Coordination to divalent cations protects the human host-defense protein calprotectin from proteolytic degradation. ii) ions to modulate its quaternary structure, transition metal binding properties, and antimicrobial activity. In this work, we report the discovery that Ca(ii)-induced self-association of human CP to afford heterotetramers protects the protein scaffold from degradation by host serine proteases. We present the design and characterization of two new human CP-Ser variants, S100A8(C42S)(I60E)/S100A9(C3S) and S100A8(C42S)(I60K)/S100A9(C3S), that exhibit defective tetramerization properties. Analytical size exclusion chromatography and analytical ultracentrifugation reveal that both variants, hereafter I60E and I60K, persist as heterodimers in the presence of Ca(ii) only, and form heterotetramers in the presence of Mn(ii) only and both Ca(ii) and Mn(ii). Coordination to Fe(ii) also causes I60E and I60K to form heterotetramers in both the absence and presence of Ca(ii). The Ca(ii)-bound I60E and I60K heterodimers are readily degraded by trypsin, chymotrypsin, and human neutrophil elastase, whereas the Ca(ii)-bound CP-Ser heterotetramers and the Ca(ii)- and Mn(ii)-bound I60E and I60K heterotetramers are resistant to degradation by these host proteases. The staphylococcal extracellular protease GluC cuts the S100A8 subunit of CP-Ser at the C-terminal end of residue 89 to afford a ΔSHKE variant. The GluC cleavage site is in close proximity to the His3Asp metal-binding site, which coordinates Zn(ii) with high affinity, and Zn(ii) chelation protects the S100A8 subunit from GluC cleavage. Taken together, these results provide new insight into how Ca(ii) ions and transition metals modulate the chemistry and biology of CP, and indicate that coordination to divalent cations transforms human CP into a protease-resistant form and enables innate immune function in the hostile conditions of an infection site.Calprotectin  is a protein component of the innate immune system that contributes to the metal-withholding response by sequestering bioavailable transition metal ions at sites of infection. Human CP employs Ca( Staphylococcus aureus.iii)-bound form.Calprotectin  is an antimicrobial protein and important player in the human innate immune response and inflammation.ii)-binding S100 proteins S100A8  and S100A9 .ii) ion binding results in formation of the CP heterotetramer (α2β2).ii) ions also modulate the transition metal binding properties and antimicrobial activity of CP.ii) levels are high, the CP heterotetramer is expected to be a relevant and abundant extracellular form.ii) binding to CP and consequent formation of the heterotetramer protects the scaffold against degradation by host and bacterial proteases. The proteolytic stability of CP is largely unexplored, and some of the results from published studies appear to be conflicting. Two independent studies demonstrated that the CP heterooligomer is more protease resistant than the S100A8 and S100A9 homodimers.ii) and Zn(ii) supplement.i.e. oligomeric state, metal-free versus metal-bound). To clarify whether CP resists protease attack and test our hypothesis that speciation plays a role, we sought to systematically evaluate how metal ions and quaternary structure influence proteolytic stability.Human CP exhibits complex oligomerizationii)-induced tetramerization protects CP from host serine proteases. Moreover, we establish that Zn(ii) complexation protects the S100A8 C-terminus from the staphylococcal serine protease GluC. These data support a new dimension to how Ca(ii) ions and transition metals modulate the function and fate of extracellular CP, indicating that Ca(ii) binding allows CP to sequester transition metals and resist attack by host proteases.In this work, we report that Ca(ii)-induced tetramerization of human CP affords protease resistance, we sought to compare the stabilities of Ca(ii)-bound heterodimers and Ca(ii)-bound heterotetramers in protease degradation assays. These experiments require CP variants that remain heterodimeric following Ca(ii) complexation. The crystal structure of Mn(ii)-, Ca(ii)-, and Na(i)-bound CP-Ser  but cannot undergo Ca(ii)-induced tetramerization. We mutated (A8)Ile60 to Glu and Lys as a case study, and focus on these variants herein. We reasoned that introduction of charged residues at position 60 of S100A8 should disfavor tetramerization by