The RV144 Phase III clinical trial with ALVAC-HIV prime and AIDSVAX B/E subtypes CRF01_AE (A244) and B (MN) gp120 boost vaccine regime in Thailand provided a foundation for the future development of improved vaccine strategies that may afford protection against the human immunodeficiency virus type 1 (HIV-1). Results from this trial showed that immune responses directed against specific regions V1V2 of the viral envelope (Env) glycoprotein gp120 of HIV-1, were inversely correlated to the risk of HIV-1 infection. Due to the low production of gp120 proteins in CHO cells (2–20 mg/L), cleavage sites in V1V2 loops (A244) and V3 loop (MN) causing heterogeneous antigen products, it was an urgent need to generate CHO cells harboring A244 gp120 with high production yields and an additional, homogenous and uncleaved subtype B gp120 protein to replace MN used in RV144 for the future clinical trials. Here we describe the generation of Chinese Hamster Ovary (CHO) cell lines stably expressing vaccine HIV-1 Env antigens for these purposes: one expressing an HIV-1 subtype CRF01_AE A244 Env gp120 protein (A244.AE) and one expressing an HIV-1 subtype B 6240 Env gp120 protein (6240.B) suitable for possible future manufacturing of Phase I clinical trial materials with cell culture expression levels of over 100 mg/L. The antigenic profiles of the molecules were elucidated by comprehensive approaches including analysis with a panel of well-characterized monoclonal antibodies recognizing critical epitopes using Biacore and ELISA, and glycosylation analysis by mass spectrometry, which confirmed previously identified glycosylation sites and revealed unknown sites of O-linked and N-linked glycosylations at non-consensus motifs. Overall, the vaccines given with MF59 adjuvant induced higher and more rapid antibody (Ab) responses as well as higher Ab avidity than groups given with aluminum hydroxide. Also, bivalent proteins (A244.AE and 6240.B) formulated with MF59 elicited distinct V2-specific Abs to the epitope previously shown to correlate with decreased risk of HIV-1 infection in the RV144 trial. All together, these results provide critical information allowing the consideration of these candidate gp120 proteins for future clinical evaluations in combination with a potent adjuvant. In Thailand, HIV-1/AIDS was first reported in the 1980s, and since then, over one million Thai adults have become infected, with nearly 600,000 having died from AIDS-related diseases, with an estimated 7,816 new HIV-1 infections and 20,492 AIDS related deaths in 2014 alone . While sThe HIV-1 envelope glycoprotein (Env) is a critical target for the host immune system and is currently the major focus of the rational immunogen design for the next generation of HIV-1 vaccines. The viral Env is composed of the gp120 surface and gp41 transmembrane glycoproteins that together form trimeric structures to decorate the surface of native virions and mediate interaction with its host cell surface receptor (CD4), which occurs via the CD4 binding site (CD4bs) domain of the gp120 protein. The CD4bs and other regions of gp120 represent critical epitopes recognized by the host’s immune system and are targets for broadly reactive neutralizing antibodies (nAbs) as well as other functional antibody (Ab) responses –11.RV144 was a double-blind and randomized Phase III clinical trial conducted in Thailand evaluating a prime-boost vaccine strategy for the prevention of infection and amelioration of disease course in HIV-1-negative volunteers who were at community-risk of HIV-1 infection. This trial showed 31.2% efficacy 42 months after the first immunization and 60% efficacy at 12 months . The vacZM96 (subtype C) and a new bivalent protein boost composed of gp120s from subtype C, HIV-1TV1 and HIV-11086 [A comprehensive immune correlates of risk analysis in RV144 revealed that Ab responses to variable loops 1 and 2 (V1/V2) on gp120 correlated inversely with infection risk –17, and IV-11086 , 26. TheIV-11086 , 28, in IV-11086 .MN, and subtype CRF01_AE, HIV-1A244) should be substituted with gp120s from other strains. An evaluation of the antigenicity of these proteins demonstrated that the V2 loop was better exposed on the A244 gp120 Env compared to MN gp120 when assessed with 697-D mAb that recognizes a conformational V2 epitope [In addition to the P5 efforts in the southern Africa region, a Product Development Advisory Group (PDAG) recommended the need to optimize protein (antigen) and adjuvant selections for the next vaccine candidates in Thailand. Because two gp120 proteins, one from subtype B and one from subtype CRF01_AE were used in the RV144 trial, the group decided to proceed with a similar bivalent composition of monomeric gp120. Importantly, the group also considered if the gp120 proteins used in RV144  protein and one expressing the subtype CRF01_AE A244 gp120 protein (A244.AE) at sufficient levels for future development and manufacturing. We reported results of a comprehensive evaluation of antigenicity of the purified proteins using a panel of well-characterized HIV-1-specific mAbs, glycosylation mapping and profile analyses by mass spectrometry, and an evaluation of immunogenicity using monovalent and bivalent combinations of the gp120s in guinea pigs with either AH, the adjuvant used in RV144, or MF59, the adjuvant being evaluated in the HVTN100/702 clinical trials, the latter with the goal of potentially improving the titer and the kinetics of the immune responses. Since RV144 provided a baseline for potentially protective efficacy based on the Ab responses, we focused on evaluating Ab responses for the HIV-1 vaccine candidates in this study.Codon-optimized A244.AE and 6240.B gp120 coding sequences with 11 amino acids deleted from N-terminus (Δ11 gp120) were generated synthetically using a heterologous signal leader sequence (Life Technologies). The expression cassettes were cloned into a plasmid vector containing two selectable markers, a Neomycin resistance marker and mutated dihydrofolate reductase gene (DHFR), for Chinese Hamster Ovary (CHO-K1) stable cell line development. The CHO-K1 was obtained from American Type Culture Collection (ATCC).CD4-IgG2 (sCD4) was purchased from Progenics Pharmaceuticals. The monoclonal antibodies (mAbs) CH01 , CH58 anExpression plasmids were transfected into the Novartis proprietary CHO K1PD parental cell line, which was derived originally from CHO-K1 (ATCC), by electroporation. Transfected CHO cell pools were subjected to two sequential selections, 0.8 mg/ml geneticin (G418) for ~ 14 days and 1 μM methotrexate (MTX) for an additional 14 days. The CHO transfected pools were then evaluated and selected based on cell viability and gp120 expression (A244.AE or 6240.B). Proteins were purified from culture media of CHO transfected pools by lectin affinity chromatography and analyzed by RP-HPLC, SEC-HPLC and SDS-PAGE as previously described [2 and shaken at 150 rpm for seven days. Cell cultures were harvested by centrifugation and the supernatants were sterile filtered prior to protein purification.Single CHO clones were then isolated from transfected cell pools by serial dilutions in 96-well plates. Positive clones were selected based on the cell viability and level of gp120 expression. The top ~ 20 clones for each construct were subjected to 12-week stability and 3 consistency cultures to select for the best-performing clones. In the stability study, a continuous parent culture was run for each clone with passaging every 4 days for 17 passages. The cells from the continuous culture were used to inoculate 3 intermittent terminal batch (TB) bioreactors, which were cultured for 7 days before harvest. Top CHO cell lines were selected based on the quantity and consistency of gp120 expression. The final cell lines for A244.AE or 6240.B gp120s were seeded at a density of 0.26 cells/mL in culture media and incubated at 36.5°C, 10% COThe harvested media were concentrated 5-fold and buffer exchanged into 20 mM Tris-HCl (pH 8.0 for A244.AE and pH 8.5 for 6240.B gp120) with 5 mM NaCl and loaded on a DEAE column (GE Life Sciences). The gp120 protein was eluted from the DEAE resin by 90 mM NaCl for A244.AE or 65 mM NaCl for 6240.B gp120. The eluted proteins were then buffer exchanged into 10 mM NaCl, 20 mM Tris-HCl (pH 8.0 for A244.AE and pH 8.5 for 6240.B gp120) and loaded onto a Fractogel SO3 (EMD) column. The Fractogel SO3 column captured additional contaminants and the purified gp120 product flowed through. The purified recombinant proteins were concentrated and buffer exchanged to 150 mM NaCl, 20 mM Tris-HCl pH 7.5 and finally, sterile filtered through a 0.22 μm membrane. The molecular weights and purity of the gp120 proteins were assessed by SEC-HPLC (Agilent SEC 3) and SDS-PAGE gel analysis. Throughout the purification process gp120 protein concentration was monitored by RP-HPLC. For this, media and intermediate purification products were injected on a Poros R1/10 column (PerSeptive Biosystems) equilibrated with 0.1% TFA in 5% ACN. gp120 proteins were eluted by a 30–35% ACN gradient in 0.1% TFA. gp120 elution peaks were selected and measured against standard gp120 A244.AE or 6240.B protein of known concentration. To evaluate the reproducibility of the protein production and purification processes, at least three independent batches were run and analyzed for each gp120 antigen.For early analytical purposes, gp120s were purified using lectin affinity purification using agarose bound Galanthus nivalis lectin resin . The resin was initially washed 3 times with phosphate buffered saline pH 7.4  using 3 times the resin volume for each wash. Resin was resuspended at 50/50 (v/v) resin/PBS, and added directly to harvest culture media. 