases and controls could be clustered into six supertypes , DP2 (GEG), DP3 (LKD), DP4 (GKG), DP6 (LED), and DP8 (GED).The majority (90%) of >30 pertypes , consistn=895) and the newborn controls (n=864), we identified 14 DPβ69E alleles, of which four are DP2 (GEG), seven are DP6 (LED), and three are DP8 (GED). Of 15 DPβ69K alleles, six are DP4 (GKG), six are DP3 (LKD), and three are DP1 (GKD). In global χ2 analysis, the supertype frequency in the total leukaemia series was significantly different (P<10−6) from the controls (P=0.04). In univariate analysis, DP2 (GEG) : 1.6, 95%, CI, 1.2–2.0; 2 sided P=0.0002) and DP8 (GED)  were significantly more frequent in leukaemia cases than controls. DP6 (LED)  was only marginally significant without correction for six supertypes, while DP2 and DP8 were significant after correction.In the total leukaemia case series  and non-BCP acute leukaemia (n=208) revealed that DP supertypes in BCP ALL differed significantly from the newborn controls (P<10−6), but non-BCP leukaemia was only marginally significant (P=0.04) (DP2 (GEG)  and DP8 (GED)  were significantly more frequent, after correction for six supertypes, than controls. DP6 (LED) was not significant in BCP ALL, but was significant in non-BCP leukaemia . DP1 (GKD) was significantly less frequent, after correction, than controls in BCP ALL , but not in non-BCP leukaemia.Stratification of the leukaemias into BCP ALL ((P=0.04) . In univDP2 and DP8 raised the possibility of a chance finding. To test this, supertype frequencies in four BCP ALL case series were compared with controls: (1) cases included in our previous study ; (3) half of the cases in the previous study combined with the ‘new’ cases (n=343); (4) the ‘new’ cases (n=128) alone. DP2 and DP8 were significant in all four case series, though only DP2 remained significant after correction , but was not significant in BCP ALL diagnosed <3 or >6 years. DP4 was significantly increased in BCP ALL diagnosed <3 years, though not after correction. DP8 was not significant after correction, while DP1 protected from BCP ALL in all age groups.To determine the relationship between the age at diagnosis of BCP ALL and DPB1 genotypes, such as DPB1*0201/0202 can have a homozygous supergenotype (DP2/DP2:GEG/GEG) (DP2/DP4), was associated with a significantly increased risk (110%), after correction, of BCP ALL . DP2/DP4 was associated with an increased risk (130%) of BCP ALL arising between 3 and 6 years of age , but not <3 or >6 years of age. No DP2 supergenotypes were significant in non-BCP leukaemia or solid tumours. DP4/DP6 (GKG/LED) was significant in non-BCP leukaemia after correction , but not in BCP ALL or solid tumours. Homozygous DP1 (GKD/GKD) significantly protected, after correction, against BCP ALL . No other DPB1 supergenotypes were significant at any of the diagnostic ages.To determine which combination of supertype alleles was associated with BCP ALL, we compared the frequency of all 21 possible supergenotypes  in BCP ALL, non-BCP leukaemia, and solid tumours with newborn controls . Note thGEG/GEG) . Of the DP supertype associations could be explained by LD with HLA-DQ alleles, we analysed the co-occurrence of DP and DQ alleles in 451 BCP ALL cases, using POPGENE. We detected only one DP allele, 1601, in LD with DQ . Five BCP ALL cases (0.4%) typed for DPB1*1601, a frequency not significantly greater than in the controls, indicating that the DP-supertype results cannot be explained by LD between DP and DQ alleles.To test whether the β1 domain was associated with BCP ALL , 69 (P4 pocket), and 11 (P6 pocket) represents an expanded version of the scheme proposed by DP1 (GKD), DP2 (GEG), DP3 (LKD), DP4 (GKG), DP6 (LED), and DP8 (GED) since they broadly resemble those defined in the primed lymphocyte test (PLT) as DPw specificites . In a rare disease such as BCP ALL in which there are likely to be multiple aetiological factors, weak HLA associations potentially require hundreds of cases and controls to allow for correction for multiple testing. Supertype analysis, in which alleles are clustered according to common functional  properties, overcomes this problem. DPB1 alleles comprise combinatorial series of six variable regions (A-F) encoded by exon 2 , LED (DP6), GED (DP8)), and to compare these with three homologous β69K series (GKG (DP4), LKD (DP3), GKD (DP1)).The DP2 (GEG), a 98% increase in DP2-associated risk between 3 and 6 years of age, and a 130% increased risk associated with a single supergenotype, DP2/DP4. This association was not present in BCP ALL diagnosed <3 or >6 years of age, and leads us to conclude that the peak of BCP ALL . There is no evidence that childhood BCP ALL is caused by an oncogenic virus (DP2 suggests that binding of specific peptide(s) and T-cell activation are involved in causation, which is inconsistent with immune evasion by an oncogenic virus. The negative association of DP1 with BCP ALL may be due to the binding and recognition of TEL-AML1 peptide(s) in children with pre-ALL with this supertype, as discussed elsewhere (DPB1*0501-restricted (DP1) CD4+ T cell response  of 15 heterozygous supergenotypes (GEG/GKG) is associated with BCP ALL fits this model.The delayed response to infection hypothesis for BCP ALL  proposesozygotes , a recenDPB1*0201 peptide-binding data and molecular modelling (β84) and pocket 6 (β11) deep and hydrophilic, preferentially-binding hydrophobic and aromatic amino acids, notably phenylalanine (F), and tyrosine (Y)  are likely to bind to DP2.Using sine (Y) . This pr162D-N179 containing two overlapping T-cell epitopes, 163FHFEVFNFV171 and 165FEVFNFVPC173 that are restricted by DPB1*0401 (DP4), and DPB1*0201 (DP2) (DP2) and GKG (DP4), consistent with the association of BCP ALL with DP2/DP4. While this conclusion is speculative it points to a need for detailed sero-epidemiological studies of RSV in BCP ALL.In this context,


NADPH-cytochrome-P450 oxidoreductase (CPR) is a ubiquitous enzyme that belongs to a family of diflavin oxidoreductases and is required for activity of the microsomal cytochrome-P450 monooxygenase system. CPR gene-disruption experiments have demonstrated that absence of this enzyme causes developmental defects both in mouse and insect.D. discoideum revealed the presence of three genes  that encode putative members of the diflavin oxidoreductase protein family. redA transcripts are present during growth and early development but then decline, reaching undetectable levels after the mound stage. redB transcripts are present in the same levels during growth and development while redC expression was detected only in vegetative growing cells. We isolated a mutant strain of Dictyostelium discoideum following restriction enzyme-mediated integration (REMI) mutagenesis in which redA was disrupted. This mutant develops only to the mound stage and accumulates a bright yellow pigment. The mound-arrest phenotype is cell-autonomous suggesting that the defect occurs within the cells rather than in intercellular signaling.Annotation of the sequenced genome of redA implicates CPR in the metabolism of compounds that control cell differentiation.The developmental arrest due to disruption of  NADPH-cytochrome-P450 oxidoreductase CPR, EC 1.6.2.4) is a ubiquitous enzyme that is required for activity of the microsomal cytochrome-P450 (CYP) monooxygenase system .6.2.4 is,4. CPR b5 dATP and [α-32P]dCTP by the random hexanucleotide priming method .Total RNA was isolated from 5 × 10escribed . Probes D. discoideum total RNA primed with a mixture of oligo dT and random hexamers using SuperScript First-Strand Synthesis System (Invitrogen). A 20 μg amount of the resulting cDNA were subjected to quantitative PCR using Platinum SYBR Green qPCR SuperMix UDG (Invitrogen) on a GeneAmp 5700 System (Applied Biosystems) using the default thermocycler program for all genes. Threshold values were normalized according to Ct of D. discoideum mitochondrial large subunit rRNA (IG7), which is expressed at similar levels throughout the D. discoideum development [-ΔCt method [redA (5'-CCTATGGTGATGGTGTTCCACCAAC-3' and 5'-CCCCACTAAATTGAATATGTGAAAGATTTAAACGA-3'), redB (5'-GCAACCGAAGAAGCAAACGAAGAATACAAT-3' and 5'-CAAAGGTTGAAGACCTGGGAAAGATTCTAA-3'), redC (5'-AGGTGGAGTCTTTGAAAGATGTTGTAAAAATCC-3' and 5'-GGTCCAGGTACTGGTGTTGCAC-3'), ecmA (5'-AGCTGATAGTTGCGATTCCA-3' and 5'-TACCTCCTGTACCACCACCA-3') and rnlA (IG7) (5'-GTGGTTCGGCACCTCGAT-3' and 5'-CACCCCAACCCTTGGAAACT-3').Reverse transcription was carried out with 5 μg of elopment . The folt method . qPCR as8 AX4 and redA- cells developed on filters for 48 hours were extracted with 1 ml chloroform and the organic phase was collected by centrifugation at 3000 × g for 5 min at 4°C. UV/Visible spectra of the chloroform extracts were obtained in UV-2401PC Shimadzu spectrophotometer.5 × 10K. aerogenes [Mutants and wild-type cells were allowed to develop on nitrocellulose filters. At 0, 8, 12, 16, 20, 24 and 48 hours the cells were harvested from the filters with 20 mM phosphate buffer (pH 6.4) and sporulation efficiency was determined by detergent and heating treatment of the cells following plating on SM agar in association with erogenes . The numCPR, NADPH-cytochrome P450 oxidoreductase; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide.DCGK carried out most of the experimental work and drafted the manuscript. LF performed RT-qPCR assays and helped with chloroform extractions. LCF was involved in the initial steps of this work and helped with cDNA cloning and sequence analyses. WFL isolated strain DG1047, participated in the design of the study and in writing the manuscript. AMDS coordinated the study, participated in its design and wrote the manuscript. All authors read and approved the final manuscript.


