Notably, pradimicin S has good pharmaceutical properties and enables cure of an acute form of the disease in mice. By inducing resistance in vitro we have established that the composition of the sugars attached to the variant surface glycoproteins are critical to the mode of action of pradimicins and play an important role in infectivity. The compounds identified represent a novel approach to develop drugs to treat HAT.Current treatments available for African sleeping sickness or human African trypanosomiasis (HAT) are limited, with poor efficacy and unacceptable safety profiles. Here, we report a new approach to address treatment of this disease based on the use of compounds that bind to parasite surface glycans leading to rapid killing of trypanosomes. Pradimicin and its derivatives are non-peptidic carbohydrate-binding agents that adhere to the carbohydrate moiety of the parasite surface glycoproteins inducing parasite lysis  Trypanosoma brucei, the causative agent of African trypanosomiasis, is coated with a dense layer of the variant surface glycoprotein (VSG), which plays an essential role in antigenic variation and the ability of the parasite to evade the immune system. VSGs are N-glycosylated with complex and/or mannose-rich N-glycans by two oligosaccharyltransferase activities in a site-specific manner. Here, we describe that a series of non-peptidic carbohydrate binding agents, previously reported to display antifungal and antiviral properties, exhibit a marked trypanocidal activity. Pradimicin A (PRM-A) and pradimicin S (PRM-S) show specific binding to VSGs and a pronounced cytocidal effect by inducing defects in endocytosis and cytokinesis. Remarkably, PRM-S produces parasitological cure in two different models of acute sleeping sickness. Information on the mode of action was provided by generating PRM-A-resistant parasites, which suffered from defective glycan composition as a consequence of down-regulation of oligosaccharyltransferase genes. Aberrant glycosylation of VSGs resulted in decreased binding of pradimicins but reduced parasite infectivity underscoring the role of glycosylation in virulence. Our findings identify antibiotics with the ability to effectively bind to glycans of the parasite surface as agents capable of affording parasitological cure, thereby providing a novel avenue for design of highly specific drugs to combat African trypanosomiasis. Trypanosoma brucei. Treatments are largely insufficient and unsatisfactory and new approaches for drug design are highly necessary.Human African trypanosomiasis or sleeping sickness is a neglected disease caused by the protozoan parasite T. brucei parasites living in the mammalian host rely on antigenic variation to evade the immune system of the host. They are mainly covered by only one kind of a variant surface glycoprotein (VSG) that constitutes an effective barrier that protects from effectors of the host immune system. In the formation of this protective barrier the N-glycosylation of VSGs is of major importance [portance . The VSGportance –4. DurinN-glycan composition of VSGs by changes in the expression of oligosacharyltransferases. Thus, CBAs appear to exert their mode of action against Trypanosoma by specifically binding to surface glycans [Recently, we have reported a series of carbohydrate-binding agents (CBAs) that bind to parasite surface glycoproteins and exhibit a strong trypanocidal activity against the clinically relevant bloodstream form, presenting activity in the nanomolar range . Analysi glycans , 6.However, the previously reported antitrypanosomal CBAs were proteins with molecular masses ranging between 8,700 Da  and 50,000 Da  or even higher. Proteins present a series of disadvantages to become potential drugs, including efficient scale-up, poor, if any, oral bioavailability and/or potential generation of an immune response.Actinomadura hibisca [Actinomadura sp [Aspergillus) [2+-dependent manner [Non-peptidic, low-molecular-weight antibiotics designated PRM-A and benanomycin A have been discovered in the culture fluid of  hibisca  and Actiadura sp , respectrgillus)  and virargillus) . It has rgillus) . Memberst manner . PRM-S it manner . In addit manner , 11–13, t manner , PRM-FA-t manner , BMS1811t manner  and BMY2t manner  were repT. brucei bloodstream forms at low micromolar concentrations by perturbing cytokinesis and endocytosis and consequently inducing parasite cell lysis. We provide information on their mode of action by generating mutant parasite cells resistant to the drug and examining binding efficiency of the pradimicins to parental and resistant parasitic VSGs and the glycan composition. Furthermore, we found that treatment at 50 mg/kg with PRM-S cures T. brucei brucei and T. brucei rhodesiense infection in mice. We propose that pradimicins and carbohydrate-binding agents in general may provide a unique and highly novel avenue for the development of an efficient treatment of parasitic diseases.Here we report that pradimicins inhibit the growth of in vitro trypanocidal activities of PRM-A, PRM-S and the derivatives BMY28864, PRM-FS, PRM-FA-1 and BMS181184 were evaluated against the bloodstream forms of T. brucei. All of them exhibited 50% effective concentration (EC50) values in the low micromolar range  for 48 h . The resistance selectivity index  was determined as an estimate of the degree of drug resistance. A value of 25 was obtained for PRM-A25 and PRM-A50, and of 40 for PRM-A100 . PRM-A100 cells exhibited significant resistance to PRM-S, HHA and EHA and low resistance to UDA, NPA and GNA -α Man), EHA (Man), GNA  Man), NPA  Man), UDA (GlcNAc oligomers) and PRM-S  Man) were tested. Cross-resistance indices (R-index) were calculated as ratios of EC and GNA .N-glycosylation profile [N-glycosylation nature were analysed in parasites resistant to PRM-A. First, indirect immunofluorescence using an anti-TbVSG221 polyclonal antibody [N-glycan nature of the resistant parasite strains. Endo H or PNGase F, which remove conventional triantennary oligomannose and hybrid N-glycans or all types of N-glycans respectively, were used to confirm this. Parental sVSG harbours an Endo H-resistant (Asn263) and an Endo H-sensitive (Asn428) N-glycosylation site, coming from the action of different oligosaccharyltransferase activities, such as TbSTT3A and TbSTT3B which transfer Man5GlcNAc2 or mainly Man9GlcNAc2 structures, respectively [T. brucei BSF strain used in this study ruling out the possibility that changes in glycosylation are due to mutations in the N-glycosylation sites Manα1-specificity [N-glycans containing mainly one or several N-acetyl lactosamine structures. However, the additional presence of any abbreviated core structures derived from paucimannose N-glycans cannot be discarded. In contrast, the binding of ConA to sVSG was unaltered in the PRM-A-resistant cells. When whole cell extracts were probed with TL, ECL and ConA, moderate changes in the glycosylation patterns were observed.Blotting with lectins of different specificities was performed to further characterize the nature of the tosamine  or the Mtosamine , ECL distosamine  but alsocificity –26. In aN-glycosylation were also assessed by labelling with lectin-FITC conjugates with different glycan binding specificities: CV-N-FITC  Man), HHA-FITC - α Man) and UDA-FITC (GlcNAc oligomers). Thus, resistant cells had a strongly reduced ability to bind to all of the lectins tested, CV-N -high performance liquid chromatography-fluorescence coupled to mass spectrometry (UPLC-FLD/MS). Assignment of peaks was based on exact mass and previous biochemical knowledge of the trypanosome glycome or via diagnostic fragment ions [Tb BSF free glycans at m/z 932.877 and 1905.687, were assigned to paucimannose Man3GlcNAc2 (H3N2 with m/z 932.877) and triantennary oligomannose Man9GlcNAc2 , respectively. In line with previously published trypanosoma VSG glycosylation profiles, further peaks were tentatively assigned to the oligomannoses structures such as the biantennary H4N2  or the triantennary H5N2 , H6N2 , H7N2  and H8N2 . In addition, we observed peaks corresponding to the biantennary hybrid N-glycans H3N3 , H4N3 , H5N3  and H6N3 . Further peaks in the glycan profile were assigned to the poly-N-acetyl lactosamine complex N-glycans Gal2Man3GlcNAc4  and Gal4Man3GlcNAc6   . The anaN-glycans  and complex N-glycans (H5N4 and H7N6) were identified. The two new H4N4 (m/z 1501.401) and H6N5 (m/z 2028.827) species present in the profile were assigned to GalMan3GlcNAc4 and Gal3Man3GlcNAc5 structures, respectively  and a significant increase in the hybrid  and complex N-glycans with poly-N-acetyl lactosamines , whereas paucimannoside levels  remained unaltered  or α-α bonds and the emergence of complex glycans with terminal poly-N-acetyl lactosamine motifs, which would be responsible for the decreased binding affinity of pradimicins.In the PRM-A100 resistant cell line, the oligomannose structures H3N2, H4N2, H5N2, H6N2 and H9N2 were found whereas H7N2 and H8N2 were absent. In addition, peaks corresponding to hybrid ectively . In-sour species –S7 Figs.naltered . TherefoT. brucei cell lines. Both VSGs were immobilized on a sensorchip. Parental Tb BSF VSG221 was bound at low and high density  and PRM-A100 VSG221 derived from the drug-resistant parasites at high density    were performed using VSG221 derived from parental (BSF) and drug (PRM-A100)-resistant ,070 RU) . PRM-A bmplitude . Insteadobserved . Interesctively) . A similctively) . A signiD) of the pradimicins, as previously reported for HIV gp120 [on and koff rates failed, mainly due to the lack of 1:1 stoichiometric kinetics of the binding. Indeed, given the small size of the pradimicins, it might be assumed that several pradimicin molecules can bind on one single VSG molecule given the high amount of glycans present on VSG. In addition, the pradimicin antibiotics are known to internally staple (associate) at higher (micromolar) concentrations, further compromising relevant calculations of the KD values for the parental and resistant VSGs. Nevertheless, the SPR-based binding study of the pradimicins to parental and PRM-A-resistant VSG convincingly revealed that PRM-A and PRM-S concentration-dependently bind to VSG and that both PRM-A and PRM-S showed a compromised binding efficiency for the PRM-A100-resistant versus the parental Tb BSF VSG. These findings confirm the specific binding of the pradimicins to parasitic VSGs, and the poorer binding of PRM-A and PRM-S to PRM-A-resistant VSG than parental VSG. In addition, they support our view and provide further evidence that resistance against pradimicins is due to the glycan changes in the parasitic VSG.Attempts to calculate the binding affinities  and TbSTT3B (2.9-fold) in PRM-A-resistant cells compared to the parental line was observed, even when PRM-A pressure was removed for up to 3 