decreasing the hydrophobic driving force and introducing electrostatic repulsion. Moreover, the parity of charges introduced by the Glu and Lys mutations provides a control to minimize the likelihood that any biophysical or functional differences between CP and the variants are artifacts of a given amino acid substitution. Lastly, it was important to preserve the metal-binding properties of CP. Because (A8)Ile60 does not contribute to Ca(ii)-ion coordination and is distant from both the transition metal binding sites , we reasoned that mutating this residue would result in negligible perturbation to the metal-ion coordination spheres. We abbreviate the CP-Ser variants bearing the (A8)Ile60Glu and (A8)Ile60Lys mutations I60E and I60K, respectively.In order to investigate whether Caii) with high affinity when excess Ca(ii) is included in the buffer. Like CP-Ser, both variants outcompeted ZP1 for Mn(ii) in the presence of excess Ca(ii), which indicated that the apparent Kd,Mn value of each variant is less than 550 nM  into a solution containing ZP1, Mn(ii), and protein demonstrated that CP-Ser, I60E, and I60K show comparable Ca(ii)-dependent Mn(ii)-binding properties. Each protein required ≈20 equivalents of Ca(ii) to fully sequester Mn(ii) from ZP1 -binding properties of I60E and I60K are Ca(ii)-dependent, and that these variants behave like CP-Ser, at least in the context of ZP1 competition assays.We prepared pET41a-S100A8(C42S)(I60E) and pET41a-S100A8(C42S)(I60K) plasmids by site-directed mutagenesis. We overexpressed and purified I60E and I60K as described previously for CP-Ser.M Fig. S4. Moreove1 Fig. S5. Taken tii), we employed analytical size-exclusion chromatography (SEC) and determined the elution volumes and corresponding molecular weights of the proteins in the absence and presence of excess Ca(ii) (ii) (600 μM) was included in the running buffer . This behavior was in agreement with our prior analytical SEC studies of CP-Ser,ii)-dependent formation of the α2β2 tetramer (ii) (ii) (600 μM) was included in the running buffer (ii), which is in agreement with the competition titrations described above; (ii) Ca(ii) complexation causes the hydrodynamic radii of I60E and I60K to decrease relative to the apo proteins; and (iii) Ca(ii)-bound I60E and I60K do not form α2β2 heterotetramers under these experimental conditions. To probe whether the protein concentration influences the oligomerization properties, we increased the I60K and Ca(ii) concentrations to 500 μM and 10 mM, respectively, and observed no evidence for heterotetramer formation  . In thistetramer . Apo I60mer (ii) . In contg buffer . This ben Fig. S6.ii)-induced tetramerization, we employed analytical ultracentrifugation (AUC) and determined sedimentation coefficients for CP-Ser, I60E, and I60K in the absence and presence of 20 equivalents of Ca(ii) . We obtained theoretical sedimentation coefficients  using HYDROPRO1XK4,4GGF,4XJK45To confirm that the I60E and I60K mutations disrupted Ca(ii) were added to the sample, the sedimentation distribution for CP-Ser was dominated by a single species at 3.7 S, which corresponds to the predicted S value for the α2β2 heterotetramer (ii). In agreement with the SEC experiments, the AUC data demonstrate that I60E and I60K remain heterodimeric with Ca(ii). Analysis of the AUC data using DCDT+ afforded the same conclusions  and Fe(ii) with high affinity at a hexahistidine site comprised of (A8)His17, (A8)His27, (A9)His91, (A9)His95, (A9)His103, and (A9)His105.ii) and Fe(ii) affinities at this site depend on the presence of Ca(ii) ions, and stoichiometric binding of Mn(ii) and Fe(ii) is observed at the His6 site when excess Ca(ii) is present.ii) or Fe(ii) to CP-Ser results in conformational change.ii) results in a new peak  that elutes between the apo heterodimer and Ca(ii)-bound heterotetramer  and no Ca(ii) afforded peaks with the same peak elution volumes as the apo heterodimer . Nevertheless, the peaks markedly broadened under these conditions, indicating that the samples contain a mixture of heterodimers as well as heterotetramers  and Ca(ii) on the elution properties of I60K and I60E, the proteins (30 μM) were pre-incubated with 20 equivalents of Ca(ii) and 1.1 equivalents of Mn(ii), and excess Ca(ii) (600 μM) was included in the running buffer. Under these conditions, I60E and I60K afforded peaks with elution volumes of ≈11 mL (ii) and