Proper development of plastids in embryo and seedling tissues is critical for plant development. During germination, plastids develop to perform many critical functions that are necessary to establish the seedling for further growth. A growing body of work has demonstrated that components of the plastid transcription and translation machinery must be present and functional to establish the organelle upon germination.Arabidopsis thaliana mutants in a gene that encodes a plastid-targeted elongation factor G (SCO1) that is essential for plastid development during embryogenesis since two T-DNA insertion mutations in the coding sequence (sco1-2 and sco1-3) result in an embryo-lethal phenotype. In addition, a point mutation allele (sco1-1) and an allele with a T-DNA insertion in the promoter (sco1-4) of SCO1 display conditional seedling-lethal phenotypes. Seedlings of these alleles exhibit cotyledon and hypocotyl albinism due to improper chloroplast development, and normally die shortly after germination. However, when germinated on media supplemented with sucrose, the mutant plants can produce photosynthetically-active green leaves from the apical meristem.We have identified sco1 alleles reveals differences in chloroplast formation during seedling germination compared to chloroplast differentiation in cells derived from the shoot apical meristem. Our identification of embryo-lethal mutant alleles in the Arabidopsis elongation factor G indicates that SCO1 is essential for plant growth, consistent with its predicted role in chloroplast protein translation.The developmental stage-specific phenotype of the conditional-lethal  Arabidopsis thaliana) and rapeseed (Brassica napus), developing embryos are green and cells in these embryos develop functional chloroplasts  mutant  mutant . Two allsco1 (sco1-2 and sco1-3) isolated from the Salk T-DNA insert collection have T-DNA inserts at the C-terminal end of the gene  with a Salk T-DNA insert located 14 base-pairs upstream of the SCO1 ATG start site was found to have a similar seedling-lethal phenotype. However, sco1-4 plants could also be rescued when germinated on medium supplemented with sugar .We identified one EMS-derived t et al. , where a1 Figure . Seedlin1 Figure , sco1-4 s Figure . When gesco1-1 cotyledons appear completely white, fluorescence microscopy revealed some red autofluorescence is present in all mutant cotyledons, especially along the vasculature . Leaves and other photosynthetic tissues derived from the apical meristem in sco1-1 were green and visually indistinguishable from wild-type plants . Consistent with the role of amyloplasts in gravity perception  and At2g45030 [GenBank:NM_130067]) contain predicted mitochondrial-targeting sequences . We subcloned BAC F23N19 because it contained the bulk of the genetic interval. The pBeloBAC plasmid containing BAC F23N19  was partially digested by Sau3AI  for 30 minutes at 37°C to obtain fragments approximately 10–15 kilobase in size. These fragments were ligated  at 15°C overnight to the BamHI-digested  binary vector pCLD04541 . Plasmid DNA was introduced into Escherichia coli (DH5α) using a Gigapack III XL cosmid packaging kit . E. coli colonies were screened using BAC F23N19 specific markers located every 10 kilobases to identify clones that provided coverage of the entire BAC. These were mated into Agrobacterium tumefaciens (GV3101) using the E. coli helper strain pRK2013. sco1-1 plants were transformed by floral dipping [sco1-1 phenotype and were confirmed in the T2 generation. Sequencing of the seven genes in the identified interval revealed a mutation in a Tu family protein translation EF-G (At1g62750).An F2 mapping population was established between  dipping  and selesco1-1 plants that were similar in appearance, staged embryos were obtained from siliques formed from flowers that were dated upon reaching anthesis. Ovules were collected from the central portion of siliques that were from 5 to 15 d post anthesis (DPA) and the embryos were dissected from the ovules. Ovules of sco1-2 and sco1-3 were examined in 9-d-old siliques that were cut open and photographed using a Nikon SMZ1500 dissecting scope with a Nikon Digital Camera DXM1200 .Using wild-type and 4 at 4°C overnight. The samples were then washed, dehydrated, and embedded in spurs resin . For the cotyledon cross-sections, the embedded pieces were sectioned using an automated ultra-microtome and a glass knife. The sections were stained with bromophenol blue  to reveal chloroplasts and cell walls. Images were captured using the brightfield function on a Nikon E800 microscope . For the transmission electron microscope images, cotyledon pieces were sectioned using an automated ultra-microtome with a diamond knife . The sections were stained with a 2% uranyl acetate solution and lead citrate as previously described [Plant materials were cut and placed into a 3% formaldehyde/gluteraldehyde solution in 0.1 M sodium cacodylate buffer, pH 7.4  and fixed overnight at 4°C. The fixed samples were washed and post-fixed in 2% OsOescribed . StainedSCO1 gene specific primers used for amplification were (For – 5'AAAAACAAAAGCAGACATCGAC3') and (Rev – 5'GGATCCTTAAGCAGCAACTTCTTGATCC3').RT-PCR was performed using the SuperScript III One-Step RT-PCR with Platinum Taq . A total of 50 nanograms starting RNA concentration were used in each reaction. SCO1 was fused to a 35 S driven, C-terminal GFP construct utilizing the Gateway vector system . Gene specific primers with flanking attB sites were used to amplify the gene (5'GGGGACAAGTTTGTACAAAAAAGCAGGCTTCAACAATGGCGGCGGATGCTCTGAG3' and 5'GGGGACCACTTTGTACAAGAAAGCTGGGTCAGCAGCAACTTCTTCTTGATCCTTG3'). The expression clone was inserted into the donor vector pDONR201, and subsequently transformed into One Shot TOP10 chemically competent cells . Transformed clones were identified on Luria-Bertani (LB) medium containing 50 μg/ml kanamycin and tested with gene specific primers. A miniprep purification  was then done on a positive clone and used in a recombination reaction with the destination vector pVRGFP (provided by Vincente Rubio and Xing Wang Deng). The ligated plasmid was subsequently transformed into One Shot TOP10 chemically competent cells. Transformed clones were identified on LB medium containing 50 μg/ml spectinomycin and tested with gene specific primers. A positive clone was mated into Agrobacterium tumefaciens (GV3101) using the E. coli helper strain pRK2013 and used to transform wild-type Arabidopsis plants (ecotype Columbia) by floral dipping [ dipping . Rescuedsco1, At1g45332, At2g45030, and the E. coli EF-G (fusA). Outputs were designed by BOXSHADE [The publicly available program TCOFFEE was used to produce sequence alignments of BOXSHADE .NJR participated in the design of the study, carried out all experiments, and drafted the manuscript. RPH conceived of the study, participated in its design and coordination, and assisted with manuscript preparation.


The use of shRNAs to downregulate the expression of specific genes is now relatively routine in experimentation but still hypothetical for clinical application. A potential therapeutic approach for HIV-1 disease is shRNA mediated downregulation of the HIV-1 co-receptor, CCR5. It is increasingly recognized that siRNAs and shRNAs can have unintended consequences such as cytotoxicities in cells, particularly when used for long term therapeutic purposes. For the clinical use of shRNAs, it is crucial to identify a shRNA that can potently inhibit CCR5 expression without inducing unintended cytotoxicities.Previous shRNAs to CCR5 identified using conventional commercial algorithms showed cytotoxicity when expressed using the highly active U6 pol III promoter in primary human peripheral blood derived mononuclear cells. Expression using the lower activity H1 promoter significantly reduced toxicity, but all shRNAs also reduced RNAi activity. In an effort to identify shRNAs that were both potent and non-cytotoxic, we created a shRNA library representing all potential CCR5 20 to 22-nucleotide shRNA sequences expressed using an H1 promoter and screened this library for downregulation of CCR5. We identified one potent CCR5 shRNA that was also non-cytotoxic when expressed at a low level with the H1 promoter. We characterized this shRNA in regards to its function and structure. This shRNA was unique that the use of commercial and published algorithms to predict effective siRNA sequences did not result in identification of the same shRNA. We found that this shRNA could induce sequence specific reduction of CCR5 at post transcriptional level, consistent with the RNA interference mechanism. Importantly, this shRNA showed no obvious cytotoxicity and was effective at downregulating CCR5 in primary human peripheral blood derived mononuclear cells.We report on the characterization of a rare shRNA with atypical structural features having potent RNAi activity specific to CCR5. These results have implications for the application of RNAi technology for therapeutic purposes. A finding with critical bearing upon HIV-1 disease was the fact that individuals homozygous for a defective CCR5 gene, CCR5Δ32, are protected from HIV infection and heterozygous individuals have a substantially prolonged course of disease[Silencing of genes through homologous double stranded RNA is a sequence specific, highly conserved mechanism. It serves as an antiviral defense mechanism and protWe previously identified several shRNAs that could effectively downregulate CCR5 -20. HoweHere, we demonstrate the selection of an shRNA from a library specific to CCR5 that maintains both potency and lack of cytotoxicity when expressed within primary human PBMC. We characterize this shRNA in regards to its downregulation of CCR5 and target sequence specificity.Using published algorithms to prediSince screening for several shRNAs predicted by commercially available algorithms were ineffective to downregulate CCR5 without causing cytotoxicity, we adopted a different approach – constructing an shRNA library represenOur previous results indicate that the higher levels of shRNA expression driven by the U6 promoter resulted in cytotoxicity. Since CCR5 shRNA 1005) is expressed by the H1 promoter, we wanted to know whether