months  activities coded by three genes: STT3A, STT3B and STT3C , 6. OSTsc manner , 27, 28.analysis . The occ3 months . In ordeormation . Three nnd L705P . In the Parasites from the parental, PRM-A25, PRM-A50 and PRM-A100-resistant cell lines were used to infect mice and survival was monitored. We observed a strong parasite fitness cost for the three resistant cell lines that resulted in reduced infectivity. Whereas the parental line exhibited a median survival days (MSD) of 6.0 ± 0.0 days, the four mice infected with PRM-A25-resistant parasites and two mice out of the seven infected with the PRM-A100-resistant parasites died showing an MSD of 38 ± 7 days and 34.5 ± 0.7 days, respectively. All the mice infected with PRM-A50 were alive after 50 days (MSD >50 days) . Thus, tTbSTT3A, TbSTT3B and TbSTT3C in the parental line were accomplished  thus pointing towards a major role for this OST in the resistance mechanism. Moderate overexpression of TbSTT3A did not result in sensitization to PRM-S, thus curtailing its role in the resistance phenotype   . These rTo evaluate whether PRM-A resistance involved modifications in endocytosis, PRM-A100-resistant and parental parasite strains were probed with ConA as a marker for membrane-bound endocytic activity , transfeT. brucei rhodesiense EATRO3 ETat1.2 TREU164 or T. brucei brucei single-marker 427 strains. PRM-S exhibited a dosage-related efficacy at intraperitoneal dosages of 25 mg/kg and 50 mg/kg per day administered on four consecutive days. At 25 mg/kg PRM-S, two of five mice infected with T. brucei rhodesiense were cured  group  group . Notablyi brucei .T. brucei brucei-infected mice treated with 25 mg/kg PRM-S were isolated just before animal sacrifice, inoculated into HMI-9 medium and exposed to increasing concentration of PRM-S for determination of the EC50 value. The EC50 obtained (5.0 ± 0.2 μM) was similar to the control, showing that short term PRM-S exposure in vivo did not generate resistance to PRM-S.Parasites from T. brucei brucei at 30 min, 1 h and 2 h after drug treatment (50 mg/kg) in order to provide an insight into the mechanism of action in vivo. PRM-S provokes a rapid parasite clearance , while the MSD was extended to 12.8 ± 3.1 days, thereby doubling the survival of the control group (MSD of 6.25 ± 0.5 days). BMY28864-treated animals died at the same time as the control group, and therefore, proved not to be active in vivo  that exhibit antiviral and antifungal properties mediated by lectin-mimic binding to surface glycans , 17, 31.. brucei . Our obstoxicity . Althougtoxicity .N-glycosylation pathway that resulted in an altered N-glycosylation of VSGs and other glycoproteins which presumably lead to a reduced binding of the CBA. Indeed in the resistant mutant parasites, lectin blotting analysis together with the observation that CV-N, HHA and UDA uptake and binding are impaired suggested profound modifications in surface glycans. Given that this phenotype remained after withdrawal of drug pressure, we concluded that the resistance phenotype was genetically encoded and stable. On the other hand, defects in endocytosis in the resistant mutants were minor and reversible upon drug withdrawal. Transferrin and ConA uptake reduction was reversed after culture in the absence of PRM-A while no defects were observed in fluid-phase endocytosis thereby establishing that this process is not relevant to the resistance phenotype.Studies on the molecular mechanisms of resistance to the pradimicins were designed in order to shed light on the mode of action of these compounds. Thus resistance was generated by a step-wise selection to PRM-A and a high resistance index was achieved for mutant cells that also exhibited cross-resistance to PRM-S and to other mannose-binding lectins. The resulting resistance phenotype was characterized by defects in the 2+ [2+ concentration is not excessive has also been reported [D) of ~ 0.4 μM and hence exhibit promising antiviral properties [N-glycans of the trypanosome surface glycoproteins. Moreover while PRM-A/S is able to bind VSGs, affinity is strongly dependent on N-glycan structures and was markedly reduced in PRM-A-resistant parasites. Moreover, binding competition experiments with HHA and CV-N indicate clearly that pradimicins compete with these lectins in the interaction with VSGs. On the other hand, direct evidence for efficient pradimicin binding was provided by SPR analysis. Indeed, VSGs from resistant parasites exhibit a lower capacity to bind pradimicin than parental VSGs further confirming changes in glycan composition in order to overcome the anti-parasitic CBA suppressive effects. Definitive evidence for changes in VSG glycosylation was obtained by analysis of free and procainamide labelled glycans by mass spectrometry and liquid chromatography. Mutant resistant cells exhibited a significant reduction in the proportion of oligomannose type glycans, namely H8N2 and H9N2, while hybrid and complex species accounted for 47% of total glycans versus 23% in the parental cell line. In summary, studies on VSG endoglycosidase treatment, lectin binding, lectin blotting and glycan composition show that parasites overcome PRM-A pressure by an altered N-glycan processing leading to an enrichment in hybrid and complex N-glycan structures presenting lower numbers of α-mannose residues prone to bind this CBA.Pradimicins and benanomicins comprise a unique family of antibiotics with a lectin-like ability to bind D-mannose (D-Man) in the presence of Ca2+ , 9, 34. 2+ . The evireported . Both PRoperties . Here, w5GlcNAc2-PP-Dol to asparagines flanked by an acidic sequence yielding paucimannose structures, and STT3B relocates Man9GlcNAc2-PP-Dol to any remaining asparagine rendering oligomannose N-glycans, respectively [TbSTT3A and TbSTT3B mRNA levels that prompts hypoglycosylation and changes in the N-glycan nature directed towards a reduction of oligomannose and an increase in paucimannose structures. These modifications clearly minimize their accessibility and the ability for binding pradimicins, and consequently are responsible for the appearance of resistance. In this process TbSTT3B appears to be a major player since overexpression of the enzyme in resistant parasites reversed resistance to pradimicins while conversely RNAi mediated depletion in wild type parasites resulted in high levels of resistance. Thus down-regulation of TbSTTB appears to be the main mechanism involved in pradimicin resistance. A question that remains to be addressed is how long term down-regulation of TbSTT3A and TbSTT3B mRNA is achieved. It is well-established that trypanosomatids lack the ability to regulate RNA-polymerase II transcription initiation, and the control of mRNA abundance and protein profiles depend largely on RNA-binding proteins [STT3 genes as potentially responsible of STT3 mRNA down-regulation. No modifications were identified in the UTRs yet a series of mutations were found in the ORFs of STT3A and STT3B. The possibility that mutations within the coding region are related with the modification of mRNA levels has not been established. RNA-binding proteins that bind within the coding region of mRNAs have been described although their major role is modulation of translation [STT3 genes are regulated in mutant cells remains to be understood.We sought to establish how changes in glycan composition occur in pradimicin-resistant parasites. It is well-known that VSG glycosylation is accomplished in a site-specific manner by the action of two catalytic OSTs: STT3A activity transfers Manectively , 27. Theproteins . For exaproteins . We havenslation . In addiin vivo further demonstrates that we can obtain highly potent and efficient trypanocidal agents by designing compounds that interact with surface glycans. This is an entirely novel concept that warrants further investigation. Indeed, our data revealed that small-size non-peptidic CBA molecules are emerging as a promising strategy for parasite suppression although further studies will be required to improve the pharmacokinetic properties of this kind of compounds and to achieve sufficient central nervous system penetration and efficacy in the late stage of the disease. Pharmacokinetic data with pradimicin derivatives obtained in previous studies have shown that drug levels in brain tissue and cerebrospinal fluid were lower than those measured in other tissues but detectable at concentrations exceeding 1 μg/g after multiple dosing [The striking efficacy e dosing .in vivo appears to result from direct interaction of the CBA with bloodstream forms since after treatment the morphology of parasites isolated from the blood would suggest a similar mechanism of action to that observed in vitro. Interestingly, an important consequence of glycosylation changes was the strong fitness cost observed in mice models. Resistant parasites were either not infective or exhibited a highly attenuated virulence. These findings are in agreement with previous work that has shown that a correct glycosylation of VSG is critical for optimal and efficient host-parasite interaction [Here the trypanocidal activity eraction , 6, 28.T. brucei and render parasitological cure in vivo using an acute model of sleeping sickness. By binding to surface glycans, pradimicins lead to defects in cytokinesis resulting in cell lysis. While specific binding to surface VSGs has been demonstrated, interaction with other glycoproteins cannot be ruled out. All this evidence allows us to propose the development of lectin-mimetic agents, such as the non-peptidic pradimicins, as a novel approach for the design of antitrypanosomal agents.In conclusion, pradimicins exhibit a highly cytotoxic activity against bloodstream forms of Trypanosoma brucei brucei single-marker bloodstream forms (BSF)  strain 427, harbouring T7 RNA polymerase and the tetracycline repressor [Trypanosoma brucei rhodesiense EATRO3 ETat1.2 TREU164 [2 in HMI-9 with 10% (v/v) or 20% fetal bovine serum, respectively.epressor  and Tryp TREU164  were useActinomadura hibisca) [Actinomadura spinosa strain A A08 51) [Actinomadura spinosa strain A A08 51 grown in presence of D-serine) [Actinomadura hibisca P157-2 grown in presence of D-serine) [Actinomadura sp) [Hippeastrum hybrid agglutinin (HHA) [Urtica dioica) [Epipactis helleborine) [Galanthus nivalis) [Narcissus pseudonarcissus) [Nostoc ellisporum) [Erythrina cristagally lectin (ECL) were obtained from Vector Laboratories, Inc and ConA from Sigma.The following non-peptidic mannose-specific CBAs of prokaryotic origin have been used: pradimicin A  ; pradimi A08 51) ; pradimi-serine) ; pradimidura sp) ; and BMYdura sp) . The Amain (HHA) , stingin dioica) , broad-leborine) , snowdronivalis) , daffodircissus)  and cyanisporum) . Tomato T. brucei bloodstream forms by exposure to increasing concentrations of compound. The process started with a pradimicin