excess Ca(ii) causes I60E and I60K to completely form heterotetramers.CP coordinates Mn coordination influences the quaternary structure of CP-Ser, I60E, and I60K in the absence or presence of Ca(ii). The sedimentation distributions for samples containing CP-Ser and one equivalent of Mn(ii) reveal two main species at 2.4 S and 4.1 S , CP-Ser coordinates Mn(ii) with relatively low affinity and the reported dissociation constant at the His6 site is in the micromolar range (Kd1 ≈ 5 μM by room-temperature electron paramagnetic resonance spectroscopy).Kd value and the CP-Ser and Mn(ii) concentrations employed for the AUC sample, we calculate that the sample contained ≈34% apo and ≈66% Mn(ii)-bound CP-Ser. On the basis of this analysis, we assign the 2.4 S species to the apo CP-Ser heterodimer and the 4.1 S species to a Mn(ii)-bound heterotetramer. This analysis informs our prior SEC results that revealed addition of Mn(ii) to CP-Ser causes a shift in peak elution volume (vide supra), and we conclude that apo heterodimers and Mn(ii)-bound heterotetramers are present in the sample. Moreover, when I60E and I60K were pre-incubated with one equivalent of Mn(ii), the sedimentation distributions exhibited the same dimer (≈2.3 S) and tetramer species (≈4.0 S) -induced tetramerization is disrupted in the I60E and I60K variants. This analysis is consistent with the SEC results, which showed a peak elution volume consistent with heterodimers and marked peak broadening indicative of heterotetramers.We employed AUC to further investigate how Mn Fig. S8; howeverii) and one equivalent of Mn(ii) were added to the buffer, CP-Ser, I60E, and I60K exhibited similar sedimentation distributions (ii)- and Mn(ii)-bound heterotetramer. The CP-Ser distribution also contained a less abundant species at 6.5 S. The theoretical maximum sedimentation coefficient for the CP tetramer is ≈4.5 S, suggesting that a higher order oligomer forms under these conditions. This species warrants further investigation, and is reminiscent of prior SEC studies that revealed low-abundance peaks with lower elution volumes (higher molecular weight) than the α2β2 heterotetramer.ii) and Mn(ii), (ii) heterotetramerization is not restricted to Ca(ii) because the CP-Ser samples containing only Mn(ii) also form heterotetramers, and (iii) the I60E and I60K mutations do not preclude formation of tetramers when Ca(ii) and Mn(ii) are present. Moreover, crystal structures of Ca(ii)- and Mn(ii)-bound CP-Ser reveal heterotetramers,When 20 equivalents of Ca(ibutions . The majii) on the oligomerization properties of CP-Ser, I60E, and I60K in the absence and presence of excess Ca(ii). In agreement with our previous work, we observed a shift in peak elution volume following addition of Fe(ii) to CP-Ser.ii), CP-Ser, I60E, and I60K (30 μM) shifted to an earlier peak elution volume  -only I60E and I60K samples -only samples exhibited a complete shift to earlier elution volumes and no broadening, which indicated that addition of Fe(ii) favored tetramerization more than Mn(ii) alone. Moreover, preincubation of the proteins with 1.1 equivalents of Fe(ii) and 20 equivalents of Ca(ii) afforded the same peak elution volume of 11.1 mL (ii) than Mn(ii) at the His6 site may explain why Fe(ii) causes more tetramerization of I60E and I60K than Mn(ii) in the absence of Ca(ii).ii)-induced formation of heterotetramers by mass spectrometry, albeit the Zn(ii)/CP stoichiometries obtained from this analysis are unlikely to be relevant because the number of bound Zn(ii) ions reported exceeds the number of Zn(ii)-binding sites per CP heterotetramer.ii)-induced tetramerization, but allow tetramerization to occur via transition metal binding. Nevertheless, the molecular basis for the metal-dependent oligomerization properties of CP-Ser and the I60 variants is unclear from standpoints of Ca(ii) coordination at the EF-hand domains as well as transition metal binding at the His6 site. Further characterization of these metal-dependent self-assembly pathways is an avenue for future work.We also employed SEC to examine the effect of Fe Fig. S7. In conts Fig. S6, the chr 11.1 mL . The higii)-induced tetramerization of CP-Ser confers resistance to proteolysis, we evaluated trypsin, chymotrypsin, and human neutrophil elastase (HNE) as host serine proteases in degradation assays. We selected trypsin, chymotrypsin, and HNE because we reasoned that