it also exhibited reduced cytotoxicity. We determined whether CCR5 shRNA (1005) had toxicity to primary T-cells  was selected by a non-conventional means we further determined whether its functional and structural properties resembled that of typical shRNAs.The use of commercial  predictThe stringency of target sequence selection for RNAi activity of CCR5 shRNA (1005) was further demonstrated by constructing an shRNA with deletion of a single nucleotide from the 3' end of the CCR5 shRNA (1005) sense sequence. This resulted in complete loss of activity (data not shown).shRNAs and siRNAs act principally by degradation of the target mRNA -33. We dThe double stranded stem of the CCR5 shRNA (1005) sequence joined by a 9 nt loop is the structure typically used for shRNA constructs. In some studies, the sense and antisense of the double strand effector siRNA can be expressed independently within the same cell, although the level of activity is generally lower. We testWe confirmed that the CCR5 shRNA (1005) acts specifically upon its homologous target sequence by constructing a vector which expresses a chimeric mRNA consisting of an EGFP reporter gene followed immediately by CCR5 sequences of the 20-nucleotide predicted target sequence Figure . ExpressWe further assessed the specificity of CCR5 shRNA (1005) by mutating the shRNA within its core domain, disrupting the complete homology with the target sequence  cell surface marker gene in place of the HIV-1 accessory gene Vpr, which allows the detection of HIV-1 infected cells (HSA+) by flow cytometry. We stained HIV infected cells with monoclonal antibodies against HSA and examined HSA expression in EGFP+ population. HSA expression was inhibited in the EGFP+ population in the CCR5 shRNA (1005) vector transduced PBMC, indicating CCR5 reduction induced by CCR5 shRNA (1005) was sufficient to inhibit HIV infection. In contrast, HSA expression was not inhibited in the EGFP+ population in the non-shRNA expressing control vector (FG11F) transduced PBMC. These results demonstrated inhibition of CCR5 tropic HIV-1 by CCR5 shRNA (1005).To examine HIV-1 inhibition by CCR5 shRNA (1005), we transduced PHA/IL2 activated, CD8+ cell- depleted PBMC with the vector expressing CCR5 shRNA (1005) from the H1 promoter. CCR5 shRNA (1005) efficiently reduced CCR5 expression in the EGFP+ population Figure . TransduIn this report, we demonstrate that a potent shRNA directed to CCR5 with minimal cytotoxicity can be selected from a library of CCR5 target sequences. This shRNA is relatively rare, identified after screening 380 sequences, as opposed to a frequency of approximately 1 out of 3 effective shRNAs identified through conventional shRNA prediction algorithms. Similar to other shRNAs selected by more conventional means, this shRNA is dependent upon the double strand structure of the RNA and has specific target site specificity. Most importantly, this shRNA shows no obvious cytotoxicity and is effective at downregulating CCR5 in primary cells.The frequency at which we obtained CCR5 shRNA (1005) was considerably lower than that using conventional algorithms. These algorithms predict potentially potent shRNA expressed using efficient expression vectors or direct transfection of chemically synthesized siRNA; in both cases, high levels of siRNA are present within the cells. Under those conditions, in our hands, all RNAi sequences identified have some cytotoxicity in sensitive primary human lymphocytes when expressed from a U6 promoter. We deliberately screened for potent shRNAs that have low levels of expression, hypothesizing that those RNAs should have lower cellular toxicity. It is noteworthy that the sequence of CCR5 shRNA (1005) does not fit most of the favored rules for identification of effective shRNAs, suggesting that shRNAs obtained by functional screening for potency at lower levels of expression may have different sequence requirements. We have not characterized the specific siRNA species derived within the cells by DICER processing of the shRNA. A related shRNA bearing a single nucleotide substitution and with the same loop sequence was processed correctly to a 22 nucleotide species in rhesus macaque PBMC.Thus, under experimental conditions where cytotoxicity may be an issue, identification of shRNAs that maintain potency, but with reduced cytotoxicity is possible, but considerably more sequences will need to be assayed. Nevertheless, these studies provide confidence that effective shRNAs can be obtained for long term therapeutic purposes such as for use in stem cell gene therapy.We characterized the function and structure of a potent shRNA against CCR5 selected by library screening. This shRNA has unique characteristics in regards to its function and structure. The sequence of CCR5 shRNA (1005) did not fit most of the favored rules for identification of effective shRNAs, suggesting that shRNAs obtained by functional screening for potency at lower levels of expression may have different sequence requirements.The lentiviral vector encoding CCR5 shRNA (1005) under the human H1 RNA polymerase III promoter was previously described.BbsI and XhoI sites downstream of the U6 promoter of pBS-hU6 plasmid DNA[XbaI/XhoI digestion and cloned into the same sites of FG12[To express CCR5 shRNA (1005) from the human U6 RNA Polymerase III promoter, two complementary DNA oligos, sense 5'-GATCCCCGAGCAAGCTCAGTTTACACCTTGTCCGACGGTGTAAACTGAGCTTGCTCTTTTTC-3', antisense 5'-TCGAGAAAAAGAGCAAGCTCAGTTTACACCGTCGGACAAGGTGTAAACTGAGCTTGCTCGGG-3', were synthesized, annealed, and inserted between asmid DNA {designes of FG12.XbaI and XhoI and cloned into the corresponding sites of pBS-SKII vector (Stratagene) and FG12.To express either sense or antisense strand of CCR5 siRNA (1005) by the U6 promoter, U6 promoter containing either strand was amplified by PCR using following primer pairs: for the antisense strand of CCR5 siRNA (1005); 5'-CTCGAGTCTAGAGAATTCCCCCAGTGGAAAGAC-3' and 5'-GAATTCCTCGAGGCTAGCAAAAAGAGCAAGCTCAGTTTACACCGGTGTTTCGTCCTTTCCAC-3', for the sense strand of CCR5 siRNA (1005); 5'-CTCGAGTCTAGAGAATTCCCCCAGTGGAAAGAC-3' and 5'-ACTAGTCTCGAGAAAAAGGTGTAAACTGAGCTTGCTCGGTGTTTCGTCCTTTCCAC-3'. The amplified PCR fragments were digested with BamHI and EcoRI and cloned into the corresponding sites of the FG12 lentiviral vector. For the generation of 3-nt mismatch mutant of CCR5 shRNA (1005) {designated as a mutant (1005)}, entire human H1 promoter DNA in the pBS hH1-3[EcoRI and XhoI sites.The lentiviral vector expressing EGFP-CCR5 target chimeric mRNA (EGFP-CCR5 target/FG11F), which contains the 20 nucleotide predicted shRNA target sequence (5'-GAGCAAGCTCAGTTTACACC-3'), was prepared by PCR amplification using the following primers: 5'-gatcggatccccgggtaccggtcgccaccatggtga-3' and 5'-GCATgaattcgatcgggtgtaaactgagcttgctcgcttacttgtacagctcgtccatgcc-3'. The amplified PCR fragment was digested with pBS hH1-3 was amplMAGI-CCR5 cells  were maihuCCR5-293T cells were created by infecting 293T cells with a VSV-G pseudotyped human CCR5 expressing pBABE-huCCR5 retroviral vector  followedHuman primary PBMCs were isolated from leukopacks by Ficoll-Paque PLUS  purification and stimulated by 2.5 μg/ml of PHA for 2 days. PBMCs were cultured in RPMI 1640 medium containing 20% FCS and 20 units/ml IL-2 (Roche).All lentiviral vectors were produced by calcium phosphate transfection of 293T cells as previously described. The cul5) were plated into 96 well plates. Cells were infected with lentiviral vectors at different MOI depending on experiments for 2 hr in the presence of 8 μg/mL polybrene (Sigma). The infected cells were collected 3–12 days after infection and analyzed for CCR5 and EGFP expression by flow cytometry or infected with reporter HIV-1 as described previously[huCCR5-293T cells and MAGI-CCR5 cells were plated into 24 well plates one day before infection. PHA/IL-2 activated PBMCs , mouse IgG2a/κ isotype control conjugated with either PE-Cy5 or APC according to the manufacture's instructions. For measuring HIV-1 reporter virus infection, a PE-labeled anti murine HSA mAb  was used. The cells were then fixed with 2% formaldehyde, and EGFP and CCR5 expression were monitored on FC500 (Becton Dickinson). The data was analyzed by CELLQUEST (Becton Dickinson) or FLOWJO (Tree Star) software.5) in a 12-well plate. FuGENE (Roche) was used for cotransfection according to the manufacturer's protocol. Forty-eight hours posttransfection, cells were analyzed for EGFP expression by flow cytometry.Lentiviral vector encoding EGFP-CCR5 target (1 μg) was cotransfected with either 3 μg of CCR5 shRNA (1005) or LacZ in vitro transcribed human CCR5 RNA using T7 RNA polymerase . Following primers and proves were used for RT-PCR reactions; For Human CCR5; sense primer: gtccccttctgggctcactat, reverse primer: ccctgtcaagagttgacacattgta, probe: FAM-tccaaagtcccactgggcggcag-BHQ1, For β actin; sense primer: cgagcgcggctacagctt, reverse primer: ccttaatgtcacgcacggatt, probe: HEX-accaccacggccgagcgg-BHQ1.Total RNAs from the infected CEM NKR-CCR5 cells were isolated using the Qiagen RNeasy extraction kit following manufacture's instruction. Quantification of mRNA was performed using IQ5 (BioRad) with iScript one-step RT-PCR kit and the following conditions . RNA standards for CCR5 mRNA quantitation was made by serial dilution of The authors never received reimbursements, fees, funding, or salary from an organization that may in any way gain or lose financially from the publication of this paper. The authors never have any stocks or shares in an organization that may in any way gain or lose financially from the publication of this paper. The authors have no competing interests to declare in relation to this paper.SS and DSA designed and performed cytotoxicity experiments, construction of shRNA and siRNAs, infection, flow analysis and wrote the manuscript. PK performed cytotoxicity experiments and constructed shRNA and siRNAs. JB performed HIV infection experiments. QFX constructed shRNAs. MK constructed CCR5 shRNA 1005-mutant, performed flow analysis and qRT-PCR. KC and SK performed flow analysis, SP examined shRNA sequences using commercial and published algorithms. ISYC designed study and drafted the manuscript. Authors read and approved the final manuscript.