concentration equal to the EC50 (3.20 ± 0.04 μg/ml) and then stepwise selection was performed, obtaining several strains at escalating PRM-A concentrations of 3.2, 3.6, 10, 25, 50 and 100 μg/ml. Parasites were exposed to a higher drug concentration when the generation time (6–8 hours) had equalled that of the parental line, a process which took around 15–25 days. Resistance stability was checked at 1, 2 or 3 months on PRM-A50 and PRM-A100 strains after removal of the drug pressure.PRM-A, a non-peptidic CBA with α mannose specificity, was used to generate resistant cell lines of The coding sequences for PRM-A50 and PRM-A100 VSG221 were amplified by PCR using cDNA as template, cloned in the pGEM-T vector (Promega) and finally sequenced.p-formaldehyde, washed twice (PBS and 0.2% Tween 20) and blocked during 30 min with Blocking Reagent 1% (Roche). Subsequently, samples were incubated with anti-VSG221 polyclonal antibody for 1 h, labelled with FITC-conjugated anti-rabbit secondary antibody for 1 h, washing before and after labelling. Slides were then dehydrated in methanol for 1 min and finally stained and mounted with Vectashield-DAPI . The microscopy and digital image acquisition were performed using a Zeiss Axiophot microscope VSG221 expression was evaluated by immunofluorescence using an anti-VSG221 polyclonal antibody on PRM-A-resistant cells as described . BrieflyTbSTT3A, TbSTT3B and TbSTT3C genes was measured as described [myosin 1B gene (Tb927.11.16310) as reference, which was kindly provided by Dr. Navarro [Total RNA of parental and PRM-A-resistant cell lines was extracted using TRIzol reagent (Invitrogen), and treated with DNase to avoid a genomic DNA contamination using the RNeasy Micro kit (Qiagen). cDNA was obtained by reverse transcription using iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR assays were carried out in an iCycler IQ real-time PCR detection system (Bio-Rad) using SsoFast EvaGreen Supermix (Bio-Rad). All procedures were performed according to the manufacturer’s instructions. Relative expression of the escribed  using th Navarro . Three iet al [8 cells were lysed in 300 μl of hypotonic lysis buffer ) for 5 min at 37°C. The supernatant containing VSG was collected by centrifugation at 14,000 x g for 5 min, loaded onto 0.2 ml of a DE52 (Whatman) and eluated with 10 mM sodium phosphate buffer, pH 8.0. Finally VSG was diluted in water after concentrating and diafiltering on a Nanosep 10K Omega .The sVSG isolation of PRM-A-resistant strains was performed following the protocol described by Cross et al , 51 withsVSGs isolated of parental and PRM-A-resistant cell lines were subjected to endoglycosidase digestion. For each enzyme digestion, 1 μg of sVSG was denatured in 10 μl of 0.5% SDS and 0.1 M dithiothreitol for 10 min at 100°C, followed by overnight treatment at 37°C with 500 units of Endo H or PNGase F (New England Biolabs) in the corresponding buffer supplied by the manufacturer.p-formaldehyde on poly-L-lysine-coated slides at RT for 20 min, washed (PBS and 0.2% Tween 20) and blocked with Blocking Reagent 1% (Roche) for 30 min. Then, samples were incubated with anti-TbVSG221 for 1 h, washed, probed with FITC-conjugated anti-rabbit antibody for 1 h and washed again. Slides were finally stained and mounted with Vectashield-DAPI  after dehydrating in methanol. The microscopy and digital image acquisition were carried out with a Zeiss Axiophot microscope To assess the VSG nature in PRM-A-resistant cells, an immunofluorescence analysis using an anti-TbVSG221 polyclonal antibody was carried out. Trypanosomes were fixed in 4% 2. Interaction studies with PRM-S were performed in the same buffer without DMSO (due to a markedly higher solubility of PRM-S versus PRM-A). A reference flow cell was used as a control for non-specific binding and refractive index changes. Several buffer blanks were used for double referencing. A variety of PRM-A and PRM-S concentrations were injected for 2 min at a flow rate of 30 μl/min and followed by a dissociation phase of 5 min. The CM5 sensor chip surface was regenerated with a single injection of 10 mM NaOH.Binding of PRM-A and PRM-S to VSG221 expressed in parental or PRM-A-resistant cell lines was evaluated using SPR on a Biacore T200 instrument . TbBSF VSG221 was covalently immobilized on a CM5 sensor chip in 10 mM sodium acetate, pH 5, using standard amine coupling chemistry, resulting in chip densities of 826 (low-density) and 6,500 (high-density) RU. The same coupling chemistry was used to immobilize 6,070 RU of PRM-A100-resistant VSG221 to the sensorchip. Interaction studies with PRM-A were performed at 25°C in HBS-P  containing 5% DMSO and 10 mM CaClN-glycans containing α and/or α mannose forming a VSG-HHA complex which is rapidly endocytosed [T. brucei cells (1.5 x 106 parasites) were washed once with Voorheis PBS (PBS containing 10 mM glucose and 79 mM sucrose), resuspended in 1 ml of serum-free HMI-9 medium containing 1% BSA and preincubated for 20 min at 37°C. Samples were incubated with HHA-FITC (1 μg/ml) or CV-N-FITC (0.6 μg/ml) in the presence or absence of PRM-S, washed twice with cold PBS and resuspended finally in PBS. FACS analysis was carried out using a Becton Dickinson FACSCalibur and BD CellQuest Pro version 4.0.2 software. For microscopy analysis, cells were fixed after labelling with 2% p-formaldehyde for 1 h at 4°C, washed, adhered on poly-L-lysine coated slides, dehydrated in methanol and stained with Vectashield-DAPI . Vertical stacks of 10–15 slices (0.2 μm steps) were captured using an Olympus microscope and Cell R IX81 software. Deconvolution and pseudo-colouring of images was performed using Huygens Essential software  and Image J software , respectively.An HHA-FITC conjugate binds to VSG ocytosed . A CV-N-N-acetylglucosamine residues of VSG N-glycans [T. brucei cells were prepared and labelled using CV-N-FITC (0.6 μg/ml), HHA-FITC (1 μg/ml) and UDA-FITC (5 μg/ml).UDA-FITC conjugates that bind to -glycans , CV-N-FIEndocytosis dynamics in the parental line upon PRM-S supplementation and in the presence or absence of HHA (1 μg/ml) was determined by uptake of Alexa Fluor 488-dextran 10,000 conjugates (1 mg/ml) in 50 μl of final sample volume. To analyse endocytosis in PRM-A-resistant cell lines, AlexaFluor 594-ConA (100 μg/ml), FITC-transferrin (50 μg/ml), and Alexa Fluor 594-dextran 10,000 (1 mg/ml) conjugates were used as described [−6 cell equivalents/sample) coming from hypotonic lysis of PRM-A100 cells were denatured in SDS-sample buffer containing 8 M urea and 50 mM DTT, analysed using MOPS electrophoresis on NuPAGE Bis-Tris 4–12% gradient gel (Invitrogen) and transferred to a nitrocellulose membrane. Proteins were stained with Ponceau S (Sigma) as loading control and blocked with 3% BSA in PBS, previously probing with either biotinylated TL  in a solution containing 50 mM Tris-HCl pH 7.4, 0.5 M NaCl, 0.05% IGEPAL and 0.25% BSA, biotinylated ECL  in a solution containing 10 mM HEPES pH 7.4, 0.15 M NaCl, 1 mM CaCl2, 0.05% IGEPAL and 0.25% BSA, or biotinylated ConA  in PBS containing 1 mM MgCl2, 1 mM CaCl2, 1 mM MnCl2, 0.05% IGEPAL and 0.25% BSA. In all cases, specific inhibitors of lectin binding such as chitin hydrolysate  for TL, D-lactose  for ECL and methyl α-D- mannopyranoside  for ConA were used as carbohydrate-specific binding controls. Finally, glycoproteins were detected with Extravidin-peroxidase conjugated (Sigma) by chemiluminescent detection ECL Western Blotting Detection Reagents .In order to study the glycosylation profile of the PRM-A-resistant strains, lectin blotting using TL, ECL and ConA was performed. Both, sVSG (2 μg) and cell pellets (1 x 10T. brucei VSG221 samples (50 μg) of parental (Tb BSF) and resistant (PRM-A100) cells were denatured and enzymatically deglycosylated with PNGase F according to the manufacturer´s instructions . After deglycosylation, the protein fraction was removed by filtration on a 10 kDa spin filter , recovering the free glycans in the filtrate. The released glycans from 50 μg of VSG221 protein were purified over C18 loaded Zip-Tips to remove polymers, the sample was treated with 1 μl of sodium citrate tribasic (1 mM) to favour the exclusive production of sodium adducts and subjected to MALDI-TOF MS analysis. Super-DHB was used as MALDI matrix as a 20 mg/ml solution in ACN. 1 μl of both sample and matrix solution were spotted to the MALDI sample plate into the same spot. The spectra were recorded in reflector positive mode in the 700–3000 Da range, on an UltrafleXtreme III MALDI-TOF, Bruker Daltonics, Germany. Further information on glycan structure was obtained by fragmentation by MALDI-TOF MS selecting the ions 2393 m/z, 2028 m/z and 1663 m/z, and employing in-source fragmentation routine . Glycan fragments were identified using the Flexcontrol Ultraflex TOF/TOF software application and GlycoWorkBench 2.1 free software.Glycan profiling by mass spectrometry and liquid chromatography was performed to gain a more detailed view of VSG glycosylation including the relative quantification of glycan distribution. A relative quantification of the glycan profile was performed by analysing procainamide labelled glycans by UPLC-FLD-MS. Glycans were enzymatically released from 50 μg of filtered VSG221, dried and labelled with procainamide (procainamide/cyanoborohydride in DMSO/AcOH) for 3h at 60°C. The labelled glycans were dried, redissolved in ACN, and analysed by UPLC-FLD on an Acquity UPLC Glycan BEH amide column  in ammonium formate/ACN. Fluorescent labelled glycans were detected with a FLD and an ESI-TOF mass analyser.in vivo of CBAs. Groups containing between three to six C57BL/6 or Balb/C mice (6–8 weeks old)  were infected intraperitoneally with 5 x 103 monomorphic T. brucei brucei (PRM-S treatment) or 1 x 104T. brucei rhodesiense parasites , respectively. Different dosages of 25 mg/kg and 50 mg/kg of PRM-S (four dosages) and 50 mg/kg of BMS181184 and BMY28864 (three dosages), were administered once a day intraperitoneally (0.2 ml) starting on the third day post-infection. Compounds were initially dissolved in PBS at 12.5 mg/ml, followed by dilution in 0.5% w/v hydroxypropylmethylcellulose, 0.4% v/v Tween 80 and 0.5% v/v benzyl alcohol used as dosage formulation. One group of mice in each case was also infected and treated with the vehicle as control. Parasitaemia was monitored daily in a haematocytometer and morphology was examined under a microscope from the third day post-infection and after tail blood extraction.PRM-S, a more soluble analogue of PRM-A, BMS181184 and BMY28864, were used to evaluate the trypanocidal activity T. brucei brucei parasites of parental, PRM-A25, PRM-A50 and PRM-A100 strains were used to infect intraperitoneally between four and seven female C57BL/6 mice per group, respectively (6–8 weeks old). Parasitaemia was monitored daily in a haematocytometer under a microscope from the fourth day post-infection and after tail blood extraction.To study the effect of the glycosylation changes observed in PRM-A-resistant parasites on infectivity, 600 monomorphic The animal research described in this manuscript complied with Spanish (Ley 32/2007) and European Union Legislation (2010/ 63/UE). The protocols used were denoted as 1646/13 (PRM-A100 infection), 511/15.2 (PRM-A25 and PRM-A50 infection), 1947/13.A.1, 1947/13.B.1 and DGP.2/2014/CEEA (PRM-S treatment) and 2738/13.A.1 (BMS181184 and BMY28864 treatment) and approved by the Animal Care Committee of the Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC.t-tests using a commercially available, computer-based statistical package (GraphPad Software Inc.) for all calculations. A p value ≤0.05 was considered statistically significant.We expressed the results as the mean ± SD for each group, and comparisons between groups were performed using Student’s S1 FigT. brucei brucei single-marker strain 427 bloodstream forms treated with different pradimicin derivatives. Cells were cultured in triplicate for 48 hours with varying concentrations of pradimicins.Growth inhibition profile of (TIF)Click here for additional data file.S2 FigThe alignment was obtained with ClustalW2 (EMBL-EBI).