co-localization of these proteases with CP is likely to occur in vivo. CP is expressed by the intestinal epithelium and serves as a biomarker for inflammation in the gut, where trypsin and chymotrypsin are abundant.ii)-bound CP-Ser, I60E, and I60K in these studies to compare the proteolytic stabilities of the Ca(ii)-bound heterotetramer (CP-Ser) and heterodimers (I60E and I60K). Because proteases such as trypsin and chymotrypsin are activated/stabilized by Ca(ii) ions, a degradation study of the apo CP-Ser heterodimer degradation would necessitate utilizing incompletely active proteases and direct comparison with the Ca(ii)-bound heterotetramer would not be possible. With the tetramer-deficient variants, it is possible to compare the stability of the Ca(ii)-bound dimer and the Ca(ii)-bound tetramer under the same experimental conditions that provide equally active proteases.To ascertain whether Ca(ii)-bound CP-Ser display resistance to trypsin whereas both subunits were rapidly degraded for Ca(ii)-bound I60E and I60K (ii)-bound I60K and I60E more readily than Ca(ii)-bound CP-Ser. We also observed that HNE digested I60E more rapidly than I60K. The reason for the difference is unclear because HNE preferably cleaves after small hydrophobic residues, so the I60E and I60K mutations are not expected to affect the HNE cleavage pattern.ii) and Ca(ii) to I60E and I60K, which causes heterotetramer formation (vide supra), resulted in enhanced stability to digestion by trypsin, chymotrypsin, and HNE. The corresponding chromatograms indicate negligible loss of full-length S100A8 and S100A9, and new species were not observed  and S100A9 (39.8 min) subunits on a C4 column. We confirmed the identity of each peak by LCMS Fig. S10, and useand I60K  and S11†and I60K . Similarand I60K  and S13†ii)-dependent manner by sequestering essential transition metal ions in the extracellular space.ii) and transition metal binding sites at the S100A8/S100A9 interface remain intact and that the coordinated transition metals remain bound and inaccessible to an invading microbe. In accord with this model, it is reasonable to expect that proteolytic destruction of the metal-binding sites and heterooligomeric assembly results in attenuated antimicrobial activity.The current model states that CP inhibits microbial growth in a Ca-bound I60E and I60K were degraded into many proteolytic fragments by these enzymes , incubated at 37 °C for ≈20 h, and subsequently employed in antimicrobial activity assays. Under standard conditions using the assay medium employed in this work, CP-Ser exhibits full growth inhibition of E. coli 25922 at 250 μg mL–1.–1. The untreated samples exhibited antimicrobial activity, demonstrating that CP-Ser, I60E, and I60K were stable and active after the overnight incubation -bound heterodimers, GluC exhibited no preference for either Ca(ii)-bound CP-Ser or the I60E and I60K variants; each substrate was cleaved in a similar manner .Many bacterial pathogens produce extracellular proteases that are capable of degrading proteins central to the immune response.r manner  and S16†r manner . LCMS an3Asp metal-binding site, and the GluC cleavage site is in close proximity to His87  with high affinity, and we questioned whether Zn(ii) coordination at this site would afford GluC resistance (ii) sites,ii). Under these conditions, both S100A8 and S100A9 HPLC peaks exhibited negligible change and the S100A8 cleavage product was not observed (ii) coordination to the His3Asp site provided protection against GluC, we took advantage of the different properties of the two transition metal binding sites and performed an experiment where the protein substrate was pre-incubated with one equivalent of Mn(ii). Because the Mn(ii) affinities of the His3Asp and His6 vary substantially and Mn(ii) preferentially populates the His6 site, the His3Asp site remains vacant when CP-Ser (+Ca) is incubated with one equivalent of Mn(ii).ii) experiments, GluC cleaved S100A8 between Glu89 and Ser90 (ii) to the I60E and I60K variants afforded no protection of the GluC cut site  , Fe(ii), and Zn(ii), contributes more to growth inhibition of E. coli and S. aureus than the His3Asp site under these assay conditions.3Asp site sequesters Zn(ii) but not Fe(ii) or Mn(ii), and this site is essential for antibacterial activity against L. plantarum.ii) restriction under the assay conditions; thus, growth