3 ml total resin was used per 100–150 ml cultures. The mixture was incubated at room temperature for 30 min with gentle rocking, and then poured over a gravity flow column (Bio-Rad), and media was allowed to flow through. Accumulated resin was washed with 5 column volumes PBS followed by gp120 elution using 3 column volumes of 1 M Methyl-alpha-D-Mannopyranoside added manually at approximately 1 ml/min. Eluted gp120 was dialyzed into 20 mM Tris pH 8.0, 150 mM NaCl buffer for analysis.The gp120 proteins were denatured and reduced at 100°C for 10 min with RapiGest (Waters) and Dithiothreitol (DTT), at 0.1% (w/v) and 10 mM respectively, followed by alkylation with 20 mM Iodoacetamide (IAA) at room temperature for 30 min in the dark. Excess of IAA was quenched by adding DTT to a final concentration of 25 mM and incubated at room temperature for 15 min. The gp120 proteins were then de-N-glycosylated with PNGase F (New England Bio Labs) for 15 min, 60 min or overnight, or Endo H (New England Bio Labs) or Endo F3 (Sigma-Aldrich) for 8 hours at 37°C, as described by the manufacturer.Deglycosylated proteins were digested with trypsin or pepsin using an enzyme/substrate ratio of 1/20 (w/w) and incubated overnight at 37°C. Prior to pepsin digestion, 1% (v/v) final concentration of formic acid (FA) was added (pH 2.0). Each digestion was subjected to an off-line desalting procedure using OASIS cartridges (Waters) following the manufacturer’s protocol and analyzed by LC-MS/MS on an ESI Synapt G2 or an ESI QTOF Premier mass spectrometer coupled to a NanoAcquity UPLC system (Waters). Samples were loaded onto a NanoAcquity 5 μm Symmetry C18 trapping column , using full loop injection, for 3 min at a flow rate of 5 μl/min with mobile phase A (2% (v/v) ACN, 0.1% (v/v) FA) and were then separated on a NanoAcquity 1.7 μm BEH130 C18 analytical column  using a 30-min gradient of 2–40% mobile phase B (98% (v/v) ACN, 0.1% (v/v) FA) at a flow rate of 300 nl/min.m/z 204.1 for HexNAc, m/z 366.1 for HexNAcHex, m/z 292.1 for NeuAc, m/z 274.1 for NeuAc-H2O) and manually validated. The presence of sulfated residues was analyzed by using sulfation  as variable modifications in the program MASCOT.Peptide identification was carried out from the generated peak list using the Mascot software 2.4  run on a custom database comprising the sequences of the two recombinant gp120 proteins. The Mascot search parameters were set as follows: (a) enzyme: trypsin or unknown (for pepsin search); (b) missed cleavage: 1; (c) fixed modification: carbamidomethyl (C); (d) variable modifications: oxidation (M) and deamidation  associated with HexNAc (N) or HexNAc(1)dHex(1) (N) or Hex(1)HexNAc(1)  or Hex(1)HexNAc(1)NeuAc(1)  or Hex(1)HexNAc(1)NeuAc(2) ; (e) peptide tolerance 0.3 Da and (f) MS/MS tolerance of 0.3 Da. Glycopeptides assigned by Mascot search were confirmed by the MS/MS signal of characteristic sugar ions  treated with PNGase F and trypsin were completely dried in a Concentrator plus (Eppendorf) and resuspended with 10 μl of 0.1% TFA in 3% ACN. Samples (1 μl) were mixed on a ground steel target in a 1:1 ratio (vol/vol) with the matrix consisting of 2,5-DHB dissolved at 20 mg/ml in 0.1% (v/v) TFA in 30% (v/v) ACN supplemented with 1 mM NaCl and left to dry by air. The matrix crystals were made uniform by addition of 0.2 μl ethanol, causing rapid recrystallization.m/z. Spectra were externally calibrated by using a peptide calibration standard  spotted on the target. MS spectra were analyzed with FlexAnalysis 2.4  using default parameters and manually revised. The obtained peak list was analyzed by GlycoWorkbench software 1.2.4105 a tool for the computer-assisted annotation of glycan mass spectra using in this case the internal database of CHO cells and literature match.Mass spectra were acquired on an MALDI-TOF/TOF Ultraflex mass spectrometer  in reflectron positive mode in the mass range of 700–3500 Binding of the gp120 proteins to various analytes (sCD4 and mAbs) was determined using direct ELISA and Surface Plasmon Resonance (Biacore T200). For direct ELISA, either A244.AE or 6240.B gp120 protein was coated onto 96-well plates for 12 hours at room temperature. The wells were blocked with 1% (w/v) BSA in PBS for 60 min. Serial dilutions of human anti-gp120 IgG mAbs (with blocking buffer and 0.1% (w/v) Triton X-100 as diluent) were added to wells and incubated for 60 min, followed by a 3x rinse with PBS containing 0.05% Tween. For detection, a secondary anti-human Ab coupled with HRP (in diluent used above) was incubated for 60 minutes on the wells, followed by a 3x rinse with PBS containing 0.05% Tween. For development, TMB substrate (Rockland) was used, and the reaction stopped by adding 2.0 N sulfuric acid. All steps were carried out at room temperature. The optical density was determined spectrophotometrically at 450 nm wavelength using a microplate reader. EC50 values were calculated using Graphpad Prism software employing variable slope dose-response curves with a shared top and bottom to fit the data.2). For quality control, every kinetic assay required the following: mass transfer (t >108), closeness of the fit (percentage of chi2/Rmax < 5%), and the uniqueness of the calculated rate constants and Rmax U value (< 25).For the Biacore T200  antigenicity measurements, immobilization procedures were carried out using a standard amine coupling method with 10 mM Hepes, 150 mM NaCl, and 0.0025% Tween-20, pH 7.4 (HBS-0.0025%T). The CM5 chip surface was activated by a 1:1 mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.1 M N-hydroxysuccinimide (NHS) for 600 s with a flow rate of 10 μl/min. Next, 10 μg/ml of either anti-human IgG Fc (Bethanyl) or anti-RSV Synagis Palivizumab for a reference flow cell  was immobilized to obtain 5,000–9,000 RU. The immobilized surface was then deactivated with 1.0 M ethanolamine-HCl pH 8.5 for 600 s. Different mAbs were captured , followed by injection of different concentrations of A244.AE or 6240.B gp120 proteins. The bound surface was regenerated with 125 mM HCl for 45–60 s. Each kinetic assay was repeated at least three times. The data were analyzed using the Biacore T200 Evaluation software v1.0  and the data were fitted into a 1:1 Langmuir model using double subtraction (reference flow cell and the running buffer). Parameters including association rate constant (ka), dissociation rate constant (kd), maximum response (Rmax) and mass transfer constants (tc) were fitted globally. Closeness of the fit was assessed by chi-square  in compliance with the Animal Welfare Act and in accordance with the principles set forth in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animals resources, National Research Council, National Academy Press, 2011 edition.Female Hartley Guinea pig (Charles River Labs) with body weights of 250–275 g  were immunized using bilateral intramuscular injections (250 μl (each) in the quadriceps on weeks 0, 4, 8, and 12 with 25 μg of each gp120 protein formulated with adjuvant MF59 (1:1 volume ratio) or adsorbed to aluminum hydroxide . These included Group 1 (6240.B + MF59), Group 2 (A244.AE + MF59), Group 3 (A244.AE + 6240.B + MF59), Group 4 (6240.B + AH), Group 5 (A244.AE + AH) and Group 6 (A244.AE + 6240.B + AH). Serum samples were collected prior to the first immunization (pre-bleed) and four weeks following the first, second, third and fourth immunizations . The method of anesthesia used during this study was Isoflurane and animals were monitored during treatment for respiratory rate, body temperature, heart rate and absence of pedal reflex. The method of sacrifice was C02 overdose with confirmation by exsanguination. Methods to reduce any pain or suffering during the study included the following procedures: Animals were monitored regularly for clinical signs and symptoms, at least once weekly from the time of receipt at facility; directly after the vaccination, then one day after the vaccination and then once a week, and during recovery from anesthesia, at one day post-procedure and weekly thereafter, and appropriate treatment was administered as required.gp120- and scaffolded gp70-V1V2-specific binding Ab titers were measured by a standard ELISA format as previously described , 45. BriBinding Abs reactive with cyclic V2 and V3 peptides were measured using N-terminal biotinylated peptides  using SPR Biacore 4000 . The surface of sensor chip CM5 was activated using EDC/NHS amine coupling followed by immobilization of 1 μM Streptavidin  in 10 mM sodium acetate pH 4.5 to obtain 4,000–5,000 RU. The immobilized surface was then deactivated with 1.0 M ethanolamine-HCl pH 8.5 for 600 s. Spot 3 in each flow cell was left unmodified to serve as a reference. After the surface deactivation, the following cyclic biotinylated peptides were captured to obtain 1,200–2,200 RU:CSFNMTTELRDKKQKVHALFYKLDIVPIEDNTSSSEYRLINCV2.AE (92TH023): CTRPSNNTRTSINIGPGQVFYRTGDIIGDIRKAYCV3.AE (92TH023): CSFNITTGIGNKMQKEYALFYKLDVVPIDSNNNSDNTSYRLISCV2 (6240.B): CTRPNNNTRKGIHIGLGQALYATGDIIGDIRQAHV3 (6240.B): For SPR measurements evaluating gp120-specific binding Abs in guinea pig immune sera, the A244.AE or 6240.B gp120 proteins were immobilized directly on the activated EDC/NHS CM5 chip to obtain 250–5,000 RU. Following the surface preparation, control mAbs and individual guinea pig serum samples were diluted (1:300) in 10 mM HEPES, 300 mM NaCl and 0.005% Tween 20. Quadruplicate samples were injected at a flow rate of 10 μl/min at 25°C onto the protein captured surface for 250 s followed by a dissociation period of 1800 s. The signal was then enhanced with a 200 s injection of 30 μg/ml goat anti-guinea pig IgG Ab. To regenerate the bound surface, 125–250 mM HCl was injected for 60 s and repeated twice. Data analysis was performed using Biacore 4000 Evaluation Software 4.1 . The binding Ab responses were then normalized using double subtraction to the unmodified reference surface and the blank buffer injection). For avidity analysis, the 1:1 (Langmuir) dissociation fitting was applied, given by the formula, R0 * exp(-kd * (t—t0)) + Offset, where kd is the dissociation rate constant (1/s); R0 is the response units at the start of the fitted data; t0 is time at the start of the fitted data; and offset is the residual response units at infinite time.Linear epitope mapping was performed as previously described , 47 withVirus neutralizing Ab activity was quantified using the well-standardized assay employing HIV Tier 1 and 2 pseudoviruses and a luciferase reporter gene assay in TZM-bl cells as reported previously .th and 75th percentiles. The whiskers denote the most extreme data points that are no more than 1.5 times the interquartile range . As necessary, the plots are drawn on the base 10 log scale. Only significant p values less than 0.05 are displayed above the boxplots when comparing two groups. To determine a difference in response magnitude between groups at specific time points the Wilcoxon rank-sum test was used based on the exact distribution and using the mid-ranks method for ties [Graphical distributions of response magnitudes are plotted for each group and time point with box plots superimposed on the distributions magnitudes. The mid-line of the box denotes the median and the ends of the box denote the 25for ties . To detefor ties . All tesFollowing a consultation with HIV vaccine experts assembled by the US Military HIV Research Program and the National Institute of Allergy and Infectious Diseases in November, 2011, recombinant gp120s from the HIV-1 subtypes CRF_01 AE A244 (A244.AE) and B 6240 (6240.B) were selected as potential candidate protein boosts for consideration for future clinical trials in Thailand [ lanes 1, 2), and three bands, one dominant intact gp120 band and two cleaved gp120 bands by SDS-PAGE under reduced conditions . N-terminal sequencing  of the cleaved bands showed that the cleavage site was in the V2 loop of gp120 and consensus to that for aspartic acid protease , which is known to be active at acidic pH. To inhibit the aspartic acid protease activity, the A244.AE gp120 was purified from the CHO cell culture media while maintaining a neutral pH (pH 7.0 +/- 0.2) resulting in the purification of intact protein without any cleavage products ( lanes 5–8).Initial SDS-PAGE evaluation of A244.AE gp120 purified from the culture media of transfected CHO cell pools showed a single gp120 band under non-reducing conditions , but no or very little intact gp120 and two smaller MW bands under reduced conditions , indicating that the 6240.B gp120 protein was also subjected to protease cleavage. N-terminal sequencing of the cleaved bands showed that the cleavage site was in the V3 loop of gp120 and consensus to a trypsin-like protease cleavage site . In contrast to the A244.AE gp120 situation, pH control during purification did not appear to prevent this cleavage effectively with preparations of this molecule. Therefore, to prevent this cleavage, a variant gp120 6240.B construct was generated with a single site mutation of Arg to Gln at p1’ at the cleavage site, and transfected into CHO cells to generate CHO cell pools for screening. The gp120 purified from these transfected pools of cells appeared as a single intact gp120 band under both reducing and non-reducing conditions with no cleaved bands detectable . This confirmed that this single site mutation in the V3 region completely prevented cleavage of 6240.B gp120.Similarly, SDS-PAGE analysis of the 6240.B gp120 transfected CHO cell supernatants showed one intact gp120 band under non-reducing conditions .To derive clonal CHO stable cell lines expressing A244.AE and 6240.B gp120s, single cells were isolated from the transfected CHO cell pools and evaluated for expression levels, integrity of the product, and stability as described in Methods. The final top clones expressing each of the A244.AE and 6240.B gp120 antigens were identified that consistently produced yields of > 200 mg/L in batch cell cultures. Once top cell lines were established, methods to consistently generate intact gp120 proteins with high purity and high yields were developed with features that would be suitable for future clinical product manufacturing. For several reasons , lectin affinity purification was not considered for this purpose. The platform that was developed included two chromatography steps and three ultrafiltration/diafiltration steps (UF/DF). DEAE anion exchange chromatography captured gp120 protein while contaminants either flowed through or strongly bound to the column. The target gp120 product was eluted from the column with increased NaCl concentration. Fractogel SO3 cation exchange further separated gp120 monomer from aggregated proteins, with gp120 monomer flowing through and gp120 aggregates and other contaminants remaining bound to the column. The purification platform was applied for each of the A244.AE and 6240.B gp120s with buffer components optimized separately for each. With this platform, each of the A244.AE and 6240.B gp120 proteins were produced as monomeric gp120 with high purity (>95% as measured by SEC-HPLC) and with minimal or no detectable cleavage products following three independent production/purification (“consistency”) runs . Thirteen PNGS were exclusively modified by high mannose/hybrid glycans (substrate of PNGase and Endo H), one was exclusively modified by complex glycans (substrate of PNGase and Endo F3), six were found modified by complex and high mannose/hybrid glycans , and at last, undetermined glycans were assigned to five PNGS (substrates of PNGase). It is noteworthy that the N165 of the A244.AE gp120, a non-consensus glycan motif in the V2 region was found to carry either complex or high mannose/hybrid glycan. The modification was identified from the fragmentation of the tryptic peptide 163LINCNTSVIKQPCPK177 . From the twenty-seven 6240.B gp120 PNGS, only the N201 was found unmodified . Ten PNGS were exclusively modified by high mannose/hybrid glycans, three were exclusively modified by complex glycans, five were found modified by both complex and high mannose/hybrid glycans and undetermined glycans were assigned to eight PNGS.Initially, the potential N-linked glycosylation sites (PNGS), which fit with the N-linked glycosylation consensus motif  were assigned when an asparagine residue was identified as an aspartic acid residue after treatment with PNGase F. Because sample treatment can allow the deamination of asparagine to aspartic acid and lead to a misinterpretation of the N-linked glycosylation sites, the glycoprotein gp120s were also treated with PNGase F for a short period followed by pepsin digestion. To differentiate glycosylated sites with complex or high mannose/hybrid glycans, the glycoprotein gp120s were deglycosylated with Endo H that catalyzes the cleavage of high mannose/hybrid glycans and Endo F3, which catalyzes the cleavage of complex glycans. These two glycosidases cleave N-glycans leaving only one GlcNAc intact on the asparagine residues and the corresponding modification could be easily detected by LC-MS/MS. The N-consensus glycosylation sites on both A244.AE and 6240.B gp120s were mapped to the five variable loops (V1-V5) and the five constant regions (C1-C5). Of the twenty-five A244.AE gp120 PNGS, only the N428 was found unmodified HexNAc(1), Hex(1)HexNAc(1)NeuAc(1) or Hex(1)HexNAc(1)NeuAc(2) lead to a mass difference of +365, +656 or +947 Da, respectively, from the non-modified amino acids residues. Three peptides derived from A244.AE gp120 containing T51, T423 and T462 and two peptides derived from 6240.B gp120 containing T51 and T466 were identified as O-glycosylated. While O-glycosylation near the N-terminal (T51) and C-terminal of both gp120 molecules have been previously reported , this stFinally, the glycan fractions obtained from PNGaseF and trypsin treatment of both A244.AE and 6240.B gp120 proteins were analyzed by MALDI-TOF/TOF MS. The two mass spectra, from each of the two treated gp120 glycoproteins, were analyzed by Glycoworkbench3 software for the computer-assisted annotation of glycan mass spectra using, in this case, an internal database of predicted glycan structures for CHO cells as well as information available in the literature , 57. TheFig 3). In the ELISA assay, the 6240.B gp120 bound sCD4 as well as mAbs against CD4bs epitopes , the CD4 inducible (CD4i) region (17b), V2 apex (PG9), a glycan-specific epitope (2G12) and a V3-glycan-specific mAb (PGT128). 6240.B gp120 did not bind Abs directed against a V2 linear peptide epitope in CRF01_AE , which had been also previously shown by Liao et al., [Fig 3).Two independent assay platforms, ELISA EC50 and Surface Plasmon Resonance (SPR), were used to analyze the binding of the purified gp120s to a panel of well-characterized mAbs, b12, VRC01, 17b, CH58, CH59, PG9, PG16, 2G12, PGT128, PGT145, and CH23 and soluble CD4 (sCD4). Results from this initial analysis using ELISA showed that the A244.AE gp120 bound sCD4 , CH58 an et al.,  nor V2 aS3 and ). For mAbs tested by both SPR and ELISA  the data for each mAb are consistent. It was noteworthy that the expanded data from SPR showed that two of the four mAbs that target V2i, mAbs 697 and 1393, had different patterns with A244.AE and 6240.B in that both mAbs had slower on and off rates with the former versus the latter . As a control for non-specific binding, mAbs 1418, 2F5 and 4E10 were also employed.To further explore the kinetics of mAb binding to the A244.AE and 6240.B gp120 proteins, SPR analyses were performed using the CD4bs mAbs, VRC01 and VRC03, and an expanded panel of mAbs that binds to the conformation-dependent V2i epitope that targets the integrin binding site in V2   and the 179LDI/V181, distal to the PG9 and CH58 epitopes; these include the V2i mAbs 697-30D) [Table 1 and S3 Fig). Both CH58 and CH59 mAbs bound to A244.AE gp120 protein with high affinity; with a KD of 1.58 nM for CH58, and 0.90 nM for CH59, while no binding was observed with the 6240.B gp120 protein. As noted above, V2i mAbs 697-30D and 1393A bound to both the A244.AE gp120 and to 6240.B gp120s but with much less affinity than did V2i mAbs 830A and 2158. All four of these V2i mAbs however showed higher binding to the 6240.B gp120 as compared to the binding seen with the A244.AE gp120. . Overall, the high binding affinity was observed between all four tested V2i mAbs to both A244.AE and 6240.B antigens. Comparison binding affinity of V2i mAbs to A244.AE and 6240.B antigens, the binding of all four V2i mAbs to 6240.B exhibited higher binding affinity than that of the A244.AE antigen (Table 1). The highest binding affinity on all tested mAbs was binding between V2i 830A mAb and 6240.B antigen (KD = 0.199 nM). Moreover, all V2i mAbs bound to A244.AE and 6240.B with fast on and slow off rates.Although no broadly neutralizing Ab activity to the V2 apex was observed in RV144 vaccinees, Abs directed to the V2 region were an important correlate of reduced risk of infection in the RV144 vaccine trial –17. We t697-30D) , 830A, [697-30D) , 1393 [7697-30D) , 76, and697-30D) . V2 apexTable 1 and S4 Fig). Higher affinities were observed with the V3 crown mAb 2557 [Table 1 and S4 Fig).The V3-specific mAb 2219 (recognizing an epitope in beta-hairpin of the V3 crown), , 78 was mAb 2557  for bothmAb 2557  bound neth immunization  to A244.AE protein antigen . Animals immunized with the 6240.B gp120 protein with MF59 or AH showed high titer binding Ab responses against the strain/subtype-matched 6240.B gp120 protein . In contrast, animals immunized with the A244.AE gp120 with MF59 showed robust and nearly comparable binding Ab responses against the matched and mismatched gp120 antigens  or 6240.B gp120 proteins (Grps 1 and 4), or a bivalent combination . The gp120s were formulated with either a proprietary oil-in-water emulsion MF59 (Grps 1–3) or AH (Grps 4–6) adjuvant. Overall, robust gp120 binding Ab responses to one or both protein antigens were observed following vaccination in all groups with significant titers measurable after the second immunization and peak titers observed following the 4Fig 4). Binding Ab responses to gp70-scaffolded V1V2 proteins were measured using ELISA, and responses to cyclic V2 and V3 peptides were analyzed using SPR. While cross-clade Ab binding activity was elicited by each gp120, the responses were clade-biased in that gp120 6240.B gave higher responses to gp70 antigens scaffolded with gp70-V1V2 B.CaseA2 rather than gp70-V1V2 AE.92TH023, and vice versa. Sera from groups immunized with 6240.B gp120 with MF59 and AH (Grp1 and Grp4) showed similar high binding Ab titers to the gp70-V1V2 B.CaseA2 protein . These animals also elicited Ab responses that cross-reacted with the gp70-V1V2 AE.92TH023, but the magnitude was much lower than that measured with the gp70-V1V2 B.CaseA2 protein, and the AH group showed higher cross-reactive titers than the MF59 group . Sera from animals immunized with the A244.AE gp120 protein (Grp2 and Grp5) showed high titer responses against the gp70-V1V2 AE.92TH023 and substantial cross-reactivity to the gp70-V1V2 B.CaseA2 . Sera from the A244.AE gp120 MF59 group showed significantly higher Ab titers to gp70-V1V2 AE.92TH023 as compared to those from the AH group , consistent with the higher gp120-specific Ab responses measured in the MF59 group . Immune sera from animals receiving the bivalent gp120 formulations (Grps 3 and 6) showed substantial Ab responses to each of the scaffolded V1V2 proteins as was seen with the monovalent A244.A_E gp120 groups  as compared to the AH group (p = 0.0192). Nevertheless, sera from both groups given