(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 FigTbSTT3 genes and the likely rearrangements between TbSTT3B and TbSTT3C genes, which were observed in an HHA20-resistant cell line and reported previously [TbSTT3 genes according to the scheme shown in panel A in both PRM-A50 and PRM-A100 resistant strains.(A) Scheme of eviously . Name of(TIF)Click here for additional data file.S8 FigTbSTT3 genes. Sequencing was performed on DNA purified from agarose gels after restriction endonuclease digestion using different primers.Scheme of the DNA region containing the (TIF)Click here for additional data file.S9 Fig(A and B) Parasitaemia of animals treated with 50 mg/kg of BMY28864 (A) BMS181184 (B) and the vehicle used as control. (C) Kaplan-Meier survival analysis of mice infected and treated. † denotes the day of infection when mice died.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses.  •Designed boosting immunogens to follow a VRC01-class germline-targeting prime•Boosted Abs selectively incorporate VRC01-class somatic mutations•Sequential boosts elicit VRC01-class mAbs with broad neutralization of N276A viruses•Vaccine-elicited VRC01-class mAbs neutralize one fully native HIV isolate Development of boosting HIV envelope immunogens designed to guide the genetic and functional maturation of VRC01-class bnAbs provides a roadmap for the design of an effective HIV vaccine. Elicitation of a broadly neutralizing antibody (bnAb) response is thought to be a highly desirable feature of an effective HIV vaccine . PassiveSeveral major challenges must be overcome to meet this goal. First, potent bnAbs targeting relatively conserved epitopes on the HIV envelope (Env) trimer develop in only a minority of infected individuals, whereas narrowly neutralizing Abs targeting variable epitopes on the infecting virus develop in all or most cases of infection . FurtherVRC01-class bnAbs targeting the CD4 binding site (CD4bs) on HIV Env are attractive leads for HIV vaccine design, as they are among the most broad and potently neutralizing  and theyWe recently demonstrated that eOD-GT8 60-mer immunization primes relatively rare VRC01-class precursors in a transgenic mouse model expressing the VRC01 germline-reverted heavy chain (VRC01 gH) . In this−/− cells (or insect cells) to present oligomannose glycans, for selection of light-chain CDR1 mutations and/or deletions to accommodate the base of the N276 glycan and to allow neutralization of viruses passaged in GnTI−/− cells; and (4) native-like trimers bearing native glycans, for selection of light-chain FW3 mutations to accommodate the distal portions of the N276 glycan and to allow neutralization of viruses bearing native glycans.Based on structure-function studies of VRC01-class bnAbs , we haveIn the present work, we focused on the first two steps of this strategy. We hypothesized that the eOD-GT8 60-mer prime might fail to generate memory B cells capable of being activated by native-like N276(−) trimers. Supporting this hypothesis, mAbs induced by eOD-GT8 60-mer in the VRC01 gH mouse showed affinity for an N276(−) core gp120  but not We report the development and testing of two such boosting immunogens, BG505 core-GT3 nanoparticle (NP) and BG505 SOSIP-GT3 trimer. We show that sequential immunization schemes employing these bridging boost immunogens drove the maturation of eOD-GT8 60-mer primed B cells toward VRC01-class bnAbs and induced broad neutralization of near-native (N276A) viruses and weak neutralization of a fully native virus in VRC01 gH mice. The results demonstrate that reductionist sequential immunization can initiate and guide maturation of pre-defined neutralizing antibody specificities. Furthermore, our findings provide a foundation on which to develop a vaccine to induce VRC01-class bnAbs.We sought to develop a boost immunogen to activate eOD-GT8 60-mer-induced memory B cells, cause the formation of new germinal centers, and select for a pool of more highly mutated memory B cells that could subsequently be boosted by native-like N276(−) trimers. To achieve this goal, we designed “bridging” molecules, which, while still germline-targeting, display a more native CD4bs epitope in order to drive maturation toward mature VRC01-class bnAbs. We allowed fewer overall mutations than in eOD-GT8 while retaining native VRC01-class contact residues wherever possible. Engineered mutations at non-contact positions were selected to improve affinity for germline-reverted (GLrev) Abs via conformational stabilization or removal of occluding glycans A. Two deProtein engineering was initially carried out by yeast display directed evolution on the core gp120 platform. We generated combinatorial libraries of core BG505 gp120 containing mutations that we had previously noted in the development of core BaL-GT1 and eOD-GT8 . These lTo generate a native-like trimer variant of GT3 with more native-like epitope features and CD4bs steric access restrictions, we transferred the BG505 core-GT3 mutations onto BG505.D664 SOSIP and added a C-terminal PADRE epitope, resulting in BG505 SOSIP-GT3-PADRE, from now on referred to as BG505 SOSIP-GT3 . OverallThus, to develop a sequential immunization scheme with considerations of gradual epitope change toward a native configuration, T-help conservation, and minimizing the boosting of off-target responses, two boost candidates were designed to follow the eOD-GT8 60-mer and precede the BG505 SOSIP N276D native-like trimer.To quantify the ability of BG505 core-GT3 NP and BG505 SOSIP-GT3 to recall VRC01-class precursor B cells primed with eOD-GT8 60-mer, we sequentially immunized the VRC01 gH mouse with eOD-GT8 60-mer, BG505 core-GT3 NP, and BG505 SOSIP N276D trimer according to the immunization schedule described in For a serological probe, we developed a resurfaced HXB2 core gp120 (r1-core-N276D) with improved VRC01-class antigenicity compared to RSC3  Figure . The resFollowing the third boost, we evaluated serum antibody binding to r1-core-N276D and r1-core-KO B and S5.These differences in serum antibody responses were mirrored in the frequencies of epitope-specific memory B cells. Splenocytes and lymph nodes from immunized animals were harvested and stained for IgG memory B cells. Single cells were then antigen-sorted by flow cytometry using the baits listed in To determine whether consecutive boosting with tailored immunogens selects for productive mutations, we divided the immunized VRC01 gH mice into two test groups: (1) mice that received only the eOD-GT8 60-mer prime and a single boost of BG505 GT3 (either SOSIP or NP), and (2) mice that received the complete immunization protocol outlined in We next examined whether vaccine-induced SHM was progressing toward mature VRC01. For each VH1-2 sequence, we determined the total number of amino-acid mutations and the number of amino-acid mutations shared with a panel of VRC01-class mAbs   Figure D. In ordWe also interrogated vaccine-elicited light chains for evidence of maturation toward mature VRC01. This analysis is less straightforward than with heavy chains, because the light chains were derived from mouse germline genes that would not be expected to follow the same maturation pathway as human VRC01-class light chains. Instead, we assessed two critical features: the distribution of LCDR1 lengths and sequence convergence in LCDR3. VRC01-class bnAbs encode relatively short LCDR1 loops (2AA–8AA) by utilizing germline variable genes with short (6AA–8AA) LCDR1s and in some cases with further shortening by SHM-associated deletions  West et. AlthougFollowing immunization, purified serum IgG from each of the 12 mice receiving the entire immunization schedule was screened on an 8-virus cross-clade indicator panel of near-native (N276A) HIV isolates A. Ten ofTo verify the results of the first immunization experiment, we performed a repeat immunization with an additional 16 VRC01 gH mice, divided into two groups. All mice in the repeat experiment were primed with eOD-GT8 60-mer and boosted with BG505 core-GT3 NP according to the schedule in 50 of <1 μg/ml.We expressed 25 mAbs from eight VRC01 gH mice that were primed with GT8 and boosted with GT3 and SOSIP and tested them for neutralizing activity on the 7-virus N276A virus panel described earlier. Generally, neutralization breadth of mAbs from each mouse correlated with the breadth of serum neutralization, although one mouse (285) that showed no detectable serum neutralization produced a single mAb with moderate neutralization activity. Antibodies from several mice showed broad neutralization on the 7-virus near-native panel, and mAbs from mouse 286 neutralized select isolates with potency comparable to mature VRC01 . The mAbBecause Nem10 and Nem11 neutralized 191084 B7-19 N276A with potencies comparable to mature VRC01, we tested the ability of these two mAbs to neutralize the fully native version of 191084 B7-19, a tier 2 virus . Both anVRC01-class bnAbs are prototypical examples of the neutralizing anti-HIV response that an optimal vaccine would elicit: they are broadly and potently neutralizing; multiple VRC01-class bnAbs have been shown to be protective against infection in animal models; and VRC01-class naive B cell precursors are likely to be present at a reasonable frequency in a large fraction of the population thus offering targets to initiate vaccine elicitation. However, induction of such responses remains a massive challenge in part because VRC01-class bnAbs display exceptionally high levels of somatic mutation and GLrev versions of these antibodies have no detectable affinity for all