USP1 in chicken cells to dissect its role in a stable genetic system. USP1 ablation increases FANCD2 and PCNA monoubiquitination but unexpectedly results in DNA crosslinker sensitivity. This defective DNA repair is associated with constitutively chromatin-bound, monoubiquitinated FANCD2. In contrast, persistent PCNA monoubiquitination has negligible impact on DNA repair or mutagenesis. USP1 was previously shown to autocleave after DNA damage. In DT40, USP1 autocleavage is not stimulated by DNA damage, and expressing a noncleavable mutant in the USP1 knockout strain partially rescues crosslinker sensitivity. We conclude that efficient DNA crosslink repair requires FANCD2 deubiquitination, whereas FANCD2 monoubiquitination is not dependent on USP1 autocleavage.Monoubiquitination of FANCD2 and PCNA promotes DNA repair. It causes chromatin accumulation of FANCD2 and facilitates PCNA's recruitment of translesion polymerases to stalled replication. USP1, a protease that removes monoubiquitin from FANCD2 and PCNA, was thought to reverse the DNA damage response of these substrates. We disrupted  The ability to target DNA repair enzymes to specific sites in the genome is essential for proper and appropriate DNA repair. Once repair is completed, the repair factors have to be inactivated to avert inappropriate action and corruption of genetic information. A means by which this regulation is achieved is by the posttranslational modification of repair proteins, and it is now clear that interconnected signal transduction pathways orchestrate such modifications . RecentlUSP1 knockdown in human cells results in an accumulation of monoubiquitinated FANCD2. There is some evidence that this leads to constitutive DNA repair activity   and fromactivity . In additination . The montination . This motination . Studiesear foci . In addiBecause studies in human cells point toward an important role for USP1 in the regulation of repair by PCNA and FANCD2, the regulation of USP1 itself has become important. In particular, how do USP1 levels and activity correlate with the modification of these two substrates? Both modifications are induced during DNA replication  but are USP1 to address the role of this protein in DNA repair in a stable genetic system. We have used this to clarify whether USP1-mediated deubiquitination functions to switch off DNA repair and how specific and crucial self-destruction is for the regulation by this enzyme. The studies presented here lead us to reappraise the role of USP1 in DNA repair and mutagenesis.In this study, we created a DT40 knockout of USP1 locus in the avian B cell line DT40. The chicken USP1 gene was cloned, and its locus was identified and mapped. The last exon of the USP1 gene encodes a histidine motif that is essential for catalytic activity  from ΔUSP1 sIgM-loss variants. This revealed an average of 0.71 gene conversions per mutated sequence and 0.29 nontemplated point mutations in ΔUSP1  expression in a fluctuation analysis starting from multiple sIgM-positive subclones. Single sIgM-positive cells were sorted and expanded for 4 weeks, after which the proportion of sIgM-negative cells was assessed by flow cytometry A. Wild-tin ΔUSP1 B. These in ΔUSP1  , as well as UV irradiation and X-rays. The data in USP1 is hypersensitive to crosslinks  and marginally sensitive to UV light but not sensitive to X-rays. Cell-cycle analyses after exposure to cisplatin reveal a corresponding cell-cycle response to DNA damage by the ΔUSP1 cell line (USP1 gene results in DNA crosslinker hypersensitivity.siRNA knockdown of A damage . This moell line B. In sumUSP1 in a DT40 strain in which the endogenous PCNA locus has been disrupted and the cells rescued by ectopic expression of a point mutated version of human PCNA that cannot be monoubiquitinated (PCNA-K164R). This cell line had previously been created and characterised by USP1 in this strain shows very clearly that PCNA monoubiquitination is undetectable, whereas modified FANCD2 persists , whereas the effect on UV sensitivity is marginal. To determine the contribution of persistently ubiquitinated FANCD2 to the observed crosslinker sensitivity in ΔUSP1, we disrupted USP1 in a ΔFANCL strain. FANCL is the ubiquitin ligase component of the FA core complex , or USP1 mutated in the Gly-Gly motif (USP1 GG-AA). Strikingly, all clones obtained from transfections with either mutant USP1 isoform displayed very high levels of USP1 relative to those of clones transfected with the wild-type USP1  linker sequence, leaving an N-terminal fragment, which is approximately 15 kDa smaller than the full length . To studype USP1 A, indicaype USP1 B. This rant USP1 C. As expant USP1 .The hypersensitivity to cisplatin and UV can be complemented by the ectopic expression of wild-type USP1 D. We theUSP1 strain complemented with USP1 GG-AA can respond to DNA damage by the monoubiquitination of FANCD2, though with slightly slower kinetics , as well as the high-salt buffer , 500 mM NaCl, 1 mM MgCl2, 10% glycerol, 10 mM 2-mercaptoethanol, 3 mM NEM and protease inhibitor cocktail).Cellular subfractionations were performed as previously described , except Cell proliferation was determined by the mixing of DT40 cells with a fixed number of CaliBrite beads (Becton Dickinson). The numbers of beads and cells were counted simultaneously by FACS Scan flow cytometry. Apoptosis was measured by staining with FITC-conjugated Annexin V (Sigma) according to the manufacturer's protocol, followed by FACS analysis.2B4O7 (pH 8.5) before being stained with fluorescein isothiocyanate (FITC)-conjugated anti-BrdU (BD Biosciences). Cells were resuspended in phosphate-buffered saline (PBS) containing 5 mg/ml propidium iodide (Sigma) and analyzed by FACS Scan flow cytometry. For synchronization, cells were arrested (in G2/M) by treatment with nocodazole (25 ng/ml) for 8 hr, followed by washes and release.Cells were grown with or without drug and where indicated pulsed with 10 μM BrdU for 10 min before being harvested by centrifugation. Cells were fixed with 70% ethanol overnight at −20°C. After fixation, DNA was denatured by 30 min incubation in 2 M HCl and 0.5% Triton X-100. Samples were subsequently neutralized with 0.1 M NaCells were lysed in radioimmunoprecipitation assay (RIPA) buffer  by syringing eight times through a 25 G needle. Lysates were cleared by centrifugation. For the analysis of FANCD2, samples were separated on 8% polyacrylamide Tris-Glycine gels by SDS-PAGE (polyacrylamide gel electrophoresis). Rabbit anti-FANCD2 polyclonal and goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (Southern Biotech) were used as the primary and secondary antibody, respectively. For the analysis of EGFP-USP1 and PCNA, samples were separated by SDS-PAGE (10% Bis-Tris gels [Invitrogen]). For the detection of EGFP, rabbit anti-GFP antibody (Abcam) and goat anti-rabbit IgG conjugated to HRP (Southern Biotech) were used as the primary and secondary antibody, respectively. So that PCNA could be detected, mouse anti-PCNA (Abcam) and rabbit anti-mouse IgG conjugated to HRP (Dako) were used as the primary and secondary antibody, respectively.Mutagen sensitivity was investigated with the colony survival assay as described . UV lighA comprehensive and detailed description of the method is described previously . In brieThe chicken USP1 complementary DNA (cDNA) fused to the EGFP coding sequence was a kind gift of Dr. M. Takata. The C92S mutation in USP1 was generated by the Quikchange mutagenesis method with the following primers: forward (5′-CTTGGCAACACTAGTTACCTTAACAGCGTTCTTCAG-3′) and reverse (5′-AACGCTGTTAAGGTAACTAGTGTTGCCAAGATTATTCAA-3′). The G680A-G681A mutation (GG-AA) in USP1 was generated by the Quikchange mutagenesis method with the following primers: forward (5′-ATGGAAGCTGTGGGACTGCTAGCAGCACAGAAGAGCAAGAGCAAGTCT-3′) and reverse (5′-GCTCTTGCTCTTCTGTGCTGCTAGCAGTCCCACAGCTTCCATGT-3′).


SCE). Bulk electrolyses at +0.9 V and +1.4 V result in n values of 0.95and 3.7, respectively. Chemical oxidation of Au2(dppp)(pdt) using one equivalent of Br2 yields 1,2-dithiolane and Au2(dppp)Br2. The reactivity seen upon mildoxidation ≤ +1.0 V is consistent with formal oxidation of a thiolate ligand, followed by a fastchemical reaction that results in cleavage of a second gold-sulfur bond. Oxidation at higherpotentials (≥ +1.3 V) is consistent with oxidation of gold(I) to gold(III). Structural andelectrochemical differences between gold(I) aromatic and aliphatic thiolate oxidation processes arediscussed.The redox chemistry of mononuclear and dinuclear gold(I) phosphine arylthiolate complexeswas recently investigated by using electrochemical, chemical, and photochemical techniques. Wenow report the redox chemistry of dinuclear gold(I) phosphine complexes containing aliphaticdithiolate ligands. These molecules differ from previously studied gold(I) phosphine thiolatecomplexes in that they are cyclic and contain aliphatic thiolates. Cyclic voltammetry experiments ofAu


In this study, we have investigated gene knockdown analysis of endothelial Robos, in particular Robo1 and Robo4 knockdown cells display distinct activity in endothelial cell migration assay. The knockdown of robo4 abrogated the chemotactic response of endothelial cells to serum but enhanced a chemokinetic response to Slit2, while robo1 knockdown cells do not display chemotactic response to serum or VEGF. Robo4 knockdown endothelial cells unexpectedly show up regulation of Rho GTPases. Zebrafish Robo4 rescues both Rho GTPase homeostasis and serum reduced chemotaxis in robo4 knockdown cells. Robo1 and Robo4 interact and share molecules such as Slit2, Mena and Vilse, a Cdc42-GAP. In addition, this study mechanistically implicates IRSp53 in the signaling nexus between activated Cdc42 and Mena, both of which have previously been shown to be involved with Robo4 signaling in endothelial cells.This study identifies specific components of the Robo signaling apparatus that work together to guide directional migration of endothelial cells. RT-PCR was performed by using primer pairs described previously for hRobo1 . Real tirobo4 region corresponding from 1324 bp to 1732 bp was amplified using primers F: GCTGCAGTCACTGGTGCTGGAGCTGG and R: GGTCCCGGGCATCCGCCCCCAGCCG. The human robo1 region corresponding from 127 bp to 579 bp was amplified using primers F: TAATACGACTCACTATAGGGCCAATCCCCACCTCTGATAA and R: ATTAACCCTCACTAAAGGGAGGTTGGCATTCCATTACTGC. SiRNA was generated according to BLOCK-iTTM Complete Dicer RNAi Kit (Invitrogen) recommendations. HUVEC cells were grown to 80% confluence and transfected with 125, 250, 750 and 1000 ng of siRNA per well by LipofectamineTM 2000 Reagent. For robo1 siRNA experiments, 250 and 500 ng were used and for control lacZ siRNA comparable doses were used. For rescue experiments, zebrafish Robo4 described previously [The human eviously  was usedrobo4 siRNA. Lysates were also probed for actin (Sigma). For co-IP experiments in HEK293T cells, equal amounts of the two plasmids (5 μg) were either co-transfected or not depending on the combination desired for 36 h. Lysates were made in RIPA buffer (Sigma) and pre-cleared with protein G-agarose (Pierce) for 3 h. Precleared lysates were IP'd with myc antibody (1:1000)  for overnight. The myc antibody was captured by protein G-agarose for 3 h. All IP steps were performed at 4°C. The beads were washed three times with RIPA buffer and 2× sample buffer was added prior to boiling. The heated samples were resolved on 7% SDS-PAGE (Novex) and western blot was performed as described previously [robo4 siRNA (250 ng) or control lacZ siRNA (250 ng) endothelial cells by Lipofectamine 2000 reagent. Twenty-four hours post transfection lysates were made as described earlier. Quantitation of western blots were performed as described before [Pull down assays were performed as previously described  with a meviously . In cased before .robo4 siRNA cells for 1 h at 4°C. Cells were washed three time with 1× PBS and lysates were made with AP lysis buffer (GenHunter). The lysates were heat inactivated at 65°C for 10 min to inactivate background phosphatase activity and protein concentration was measured using bicinchoninic acid (BCA) assay (Pierce). Equal amounts of protein were used for detecting AP activity. AP activity was measured by adding AP assay reagent A (GenHunter) and absorbance read at 405 nm using a microplate reader.For AP fusion proteins, HEK293T cells were transfected with pAP-tag5 and pAP-Slit2N constructs. Forty-eight hours post transfection; supernatant was collected by centrifugation to remove cell debris. All assays with AP proteins used HEK293T supernatants directly without further purification. AP controls processed in an identical manner were used as controls. AP binding assay was performed by incubation of equal amounts of AP and AP-Slit2N proteins (25 ng/mL) with control or Migration assay was performed as described before . All migHUVEC cells were grown on cover slips, fixed with 4% formaldehyde in PBS for 1 h at room temperature, and permeabilized for 30 min with 0.1% Triton X-100 in PBS containing 1% BSA. Cells were incubated for 20 min with phalloidin conjugated to Texas Red , and cover slips were mounted in DAPI media for confocal imaging using a Zeiss LSM510 confocal system mounted on a Zeiss Axiovert 100 M microscope.SK, GVS, PWL, KP and MP performed experiment, designed experiments and interpreted results. DR suggested experiments, interpreted results and edited the paper. RR co-ordinated the study, designed experiments and wrote the paper.