bivalent vaccine with either MF59 or AH, surprisingly, showed significant reductions in their binding Ab titers against gp70-V1V2 AE.92TH023 as compared to those seen with the A244.E gp120 monovalent groups with the same adjuvants, respectively . In contrast, they showed similar Ab titers against the gp70-V1V2 B.CaseA2 compared to either monovalent group . These observations should be further explored in dose ranging studies with these gp120 antigens administered alone and in in combination.Further evaluations of epitope-specific binding Abs were performed using guinea pig sera collected following the fourth immunization when responses against gp120 were at their highest . The groups immunized with the A244.AE gp120 protein elicited 10-fold higher binding to their subtype-matched AE.92TH023 cyclic peptide than did the 6240.B gp120 groups to their sequence-matched cyclic V2 peptide, and also showed some low level cross-reactivity with the subtype B cyclic V2 peptide . The A244.AE gp120 MF59 group showed 2-fold higher Ab binding responses to AE.92TH023 cyclic V2 peptide as compared to AH group . In contrast to what was observed with gp70-V1V2 AE.92TH023 peptide , similar responses to the AE.92TH023 cyclic V2 peptide were obtained following immunization with the bivalent or monovalent formulations . However, the bivalent gp120 MF59 group appeared to show a reduced response to the 6240.B cyclic V2 peptide as compared to the responses elicited by the 6240.B gp120 monovalent vaccine ( p = 0.0288).Ab responses directed to epitopes within the V2 and V3 regions of Env were evaluated by SPR using full-length cyclic peptides corresponding to these regions. The Ab responses against the cyclic peptides were generally poorly cross-reactive and far more biased than the responses measured with the gp70-V1V2 antigens. For the subtype CRF01_AE, V2 and V3 peptides from the 92TH023 strains were used, while for subtype B, a polypeptide with the matched sequences from 6420.B HIV- strain was employed . Compared with the observed Ab binding responses against the 6240.B cyclic V2 peptide, Grp1 (6240.B gp120 MF59) and Grp4 (6240.B gp120 AH) showed 3.7- and 4.7-fold higher responses to the cyclic V3 peptide, respectively (6D). In contrast, immunizations with the A244.AE gp120 protein induced 2.4-fold and 3.7-fold lower Ab responses to V3 as compared to those against V2 for Grp2 (MF59) and Grp5 (AH), respectively (6E). However, the MF59 group (Grp2) still induced 2.4-fold higher V3 Ab binding response than Grp5 with AH , similar to the 2.1-fold higher V2 Ab binding responses . Immunization with the bivalent protein formulations elicited overall similar Ab binding responses to the two cyclic V3 peptides tested, but the MF59 group (Grp3) showed slightly higher responses (2.1 fold) against the AE.92TH023 V3 than the AH group (Grp6) .The 6240.B gp120 protein elicited Ab binding responses to the 6240.B cyclic V3 peptide in both the MF59 and AH immunization groups although at very low levels . Groups 2, 3, and 6 all developed Abs that targeted linear epitopes in C1, V2, C2, V3, C3, C4, V5, and C5 regions of gp120, with the magnitude and clade preference varying among groups. Comparison of CRF01_AE-binding profiles revealed comparable binding response to CRF01_AE C1.2, C1.3, C1/V1, C2.1, C2.2, V5/C5 and C5.2 epitopes . The bivalent Group 3 (MF59) induced significantly Ab responses to C5.2 epitope compared to bivalent group 6 (AH) . The monovalent A244.AE gp120 MF59 vaccine (Grp2) induced binding Ab responses against V2.3, V3.1, V3.2, C3 and C4.1 epitopes that were distinguishable from other groups, while the bivalent MF59 vaccine (Grp3) elicited binding Ab responses to V2.1, V2.2 and C4.2 epitopes distinguishable from other groups. Specifically, binding to the CRF01 AE V2.1 epitope was significantly higher  for the bivalent MF59 group (Grp3) compared to the A244.AE M59 group (Grp2), and binding to the clade C V2.1 epitope was significantly higher for bivalent MF59 group when compared either to the A244.AE M59 group  or the bivalent AH group . In addition, the bivalent MF59 (Grp3) generated Abs binding to subtype B C4/V5 peptides while other groups did not. Interestingly, the Abs generated in all four groups cross-recognized the subtype C peptides showing a similar pattern of reactivity as compared to that seen with the CRF01_AE peptides . The bivalent MF59 animals (Grp3) showed a unique pattern of reactivity to V2.1 and V2.2, the most critical regions associated with decreased HIV-1 risk in RV144 [S3 Table). Overall, the bivalent MF59 animals (Grp3) induced Abs targeted the broadest epitopes and the most important epitopes of V2.1, which has been shown in the HIV-1 vaccine efficacy study. Moreover, these vaccine formulations induced Abs cross-reactive multiple HIV-1 subtypes  tested in this study, manifesting considerable breadth of the binding Ab response.Ab responses to linear epitopes spanning the entire gp120 molecule were mapped using the peptide microarray assay format . The linin RV144 . StatistFig 8A–8C, S4 and ). Increased Ab avidities were also measured over the course of immunization in the groups receiving AH formulated vaccines. Ab avidities following the final immunizations  were significantly higher in the MF59 groups as compared to the AH groups for A244.AE gp120 in the comparison of Grp2 vs. Grp5 at 4wp4 (p = 0.0023) . In case of the comparison for the bivalent groups between MF59 and AH, both groups induced high avidity binding Abs directed to either of the A244.AE and 6240.B gp120 proteins; however, the bivalent group (Grp6) of AH induced higher avidity than Grp3 of MF59 at 4wp4 . Moreover, no statistically significant differences were observed between monovalent and bivalent groups using either MF59 or AH for A244.AE gp120 protein  while monovalent group induced significantly higher avidity than bivalent groups for 6240.B gp120 protein . When similar SPR analyses were performed with the 6240.B gp120 protein on the sensor chip, increased Ab avidities were also seen following sequential immunizations . As was seen with the A244.AE gp120 specific Ab responses, all groups rapidly elicited high Ab avidities specific to the subtype 6240.B gp120 protein . In conclusion, both MF59 and AH groups showed high Ab avidities that were achieved over the period of repeated immunizations and likely reflect the maturation of Abs in the lymph node germinal centers by somatic hypermutation.The avidity of Ab binding to the gp120 proteins  was measured in guinea pig immune sera collected at 4-week intervals over the course of the study by SPR using either A244.AE or 6240.B gp120 protein immobilized onto sensor chips as described in the Methods section. When the A244.AE gp120 protein was used, gp120-specific Abs reached higher avidity with statistical significance after each sequential immunization in all groups formulated with the MF59 adjuvant . Guinea pigs immunized with the A244.AE gp120 developed robust neutralizing activity against the Tier 1a CRF01_AE TH023.6 pseudovirus . Moderate neutralizing activity against the Tier 1a subtype B MN.3 and SF162. LS strains , and strong cross-subtype neutralizing activity against the Tier 1a subtype C MW965.26 pseudovirus  were also measured. Moreover, significantly higher neutralizing Ab titers were measured in animals that received the monovalent A244.AE gp120 in MF59 as compared to A244.AE gp120 AH for TH023.6, MN.3, SF162.LS, and MW965.26 . The groups immunized with the bivalent gp120 formulations showed similar virus neutralizing Ab profiles as those immunized with the monovalent A244.AE gp120 alone. In contrast, sera from animals immunized with 6240.B gp120 in either MF59 or AH showed no or very low neutralizing Ab responses against both the Tier 1a subtype B strains ; and only low titers of neutralizing Abs were seen against Tier 1a subtype C pseudovirus, MW965.26 .As a measure of the functionality of the Ab responses to the gp120 proteins, virus neutralization assays were performed with sera collected at baseline and after the fourth immunization using the standardized TZM.bl assay  used for the vector prime, can elicit responses against the HIV strains relevant to the regions where the vaccines are tested in order to provide optimal coverage. Therefore, for future post-RV144 clinical trials in key populations at risk for HIV-1 subtypes CRF01_AE and B, two gp120 candidates were evaluated from the subtypes B 6420 and CRF01_AE A244 HIV-1 strains, and stable CHO cell lines and methods were developed to produce these vaccine materials.To achieve the required high level, stable expression of homogeneous intact gp120 molecules for potential future manufacturing, the antigens were first subjected to modifications that included codon-optimization and N-terminal truncation. Subsequently, a two-step ion-exchange purification method was developed that could be used for both candidates that offered the purity, integrity, consistency and high yields necessary for cost effective vaccine production with the potential to be broadly applicable to diverse HIV gp120s. Early analysis of purified gp120 6240.B and A244.AE revealed substantial clipping in the V3 and V1/V2 regions, respectively. Elimination of the proteolytic clipping with a single amino acid substitution in the V3 region, in the case of gp120 6240.B, and with pH control during the purification process in the case of A244.AE allowed for the production of intact and homogeneous products with good recoveries.In addition, the complex glycosylation pattern of the HIV Env has highlighted the importance of characterizing these residues in vaccine preparations . TherefoThe purified A244.AE and 6240.B gp120 proteins also were shown to bind with high affinities to an overlapping and complementary set of HIV Env-specific ligands including sCD4 as well as mAbs recognizing the CD4 binding site (CD4bs) epitopes, the CD4 inducible (CD4i) region, and V1/V2 and V3-epitopes, some of which include conserved glycan residues known to play a critical role in virus neutralization. Importantly, the results shown here for the A244.AE gp120 prepared from the new stable CHO lines with new purification methods were the same as those seen with purified preparations representative of the HSVgD-tagged A244 gp120 used in RV144, and were consistent with those previously published using other preparations of the A244 gp120 antigen [Immunization of guinea pigs with either the monovalent or bivalent gp120 proteins formulated in either the MF59 or AH adjuvants was shown to elicit high titers of gp120-binding Abs against the vaccine antigens, and in the case of the A244.A-E gp120, high titers of Abs cross-reactive with the mismatched subtype B 6420.B gp120. In addition, all vaccine preparations elicited high level Ab responses to at least one of the gp-70V1V2 reagents tested, an important observation since binding to the gp70-V1V2 B.CaseA2 protein, in particular, was shown to be inversely correlated with the rate of HIV infection in RV144 , 17. In TM, in Italy in 1997, and more recently, in the USA in 2015 [Importantly, the peptide array