native-like HIV Env molecules tested thus far. A multi-step reductionist vaccine strategy has the potential to address both of these issues: an engineered germline-targeting prime can activate VRC01-class precursors and generate boostable VRC01-class memory B cells, and successive heterologous boosts with increasingly native-like immunogens can produce additive rounds of somatic mutation and gradually refine the ability of maturing antibodies to recognize native HIV Env. Development of minimally mutated variants of VRC01-class antibodies that retain broad and potent neutralizing activity has further raised expectation that a VRC01-like antibody response is achievable by vaccination .We have previously reported a germline-targeting immunogen, eOD-GT8 60-mer, capable of activating germline precursors of VRC01-class bnAbs. Because eOD-GT8 60-mer requires a highly engineered CD4bs epitope to activate VRC01-class precursors, antibodies elicited by priming with eOD-GT8 60-mer do not show any detectable affinity for native HIV Env . TherefoAlthough boosted VRC01 gH mice showed broad neutralization on a panel of N276A viruses, neutralization of fully native virus containing the N276 glycan site was limited to a single heterologous tier 2 isolate and was substantially less potent. While the weak neutralization of fully native HIV indicates that there is still significant work to be done before we are able to elicit a truly functional broadly neutralizing response, these data strongly suggest that the elicited responses are VRC01-class antibodies of intermediate maturity. Mature VRC01, in contrast to non-neutralizing mAbs that target the CD4bs, neutralizes N276A viruses much more potently than fully native viruses, so the limited activity of the elicited mAbs against fully native viruses containing the N276 glycan site may simply be a normal feature of partially mature VRC01-class antibodies . Indeed,The relatively low VRC01-class precursor frequency and substantial competition from other clones in the VRC01 gH mouse pose a relatively high bar for elicitation of VRC01-class responses. Thus, the ability to recall VRC01-class precursors and drive maturation toward mature VRC01-class function validates the reductionist sequential immunization strategy and represents a significant milestone in HIV vaccine development.schief@scripps.edu).Further information and requests for reagents may be directed to, and will be fulfilled by the corresponding author William R. Schief  were obtained from a commercial vendor (AllCells) using a protocol approved by the Institutional Review Boards of both AllCells and The Scripps Research Institute. Peripheral blood mononuclear cells (PBMCs) were isolated using gradient centrifugation.6 cells/ml. The genes were transfected using 293fectin (Invitrogen) and harvested 4-5 days later. The cells were centrifuged at 4000rpm for 15min, filtered using 0.2 μm filter (Millipore) and a protease inhibitor was added at ratio of 1ml per liter of supernatant . The supernatants were purified by nickel affinity purification using His-Trap columns (GE), starting with a wash buffer  and mixing with elution buffer  using a linear gradient. The trimers were then purified by semi-analytical size exclusion chromatography on a S200Increase 10-300 column (GE) in HBS . The trimer fractions were pooled, concentrated to 1mg/ml by using Ultracel 30K centrifugal spin concentrators (Millipore) and measuring concentration on a NanoDrop 2000c Spectrophotometer using the absorption signal at 280 nm, frozen in thin-walled PCR tubes using liquid nitrogen, and then stored at −80°C. BG505 SOSIP trimers produced by this in-house process have been thawed and analyzed by SECMALS, SPR, differential scanning calorimetry, and electron microscopy and have been found to possess the native-like antigenic profile, thermal stability and closed trimeric structure that have been reported by others for BG505 SOSIP purified by an antibody-affinity column followed by SEC (eOD-GT8 and BG505 GT3 monomers and NPs were produced and purified as described previously . BG505 Sd by SEC .1HQK), previously described for displaying eOD-GT6 . Heavy- and light-chain plasmids were cotransfected (1:1 ratio) in 293 FreeStyle cells using 293fectin (Invitrogen). Transfections were performed according to the manufacturer’s protocol, and antibody supernatants were harvested 4-5 days after transfection. Antibody supernatants were purified over Protein A Sepharose 4 Fast Flow  columns, eluted with 0.1M citric acid (pH 3.0), and dialyzed against phosphate-buffered saline.Kinetics and affinities of antibody-antigen interactions were measured as described previously . BrieflyMice spleen and lymph node samples were processed for single B cell sorting based on previously described methods , with slhttps://.github.com/briney/abstar).Reverse transcription and subsequent PCR amplification of heavy and light chain variable genes were performed using SuperScript III (Life Technologies) according to published protocols . All PCR)2 conjugated to alkaline phosphatase (Pierce), diluted 1:1000 in PBS containing 1% BSA and 0.025% Tween 20, was added to the wells. The plate was incubated at room temperature for 1 hr, washed four times, and the plate was developed by adding 50 uL of alkaline phosphatase substrate (Sigma) to 5 ml alkaline phosphatase staining buffer (pH 9.8), according to the manufacturer’s instructions. The optical density at 405 nm was read on a microplate reader (Molecular Devices). ELISA protocol for mAbs was as follows. ELISA plates were coated overnight at 4°C with 25 μl of 4 μg/ml anti-His Ab . The wells were washed 5 times with PBST (PBS with 0.2% Tween 20) and blocked with 5% milk at RT for 1 hr. The wells were washed 5 times with PBST. mAb were diluted to 10 μg/mL in 0.1% milk PBST then added to the plates and incubated at RT for 1 hr. After washing 5 times in PBST, goat anti-human HRP (Jackson) was diluted 1:5000 in 0.1% milk PBST, then 25 μl was added to each well and the plate was incubated at RT for 1hr. After washing 5 times in PBST, the plate was developed by adding 50 μl TMB ELISA solution (Thermofisher) and then 50 μl sulfuric acid stop solution after 10 min. The optical density at 450 nm was read on a microplate reader (Molecular Devices).Ninety-six-well ELISA plates were coated overnight at 4°C with 50 uL PBS containing 100 ng of antigen per well. The wells were washed four times with PBS containing 0.05% Tween 20 and blocked with 3% BSA at room temperature for 1 hr. Serial dilutions of sera were then added to the wells, and the plates were incubated at room temperature for 1 hr. After washing four times, goat anti-mouse IgG F(ab’Mutations were introduced by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene) and mutants were verified by Sanger DNA sequencing.50 values, serial dilutions of mAbs were incubated with virus and the dose-response curves were fitted using nonlinear regression.To produce pseudoviruses, plasmids encoding Env were co-transfected with an Env-deficient genomic backbone plasmid (pSG3ΔEnv) in a 1:2 ratio with the transfection reagent Fugene 6 (Promega). Pseudoviruses were harvested 72 hr post transfection for use in neutralization assays. Neutralizing activity was assessed using a single round of replication pseudovirus assay and TZM-bl target cells, as described previously . BrieflyLeukopaks were obtained from two healthy, HIV-negative individuals (AllCells) and peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation. PBMCs from each donor were separated into aliquots of 500 m cells and total RNA was extracted separately from each PBMC aliquot . In quadruplicate, 10uL of each RNA aliquot was separately amplified in 100uL RT-PCR reactions  using previously reported primers  and withhttps://github.com/briney/abstar), raw sequencing reads were quality trimmed with Sickle (https://github.com/najoshi/sickle), adapters were removed with cutadapt , the probability distribution for the subsequent synthetic mutation was computed using NGS sequences that contain a naturally occurring 24A mutation. If the second mutation was 36F, then the probability distribution for the third synthetic mutation would be computed from NGS sequences with both 24A and 36F. Of note, prior mutations were excluded from the conditional probability distribution. This ensures that, for example, the 24A mutation will not happen a second time in the same sequence. It is also important to note that, due to technical limitations on sequencing length and the annealing location of amplification primers midway through the framework 1 region (FR1), mutations in the first portion of FR1 were not sampled and thus were not used in mutation probability calculations. This is evident in the lack of mutations near the start of synthetically generated antibody sequences F. Becaus3J5M) as well as inclusion of the V2 loop. Three of the five trimers formed native-like structures and antigenic profiles and were used as boosting immunogens in the VRC01-gH mice.A five member CD4bs cocktail was engineered on gp120-core by analyzing the sequence diversity of HIV strains at VRC01-class epitope positions, which includes the V5 loop. Each member of the cocktail incorporates mutations from a single strain, and these five strains were chosen to best mimic the diversity of HIV at VRC01-class epitope positions. We next created a native-like trimer cocktail (ABC) by transferring the mutations from the gp120-core cocktail and adding new mutations found proximal to the PGV04 VRC01-class bnAbs in the Env trimer structure  was used for DSC measurements. The protein samples were diluted into HEPES buffer to a final concentration of 0.25 mg/ml. The experiment scanned from 20°C to 90°C at a scan rate of 90°C/h. Data were analyzed by buffer correction, normalization, and baseline subtraction (Origin 7.0).When computing the frequency of random incorporation of VRC01-class mutations, we iterated temporally through mutations  and determined the frequency of mutations from each synthetic antibody sequence that were VRC01-class. Using the range of VRC01-class frequencies at each step, we computed the mean frequency (shown as a black line KX779470–KX779519 and KX808478–KX808481 as well as in the GitHub repository: https://github.com/briney/VRC01gH-GT3.The sequences of elicited antibodies reported in this paper have been deposited at GenBank: B.B., D.S., J.G.J., D.W.K., P.S., D.N., D.R.B., and W.R.S. planned and designed the experiments. B.B., D.S., J.G.J., D.W.K., P.S., S.M., R.J., O.K., N.d.V., F.S., K.M.L., A.R., M.J., K.L.S.-F., T.R.B., S.S., E.G., X.H., G.O., Y.A., M.K., and A.S. performed the experiments and analyzed data. B.B., D.S., J.G.J., D.W.K., D.R.B., and W.R.S. wrote the manuscript. All authors contributed to manuscript revisions. I.A.W., A.B.W., D.N., D.R.B., and W.R.S. supervised the research.