The relationship between aberrant expression of cyclin D1 and retinoblastoma (RB) protein and clinicopathological factors was investigated in 80 patients with oesophageal SCC using immunohistochemical analyses. Heterogeneous staining of cancer cell nuclei with antibody to cyclin D1 was found in 31.3% of patients (25 out of 80 patients). Nuclear staining of cancer cells with anti-RB antibody was homogeneous in 10.0% (8 out of 80 patients) and heterogeneous in 58.8% (47 out of 80 patients). Among cases with homogeneous staining for RB protein, 75% (six out of eight patients) exhibited simultaneous positivity for cyclin D1 (P < 0.05). No significant relationship was found between cyclin D1 or RB protein expression and various clinicopathological parameters. The prognosis of patients with cyclin D1-positive tumours was significantly poorer than that of the other patients (P < 0.01). In addition, when patients with cyclin D1-positive and -negative tumours were stratified according to presence or absence of lymph node metastasis and RB status, the cumulative survival rates in the cyclin D1-positive groups were significantly lower for patients without lymph node metastasis (P < 0.01) and for patients whose tumours were positive for RB (P< 0.0001). These findings suggest the possibility that cyclin D1 positivity is a useful prognostic marker related to lymph node metastasis and RB protein expression in human oesophageal SCC, in addition to clinicopathological factors.


The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development.We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs), AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model.Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines. Multiple distinct and geographically diverse genotypes of highly pathogenic avian influenza (HPAI) A H5N1 viruses now exist and continue to cause outbreaks of disease in domestic poultry on three continents Influenza A viruses are enveloped RNA viruses in the family Orthomyxoviridae possessing eight negative-sense genomic segments and are classified into subtypes based on their two surface glycoproteins, the hemagglutinin (HA) and neuraminidase (NA). There are 16 known HA and 9 NA subtypes that exist in aquatic birds, the natural reservoir of all influenza A viruses 2 fragments specific for HA has been reported to be effective in protecting animals from death from influenza, even when administrated after H5N1 virus infection Neutralizing antibodies directed against the HA glycoprotein are the primary mediator of protection against influenza virus infection The structure of influenza virus HA and location of antibody-binding epitopes were first characterized for HA of the human H3 subtype In this report, we describe for the first time the generation and characterization of two broadly cross-neutralizing recombinant human antibodies  generated by screening a Fab antibody phage library derived from a patient recovered from infection with a clade 2.3 H5N1 virus. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines.H4 and VL2 sequences, while the antibody designated AVFluFab03 represented the other 25 Fab clones that possessed distinct VH3, and VL1 sequences. To further characterize the two Fabs, the two unique Fab clones were converted into intact human IgG1 antibodies, AVFluIgG01 and AVFluIgG03.A combinatorial antibody library, prepared from a 26 year old donor who was infected with H5N1 virus 14 weeks earlier, was screened with purified AH/1/05 (clade 2.3) virus. After four rounds of panning, 43 human Fab clones were selected which demonstrated reactivity with AH/1/05 purified virus by ELISA. Sequence analysis of all 43 selected Fab clones revealed the presence of only two unique clones, both of them comprising an IgG1 Fd heavy chains and lambda light chains. As shown in in vivo by passive immunization study in BALB/c mice.The AVFluIgG01 and AVFluIgG03 binding properties were characterized using indirect immunofloresence assay (IFA), micro-neutralization assay (MN), and hemagglutination inhibition assay (HI) assays, and To further identified the relative binding specificities and binding regions, the two rhAbs previously shown to bind to AH/1/05 whole virus in ELISA were tested by IFA with MDCK cells infected with H5N1, H3N2, or H1N1 virus , or SF-9To evaluate the neutralizing activities of the two rhAbs, MN assays were performed first using clade-2.3 and clade-2.2 H5N1 viruses isolated from patients from China . With a To confirm the reactivity patterns of AVFluIgG01 and AVFluIgG03, HI activities against H5N1, H3N2, and H1N1 viruses were assessed . The res50  of AH/1/05 wild-type virus. As shown in p = 0.0006). A dose-dependent decrease in protective efficacy was observed in mice receiving 10- or 100-fold lower amounts of either rhAb. Although the lower doses of AVFluIgG01 or AVFluIgG03 did not completely prevent fatal disease, they delayed time to death (p<0.05). Nevertheless, even at a dose of 0.025 mg/kg, the AVFluIg03 protected 50% of mice from lethal disease (p = 0.045). Although lung and brain virus titers determined 6 days p.i. were reduced (≥3-fold) in mice that received 2.5 mg/kg of either rhAb compared with those receiving the control HIgG1, theses differences did not achieve statistical significance , 24 hr prior to challenge i.n. with 10 LDTo identify the potential individual amino acids essential for interaction with the two cross-neutralizing human antibodies, amino acid sequences of HA1 region of some human H5N1 isolates used above in the MN and/or HI assays were aligned  and anal126, Ser129 and Asn155 were important for the binding of AVFluIgG03.To test the impact of the above identified changes of amino acid substitutions identified in natural H5N1 strains, we generated AH/1/05 rHA gene products possessing single amino acid substitutions as identified above  and expr119 and Trp122) each selected amino acid was replaced by a corresponding variable amino acid identified in wild-type human H5N1 virus strains emerging in Asia and Africa respectively (Influenza Virus Resource: http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html). The two conserved amino acids were replaced with Glycine (G). To further delineate the binding epitope of AVFluIgG01, single amino acid substitutions around Ser123, from HA1 residues 115 to 129 were constructed. Mutations at I116H, I117P, P118S and W122G resulted in complete loss of AVFluIgG01 binding; moreover, mutations at S120N and S121A resulted in substantially reduced binding of AVFluIgG01, but had no impact on the binding of the polyclonal patient's serum , site II  and Site III  likely contribute to the non-continuous conformational epitope for AVFluIgG03.WB analysis demonstrated that AVFluIgG01 reacted with the denatured viral HA1 protein. In contrast, AVFluIgG03 showed no binding activity with denatured HA1 . The WB 's serum . In cont's serum  and the In the present study, we generated two H5N1-specific rhAbs (AVFluIgG01 and AVFluIgG03) representing the repertoire of Fab clones recovered from the blood of a convalescent H5N1 virus-infected patient . Interesin vitro against all viruses tested except one clade 2.3 virus, GD/1/06 in which one amino acid substitution (S123P) was implicated in the loss of reactivity with this virus  were not reported previously. Interestingly, mutations I116H, I117P and W122G also abolished AVFluIgG03 binding. Furthermore, by examining the positions of the AH/1/05 HA1 sequences on three-dimensional H5 HA structure as illustrated in http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html), we found that residues Lys119 and Trp122 were absolutely conserved while other residues in the proposed linear epitope were highly conserved among 266 human H5N1 viruses (96.2%–99.9%). Although our results indicated that the single mutation K119G had no direct effect on the antibody binding, the illustration of the three dimensional structure shown in 119, Ser120 and Ser121 comprise a major helix structure with Ser120 and Ser121 are positioned on the outer face of the helix (96.2% conservation), whereas Lys119 is positioned in the inner helix, which may explain the conservation of this residue among 266 human H5N1 viruses. Residue Ile117 is partially buried at the surface of the globular head of the HA1 and may contribute through stabilizing and/or positioning of the linear epitope on the globular head of the HA1 H5 molecule. We propose that this novel linear epitope defined by the broadly neutralizing human antibody AVFluIgG01 represents a highly conserved neutralization epitope that may play an important role in inducing protective humoral immune responses to H5N1 viruses in humans. In contrast to the continuous epitope identified by AVFluIgG01, the epitope recognized by AVFluIgG03, is comprised of three non-continuous sites which comprise a conformational determinant as illustrated in blue and green in 116, Ile117 and Trp122) in green, partially overlaps with the linear epitope identified by AVFluIgG01 but is not exposed from the upper surface of the H5 HA molecule, suggesting that this site may not directly interact with the AVFluIgG03 but may otherwise affect the stability of the conformational epitope on the globular head of the HA1 H5 molecule. Site II (Glu126 and Ser129) overlaps with a previously described antigenic site 3 in H5 HA based on the X-ray crystallographic structure of the clade 1 virus VN/1203/04 virus 152, Asn155) partially corresponds to the previously identified antigenic site 2 in the H5 HA 126 and Ser129 were critical residues for the binding of AVFluIgG03. Interestingly, the H5 epitope for the human antibody AVFluIgG03 overlaps with epitopes recognized by mouse mAbs on a mouse-adapted duck H5N2 virus  identified by the sequence analysis of virus escape mutants The epitopes recognized by AVFluIgG01 or AVFluIgG03 were on the head of the HA1, but not HA2 molecule and appeared to be linear or conformational, respectively . SequencIn summary, we have generated and characterized two recombinant baculovirus-expressed human neutralizing and protective antibodies directed against an H5N1 clade 2.3 virus which exhibit unique properties for intra and inter-clade virus reactivity. Importantly, localization of the epitopes recognized by the two rhAbs has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. The primary prophylactic functionality of the antibodies were addressed in this study with the mouse model. More detailed studies in vivo would help to understand the significance of the defined epitopes and the important mechanisms for prophylaxis or therapy of human infection with H5N1 viruses. The utility of the recombinant approach allows for rapid scale-up in production, as well as a means to rapidly clone and express antibodies with specificity for newly emerging H5N1 variants. More functional human antibodies could be obtained by additional screenings. The