analysis of immune sera showed that only the bivalent vaccine formation with MF59 (Grp3) showed a unique pattern of reactivity to V2.1 and V2.2, the most critical regions associated with protection in RV144 . This pr in 2015 .O-desacyl-4ʹ-monophosphoryl lipid A (MPL) and the saponin QS-21 [To further build upon this approach of using more potent adjuvants in post-RV144 clinical trials, additional Phase I studies will test other promising adjuvants such as AS01 shown to be clinically safe and effective in the setting of malaria vaccination . AS01 isin QS-21 . With thin QS-21  and hepain QS-21 . Moreovein QS-21 , 92. TheS1 Fig(A) A244.AE gp120 protein and (B) 6240.B gp120 protein from three independent rounds of production and purification, respectively. It was noticed that there were several pen marks on some lanes and they were kept with an original image.SDS-PAGE of purified (TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigS1 Table were calculated on an average from at least three replicates. (A) mAb PG9 vs. A244.AE gp120; (B) mAb PG9 vs. 6240.B gp120; (C) mAb PG16 vs. A244.AE gp120; (D) mAb PG16 vs. 6240.B gp120; (E) mAb CH58 vs. A244.AE gp120; (F) mAb CH58 vs. 6240.B gp120; (G) mAb CH59 vs. A244.AE gp120; (H) mAb CH59 vs. 6240.B gp120; (I) mAb 697 vs. A244.AE gp120; (J) mAb 697 vs. 6240.B gp120; (K) mAb 830A vs. A244.AE gp120; (L) mAb 830A vs. 6240.B gp120; (M) mAb 1393 vs. A244.AE gp120; (N) mAb 2158 vs. 6240.B gp120.Anti-human IgG Fc was immobilized on the sensor CM5 chip followed by mAb V1V2/V2 capture. Next, various concentrations of A244.AE/6240.B gp120 protein were injected onto the captured mAb V1V2/V2 surface. The sensograms were shown in color codes for different concentrations of gp120 proteins. The kinetics values shown in (TIF)Click here for additional data file.S4 FigS6 Fig). (A) mAb 2219 vs. A244.AE gp120; (B) mAb 2219 vs. 6240.B gp120; (C) mAb 2557 vs. A244.AE gp120; (D) mAb 2557 vs. 6240.B gp120; (E) mAb VRC01 vs. A244.AE gp120; (F) mAb VRC01 vs. 6240.B gp120; (G) mAb VRC03 vs. A244.AE gp120; (H) mAb VRC03 vs. 6240.B gp120.Anti-human IgG Fc was immobilized on a CM5 sensor chip followed by mAb V3 or CD4bs capture. Various concentrations of A244.AE/6240.B gp120 protein were injected onto the captured mAb V3/CD4bs surface. Sensograms are shown with a single run for each concentration while the kinetics values were computed from at least three independent replicates ((TIF)Click here for additional data file.S5 FigThe amino acid sequences are highlighted in blue color. Amino acid residues that differ in the alignment are depicted as “.” or “:” while identical amino acids are shown as “*”.(TIF)Click here for additional data file.S6 FigA) comparison of kd off-rate for animal groups immunized with MF59 and (B) comparison of kd off-rate for animal groups immunized with AH. Differences between groups  were compared using Wilcoxon rank-sum test with confidence level of p<0.05 with significant differences indicated.((TIF)Click here for additional data file.S7 FigEach pie slice represents the medium binding intensity of the Groups 2, 3, and 6 to the specified epitope, with the sum of intensities to all epitopes  of the clade indicated beneath the chart.(TIF)Click here for additional data file.S8 Figth immunization as described in Methods against: (A) Tier-1A subtype CRF01_AE TH023.6; (B) Tier-1A subtype B MN.3; (C) Tier-1A subtype B SF162.LS; (D) Tier-1A subtype C MW965.26. Each symbol represents the ID50 titer for an individual animal with geometric mean for each group indicated by the bar and standard error by the box. Differences between groups (MF59 vs. AH) were compared using Wilcoxon rank-sum test with confidence level of p < 0.05 with significant differences shown where applicable.Virus neutralizing Ab responses were measured using sera collected at 4 weeks post-4(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S3 TableValues were compared using Wilcoxon rank-sum test, with confidence level of p < 0.05. Tests were not available (NA) when there was no detectible signal in either group being compared.(TIF)Click here for additional data file.S4 TableValues were compared using Wilcoxon rank-sum test when considering testing between groups at specific time points and Wilcoxon signed-rank test when considering testing between time points at specific groups, with confidence level of p < 0.05.(TIF)Click here for additional data file.S5 Table(TIF)Click here for additional data file.


In contrast, stallions not possessing the CD3+ T lymphocyte susceptible phenotype are at less risk of becoming long-term virus carriers. A genome wide association study (GWAS) using the Illumina Equine SNP50 chip revealed that the ability of EAV to infect CD3+ T lymphocytes and establish long-term carrier status in stallions correlated with a region within equine chromosome 11. Here we identified the gene and mutations responsible for these phenotypes. Specifically, the work implicated three allelic variants of the equine orthologue of CXCL16 (EqCXCL16) that differ by four non-synonymous nucleotide substitutions  within exon 1. This resulted in four amino acid changes with EqCXCL16S (XP_001504806.1) having Phe, His, Ile and Lys as compared to EqCXL16R having Tyr, Asp, Phe, and Glu at 40, 49, 50, and 52, respectively. Two alleles  encoded identical protein products that correlated strongly with long-term EAV persistence in stallions (P<0.000001) and are required for in vitro CD3+ T lymphocyte susceptibility to EAV infection. The third (EqCXCL16R) was associated with in vitro CD3+ T lymphocyte resistance to EAV infection and a significantly lower probability for establishment of the long-term carrier state  in the male reproductive tract. EqCXCL16Sa and EqCXCL16Sb exert a dominant mode of inheritance. Most importantly, the protein isoform EqCXCL16S but not EqCXCL16R can function as an EAV cellular receptor. Although both molecules have equal chemoattractant potential, EqCXCL16S has significantly higher scavenger receptor and adhesion properties compared to EqCXCL16R.Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of horses and other equid species. Following natural infection, 10–70% of the infected stallions can become persistently infected and continue to shed EAV in their semen for periods ranging from several months to life. Recently, we reported that some stallions possess a subpopulation(s) of CD3 + T lymphocyte susceptibility phenotype to in vitro EAV infection are at higher risk of becoming persistently infected carriers compared to those that lack this phenotype. Here genetic and experimental studies were used to demonstrate that CXCL16 in the horse codes for two proteins, one associated with resistance and the other associated with susceptibility of CD3+ T lymphocytes to EAV infection. The two proteins are the result of four nucleotide substitutions in exon 1 of the equine CXCL16 gene. These alleles determine the outcome of in vitro infection of CD3+ T lymphocytes with EAV and are strongly associated with the establishment and maintenance of long-term carrier state in stallions. In vitro studies demonstrated that one form of CXCL16 protein (CXCL16S) is one of the cellular receptors for EAV and has higher scavenger activity and adhesion ability as compared to the form of the protein associated with resistance (CXCL16R).A variable proportion of EAV infected stallions (10–70%) may become persistently infected and continuously shed the virus exclusively in their semen after recovery from acute infection. Previous studies in our laboratory have shown that stallions with the CD3 Arteriviridae in the order Nidovirales  and Standardbred-22 [STB22]) possessed CD3+ T lymphocytes with the EAV susceptible phenotype while the corresponding cells in Thoroughbred-3 (TB03) were resistant. Sequencing of 500 bp paired end libraries was accomplished using an Illumina HiSeq2000 platform to a depth of approximately 30× coverage. Following alignment to the reference genome (Ecab 2.0), the distribution of SNPs within ECA11: 48M-51M was compared to identify differences between susceptible and resistant horses. SNPs of interest were those which occurred in both susceptible horses but not the resistant horse. The analysis focused on identification of SNPs causing non-synonymous mutations within exons for genes annotated by Ensembl as these were considered to have a high likelihood of influencing the phenotype. In total, 12 non-synonymous SNPs were found in exons for eight annotated genes within the target region  associated with t region . No tran+ T lymphocyte EAV susceptibility phenotype were determined by sequencing PCR amplified fragments spanning each of the 12 SNP locations from a subset of the 240 horses (resistant [n = 2] and susceptible [n = 8]) (CXCL16 (EqCXCL16) showed a complete association; the two resistant horses were homozygous for the A, G, T, and G SNPs at (XM_00154756) c.715, c.801, c.804, and c.810, while all eight susceptible horses were either homozygous for the SNPs T, C, A, and A or were heterozygous A/T, G/C, T/A, and G/A at those positions, respectively, This observation is consistent with genetic dominance for the EAV CD3+ T lymphocyte susceptibility phenotype as reported previously [Potential associations between SNPs identified by genomic sequencing and the CD3[n = 8]) . Only theviously .EqCXCL16 was PCR amplified and sequenced from 240 horses  or susceptible (n = 155). Based on the annotation for the equine reference CXCL16 (XM_001504756) sequence, all resistant horses (n = 85) were homozygous for the SNPs A, G, T, and G at positions 715, 801, 804, and 810. However, all susceptible horses were homozygous or heterozygous at those positions and possessed the SNPs T, C, A, A or T, C, G, A (n = 154) at the respective positions. A single (n = 1) homozygote was observed for a putative haplotype possessing the combination T, C, G, A; that individual was phenotyped as susceptible. These two susceptibility alleles  were designated EqCXCL16Sa and EqCXCL16Sb, respectively. The predicted amino acid sequences were identical for EqCXCL16Sa and EqCXCL16Sb as described below. The allele for resistance was recessive and was characterized by A, G, T, and G at positions 715, 801, 804, and 810. This allele was designated EqCXCL16R and was identical to the reference genome sequence (Ecab 2.0). No additional alleles were found in exon 1 of EqCXCL16 among 47 horses of 20 other breeds tested during the study. The gene frequencies of the three CXCL16 alleles are shown in To investigate the distribution of the four SNPs described above in a larger, more representative sample of the horse population, exon 1 of EqCXCL16R were tyrosine (Tyr), aspartic acid (Asp), phenylalanine (Phe), and glutamic acid (Glu). These are non-conservative substitutions.The originally identified SNPs were non-synonymous mutations, predicting a change of amino acids in the protein . The pre+ T lymphocyte phenotypes for resistance or susceptibility to equine arteritis virus infection. As can be seen in The distribution of the two predicted EqCXCL16 proteins among the 240 Thoroughbred, Quarter