Type-C dose algorithms provide more accurate dosimetry for lung SBRT treatment planning. However, because current dosimetric protocols were developed based on conventional algorithms, its applicability for the new generation algorithms needs to be determined. Previous studies on this issue used small sample sizes and reached discordant conclusions. Our study assessed dose calculation of a Type-C algorithm with current dosimetric protocols in a large patient cohort, in order to demonstrate the dosimetric impacts and necessary treatment planning steps of switching from a Type-B to a Type-C dose algorithm for lung SBRT planning.95% = prescription dose using a Type-B algorithm. These were compared against three Type-C plans: re-calculated plans , re-normalized plans (D95% = prescription dose), and re-optimized plans. Dosimetric endpoints were extracted and compared among the four plans, including RTOG dosimetric criteria: , PTV Dmin, Dmax, Dmean, V% and D90%, PTV coverage (V100%), homogeneity index (HI), and Paddick conformity index (PCI).Fifty-two lung SBRT patients were included, each planned using coplanar VMAT arcs, normalized to Dmin with a mean difference of 5.2% and increased Dmax with a mean difference of 3.1%, similar or improved RTOG dose compliance, but compromised PTV coverage . Seven plans had >5% D95% reduction (maximum reduction = 16.7%), and 18 plans had >5% V100% reduction (maximum reduction = 60.0%). Re-normalized Type-C plans restored target coverage, but yielded degraded plan conformity (average PCI reduction 4.0%), and RTOG dosimetric criteria deviation worsened in 11 plans, in R50%, D2cm, and R100%. Except for one case, re-optimized Type-C plans restored RTOG compliance achieved by the original Type-B plans, resulting in similar dosimetric values but slightly higher target dose heterogeneity (mean HI increase = 13.2%).Re-calculated Type-C plans resulted in decreased PTV DType-B SBRT lung plans considerably overestimate target coverage for some patients, necessitating Type-C re-normalization or re-optimization. Current RTOG dosimetric criteria appear to remain appropriate. Lung cancer is the second most common type of cancer for both men and women, and the leading cause of cancer death, making up about 1 in every 4 cancer-related deaths –4. It is® XB dose calculation algorithm, implemented for treatment planning in Eclipse , is based on a linear Boltzmann equation solver to solve radiation transport equation, rather than simulation of particle transport implemented in Monte Carlo. It is considered a Type-C algorithm due to the comparable level of dose modeling accuracy [Lung SBRT conventionally used multiple coplanar or non-coplanar conformal beams . In receaccuracy , 34–36.Many studies have investigated the dosimetric differences between Type-A or Type-B and Type-C dose algorithms, and have shown that Type-C algorithms more accurately calculate the dose distribution for SBRT plans of lung patients , 32, 35.50%) because their values were increased with the Type-C algorithm and the corresponding compliance was worse. In contrast, Rana et al. published a study on 14 patients evaluating the effect of switching from a Type-B algorithm  to a Type-C algorithm (AXB) in Eclipse for lung SBRT plans using RTOG0813 dosimetric criteria [Several authors have previously examined the dosimetric compliance of Type-C calculated lung SBRT plans to the current protocols , 39, 40.criteria . They foThe aim of this study, therefore, was to systematically investigate the necessity and dosimetric impacts of switching from Type-B to Type-C dose algorithms for lung SBRT planning on a large patient cohort. On fifty-two patients, the target dose parameters as well as RTOG dosimetric criteria were compared among the following plans: original Type-B plans, re-calculated Type-C plans with identical beam settings, re-normalized Type-C plans (to ensure target coverage), and re-optimized Type-C plans .As will be detailed in the following sections, the dosimetric impacts of switching from Type-B to Type-C calculation were comprehensively evaluated using various dose-volume endpoints and protocol dosimetric constraints on 52 lung SBRT patient plans. We compared the original Type-B plans with the re-calculated, re-normalized, and re-optimized Type-C plans.With the approval of the University of Nebraska Medical Center institutional review board, treatment data for 70 patients with NSCLC, treated with lung SBRT at our institution between April 2014 and August 2016, were collected. From these, 52 plans treated with VMAT were selected for this retrospective study. The remaining cases were excluded because they used 3D conformal treatment techniques.According to our institutional protocol, each patient received a free-breathing three-dimensional computed tomography (3D CT) followed by an eight-phase four-dimensional computed tomography (4D CT) acquired using a Sensation Open CT simulator  and the Real-time Position Management system  as the respiratory surrogate. A slice thickness of 2 mm was used for both CT scans. Patients were simulated in treatment position and immobilized with the BlueBAG™ immobilization system . The 3D and 4D CT images were fused using the common frame of reference if visually confirmed with no relative movement, or otherwise using rigid registration to the spine. For each patient, the gross tumor volume (GTV) was delineated by the attending radiation oncologists as the visible tumor on the 3D CT, and the internal target volume (ITV) was contoured using the 3DCT, maximum intensity projection (MIP) from the 4DCT, and each phase of the 4DCT, as the union of the gross tumor seen on these images excluding possible nearby soft-tissue. The planning target volume (PTV) was generated by adding an isotropic expansion of 5–6 mm to the ITV.95% = prescription dose).For treatment planning, one to two coplanar VMAT arcs were used, with partial arcs on the ipsilateral side to spare the contralateral lung for some peripheral lesions and full arcs for the rest. Fifty plans used flattening-filter-free 6 MV and/or 10 MV beams, and the rest used flattened beams of these energies. The plans were optimized to best comply with the dosimetric criteria and normal tissue constraints specified in RTOG0813, RTOG0915, other guidelines , 38, 41,The above plans were re-calculated using a Type-C algorithm, Acuros XB or AXB in Eclipse 13.5. Identical beam parameters including MLC patterns and monitor units were used for the re-calculation as in the original Type-B, or AAA plans. For AXB calculations, dose-to-medium was reported.95% = prescription dose) as in the original clinical Type-B plans. In other words, these plans used identical MLC patterns and relative beam weighting as the original plans, but with different MUs so that the Type-C plans’ PTV doses were re-normalized to match the original Type-B, or AAA plans (D95% = prescription dose).Another set of treatment plans was generated by re-calculating the original clinical treatment plans with the Type-C algorithm (AXB), but specifying plan normalization so that 95% of the target volume received the full prescription dose , ratio of 50% prescription isodose volume to PTV (R50%), maximal dose 2 cm from the PTV in any direction as a percentage of prescription dose (D2cm), the percentage of lung receiving dose equal to or larger than 20 Gy (V20), and the volume of 105% isodose outside the PTV (V105%). The optimization objectives were adjusted to create plans that optimize these dosimetric values with intermediate and final Type-C dose calculation.For the re-normalized Type-C plans that did not meet the level of RTOG dosimetric compliance seen in their respective original Type-B plans, re-optimization was performed using the Type-C algorithm, keeping the normalization at Dmin), maximum (Dmax), and mean (Dmean) doses of the PTV were recorded, along with the doses received by 95% (D95%) and 90% (D90%) of the PTV volume. Volume-based dosimetric parameters such as D95% were included and used for plan normalization because single-point-based doses such as Dmin are associated with higher uncertainties. The PTV coverage, referring to the percent volume of PTV covered by prescription dose or V100%, was also recorded. RTOG criteria were used to calculate the values for R100%, R50%, D2cm, V20, and V105%. Additionally, the Paddick Conformity Index (PCI) [PIV)2/(TV x PIV), with TVPIV being the PTV volume receiving full prescription dose, TV being the total PTV volume, and PIV being the total irradiated volume receiving full prescription dose. For any plan, the PCI would be within the range of 0 to 1.0, with PCI = 1.0 for an ideal plan. The HI was defined as the ratio of the maximum dose over the prescription dose. The above dosimetric parameters were used to compare the four types of plans as described in II.A.