rhAbs described here alone or in combination with other functional human antibodies may provide a promising for broadly neutralizing passive immunotherapy treatment that could supplement existing antiviral strategies against human H5N1 virus infection.50) titers were determined by serial titration of virus in eggs and calculated by the method of Reed and Muench Influenza viruses used in this study were propagated at 37°C in the allantoic cavity of 10-day-old embryonated hens' eggs for 26 hours (H5N1 virus) or 48 hours (H3N2 and H1N1 viruses), and were aliquoted and stored at −70°C until use. Fifty percent egg infectious dose  and then expressed in SF-9 insect cells as previously described IFAs were performed on different cells according to the experimental design. To measure rhAbs reactivity for viral antigens, Madin Darby Canine Kidney (MDCK) cells were infected respectively with AH/1/05 (H5N1), A/Hubeihongshan/53/2005 , and A/Yunnan/1145/2005  viruses. To measure rhAbs reactivity for viral recombinant HA (rHA), rHA1, or rHA2, SF-9 cells were infected with recombinant baculoviruses expressing HA, HA1 or HA2 products from AH/1/05 virus, respectively. To evaluate rHA containing site-directed mutations, rHAs were transiently expressed in 293T cells. Cells were grown in 24 wells plates and were then either directly stained in the wells or were prepared as monolayer on glass slides followed by acetone fixation. Bound antibodies were detected by using FITC-conjugated anti-human antibodies and observation under an immunofluorescence microscope.50) of virus in MDCK cells.To verify the neutralizing ability of AVFluIgG01 and AVFluIgG03, the micro-neutralization (MN) assay was performed as previously described To verify the HI ability of AVFluIgG01 and AVFluIgG03 against avian H5N1 or human H3N2 and H1N1 viruses, the HI assays were performed using 0.5% turkey red blood cells for detecting reactivity with human H3N2 and H1N1 virus 50) and 50% lethal dose (LD50) of AH/1/05 virus were determined as previously described 2 and challenged intranasally (i.n.) with 10 LD50  of AH/1/05 virus in 50 µl. Mice were monitored daily for sickness, weight loss and death for 21 days.The fifty percent mouse infectious dose (MIDPurified virus of AH/1/05 were applied to 10% SDS–PAGE and transferred to a PVDF membrane with a mini-protean apparatus (Bio-Rad). The denatured antigens were probed with patient sera (1:150 dilution) as a PC or 1 µg/ml of an anti-SARS IgG as a NC or the two rhAbs at 1 µg/ml. A goat anti-human IgG conjugated to HRP was used as the secondary antibody. The reaction was detected by ECL reagent (Pierce) according to the manufacturer's instructions.Mutagenic primers were designed after aligning sequences of HAs region of MN and HI tested human H5N1 isolates including A/Anhui/1/2005 (Genebank accession DQ371928), A/Guangxi/1/2005 (DQ371930), A/Hunan/1/2006 (FJ492879), A/Zhejiang/1/2006 (FJ492880), A/Sichuan/1/2006 (FJ492881), A/Fujian/1/2005 (FJ492882), A/Fujian/1/2007 (FJ492883), A/Guangdong/1/2006 (FJ492884), A/Jiangxi/1/05 (FJ492885), A/Xinjiang/1/2006 (FJ492886), A/VietNam/1203/2004 (AY818135), A/HongKong/156/1997 (AF023709), A/Turkey/15/2006 (EF619989) and A/Indonesia/5/2005 (EU146622), and the DNA fragments of site-directed mutagenesis of the HA gene of AH/1/05 were amplified by PCR, cloned into XhoI and BamHI sites of pCDNA3.0, and confirmed by DNA sequencing to exclude secondary mutation. All constructs were transfected to 293T cells for transient expression. The resulting mutated rHAs were assayed by IFA described above for binding activity for AVFluIgG01 and AVFluIgG03.The H5 AH/1/05 HA1 structure was based on the crystal structure of the highly related the H5 A/Viet Nam/1194/2004 HA1 (PDB accession number 2IBX) by DS MODELER (Discovery Studio 2.0). Images were created with the software Discovery Studio™ 2.0 .Kaplan-Meier survival curves and logrank tests were used to measure differences between rhAb treated mice and NC mice.Table S1(0.04 MB DOC)Click here for additional data file.Table S2(0.03 MB DOC)Click here for additional data file.Figure S1(2.25 MB TIF)Click here for additional data file.


H1)-biased cytokines such as IFNγ, TNF and IL-2, and broadened serological responses against drifted A/H1N1 and A/H3N2 influenza variants. These results suggest that synthetic TLR4 adjuvants can enhance the magnitude and quality of protective immunity induced by influenza vaccines.Safe, effective adjuvants that enhance vaccine potency, including induction of neutralizing Abs against a broad range of variant strains, is an important strategy for the development of seasonal influenza vaccines which can provide optimal protection, even during seasons when available vaccines are not well matched to circulating viruses. We investigated the safety and ability of Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE), a synthetic Toll-like receptor (TLR)4 agonist formulation, to adjuvant Fluzone® in mice and non-human primates. The GLA-SE adjuvanted Fluzone vaccine caused no adverse reactions, increased the induction of T helper type 1 (T Seasonal influenza epidemics are responsible for an average 36,000 deaths and 226,000 hospitalizations annually in the United States alone Vaccination is the best preventative measure against influenza, and acts by increasing neutralizing Abs directed against the HA, the major surface glycoprotein Adjuvants provide an important means of improving influenza vaccines. To be effective, these adjuvants should increase the levels of hemagglutination-inhibition (HI) Ab titers against the HA components included in the vaccine and ideally also against variant viruses possessing drifted HA proteins. Emulsion-based adjuvants are currently used in certain licensed seasonal influenza vaccines. MF59 ™ (an oil-in-water adjuvant) combined with a seasonal influenza vaccine,  has been approved in some European countries since the late 1990's Salmonella minnesota which is known as Monophosphoryl Lipid A (MPL®). MPL® in a variety of formulations has been licensed in Europe as a component of the hepatitis B vaccine Fendrix® The most advanced TLR4 agonist is an adjuvant derived from the LPS of Expanding on the beneficial effects of MPL®, we have developed a series of formulations containing a synthetic molecule called Glucopyranosyl Lipid A (GLA), which is optimized for binding to the human MD2 molecule of the TLR4 complex. GLA is active on mouse and human antigen presenting cells (APC) (H1)-biased immune response). The GLA-SE adjuvanted vaccine caused no adverse side effects in animals and enhanced the potency of the Fluzone vaccine both in terms of Ab and T cell responses and in broadened serological responses against drifted A/H1N1 and an A/H3N2 influenza variant(s) compared to vaccination with Fluzone alone.Here we evaluate the safety and immunogenicity of GLA in a stable-emulsion (GLA-SE) combined with seasonal influenza vaccines in mice and cynomolgus monkeys. The results demonstrate that the adjuvanted Fluzone vaccine had increased humoral and cellular immunogenicity  dose escalation studies examining the immunogenicity and efficacy of Fluzone+GLA-SE. Two IM administrations of 1 to 50 µg of GLA-SE in combination with Fluzone over 30 days were well tolerated by cynomolgus macaques .There was no evidence of injection site reactions or systemic toxicity in the animals receiving the Fluzone+GLA-SE vaccine or adjuvant alone. No definitive test-article-related changes in hematology were identified. Serum chemistry changes were limited to a mild display of a hepatic acute phase response (mild increases in both CRP and circulating fibrinogen concentrations) after each injection in groups injected with Fluzone+GLA-SE .We recently reported the immunological effects of combining Fluzone with a research-grade GLA in an oil-in-water emulsion in BALB/c mice In mice administered a single immunization, only Fluzone formulated with GLA-SE significantly increased HI titers against the vaccine and drifted A/H3N2 strains compared to immunization with Fluzone alone  and antigenically drifted strains  were measured.In human clinical studies, the criteria commonly used to assess vaccine immunogenicity include a) rates of seroconversion (a four-fold or greater increase in post-vaccination HI titer compared with pre-vaccination titer) or b) rates of achievement of an HI titer of ≥40. An HI titer of ≥40 is a serologic endpoint generally considered to be associated with at least a 50% reduction in the risk of infection or disease within a population In contrast to the observations made in mice, the serum HI Ab titers of NHP following one (day 30) or two immunizations (day 58) with Fluzone+SE were not significantly different compared to the group administered the Fluzone vaccine alone . NeverthA desirable feature of an adjuvanted seasonal influenza vaccine is the ability to induce more broadly cross-reactive Abs against antigenically drifted variants or homosubtypic heterovariant influenza strains that are not included in the vaccine for a given season NHP that were primed and boosted with escalating doses of Fluzone+GLA-SE exhibited higher HI titers not only against the vaccine strain but also to the most recent drifted influenza variant (A/H1N1/Brisbane), a component of the subsequent years vaccine formulation . HI respIn addition to testing HI responses to drifted H1N1 strains, ,HI titers were measured against the vaccine strain (A/H3N2/Wisconsin)  and one H1-type responses, play a critical role in the clearance of influenza viruses in animal models and are associated with protection against influenza-related illness in the elderly. We, therefore, assessed the ability of the GLA-SE- and SE-formulated Fluzone vaccines to elicit influenza-specific cellular responses. In initial studies, spleen cells from mice immunized with Fluzone alone or with either SE or GLA-SE were restimulated in vitro with the vaccine strain or with the antigenically drifted A/H3N2 influenza The levels of cytokine produced in response to stimulation by any A/H3N2 strain were comparable, suggesting that the cellular responses are most likely directed towards conserved proteins within the antigen mixture  vaccines produced increased H1N1- and H3N2-specific IFNγ responses four weeks after the second immunization compared to NHPs that were immunized with the Fluzone alone, Fluzone with SE, or Fluzone with 1 µg GLA-SE  revealed that NHP boosted with Fluzone alone and Fluzone+SE had specific H1N1 and H3N2 responses marked by lower TH1:TH2 cytokine ratios than those immunized with Fluzone+GLA-SE at 5, 25 and 50 µg  to assess antigen-specific cytokine responses of immunized and non-immunized monkeys . To evalg GLA-SE . All anig GLA-SE . Thus, ond 50 µg . The hignd 50 µg , as wellnd 50 µg .Next generation influenza vaccines that potentiate immune responses and elicit protection against a broader range of antigenically