Horses, Saddlebred, and Standardbred was compared to the distribution of their CD3The 5 carriers with the resistance genotype came from 5 different breeds  while the 11 non-carriers with susceptibility genotypes came from 8 different breeds , Paint, Friesian, Hanoverian, and Quarter Horse.CXCL16 was compared for seven species using the reference genome sequences from white rhinoceros (Ceratotherium simum), horse , dog (Canis lupus familiaris or Canis familiaris), human (Homo sapiens), domestic rat (Rattus norvegicus), cattle (Bos taurus), and African elephant (Loxodonta africana)  . As noteqCXCL16R . AlthougqCXCL16R  or a C rqCXCL16R . In cont+ T lymphocytes to infection and the ability of EAV virus to establish the long-term carrier state in stallions (7). Following natural infection, some stallions continue to shed virus in the semen for long periods (more than one year to lifelong) after EAV is no longer present in blood and nasal secretions ) cell line according to the OIE described protocol . Serum nin vitro EAV-infected CD3+ T lymphocytes as described previously [in vitro EAV infection based on the in vitro susceptibility or resistance of their CD3+ T lymphocytes.The susceptible or resistant phenotype of each animal was defined by dual-color flow cytometric analysis of eviously . Horses Genomic DNA (gDNA) was obtained from PBMCs or semen of each animal by using the Puregene whole-blood extraction kit  in accordance with the manufacturer’s instructions as previously described . DNA qua+ T lymphocyte susceptibility (S) or resistance (R) phenotype based on dual-color flow cytometric analysis in the previous study [SAMN03838869/SRX1097022, SAMN03838867/ SRX1097495 and SAMN03838868/SRX1097492 for CXCL16 of TB10, TB3, and ST22, horses respectively. The variant discovery and genotyping were done with the Genome Analysis Toolkit UnifiedGenotyper using arguments -nt 4 -gt_mode DISCOVERY—validation_strictness LENIENT.[Equus caballus. EquCab2.75.Three horses were selected from those SNP genotyped and phenotyped for the CD3us study ; specifius study  using CLCXCL16, the sequence XM_001504756 was used.The primer sequences for the eight candidate genes found using the target region (ECA11: 48M-51M) are shown in Ceratotherium simum; XM_004433427), horse , dog , human , domestic rat , cattle  and African elephant .The NCBI database was used to obtain and compare reference genome predicted-protein sequences of CXCL16 for white rhinoceros  of both versions of EqCXCL16 (S and R). Following digestion with BamHI and XhoI or PstI and XhoI , fragments of amplicon and synthetic DNA were separated in E-Gel EX 1% agarose (Life Technologies) and extracted from gel using Zymoclean Gel Recovery Kit . Purified fragments encoding aa 17–247 and aa 25–199 of EqCXCL16 and entire EqCXCR6 were ligated into pET15b  followed by transformation into E. coli NovaBlue (Novagen). Ligation and transformation were performed using Rapid DNA Ligation and TransformAid kits (Thermo Scientific), respectively. Recombinant plasmids p15-16A (aa 17–247), p15-16B (aa 25–199), and p15-R6 were isolated from ampicillin-resistant clones using ZR Plasmid Miniprep™ Kit (Zymo Research). Plasmids p15-16R and p15-16S encoding R and S versions of full EqCXCL16 were produced by substitution of the smaller PstI/XhoI fragment of p15-16A to synthetic sequences encoding an N terminal half of R and S versions of this protein, respectively. In order to express recombinant polypeptides, plasmids were transformed into E. coli BL21(DE3) expression vector (Novagen). Several ampicillin-resistant clones were grown overnight in 1 ml of MagicMedia (Life Technologies) supplemented with 50 μg/ml of ampicillin (Sigma-Aldrich). Production of polypeptides was confirmed by SDS-PAGE electrophoresis following mini-scale isolation of recombinant proteins using Talon Metal Affinity Resin (Clontech Laboratories Inc.). For large scale production of recombinant EqCXCL16 (S and R) and EqCXCR6, 500 ml cultures of BL21(DE3) strain of E. coli with respective plasmids were grown overnight at 37°C in MagicMedia supplemented with 50 μg/ml of ampicillin. Following centrifugation at 6000 × g for 15 min, the cell pellet was resuspended in Buffer A  and subjected to several short cycles of sonication to reduce viscosity. The lysate was centrifuged at 16,000  × g for 30 min at 4°C to remove debris. The His-tagged recombinant EqCXCL16 and EqCXCR6 proteins were purified from the supernatant by affinity chromatography using Talon Superflow Metal Affinity Resin (Clontech Laboratories Inc.) in combination with an FPLC apparatus (Amersham Pharmacia Biotech Inc.). Columns were equilibrated and washed with Buffer A, while proteins were eluted using Buffer B . Eluted recombinant protein was dialyzed against PBS using Slide-A-Lyzer Dialysis Cassettes (Thermo Scientific). The purity and integrity of recombinant EqCXCL16S, EqCXCL16R, and EqCXCR6 proteins were evaluated by subjecting them to electrophoresis on a 4–20% gradient gel SDS followed by staining with PageBlue Protein Staining Solution (Thermo Scientific) and WB analysis. Protein concentration was determined with BCA Protein Assay (Thermo Scientific) using BSA as the standard.Recombinant plasmids for cloning EqCXCL16 and EqCXCR6 were designed by CLC Main Workbench 7 programs using horse genome sequences obtained as part of this study. Synthetic sequences encoding the predicted exposed part of EqCXCL16 (aa 25–199)  and the E.coli as previously described [2 fragment of goat anti-mouse IgG1  was used as the secondary antibody. Mouse EAV α-GP5 and mouse EAV α-nsp-1 monoclonal Abs  have been described previously [Protein-specific rabbit antipeptide sera , and guinea pig polyclonal antibody (Gp α-EqCXCL16 pAb) to detect EqCXCL16 proteins (EqCXCL16S and EqCXCL16R) were generated by immunizing rabbits with two synthetic peptides and guinea pigs with recombinant EqCXCL16 expressed in escribed . For thieviously , 71. Deteviously , 70. GoaE. coli DH10B cells (Life Technologies).Stable HEK-293T transfectants expressing EqCXCL16S (HEK-EqCXCL16S cells) and EqCXCL16R (HEK-EqCXCL16R cells) were generated as described earlier . Briefly6 cells/well) and transfected with 3 μg of codon-optimized pJ609-EqCXCL16R plasmid DNA (DNA2.0) using lipofectamine 3000 (Life Technologies) following the manufacturer’s instructions. At 24 h post transfection, the medium was replaced with fresh medium containing 4 μg/ml of puromycin (Clontech Laboratories Inc.) and cells were incubated at 37°C in a 5% CO2 incubator. This process was repeated every other day until only puromycin-resistant colonies remained. These puromycin resistant cells were cloned by limiting dilution in 96-well plates and screened by IFA, after which clones showing the highest level of EqCXCL16S and EqCXCL16R protein expression were frozen in commercial cell-freezing medium  and stored in liquid nitrogen until needed. At every 5th passage and up to the 50th serial passage, cells were analyzed by IFA using Gp α-EqCXCL16 pAb to confirm the expression of EqCXCL16R. All the experiments in HEK-EqCXCL16S and HEK-EqCXCL16R cells were performed within passage levels 5 to 10.For the establishment of stable cells, the HEK-293T cells were seeded in 6-well plates  and fixed in 4% paraformaldehyde  for 30 min at room temperature (RT). Cells were then stained as described previously . FollowiCells were lysed in RIPA lysis buffer  in Halt protease and phosphatase inhibitor cocktails (Thermo Scientific). The solubilized proteins were mixed with Pierce lane marker reducing 5X sample buffer containing 100 mM dithiothreitol  and heated for 5 min at 95°C. Samples were resolved in SDS-polyacrylamide gel  at 200 V for 45 min and then transferred onto a PVDF membrane  at 100 V for 1 h using the trans-blot transfer system (Bio-Rad) , 74. The5 HEK-EqCXCL16S, HEK-EqCXCL16R, and naïve HEK-293T cells were plated in a 96-well plate. After 24 h incubation, the cells were washed in 37°C PBS. Cells were then incubated with 0.5M EDTA for 10 min at 37°C following which they were washed again with PBS and fixed with 4% PFA for 15 min at RT. Cells were then washed in distilled water and stained with 0.1% crystal violet solution for 20 min at RT, visualized with an inverted light microscope and photographed. After washing with distilled water, the cells were air-dried, then incubated with 10% acetic acid for 20 min with shaking prior to the transfer of 50 μl of the lysate to a new 96-well ELISA plate for determination of OD595nm values using a Synergy H1MD microplate reader .The cell adhesion assay was performed in accordance with a published protocol . Approxi+ T lymphocytes (5 x 105 cells/ml) labelled with Calcein-AM (Life Technologies) was added. After 6 h of incubation at 37°C in 5% CO2, media from the inside of the insert was removed and insert was placed in a clean well with 400 μl of Cell Detachment solution and incubated at 37°C for 30 min. After complete dislodging of the cells from the underside of the insert, 400 μl of the cells containing Cell Detachment solution was mixed with 400 μl of the migratory cells from the original well. Then 50 μl of the mixed cell solution was added onto a glass slide, and cells that passed through the filter were counted under a fluorescent microscope (40× objective) and represented in a bar diagram (averages of six different fields were included).Cell migration in response to EqCXCL16S and EqCXCL16R proteins was determined using a Chemotaxis assay kit . Briefly, FPLC purified soluble recombinant EqCXCL16S and EqCXCL16R proteins were diluted in serum-free RPMI medium with 0.5% BSA (cell culture grade) at a concentration of 2 μg/ml in 500 μl of RPMI and were added to the lower well of a 24-well chemotaxis chamber; the lower and the upper wells were separated by a polyvinylpyrrolidone-free polycarbonate filter insert with the pore size of 3 μm. To the upper wells of the chamber, 100 μl of purified equine CD3Stable HEK-293T cells expressing EqCXCL16S and EqCXCL16R and naïve HEK-293T cells were seeded in 12-well plates. At about 85% confluency, cells were washed with warm (37°C) PBS (pH 7.4) and replenished with warm (37°C) growth medium containing Dil-OxLDL at 10 μg/ml  and incubated at 37°C for 3 h. Cells were washed with ice-cold PBS and examined under an inverted epifluorescence microscope for the uptake of Dil-OxLDL by cells.Approximately 100 μg of total protein lysate from naïve HEK-293T cells or stable HEK-EqCXCL16 cells were separated in 12% SDS-PAGE and transferred onto a PVDF membrane for Far-WB analysis following a modification of the published protocol . The bouEAV VBS was purified by ultracentrifugation  through a 20% sucrose cushion and protein concentration determined using the BCA protein assay kit. About 2 mg of purified EAV was biotinylated using EZ-Link Sulfo-NHS-Biotin (Thermo Scientific) following the manufacturer’s protocol. Excess unbound biotin was removed by filtering through a Zeba desalt spin column  equilibrated in PBS (pH 7.4). The naïve HEK-293T and HEK-EqCXCL16 cells were washed in cold PBS (pH 7.4) and removed from the culture dish