-II.D. for each of the 52 patients.For all above plans, the minimum (Dex (PCI)  and the ex (PCI)  were calPatient and tumor characteristics of the 52 patients are described in Table 100%), PCI, and HI. The numbers of cases with deviations to RTOG dosimetric criteria are listed in Table For the 52 patients, the average values and standard deviations are summarized in Table 50% and 16 had minor deviations for D2cm. None had deviations for R100%, V20, or V105%, and no major deviations were observed for any of the criteria. After re-calculating with Type-C algorithm, the level of deviation was reduced from minor to none for 3 plans for R50% and similarly in 5 plans for D2cm. On the other hand, these re-calculated Type-C plans revealed the target coverage dose overestimation by the Type-B algorithm, as can be seen in the reduced PTV Dmin, D95%, and D90% in Table 100% in Table 95% reduction may not seem clinically significant, a wide range of variation (standard deviation = 4.4%) was observed from patient to patient, with a maximum reduction of 16.7%. Of the 52 patients, 7 patients (13.5%) showed PTV D95% a reduction over 5%, and 4 patients (7.7%) showed a reduction over 10%. Figure 95% reduction. Similarly, PTV V100% was reduced on average by 7.4% from 92.2 ± 3.3% to 84.8 ± 14.6%. Of the 52 patients, 18 patients (34.6%) showed a PTV V100% reduction over 5%, and 15 patients (28.8%) showed a reduction over 10%. The maximum V100% reduction was 60% for one patient. Figure 95% and V100% reductions for all patients over PTV size.As shown in Table 95% reduction was 4.2%, with a maximum reduction of 15.1%. We therefore designed re-normalized Type-C plans to restore the dose coverage by ensuring D95% = prescription dose through inflated MUs. Not surprisingly, the average values for the RTOG dosimetric criteria increased on the re-normalized plans for R100%, R50%, and D2cm compared with the original Type-B plans. On 11 out of the 52 plans (21.2%), protocol compliance degraded on one or more dosimetric criteria when compared with the original Type-B plans. For R50%, 5 plans changed from minor to major deviation, and 4 plans from no deviation to minor deviation. For D2cm, 1 plan changed from minor to major deviation, 6 plans from no deviation to minor deviation, and 5 plans from minor deviation to no deviation. For R100%, 3 plans changed from no deviation to minor deviation. No plans showed changes for V105% or lung V20.The PTV coverage reductions on the re-calculated Type-C plans suggested that simply re-calculating the plans with the Type-C algorithm might not provide fair comparisons. For example, on the 8 plans with improved RTOG dose compliance on the re-calculated Type-C plans when compared with the original Type-B plans, the average PTV D100%, R50%, D2cm, and lung V20, respectively. It is apparent that the ratios for the re-calculated plans are more clustered around or below 1.0 and those for the re-normalized plans are more spread out and above 1.0. This indicates that while re-calculated Type-C plans achieved similar or even better RTOG dosimetric criteria compliance as the original Type-B plans, the compliance is worsened on the re-normalized Type-C plans in which adequate target dose coverage was restored.The ratios of the re-calculated and re-normalized Type-C plans over the original Type-B plans are plotted against PTV volume in Fig. 50%, D2cm and/or R100%, re-optimization was performed using the Type-C algorithm. While keeping the original beam arrangements and collimator angles, new optimization objectives were necessary for Type-C optimization, different from those used for the original Type-B optimization. On these 11 patients, the ratios of the re-optimized Type-C plan over the original Type-B plan were listed for PTV dosimetric parameters, RTOG criteria, and other plan quality indexes in Tables 95% reduction revealed by the re-calculated plans are also listed alongside as a reference. The original levels of compliance on the original Type-B plans were restored by the re-optimization for all but one patient, Patient #11, who continued to have a minor R50% deviation after re-optimization while the original Type-B plan had no deviation.For the 11 patients whose re-normalized Type-C plans showed worsened RTOG dosimetric criteria compliance for R100%, R50%, and D2cm values. As a result, R100% deviation increased from no deviation to minor deviation, R50% deviation increased from minor to major deviation, and D2cm deviation increased from minor to major deviation, which necessitated re-optimization using the Type-C algorithm. On the re-optimized Type-C plan, the target conformity was restored, and the compliance to the relevant RTOG criteria was also restored to the levels previously achieved by the original Type-B plan.As an example of comparing the 4 plans of the same patient, the isodose distributions in the axial view at the isocenter for Patient #3  compared with the original Type-B plans. However, re-optimization using the Type-C algorithm was able to restore the original RTOG compliance for all but one of these patients. Current RTOG dosimetric criteria hence appear to remain appropriate for lung SBRT plans calculated with Type-C algorithms, provided that such algorithms are employed for plan optimization.Our study shed some light on the conflicting results above. Similar to Rana et al.’s study , the re-100%, R50%, and D2cm, by about 10% in order for their Type-C plans to comply with the criteria [100%, 9 plans recorded minor deviations for R50%, 4 plans recorded minor deviations for D2cm, and 1 plan recorded minor deviation for V20. Similarly, in Li et al.’s series of 15 patients, Type-B plans also resulted in deviations on these RTOG criteria for some of them, such as the 5 minor deviations and 2 major deviations on D2cm [Pokhrel et al. recommended up-adjustments on the values for RTOG dosimetric criteria, such as Rcriteria , 40. How on D2cm .By designing a comprehensive comparison between original Type-B plan, the re-calculated Type-C plan, the re-normalized Type-C plan, and the re-optimized Type-C plan, we have clearly outlined in our study the dosimetric necessity as well as impact of re-calculation, re-normalization, and re-optimization using Type-C algorithms. While Li et al.’s study also considered re-optimization using the Type-C algorithm, the conventional conformal beam treatment technique was used in their study, and their re-optimization involved only adjusting the relative beam weights while keeping all other beam parameters the same as the original Type-B plans . Further95% reduction over 5%. For the remaining 4 patients, 3 had a PTV D95% reduction over 4% and 1 had a reduction of 2%. This indicates that for those patients on whom Type-B algorithms will lead to large PTV coverage dose overestimations, optimization by Type-C algorithms will likely be necessary to achieve similar levels of RTOG dosimetric criteria compliance currently achieved by Type-B algorithms. For patients on whom Type-B algorithms will lead to small PTV coverage dose overestimations, plans optimized with Type-B algorithms will likely only need to be re-normalized with Type-C calculation to ensure sufficient and accurate target dose coverage, as well as to achieve similar levels of RTOG compliance. In a small number of these patients, if the RTOG compliance of the Type-B plans are borderline, optimization with Type-C algorithms may also be necessary to achieve the same compliance goal.Of our 52 patients, 11 (21.2%) resulted in worsened RTOG dosimetric criteria compliance on the re-normalized Type-C plans and hence necessitated re-optimization with the Type-C algorithm. These patients included all 7 patients with a PTV D50% and D2cm. The re-optimized plan restored D2cm compliance to no deviation, but on R50% the re-optimized Type-C plan still had a minor deviation. Among our cohort, this patient was also the one with the highest magnitude of PTV D95% reduction on the re-calculated Type-C plan, with a reduction of 16.7%. This large discrepancy in target dose calculation between Type-C and Type-B algorithms might have partially contributed to the difficulty of re-optimizing a fully-compliant plan with the Type-C algorithm. In addition, the R50% value of the original Type-B plan for this patient, 3.4, was somewhat close to the minor deviation threshold of 3.8. The re-optimized plan had a value of 4.0, and hence scored as a minor deviation.For one patient, re-optimization using the Type-C algorithm was unable to fully restore the RTOG dosimetric criteria compliance  and the very high local control rates usually at around 90%, a very large cohort with sufficient longitudinal follow-up would be necessary to power such a study and illustrate the clinical significance of the dosimetric effects.It would be an interesting direction for future studies to assess the clinical impact of switching from Type-B to Type-C algorithms for lung SBRT. For example, on a cohort of patients treated with Type-B plans, the local failure rates can be compared between those patients with large PTV DType-B dose calculation considerably overestimates target dose coverage in lung SBRT for some patients, necessitating Type-C re-normalization or re-optimization. Current RTOG dosimetric criteria appear to remain appropriate in the setting of Type-C calculations.