drifted strains are urgently needed. To further advance development of such vaccines, we have analyzed the impact on immune responses of incorporating a stable oil-in-water emulsion and a synthetic TLR4 agonist into a standard influenza vaccine. The experiments described in this report are the first to evaluate an oil-in-water emulsion (SE) with various concentrations of this synthetic TLR4 agonist (GLA), all in combination with Fluzone, a licensed trivalent inactivated influenza vaccine for its safety and ability to significantly enhance immune responses in both mice and NHP. With the ever increasing need for safe ways to either improve vaccine efficacy and/or to provide dose sparing effects, most candidate adjuvants fail to reach the market place because of safety issues including toxicity or reactogenicity particularly at the site of injection. Though increased levels of pro-inflammatory cytokines were observed in the context of GLA-SE, there was no evidence of injection site reactions or systemic toxicity in the animals receiving the Fluzone+GLA-SE vaccine or adjuvant alone.H1 cellular immune responses. Thus, our studies clearly demonstrate that GLA-SE provides further enhancement and broadening of the immune responses already elevated by the addition of SE to Fluzone.Our results demonstrate that incorporating SE into Fluzone enhances Ab responses. Furthermore, the incorporation of GLA with SE enhances the same Ab responses, stimulates production of Abs that cross-react with antigenically variant influenza strains not present in the vaccine, and generates TThe ability of SE to adjuvant flu vaccines, as observed in our studies, is in general agreement with those previously reported for another oil-in-water adjuvant, MF59™ Our results clearly demonstrate that, compared to vaccination with Fluzone alone or Fluzone formulated with SE, the further addition of GLA increases cross-protective HI Ab responses against antigenic variants. In this study, a single administration of Fluzone with GLA-SE leads to the generation of HI titers at seroprotective levels in a majority of NHP. GLA-SE further enhanced HI titers in NHP following two immunizations, as compared to either SE plus Fluzone or Fluzone alone. Previous studies have shown that other TLR agonists, such as the TLR9 agonist CpG, enhance cellular interactions within germinal centers, leading to the selection of B cells with a high affinity for antigen binding H1 to TH2 responses H2 response induced by traditional adjuvants such as Alum and MF59 ™ Our results also show that the addition of GLA-SE to the Fluzone vaccine selectively shifts the cellular immune response toward a type-1 cellular immune response pattern, characterized by the production of IFNγ. TLR4 agonists, such as MPL, have been shown to be potent adjuvants in a range of species, including humans H1 responses The weakened immune system of elderly people presents particular challenges in the development of vaccines and adjuvants for this population. There are well recognized changes in the function of APCs, T cells and B cells in older people. Several strategies to improve vaccination in the elderly have been explored. A number of studies have shown that the use of adjuvants (e.g. emulsions and TLR agonists) could trigger more potent activation of APCs. Like other TLR4 containing adjuvants, we have preliminary data demonstrating that GLA can induce robust immune responses in aged mice and humans whose immune systems have undergone significant decline with increasing age. Recent studies suggest that T cell responses, rather than Ab, may be a better immune correlate of protection against influenza in older adults but that this population overall may have impaired TH2 responses reported to adversely affect viral clearance in animal models H1 responses.The GLA-SE-adjuvanted Fluzone vaccine described in this study elicits high IFNγ:IL-10 and IFNγ:IL-5 ratios that clearly show a dominant type-1 cellular immune response. Potent virus-specific IFNγ responses are also thought to be capable of reversing the age-related impairments of CTL activity considered essential for viral clearance Female BALB/c mice 6–7 weeks  were maintained under SPF conditions by IDRI Animal Care and Use Committee (IACUC).Twenty-one confirmed healthy male Cynomologus macaques ages 4.7 to 6.6 years old and weights of 4.81 to 6.47 kg were housed at SNBL USA  in accordance with SNBL USA's IACUC. Monkeys were pre-screened for their influenza status, randomized by body weights and assigned into study groups.The 2006–2007 Fluzone influenza vaccine formulated for the Northern Hemisphere , containing antigens from A/New Caledonia/20/99(H1N1), A/Wisconsin/67/2005(H3N2) and B/Malaysia/2506/2004 was used for the murine studies. The 2007–2008 Northern Hemisphere Fluzone influenza vaccine containing antigens from A/Solomon Islands/3/2006(H1N1), A/Wisconsin/67/2005(H3N2) and B/Malaysia/2506/2004, was used for the NHP studies. Fluzone was combined with either IDRI's oil-in-water emulsion (SE) or SE containing a synthetic TLR4 agonist (GLA-SE), before administration to animals. Adjuvant formulations were prepared as previously described Mice were immunized IM twice, 3 weeks apart. Mice received a low dose  of the 2006–2007 Fluzone vaccine with or without SE, or GLA-SE (20 µg).NHP (N = 3/group) were immunized by IM injection on days 1 and 30 with the pediatric dose, which included 7.5 µg HA/strain resulting in a total HA dose of 22.5 µg, of the 2007–2008 Fluzone vaccine alone, or adjuvanted with SE, or escalating doses  of GLA-SE.To evaluate the ability of the vaccines to induce serum Ab responses against heterovariant influenza strains, vaccine and non-vaccine drifted influenza A strains were selected for use in HI assays and splenocyte cultures. A/Wisconsin/67/2005(H3N2)(Wis/05), A/Wyoming/03/2003(H3N2)(Wyo/03) and A/Panama/2007/99(H3N2)(Pan/99) strains were provided by the CDC and Prevention  and purified viral protein from A/Brisbane/10/2007(H3N2)(Bris/07) was purchased from ProSpec . All of the viruses used were grown in the allantoic cavity of embryonated eggs and were stored at −80°C until use.Injection sites were evaluated prior to each inoculation, at 2 hours ±30 minutes and again at 24, 48, 72, 96, 120 and 144±1 hour after each dose, using a Draize dermal irritation scoring system For the NHP HI assays, the 2007 vaccine components, including A/Solomon Islands/3/2006(H1N1), A/Wisconsin/67/2005(H3N2) and B/Malaysia/2506/2004 plus all drifted A (H1N1) strains incorporated in seasonal influenza vaccines since 1998 including A/Brisbane/59/2007(H1N1), A/New Caledonia/20/99(H1N1) and A/Beijing/262/1995(H1N1) and the drifted A/Uruguay/716/2007(H3N2) influenza strains were evaluated. All viruses were provided by the Centers for Disease Control and Prevention .Antigen-specific mouse bone marrow plasma cells were quantified by ELISPOT as previously described, five weeks after the second immunization (7). Purified and concentrated A/Wisconsin/67/2005(H3N2)(Wis/05), A/Brisbane/10/2007(H3N2)(Bris/07) and A/Panama/2007/99(H3N2)(Pan/99) viral antigens were purchased from ProSpec for use in the ELISPOT.Seven weeks after the second immunization, mice were euthanized and spleens (N = 4/group) were harvested. Splenocytes were cultured with 5 HAU of inactivated A/Wisconsin/67/2005(H3N2)(Wis/05), A/Brisbane/10/2007(H3N2)(Bris/07), A/Wyoming/03/2003(H3N2)(Wyo/03) or A/Panama/2007/99(H3N2)(Pan/99) influenza virus at 37°C in 5% CO2 for 72 hrs following a method adapted from Lu et al 2 for 24 hours, 200 µL plasma was collected and IFNγ,? TNF, IL-2, IL-6, IL-5, IL-4, Granzyme B, MCP3 and IP-10 levels measured using a TH1/TH2 cytometric bead array (CBA) kit (Becton Dickinson) or a custom Luminex panel (Panomics).Monkeys were bled at day 0 and 58 to evaluate cellular immune responses by whole blood assay (WBA). Heparinized blood (450 µL) was dispensed into 48-well plates . Phytohemagglutinin , Fluzone (9 µg/mL), purified rH3 from A/Wisconsin/67/2005(H3N2) (10 µg/mL) , inactivated A/Solomon Islands/3/2006(H1N1) virus (10HAU/mL) (provided by the Centers for Disease Control and Prevention) or saline was then added to provide stimulation in a final volume of 500 µL/well in duplicate. Following incubation at 37°C, 5% COSera were collected from mice one week after each immunization. Anti-HI Abs specific to the A/H3N2 component of the 2006–2007 Northern Hemisphere Fluzone formulation, A/Wisconsin/67/2005(H3N2)(Wis/05), and to the A/H3N2 component of other seasonal influenza vaccines used since 2001 including A/Brisbane/10/2007(H3N2)(Bris/07), A/Wyoming/03/2003(H3N2)(Wyo/03) or A/Panama/2007/99(H3N2)(Pan/99) were determined as previously described using 0.5% turkey RBC Sera were collected from NHP on Day 1, 30 and 58 and evaluated for the presence of hemagglutination inhibiting antibodies specific to the 2007–2008 Northern Hemisphere Fluzone components including A/Solomon Islands/3/2006(H1N1), A/Wisconsin/67/2005(H3N2) and B/Malaysia/2506/2004 and also against drifted A (H1N1) strains incorporated in seasonal influenza vaccines since 1998 including A/Brisbane/59/2007(H1N1), A/New Caledonia/20/99(H1N1) and A/Beijing/262/1995(H1N1) and the drifted A/Uruguay/716/2007(H3N2) influenza strain by means of established methods, using four hemaglutinating units (HAU) of virus and 0.5% turkey RBCs). NHP sera were treated with receptor destroying enzyme followed by heat treatment  before use in the HI assay. Three separate HI assays were conducted on each serum sample and each individual serum sample was assayed in duplicate in each assay. The seroprotective rate was defined as the percentage of animals per vaccine group that achieved a post-vaccination HI titer of ≥ 40 HI. Preimmune titers in all animals were ≤10.Statistical analysis was performed by a standard one-way ANOVA with the Tukey Multiple Comparison Test between the groups using GraphPad Prism .Figure S1Virus-specific cytokine responses 7 weeks post boost with the 2006-2007 Fluzone influenza vaccine alone (-) or formulated with either an oil and water emulsion (SE) or a synthetic TLR4 agonist formulation (GLA-SE). Splenocytes were harvested from 4 mice/vaccine group and cultured with 5 HAU of inactived Wis/05, Bris/07, Wyo/03 or Pan/99 influenza strains. Supernatants were collected 72 hours post culture and analyzed by ELISA. Values represent the average virus-specific cytokine responses detected in cultures +SD. One asterisk represents significantly greater responses to Fluzone alone (p<0.05). Two asterisks represents significantly greater responses to Fluzone+SE (p<0.05). Three asterisks represents significantly greater responses to Fluzone +GLA-SE (p<0.05).(0.06 MB PDF)Click here for additional data file.Table S1(0.05 MB DOC)Click here for additional data file.Table S2(0.04 MB DOC)Click here for additional data file.Table S3(0.04 MB DOC)Click here for additional data file.