using a non-enzymatic cell dissociation solution . Cells were resuspended in cold PBS (pH 7.4) containing 2% FBS (PBS-F), centrifuged at 1000 × g for 5 min at 4°C and incubated with biotinylated EAV at an MOI of 100 on ice for 2 h in total darkness. Excess EAV was removed by washing three times in cold PBS-F. Subsequently, the cells were stained with Streptavidin-FITC (1:100) and incubated at 4°C for 30 min in total darkness. Cells were washed in PBS-F at 1000 × g for 5 min at 4°C, transferred onto glass microscope slides using a Shandon CytoSpin III Cytocentrifuge with Shandon single cytofunnel with white filter cards (Thermo Scientific) and incubated with DAPI solution to permit visualization of cell nuclei. Cells were then analyzed using a Nikon inverted fluorescence microscope and the percentage of cells bound to EAV was calculated.CXCL16 genotypes with phenotypes, including susceptibility and resistance as well as carrier status, were conducted using the Fisher’s Exact Test. Differences among multiple treatment groups were analyzed by statistical analysis software Sigmaplot 12.3 , by ANOVA with pairwise multiple comparison procedures by the Holm-Sidak method. P-values less than 0.05 were considered to be statistically significant.Statistical tests for association of S1 Fig+ T lymphocytes from PBMCs. T lymphocytes were enriched from PBMCs using anti-equine CD3 (Clone UC F6G) and micro magnetic beads conjugated to anti-mouse IgG1. The enriched cells were found to be > 95% T lymphocytes based on the proportion of CD4+ and CD8+ cells (top row) present in the enriched fraction. Furthermore, both CD21+ B lymphocytes and CD14+ monocytes (bottom row) were found to be a very minor component of the enriched T lymphocyte fraction. B) Expression of CXCR6 on equine CD3+ T lymphocytes. Magnetic bead purified CD3+ T lymphocytes were stained with α-EqCXCR6 antibody. Percentage of CD3+ T lymphocytes cells stained by pre-bleed rabbit sera as a negative control (shown as a). Percentage of CD3+ T lymphocytes expressing CXCR6 (shown as b).A) Enrichment of CD3(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


S100A4 (metastasin-1), a metastasis-associated protein and marker of the epithelial to mesenchymal transition, contributes to several hallmarks of cancer and has been implicated in the progression of several types of cancer. However, the impacts of S100A4 signaling in lung cancer progression and its potential use as a target for therapy in lung cancer have not been properly explored. Using established lung cancer cell lines, we demonstrate that S100A4 knockdown reduces cell proliferation, invasion and three-dimensional invasive growth, while overexpression of S100A4 increases invasive potential. In patient-derived tissues, S100A4 is preferentially elevated in lung adenocarcinoma. This elevation is associated with lymphovascular invasion and decreased overall survival. In addition, depletion of S100A4 by shRNA inhibits NF-κB activity and decreases TNFα-induced MMP9 expression. Furthermore, inhibition of the NF-κB/MMP9 axis decreases lung carcinoma invasive potential. Niclosamide, a reported inhibitor of S100A4, blocks expression and function of S100A4 with a reduction in proliferation, invasion and NF-κB-mediated MMP9 expression. Collectively, this study highlights the importance of the S100A4/NF-κB/MMP9 axis in lung cancer invasion and provides a rationale for targeting S100A4 to combat lung cancer. A majority of lung cancer patients present at an advanced stage, which often precludes treatment with surgical resection alone . Non-smaS100A4 /fibroblast specific protein (FSP1)) is a calcium binding EF-hand protein that has been implicated in carcinoma progression and is a marker of the epithelial to mesenchymal transition (EMT) . S100A4 Despite extensive investigation of S100A4 in carcinoma progression, the impact of S100A4 signaling in lung cancer is poorly defined. In this study, we investigated the contribution of S100A4 in lung cancer cell invasion, determined the clinical significance of S100A4 expression in patient-derived lung cancer tissues, and provided a rational for targeting S100A4 signaling through repurposing an FDA-approved drug.We used a panel of established lung cancer cell lines with different genetic backgrounds  to examiN = 212). A wide range of staining intensities was observed in lung carcinoma cells, which were scored using a semi-quantitative scale ranging from 0 to 3 . When all histologic subtypes were combined, there was no significant difference in median survival between patients with and without S100A4 overexpression . This observation suggests that S100A4 overexpression has a higher impact in the lung adenocarcinoma subpopulation when compared to the squamous cell carcinoma subpopulation.We constructed a lung cancer tissue microarray (TMA) and stained sections for S100A4 using immunohistochemistry in order to examine S100A4 expression in patient-derived tissues N = 12. A wid3 Figure , which w3 Figure . S100A4 3 Figure . FurtherNiclosamide affects multiple signaling pathways that are important in cancer progression and has also been shown to block S100A4 expression in colon cancer cells , 16. OurConsiderable evidence demonstrates that NF-κB signaling is constitutively activated in solid tumors and is essential for lung cancer tumorigenesis, invasion and metastasis . S100A4 TNF-α induces MMP9 expression through the NF-κB pathway . We testOur data demonstrate that S100A4 drives the invasive capacity of lung cancer cells, affects the NF-κB pathway, and acts as an essential component of TNF-α-induced and NF-κB-dependent MMP9 expression. Next, we investigated whether the NF-κB/MMP9 axis is critical to the invasive capacity of lung cancer cells. Accordingly, we pretreated A549 cells with inhibitors of either NF-κB or MMP2/MMP9, then performed Matrigel invasion assays. Inhibition of the NF-κB/MMP9 pathway not only decreased the basal invasive capacity but also inhibited cell invasion toward chemo-attractants such as TNF-α and EGF Figure . CollectThe majority of patients with advanced NSCLC die within 18 months of diagnosis, usually as a result of metastatic disease , 22. EluIt is well established that S100A4 overexpression contributes to several hallmarks of cancer. Supported by a previous study , we founMatrix metalloproteinases (MMPs) are essential in tumor invasion and metastasis due to their abilities to degrade the extracellular matrix (ECM) and also to change cellular behavior to modulate the tumor microenvironment. Downstream of the NF-κB pathway, we found that MMP9, but not MMP2, was dramatically inhibited by S100A4 depletion in response to TNF-α (data not shown). Importantly, MMP9 inhibition decreased the TNF-α- and EGF-stimulated invasiveness of A549 cells, highlighting the importance of the S100A4/MMP9 axis in S100A4-driven invasion in lung cancer cells. Given that S100A4 is an established metastasis-promoting protein, it is not surprising that this S100A4/MMP9 pathway is demonstrated in other cancer types . In suppin vivo studies are needed to confirm this concept.Currently, there is no clinically available treatment targeting S100A4 and its pleiotropic roles in cancer progression. Multiple strategies have been proposed to block S100A4 function in cancer. These strategies include targeting S100A4 with specific antibodies or interfering with the interaction of S100A4 and its targets using small molecular inhibitors , 36. HowOur study highlights the important role of the S100A4/NF-κB/MMP9 signaling axis in promoting lung cancer invasive capacity, and demonstrates that S100A4 overexpression associates with reduced overall survival among patients with lung adenocarcinoma. Importantly, our study demonstrates that niclosamide dramatically inhibits the NF-κB/MMP9 signaling axis by suppressing S100A4 to block the invasive capacity of lung cancer cells. Since S100A4 promotes metastasis, our study provides strong evidence and rationale for targeting S100A4 by repurposing niclosamide to block invasion and prevent metastasis in NSCLC.Lung cancer cell lines that are representative of different subtypes of lung cancer were obtained from ATCC. Lung adenocarcinoma , large cell lung carcinoma , NCSLC  and small cell lung cancer (H82) cell lines were used in this study. A549, H1299, H358 and H460 cells used for biological functional analysis were authenticated with short tandem repeat (STR) profile analysis by Genetica DNA laboratories in May 2015. Niclosamide and Wedelolactone were obtained from Sigma-Aldrich . MMP2/9 inhibitor I was from Calbiochem/EDM Millipore . The pIRES-GFP-S100A4 construct was obtained from Dr. Masashi Fukayama  .Cells were harvested and lysed in RIPA buffer . Total cell lysates (80 μg) were subjected to 10% or 15% SDS-PAGE, transferred and immunoblotted with rabbit anti-S100A4 antibody , p65  or p84 . Tubulin and β-actin antibodies were from Sigma-Aldrich and were used as the loading controls.4) in 200 μl growth media with 2% FBS were seeded onto solidified growth factor-reduced Matrigel  and then covered with medium containing 10% Matrigel. The next day, DMSO or niclosamide at the indicated concentration was added to the cultures. When the control cells developed an invasive growth phenotype (approx. 5-6 days), phase contrast images of randomly chosen fields were taken with a Nikon Ti-E inverted microscope and analyzed using Nikon Elements software.Culturing lung cancer cells in 3D was performed as described previously . Briefly4) were added to the upper chamber, and medium/BSA containing 5 ng/ml EGF, 5 ng/ml TNF-α or 1% FBS (control) was added to the bottom chamber as indicated; cells were allowed to invade overnight at 37°C. Non-invaded cells were removed from the top chamber using a cotton swab; invaded cells on the bottom of the transwell membrane were fixed with 100% methanol and stained with 1% crystal violet. Four fields per well were counted and averaged, and the data were presented as the mean number of cells invaded per mm2 +/− standard deviation from triplicate determinations.Matrigel  was dried onto the upper well of transwell chambers . One hour before the assay, Matrigel was reconstituted with 100 μl of serum-free medium and the bottom chamber was coated with 5 μg/ml fibronectin. Cells (70% confluent) were trypsinized and rinsed three times with serum-free medium plus 250 μg/ml BSA. Cells (5×105) were seeded in each well of a 12-well plate for direct cell counting or cells (2×103) were seeded in each well of a 96-well plate for MTT assay. For niclosamide treatment, fresh growth media containing niclosamide or DMSO was added starting the day after plating, and added daily thereafter. Cells were trypsinized and directly counted by using a Vi-Cell XR cell viability counter  at the indicated time points. Alternatively, MTT assay was performed as previously described [Cell proliferation was assessed by direct cell counting or by 3--2.5-diphenyltetrazolium bromide (MTT) assay. Cells . Total RNA (1μg) was used to reverse-transcribe into cDNA using the High Capacity cDNA Reverse Transcription Kit . Expression of