Sphingolipid metabolites are emerging as important signaling molecules in allergic diseases specifically asthma. One of the sphingolipid metabolite, sphingosine-1-phosphate (S1P), is involved in cell differentiation, proliferation, survival, migration, and angiogenesis. In the allergic diseases, alteration of S1P levels influences the differentiation and responsiveness of mast cells (MCs). S1P is synthesized by two sphingosine kinases (SphKs), sphingosine kinase 1, and sphingosine kinase 2. Engagement of IgE to the FcεRI receptor induces the activation of both the SphKs and generates S1P. Furthermore, SphKs are also essential to FcεRI-mediated MC activation. Activated MCs export S1P into the extracellular space and causes inflammatory response and tissue remodeling. S1P signaling has dual role in allergic responses. Activation of SphKs and secretion of S1P are required for MC activation; however, S1P signaling plays a vital role in the recovery from anaphylaxis. Several non-coding RNAs have been shown to play a crucial role in controlling the MC-associated inflammatory and allergic responses. Thus, S1P signaling pathway and its regulation by non-coding RNA could be explored as an exciting potential therapeutic target for asthma and other MC-associated diseases. Mast cells (MCs) are best known to trigger IgE-dependent/independent allergic diseases. They also play a significant role in providing immunity to host in response to various infections . MCs havTwo highly homologous sphingosine kinases (SphKs), known as sphingosine kinase 1 (SphK1) and sphingosine kinase 2 (SphK2) catalyze the synthesis of S1P from its precursor sphingosine Figure . SphK1 itrans-2-hexadecenal  dephosphorylate a broad range of lipid phosphate substrates including S1P , 9. LPPsadecenal .Till date, five S1P receptors (S1PR1–5) that bind to S1P have been identified in vertebrates . In mammAntigen-induced aggregation of IgE bound to FcεRI on MCs activates both the SphKs with a consequent increase in intracellular S1P levels . ActivatUpon engagement of FcεRI receptor, both the isoforms of SphKs get activated in MCs , 20. How+ progenitors-derived human MCs  in the respiratory system –39. In p2, mucus production, and IL-13  mice, recapitulated the ASM reactivity -sensitized mice, S1P/SphK pathway triggers airway hyperresponsiveness . Furthernd IL-13 . S1P-stind IL-13 . Furtherthe lung . In MC-dthe lung . Interesactivity . Pre-expsphatase . In a musphatase .n = 6), compared to control-group children. Similarly, upregulation (~3-fold) of miR-221 and miR-485-3p has been noted in the lungs of OVA-induced asthmatic mouse model and resulting into decreased levels of their target gene, Spred-2  protein through increasing the stability of TRPC1  emerge as a crucial regulator of MCs development and play an important role in allergic diseases and bronchial asthma. Loss of dicer function in mouse MC progenitor cells results in profound disruption of tissue MC compartments, suggesting a critical role for miRNAs in MC differentiation, growth, and migration . miR-221 Spred-2 . In a se Spred-2 . These fumigatus . miR-21 umigatus . In addiof TRPC1 . The oveof TRPC1 . Furtherof TRPC1 .Several miRNAs have been demonstrated to target the components of the S1P signaling pathway Table , 59, 60,Sphingosine-1-phosphate is a dual regulator of the systemic allergic responses. It is an essential signaling molecule generated upon IgE-FcεRI crosslinking and enhances the activation of MCs in allergic diseases such as asthma. On the other hand, S1P signaling through its receptors is required for recovery from anaphylactic shock. The role of non-coding RNAs is well explored in cancer; however, their role in allergy and inflammation is still in infancy, and their role needs to be explored as a potential therapeutic target and biomarker for asthma.RS, AK, MJ, SG, and AJ designed the manuscript; were involved in drafting/revising the manuscript; and read and approved the final manuscript. RS, AK, and AJ wrote the first draft.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


IL-28B genotype and NK cell activity.Immuno-genetic studies suggest a functional link between NK cells and λ-IFNs. We recently showed that NK cells are negative for the IFN-λ receptor IFN-λR1 and do not respond to IFN-λ, suggesting a rather indirect association between IL-28B (rs12979860) and IFNL-4 (rs368234815) genotypes were determined by rtPCR. Total PBMC, monocytes, and NK cells were stimulated with IL-29, the TLR-7/8 agonist R848, or a combination of both. NK cell IFN-γ response was analysed by FACS. IL-12 and IL-18 secretion of monocytes was studied by ELISA. In blocking experiments anti-IL-12/anti-IL-18 were used.A total of 75 HCV(+) patients and 67 healthy controls were enrolled into this study. IL-28B genotype, with carriers of a T/T genotype displaying the lowest frequency of IFN-γ(+)NK cells. When isolated NK cells were studied no such associations were observed, indicating an indirect association between IL-28B genotype and NK cell activity. Accordingly, we found R848-stimulated monocytes of patients with a T/T genotype to be significantly less effective in triggering NK cell IFN- γ production than monocytes from carriers of a non-T/T genotype. In line with these findings we observed monocytes from T/T patients to secrete significantly lower concentrations of IL-12 than monocytes from non-T/T individuals.Following stimulation of total PBMCs with R848 we found NK cell IFN- γ responses to vary with the IL-28B T/T genotype display a reduced ability to stimulate NK cell activity and, thus, provide a link between IL-28B genotype and NK functions.Our data indicate that monocytes from carriers of an  Infection with the hepatitis C virus (HCV) is a major cause of blood-borne hepatitis worldwide. The majority of patients exposed to HCV develop chronic infection which is associated with a significant risk to develop chronic liver disease, including cirrhosis and hepatocellular carcinoma.interleukin 28 B (IL-28B) gene, which encodes for the type III interferon lambda 3 (IFN-λ3), has been shown to be significantly associated with response to IFN-based therapy of chronic hepatitis C and with the natural course of acute hepatitis C  allele. Thus, we cannot exclude a functional role of the IFNL4 polymorphisms in our setting.More recently, Mei and colleagues demonstrated an association between the mulation . Of noteIL-28B T/T genotype was associated with high frequency of IFN-γ+ NK cells compared to carriers of an IL-28B C/C genotype, whereas the opposite was found in healthy controls [IL-28B CC genotype in comparison to the unfavorable genotype [In contrast to our study, Rogalska-Taranta et al. found that in patients with chronic hepatitis C carriage of an controls . These dgenotype . Furthergenotype .IL-28B-dependent IL-12 secretion by monocytes.An important question relates to the mechanism underlying IL-28B gene is located on chromosome 19 whereas the genes encoding IL-12A and IL-12B map to chromosomes 3 and 5, respectively. Thus, a strong genetic linkage seems rather unlikely. Moreover, expression of TLR-7 and TLR-8, the receptors for R848, did not differ between IL-28B genotypes.The IL-28B genetic variant has repeatedly been shown to be associated with differential expression of ISGs [Increased expression of interferon stimulated genes (ISGs) is a typical feature of chronic hepatitis C, most likely reflecting permanent stimulation by type I and/or type III IFNs . Of note of ISGs –40. In t of ISGs .IL-28B T/T genotype-associated up-regulation of ISGs in chronic hepatitis C results in exhaustion of the common pathway subsequently leading to ineffective response to type-I IFN [A possible explanation might be a setting in which pe-I IFN . In linepe-I IFN .IL-28B T/T genotype. Of note, an association between monocyte IL-12 production and IL-28B genotype was observed only in patients with chronic hepatitis C but not in healthy individuals. Again this would be compatible with the model of IL-28B genotype-dependent induction of ISGs because chronically upregulated IFN expression is considered to be essential for the observed ISG up-regulation in carriers of a T/T genotype in hepatitis C infection [Since stimulation of TLR-7 has also been shown to induce expression of ISGs  a similanfection .IL-28B genotype-dependent activation of macrophages/Kupffer cells to modulate anti-HCV immune-responses. Thus, it is tempting to speculate that a similar mechanism might play a role in monocytes. In line with this hypothesis, cross-culture experiments demonstrated that IL-28B genotype dependent IFN-γ production of NK cells was mainly attributable to increased IL-12 production of HCV(+) monocytes. Moreover, we observed higher expression of pSTAT1 and pSTAT5 in monocytes from patients with a non-T/T genotype than in carriers of a T/T genotype. However, these differences did not reach statistical significance.On the other hand, ISG expression in macrophages might be different from that observed in hepatocytes. Indeed, in Kupffer cells, the liver resident macrophages, the pre-therapy ISG levels have been shown to be opposite of those seen in hepatocytes, with patients not responding to IFN-α based treatment lacking induction of ISGs, whereas responders displayed strongly induced ISG . Of noteIL-28B rs12979860 polymorphism, thereby providing a mechanistic link between NK cell function and the IL-28B genotype.Taken together, we show that in chronic hepatitis C the extent of monocyte-induced and IL-12-mediated stimulation of NK cell IFN-γ production is associated with the IL-28B genetic variants determine the extent of monocyte-induced activation of NK cells in hepatitis C”we reported that a total of 74 HCV(+) patients and 80 healthy controls were enrolled into the study. However, numbers of participants in the present publication are different. The poster abstract had been erroneously included participant samples from preliminary experiments with different workflow.In a poster abstract presented on the “Liver meeting 2015” of AASLD with the title “S1 FigIL28-B genotypes (Non-TT vs TT), respectively.CD56+CD3- NK cells were sub-divided in CD56Bright und CD56 Dim NK cells by CD56/CD16 gating (A). PBMC from HCV patients were pre-stimulated with R848 then co-cultured with HUH7HCVreplicon cells (B) or hepatic stellate cells (C), respectively. After 5h of co-incubation IFN-γ production (B) or degranulation (C) of NK cells was studied by FACS analysis, respectively. The exemplary histograms show IFN-γ production (B) or degranulation (C) of CD56Bright (left side) and CD56Dim NK cells (right side) from HCV patients with different (PDF)Click here for additional data file.S2 FigIL-28B genotypes .Monocytes from HCV patients were pre-stimulated with Poly-I/C and then co-cultured with autologous NK cells in the HUH7HCVreplicon cells. After 5h of co-incubation IFN-γ production of NK cells was studied by FACS analysis. This figure shows IFN-γ production of NK cells from HCV patients with different (PDF)Click here for additional data file.S3 FigIL-28B genotypes .Monocytes from HCV patients (A) were pre-stimulated with R848 then co-cultured with healthy NK cells in the HUH7HCVreplicon cells and vice versa (B). After 5h of co-incubation IFN-γ production of NK cells was studied by FACS analysis. This figure shows IFN-γ production of NK cells from healthy donors (A) or HCV patients (B) with different (PDF)Click here for additional data file.S4 FigIL-28B genotypes  were pre-stimulated with R848 then co-cultured with HUH7HCVreplicon cells. After 5h of co-incubation IFN-γ production of CD56Bright NK cells was studied by FACS analysis. The figure shows the IFN-γ production of CD56Bright NK cells depending on serum alanine aminotransferase (A: ALT <40 vs. <40 and >120 vs. >120 U/l) and HCV viral load.Total PBMCs from HCV patients with different (PDF)Click here for additional data file.S1 TableThis table includes all raw data of Figs (PDF)Click here for additional data file.