The dual specificity phosphatase cdc25C was the first human cdc25 family member found to be essential in the activation of cdk1/cyclin B1 that takes place at the entry into mitosis. Human cdc25C is phosphorylated on Proline-dependent SP and TP sites when it becomes active at mitosis and the prevalent model is that this phosphorylation/activation of cdc25C would be part of an amplification loop with cdk1/cyclin B1.Using highly specific antibodies directed against cdc25C phospho-epitopes, pT67 and pT130, we show here that these two phospho-forms of cdc25C represent distinct pools with differential localization during human mitosis. Phosphorylation on T67 occurs from prophase and the cdc25C-pT67 phospho-isoform closely localizes with condensed chromosomes throughout mitosis. The phospho-T130 form of cdc25C arises in late G2 and associates predominantly with centrosomes from prophase to anaphase B where it colocalizes with Plk1. As shown by immunoprecipitation of each isoform, these two phospho-forms are not simultaneously phosphorylated on the other mitotic TP sites or associated with one another. Phospho-T67 cdc25C co-precipitates with MPM2-reactive proteins while pT130-cdc25C is associated with Plk1. Interaction and colocalization of phosphoT130-cdc25C with Plk1 demonstrate in living cells, that the sequence around pT130 acts as a true Polo Box Domain (PBD) binding site as previously identified from in vitro peptide screening studies. Overexpression of non-phosphorylatable alanine mutant forms for each isoform, but not wild type cdc25C, strongly impairs mitotic progression showing the functional requirement for each site-specific phosphorylation of cdc25C at mitosis.These results show for the first time that in human mitosis, distinct phospho-isoforms of cdc25C exist with different localizations and interacting partners, thus implying that the long-standing model of a cdc25C/cdk1 multi-site auto amplification loop is implausible. Mammalian cell mitosis is a dynamic process involving inactivation of interphase signaling pathways and activation of mitotic kinases, particularly cyclin dependent kinases (cdks) Here, we have investigated the site-specific phosphorylation of cdc25C on proline directed threonines 67 and 130, and the cytolocalization and timing of these phosphorylations in dividing human cells. Using site-specific affinity purified antibodies for these sites, we show they are phosphorylated in mitotic cells but in distinct pools of cdc25C with different cytolocalization.Anti-phospho-site antibodies are specific for single TP phosphorylation events.Human cdc25C is phosphorylated on three TP motifs  and one SP motif (S214) during mitosis in human cells To confirm that these phospho-site directed antibodies were specific for cdc25C, we next compared the protein patterns between normal asynchronous Hela extracts and extracts from Hela cells in which cdc25C had been ablated by RNA interference We next confirmed the specificity for each phospho-site on cdc25C by reacting the antibodies with Wt and alanine mutant cdc25C proteins phosphorylated in vitro by cdk1/cyclin B1. As shown in To confirm the existence of cdc25C phospho forms in other cells types, we analyzed extracts from asynchronous transformed U2OS and normal non-transformed human fibroblast. As shown in Finally, we analyzed the profile for each phospho-site anti-cdc25C in synchronized Hela cells. We next analyzed the cytolocalization of anti-pT67 and anti-pT130 in synchronized human fibroblasts  and phosphatase attenuators, PBS, beta-glycero-phosphate, sodium vanadate and fluoride c.f. . As showThe cytolocalizations observed for pT67-cdc25C and pT130-cdc25C are similar to those described for a number of mitotic factors including MPM2 antigens To confirm the centrosomal localization of cdc25C-pT130, we undertook detailed confocal analysis of Plk1 and pT130-cdc25C cytolocalization under different conditions. As shown in Finally, we examined the effects of transfecting cells with non-phosphorylatable mutant forms of cdc25C into U2OS cells and scoring mitotic progression. As shown in We show here differential spatial localization of 2 phospho-isoforms of cdc25C. We have developed mono-specific antibodies to the proline-linked phosphorylation sites on human cdc25C which are phosphorylated at mitosis. The antibodies demonstrate for the first time that different phospho-forms of cdc25C exist which have differential spatial organization during human mitosis and associate with different partner proteins. Furthermore, each phosphorylated isoform of cdc25C is not simultaneously phosphorylated on the other TP sites on the same cdc25C molecule. Finally, we show that non-phosphorylatable mutant forms of cdc25C impair mitotic progression in human cells. These data are the first to identify that separate phospho-forms of cdc25C with specific localizations and protein partners exist in human cells, thus questioning the long standing model of an auto-amplification mechanism involving cdc25C and cdk1 in mitotic activation.We have developed antibodies to the proline dependent kinase motifs T48P, T67P, T130P which we have previously identified as being phosphorylated during human mitosis We show here that human cdc25C phosphorylated on T130 is localized at the centrosomes during mitosis in both normal and transformed cells. Furthermore pT130-cdc25C phospho form physically interacts with the mitotic kinase Plk1 which is known to phosphorylate cdc25C on serine 198 The centrosomal localization of phospho-cdc25C is resistant to microtubule destabilization by cold showing true centrosomal localization of pT130-cdc25C, i.e. not dependent upon intact spindle poles. Interestingly cdc25B has also been implicated in modulation of cdk1 activity at the centrosome We show that expression of non-phosphorylatable (T67A and T130A) forms of cdc25C in U2OS inhibit cell cycle progression at mitosis in a manner similar to the dominant negative phosphatase inactive C377S form of cdc25C . For T13Finally, show here for the first time that different phospho-forms of human cdc25C exist in mitotic cells, that are not simultaneously phosphorylated on the 3 TP motifs phosphorylated during mitosis in human cells Primary immortalized human fibroblasts, T11HT, a generous gift from Dr. Vincent Mouly  and transformed Hela cells were grown in Dulbeccos MEM, sub-cultured onto glass cover slips and synchronized as described previously Synthetic peptides corresponding to residues 44–52 ), 63–71 (T67-LSGGTPKCC and T67-LSGG-*TPKCC) and T130, 127–135 (T130-LCSTPNGLD) and 127–135 (T130-LCS*TPNGLD) of human cdc25C were conjugated with keyhole limpet heamocyanin and injected into rabbits. Peptides were synthesized and coupled by Neosystems, Strasbourg, France. Before immunoblotting, immunoprecipitation or immunofluorescence, sera were double affinity purified on ECH Sepharose 4B columns  coupled with the non-phosphorylated or immune phospho-peptides, using the manufacturer's instructions. All antisera where first passed through the non immune- non –phosphorylated peptide column and unbound antibodies subsequently bound and eluted from the phospho-peptide column. Antisera were stored at 4°C, while purified antibodies at −80°C in 5% glycerol.Total extracts from cells were electrophoretically separated on 10% SDS-polyacrylamide gels and immunoblotted as previously described For immunofluorescence, cells were fixed in 3.7% (v/v) formalin in PBS or in –20°C methanol as described previously N-terminally HA-tagged human cdc25C (accession number GB:M34065) in pECE was a generous gift from Dr. Franck McKeon, Harvard Medical School. Plasmids were subjected to PCR based site directed mutagenesis using the Quick-change method  according to the manufacturers instructions. Complementary oligonucleotide pairs to:gCTCCAGTGGGCAAATTT-35-GTCCAGATGTCCCTAGA),T48A (gCtCCAAAATGTTGCCTCGATC-35-GCATTTTGTCTGGAGGA),T67A (gCTCCGAATGGTTTGGAC-35-CACAGCTTCTTTGTAGC) where used.T130A  according to the manufacturers instructions. Each plasmid was sequenced. Proteins were expressed in in Bl21-DE3 and purified by standard nickel chelating chromatography.Figure S1Antigenic Peptides and in vitro phosphorylation of cdc25C and mutant forms. Panel A: Multiple sequence alignments of the 3 proline directed threonine phosphorylation sites on human cdc25C phosphorylated at mitosis. Shown are 20 AA sequences at each site in Xenopus, rat, mouse pig and human. Right hand of each block of aligned sequences is the phospho-peptide used as antigen in this study. Panel B: Typical western blot analysis of purified human cdc25C protein using anti-pT48, anti-pT67 and anti-pT130 with (+) or without (−) phosphorylation by cdk1/cyclin B1. Lower panels show the same membranes blotted with anti-cdc25C. Panel C, Purified cdc25C Wt, T48A, T67A and T130A were phosphorylated in vitro and blotted for cdc25C or the anti-phospho-site antibodies. For each protein, the intensity of the bands recognized by the anti-phospho-antibodies was normalized to the staining for cdc25C protein and plotted as a percentage of the overall cdc25C staining. The results were obtained from 3 different experiments.(0.02 MB PDF)Click here for additional data file.Figure S2Staining for cdc25C phospho-T67 cdc25C is specifically abolished by incubation with the phosho-peptide. Asynchronous human fibroblasts were fixed and stained for DNA  or anti-pT67-cdc25C (B and D). Anti-pT67 antibodies were incubated with the non-phosphorylated peptide (B) or phosphorylated peptide (D). Shown are fluorescent photo micrographs of metaphase cells in each panel. Bar 5 µM.(0.05 MB PDF)Click here for additional data file.Figure S3Staining for cdc25C phospho-T67 cdc25C is present on chromatin in transformed cells. Asynchronous U2OS cells were fixed and stained for DNA  or anti-pT67-cdc25C . Shown are fluorescence photo micrographs of representative cells in prophase (A–B), prometaphase (C–D), prometaphase and metaphase (E–F) or anaphase B (G–H). Bar 5 µM.(0.12 MB PDF)Click here for additional data file.Figure S4Staining for cdc25C phospho-T130-cdc25C is specifically abolished by incubation with the phosho-peptide and centrosomal staining is present in transformed cells. Asynchronous human fibroblasts were fixed and stained for DNA  or anti-pT130-cdc25C (B and D). Anti-pT130 antibodies were incubated with the non-phosphorylated peptide (B) or phosphorylated peptide (D). Shown are fluorescence photo micrographs of typical staining patterns in prophase cells in each panel. Panels E and F show staining for pT130-cdc25C in late prometaphase and metaphase U2OS cells. Bar 5 µM.(0.06 MB PDF)Click here for additional data file.Figure S5Endogenous cdc25C proteins are present in the nucleus and on the centrosomes of non-transformed human fibroblasts during mitosis. Asynchronous human fibroblasts were fixed and stained for DNA , cdc25C  and tubulin . Shown are fluorescence photo micrographs of cells in early prophase, early prometaphase and late prometaphase. Bar 5 µM.(0.11 MB PDF)Click here for additional data file.Figure S6cdc25C phosphorylated on T130 remains associated with the centrosomes at low temperatures even after Plk1 localization has been lost: Asynchronous human fibroblasts were fixed and stained for DNA (panels A–E), cdc25C-pT130 (F–J) and Plk1 (K–O). Shown are fluorescence photo micrographs of cells in different mitotic phases after incubation at 37°C  or at 4°C for 60 minutes  or 120 minutes . Bar 5 µM.(0.05 MB PDF)Click here for additional data file.Figure S7Low levels of expression of mutated forms of cdc25C does not perturb the localization of endogenous phospho-cdc25C isoforms. Transformed U2OS cells were transfected with HA-tagged cdc25C T67A or T130A. 24 hours after transfection, cells were fixed and stained for the HA-tagged-cdc25C (panels A and B), cdc25C-pT67 (panel C), cdc25C-pT130 (panel D) or DNA (panels E and F). Shown are typical photo micrographs of cells expressing low levels of HA-tagged cdc25C. Bar 5µm.(0.07 MB PDF)Click here for additional data file.