studies with L. plantarum allow for interrogation of the His3Asp site. The fact that the ΔSHKE variant showed full growth inhibitory activity against L. plantarum indicated that loss of the four C-terminal residues of S100A8 does not impair the ability of the His3Asp site to sequester Zn(ii) from this organism. Moreover, these in vitro data suggest that GluC cleavage of S100A8 is not an effective staphylococcal resistance mechanism against human CP, although it is possible that synergism with other factors occurs in the complex milieu of an infection site. In light of these observations, we note that the C-terminus of human S100A8 differs from the C-termini of other mammalian orthologues. Human S100A8 is four residues longer than other sequenced mammalian S100A8 polypeptides, and these additional four residues occur in the C-terminal region. The C-terminal residues 87–93 of human S100A8 are HEESHKE, and the C-terminal residues 87–89 of other mammalian S100A8 are HKE. Whether this difference has functional significance for human CP is unclear.To evaluate the consequences of GluC truncation of the S100A8 subunit, we overexpressed the S100A8 truncation product identified from the GluC digests and the purified S100A8(C42S) (S90Stop)/S100A9 heterodimer, abbreviated ΔSHKE herein. This CP-Ser variant exhibits the expected α-helical fold and forms heterotetramers in the presence of Ca-induced heterotetramerization and Zn(ii) chelation enable CP to resist degradation by select host and bacterial serine proteases. We reason that the metal-induced structural changes enable CP to persist in the protease-rich extracellular space and thereby function in metal withholding and the inflammatory response. It will be important to determine whether these observations pertain to the fate of CP in the presence of other proteases, particularly those employed by bacterial pathogens to thwart the innate immune response. Although the current data indicate that CP exhibits remarkable metal-dependent resistance to the proteases evaluated in this work, pathogens overcome the host response to infection. We therefore expect that certain pathogenic microbes deploy degradation machinery that can disarm CP. Another important avenue for future inquiry is to consider the current observations in the context of biological concentrations, regulation, and other factors that come into play in the variable and dynamic extracellular environments of infection and inflammatory sites. In broad terms, this work provides an additional and previously unappreciated facet to how Ca(ii) ions influence CP function, and further exemplifies the fact that CP is a complex and multifaceted protein and that its functional properties will be governed by its speciation.This work provides the first comprehensive evaluation of how metal binding and changes in quaternary structure influence the proteolytic stability of human CP. Our results indicate that Ca(–1) by stirring each buffer/Chelex mixture in a polypropylene beaker for at least 1 h, and the Chelex was removed by filtration through a 0.22 μm filter. All buffers were stored in polypropylene bottles. A Tris buffer  was prepared from Tris base (J. T. Baker) and EDTA disodium dihydrate  and used for circular dichroism spectroscopy. Calcium chloride (99.99%) and manganese chloride (99.99%) were purchased from Alfa Aesar. Anhydrous zinc chloride (99.999%) and ferrous ammonium sulfate 2(SO4)2·6H2O or Mohr's salt) were purchased from Sigma. Stock solutions of CaCl2 (370 mM), MnCl2 (1 M), and ZnCl2 (100 mM) were prepared by using Milli-Q water and acid-washed volumetric glassware, and were stored in polypropylene tubes. For preparation of the Fe(ii) stock solution (100 mM), the Fe(ii) salt was weighed on the bench top, transferred to a polypropylene tube, and then brought into a glove box . The iron salt was dissolved in Milli-Q water that was degassed with Ar and stored in the glove box. All subsequent iron-containing samples were prepared in the glove box using buffers that were degassed with Ar and stored in the glove box. Working solutions of metals were prepared fresh for each experiment by diluting the stock solution into buffer  or Milli-Q water. Protein concentration was determined by optical absorbance at 280 nm using a BioTek Synergy HT plate reader outfitted with a calibrated Take3 Micro-Volume plate, and the appropriate extinction coefficient . For metal-binding experiments, HEPES buffer was prepared with Ultrol grade HEPES  and TraceSELECT NaCl (Fluka), and aqueous TraceSELECT NaOH (Sigma) was used for pH adjustments. Disposable polypropylene spatulas were used to transfer buffer components. Buffers were treated with Chelex 100 resin  and trifluoroacetic acid (TFA) were routinely purchased from EMD and Alfa Aesar, respectively. For all HPLC runs, solvent A was 0.1% TFA/H2O and solvent B was 0.1% TFA/MeCN.An Agilent 1200 series instrument equipped with a thermostatted column compartment set to 20 °C, and a multi-wavelength detector set at 220 and 280 nm (500 nm reference wavelength with 100 nm bandwidth), was used to perform analytical high-performance liquid chromatography (HPLC). A Proto C4 column  set at a flow rate of 1 mL min2O. Solvent B was 0.1% formic acid/MeCN. Protein samples (5 μM) were prepared in water and 1 μL was injected for each analysis. The S100A8 and S100A9 subunits were eluted by using a gradient 0–65% over 30 min. The resulting mass spectra were deconvoluted using the maximum entropy algorithm in MassHunter BioConfirm (Agilent).An Agilent 1260 series LC system equipped with an Agilent 6230 TOF system housing Agilent Jetstream ESI source was employed to perform high-resolution mass spectrometry. An Agilent Poroshell 300SB-C18 (5 μm pore) and denaturing protocol were utilized for all LC-MS analyses. Solvent A was 0.1% formic acid/H600) of bacterial cultures and optical absorption spectroscopy were carried out with a Beckman Coulter DU 800 spectrophotometer thermostatted at 25 °C with a Peltier temperature controller. Fluorescence spectra were collected on a Photon Technologies International QuantaMaster 40 fluorimeter outfitted with a continuous xenon source for excitation, autocalibrated QuadraScopic monochromators, a multimode PMT detector, and a circulating water bath maintained at 25 °C. This instrument was controlled by the FelixGX software package. FelixGX was used to integrate the emission spectra.The optical density  were grown from single colonies. The plasmids were isolated using a miniprep kit (Qiagen). The presence of the mutations and fidelity of the protein coding sequences were verified by DNA sequencing (Quintara Biosciences).A modified Quick-Change site-directed mutagenesis protocol was employed to generate plasmids encoding S100A8(C42S)(I60K), S100A8(C42S)(I60E), and S100A8(C42S)(ΔSHKE). The template plasmids and primers are listed in Table S1.ii), 600 μM CaCl2 was included in the running buffer and protein sample. For experiments with Mn(ii) or Fe(ii) only, 300 μM MnCl2 or Fe(NH4)2(SO4)2·6H2O was added to the sample only. For experiments with both Ca(ii) and Mn(ii) or both Ca(ii) and Fe(ii), the running buffer and sample contained 600 μM CaCl2 and 33 μM MnCl2 or 33 μM Fe(NH4)2(SO4)2·6H2O was included in the sample only. Samples were incubated for 15 min at 4 °C after adding metals and then centrifuged at 13 000 rpm for 10 min. The entire volume of each sample  was loaded onto the 100 μL sample loop. The loop was emptied with 0.5 mL of running buffer, and the protein was eluted over one column volume at a flow rate of 0.5 mL min–1 at 4 °C. Fe(ii)-containing samples were allowed to incubate for 50 min in the glove box before being centrifuged at 13 000 rpm for 10 min.An ÄKTA purifier (GE Lifesciences) housed in a 4 °C cold room and outfitted with a 100 μL sample loop was used to perform all analytical size exclusion chromatography (SEC) experiments. A Superdex 75 10/300 GL column (GE Lifesciences) equilibrated in running buffer was calibrated with a low-molecular-weight calibration mixture (GE Lifesciences) as described previously.η), buffer density (ρ), and protein partial specific volume (v) at 20 °C. The sedimentation velocity data was analyzed using previously described methods,ii)-bound CP-Ser -, Ca(ii)-, and Na(i)-bound CP-Ser  and buffer density (ρ) of water at 20 °C and a protein partial specific volume (v) of 0.7388 mL g–1.A Beckman XL-I Analytical Ultracentrifuge outfitted with an An-60 Ti rotor was employed for all sedimentation velocity (SV) experiments. The rotor housed conventional double-sector charcoal filled Epon centerpieces within the sample cells and contained quartz (absorption optics) or sapphire (interference optics) windows. The absorption wavelength for optical detection was 280 nm. The samples were centrifuged at 