target genes was assessed by Comparative Ct (ΔΔCt) using commercially available probes and master mix reagent and performed on a StepOnePlusTM 96-well instrument as described by the manufacturer (Applied Biosystems). The expression level of each gene was normalized by 18S or β-actin RNA and reported as a relative level to a specified control, as noted.For stable reduction of S100A4 expression in A549 and H460 cells, lentivirus-mediated shRNA construct pLKO.1-puro targeting human S100A4 (S100A4-#A6) or containing non-targeting sequence (Sigma-Aldrich) were packaged into virus. In brief, the control or S100A4-specific shRNA construct was co-transfected with Mission lentiviral packaging mix (Sigma-Aldrich) into 293T cells using polyethylenimine (PEI) based on 1:3 ratio of DNA: PEI. The viral supernatant was collected 48 hrs after transfection, as described previously . Then, AH1299 cells in 10-cm dishes were transfected with 8 μg GFP control or pIRES-GFP-S100A4 constructs using Lipofectamine 2000 . Transfected cells were selected with G418 (400 μg/ml) and sorted for GFP by fluorescence activated cell sorting (FACS).TM . Listing of randomly-sorted samples for allocation into the recipient TMA was generated by the MCC Biostatistics and Bioinformatics Shared Resource Facility, and then assembled into TMA blocks  by the MCC Biospecimen and Tissue Procurement Shared Resource Facility. After construction, TMA blocks were heated at 45°C for 1 hr and then cooled to complete assembly. S100A4 expression was assessed by IHC staining as previously reported [Institutional Review Board Approval 13-0692-P6H) was obtained prior to initiation of the project. Surgically resected NSCLC cases from 2006-2010 from our institution were screened for inclusion in this study. Cases were excluded if the primary tumor was not of lung origin or if inadequate pathological material existed. A total of 216 lung cancer cases were used to build the tissue microarrays (TMAs). A variety of histologic types were selected, including 83 adenocarcinomas and 102 squamous cell carcinomas  along with pRL-TK Renilla control reporter at a 50:1 ratio for 24 hrs. Then, cells were induced by 5 ng/ml TNF-α, vehicle control with or without niclosamide at the indicated concentration for 24 hrs. Then, cells were collected and luciferase activity was measured using the Dual-Luciferase Reporter Assay System . These data are presented as a relative value to internal control or fold induction as individual control.To detect NF-κB p65 nuclear translocation, cells were serum-starved overnight and then stimulated with 5 ng/ml of TNF-α for 4 hrs. For niclosamide treatment, cells were pretreated with DMSO (control) or 0.5 μM or 1 μM niclosamide for 48 hrs before serum starvation, then stimulated with 5 ng/ml of TNF-α for 4 hrs. Cell fractionation was then performed as described previously .http://cancergenome.nih.gov/) was accessed and visualized using The Oncomine™ Platform .For quantitative variables, differences between groups were analyzed using Welch's t-test or the Mann–Whitney U test, where appropriate. For categorical variables, differences between groups were analyzed using chi-squared or Fisher's exact test, where appropriate. All tests were two-tailed. Survival differences were assessed via log-rank test. Significance was reached when P < 0.05. Statistical analyses were performed using GraphPad software . For data mining, a lung cancer gene expression dataset  generate


Reassortment of 2009 (H1N1) pandemic influenza virus (pdH1N1) with other strains may produce more virulent and pathogenic forms, detection and their rapid characterization is critical. In this study, we reported a “one-size-fits-all” approach using a next-generation sequencing (NGS) detection platform to extensively identify influenza viral genomes for diagnosis and determination of novel virulence and drug resistance markers. A de novo module and other bioinformatics tools were used to generate contiguous sequence and identify influenza types/subtypes. Of 162 archived influenza-positive patient specimens, 161(99.4%) were positive for either influenza A or B viruses determined using the NGS assay. Among these, 135(83.3%) were A(H3N2), 14(8.6%) were A(pdH1N1), 2(1.2%) were A(H3N2) and A(pdH1N1) virus co-infections and 10(6.2%) were influenza B viruses. Of the influenza A viruses, 66.7% of A(H3N2) viruses tested had a E627K mutation in the PB2 protein, and 87.8% of the influenza A viruses contained the S31N mutation in the M2 protein. Further studies demonstrated that the NGS assay could achieve a high level of sensitivity and reveal adequate genetic information for final laboratory confirmation. The current diagnostic platform allows for simultaneous identification of a broad range of influenza viruses, monitoring emerging influenza strains with pandemic potential that facilitating diagnostics and antiviral treatment in the clinical setting and protection of the public health. Influenza viruses belonging to the family Orthomyxoviridae, are classified into influenzavirus A, B, and C. While influenza A and C viruses infect humans and many other species, influenza B viruses only infect humans and seals ,2,3. TheIt was reported that rapid influenza diagnostic tests showed low and variable sensitivity ,18 depenPre-titrated stocks of reference strains of influenza viruses were procured from Dr. Stephen Lindstrom  or cultured in embryonated chicken eggs at US Food and Drug Administration (FDA). Clinical nasopharyngeal swab specimens were collected from patients with symptoms of influenza-like illness, submitted during the 2010–11 and 2012–13 influenza seasons to the Clinical Virology Laboratory (CVL), Yale New Haven Hospital, New Haven, CT. These specimens were tested as requested by the patients’ physicians using a direct fluorescent antibody (DFA) immunoassay  and/or by quantitative RT-PCR (RT-qPCR), as previously described . A total5’-IAGCARAAGC -3’) and fusion primers for PCR  were designed and tested. The 13bp flanking sequences italicized were added at the 5’ end of each PCR primer to enhance the annealing temperature and achieve high fidelity and yield in PCR amplification. These primers target highly conserved regions at the 5'- and 3'- terminus of each segment of influenza virus to amplify the whole-genome concomitantly, and have coverage of sequence variants for influenza A, B, and C viruses to achieve improvements in analytical sensitivity and inclusivity.The RNA extraction, some primers, and Reverse Transcription-PCR (RT-PCR) procedures were described previously  and in SSample preparation and bioinformatics data analysis was performed as described . Brieflyde novo assembly. Contigs for individual gene segments from influenza viruses were generated using the de novo assembly module on the CLC bio software . The parameter for mapping reads back to contiguous was set on as similarity fraction 0.9; length fraction 0.5; mismatch cost 2; insertion cost 3 and deletion cost 3. For characterization of influenza whole-genomes with high confidence coverage, minimum contig length was set at 800bp to assemble the consensus sequences and minimum coverage was 1000 reads [de novo assembly , the Global Initiative on Sharing All Influenza Data  and NCBI database. The match with the highest score was retained to identify the specific influenza genome. Assembled sequences were aligned by the CLUSTAL W program, and phylogenetic analysis was performed using MEGA 5 and the neighbor-joining method [A master file containing all unique contigous sequences of each mega-amplicon was generated and used to perform an all-by-all blast search in Influenza Research Database  was calculated using formulae LN/G, where L is the read length, N is the number of reads and G is the theoretical genome length. The breadth of sequence coverage (BOC) was calculated as percentage of actual testing contig length (≥1000 reads or ≥30x of DOC) divided by the theoretical genome length ,37. TotaThe newly reported influenza genome sequences are available in GenBank under the following accession numbers: KM654599-KM654931, and KM654933-KM654980.9 TCID50/mL) were first produced followed by RT-PCR exponential amplification for whole-genome segments. As shown in −7 dilution of A(H5N1) viral RNA (1.8x103 TCID50/mL) was detected in the RT-PCR assay shown as multiple faint visible bands. A total of ten PCR mega-amplicons from the dilutions were next tested using MiSeq, multiple contigs were finally automatically generated for each mega-amplicons, and all contig sequences were correctly identified as genome of A/Viet Nam/1203/2004(H5N1) virus. The overall average of DOC was 4,715. The average of BOC for each identified contig was 96% (≥30x depth) indicating that the near full-length genome sequence was covered  represented 99% of BOC at all detected titration points with the lowest detectable level at 10−9 dilution (1.8x101 TCID50/mL) with high confidence coverage , A (H3N2), and B viruses, respectively. Fifteen and sixteen contigs were generated from the mixed A(H1N1), and A(H3N2) with B viruses supported by high confidence coverage, respectively  multiplexed detection and simultaneous discrimination of influenza virus infections in nasopharyngeal swabs and identification of genetically distinct lineages of influenza A(H3N2 and pdH1N1) and B viruses in a single sequencing run, (ii) identification of influenza virus co-infections in a single specimen/test, (iii) development of new degenerate universal primer pairs for identification of influenza viruses and demonstration of highly sensitive detection of influenza virus infections using the NGS assay. We proposed a “one-size-fits-all” approach using an NGS diagnostic platform for extensive identification of a broad range of influenza virus infections including co-infections, by revealing their comprehensive genetic context and providing matrix reporting information for final sequence-based confirmation.A total of 162 influenza-positive de-identified nasopharyngeal swab specimens collected from patients in the Connecticut were tested in a blinded fashion using the RT-PCR-NGS platform. No reassortants were found but multiple mutations were detected in the specimens tested. Sequence analysis of 123 influenza A viruses revealed that 66.7%(82/123) of A(H3N2) viruses had a single signature mutation of E627K in the PB2 protein, and 88%(108/123) of influenza A(H3N2 and pdH1N1) viruses contained the S31N mutation in the M2 protein. A mutation of PB2 E627K has been reported to confer high virulence to the virus by enhancing replication efficiency, and increasing polymerase activity and disease severity of avian influenza viruses in mammals . The S31We aligned all protein sequences of 47 influenza A viruses and compared their sequences with those in humans that have been reported as amino-acid signatures in past influenza outbreaks . The eme−9 dilution level (1.8x101 TCID50/mL) in the characterization study, and detect partial sequences of M and NS genes at 10−10 dilution level in the analytical study Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.