42 000 rpm and 20 °C until sedimentation was complete. SEDNTERP2 (540 μM), and/or MnCl2 (30 μM) were added to the reference and protein samples. The references and protein samples were allowed to incubate while the SV window assemblies were constructed (≈1.5 h). The SV window assemblies were loaded with 410 μL of reference buffer and 400 μL of protein containing sample.One day prior to an experiment, each protein sample was thawed and diluted to 27 μM in 75 mM HEPES, 100 mM NaCl at pH 7.5. The resulting samples were dialyzed against 1 L of the same buffer containing 10 g of Chelex resin at 4 °C overnight. The dialyzed samples were transferred to 1.7 mL polypropylene tubes and centrifuged  to sediment any Chelex resin. Aliquots of the dialysis buffer were centrifuged  in 50 mL polypropylene tubes and the supernatant was used for the reference samples in the SV experiment. In select experiments, EDTA (30 μM), CaCl–1 and ≈1 mg mL–1 for GluC and HNE, respectively. The solutions were stored at –20 °C (GluC) or –80 °C (HNE). Protease digestion assays were performed on a 350 μL scale at pH 7.5 . Aliquots of CP-Ser, I60E, and I60K were thawed at room temperature and diluted to 30 μM using the assay buffer in 1.7 mL microcentrifuge tubes. To select samples, calcium chloride (1.5 mM), manganese chloride (30 μM), and zinc chloride (60 μM) were added, and the resulting solutions were incubated at room temperature for at least 15 min prior to the assay. To initiate each digestion assay, an aliquot (between 5 and 10 μL as appropriate) of protease was added to the 350 μL the protein solution to bring the protease concentration to 0.45 μM (trypsin) or 0.3 μM (others). The resulting solution was immediately mixed with a pipet and incubated at 37 °C. Aliquots (45 μL) of the reaction were quenched with 155 μL of aqueous 0.77% (v/v) TFA at t = 0, 30 min, 1, 2, 3, and 4 h. The quenched solutions were centrifuged , and the resulting samples were analyzed by analytical HPLC using a solvent gradient of 10–60% B over 50 min. S100A8 eluted at ≈38 min (depending on the mutant) and S100A9 eluted at 39.8 min. Select peaks were collected manually and further analyzed by LC-MS. Controls without protease were prepared, quenched, and analyzed in an identical manner except that ubiquitin (Sigma) was added to 0.3 μM. Frozen stocks of ubiquitin  were prepared in water, stored at –20 °C, and thawed before use. Experimental details for control studies to confirm that the divalent cations do not affect protease activity are provided in Fig. S19–S22.Trypsin (Affymetrix) and chymotrypsin (Amresco) were obtained as lyophilized powders, stored at 4 °C, and dissolved in water to afford solutions of ≈50–100 μM immediately before use. Reported extinction coefficients for trypsin and chymotrypsin were used to determine the concentration Table S2.61,62 FrEscherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Lactobacillus plantarum WCFS1 (CP-Ser and ΔSHKE only) were assayed at 30 °C as described previously.2 and tryptic soy broth (TSB) with 0.25% (w/v) dextrose. For L. plantarum, de Man, Rogosa and Sharpe (MRS) broth (CRITERION) without additional dextrose was used in place of TSB. To prevent evaporation of the medium, the plates were sealed with parafilm and a beaker of water was housed in the incubator shaker.The growth inhibitory activities of CP-Ser, I60K, I60E, and ΔSHKE against 2, pH 7.5) three times using spin filter  that were sterilized by exposure to UV light for 15 min. For each protein, two aliquots  were prepared. Trypsin (≈2 mg) was dissolved in AMA buffer, the trypsin concentration was determined using the reported extinction coefficient  were assayed by modifying a literature protocol for standard antimicrobial activity assays.E. coli ATCC 25922 was inoculated into 5 mL of TSB containing 0.25% (w/v) dextrose and grown overnight at 37 °C in a rotating wheel (≈16 h). The culture was diluted 1 : 100 into 5 mL of fresh TSB with (0.25% w/v) dextrose and grown with shaking  until the OD600 reached 0.6. The culture was diluted 1 : 500 into fresh AMA medium. The antibacterial activity assay was carried out in sterile polystyrene 96-well plates (Corning). Each well contained 10 μL of the digested protein, undigested protein (no protease control), trypsin only, or buffer only and 90 μL of the diluted bacterial culture. Control wells containing 10 μL of trypsin and 90 μL of sterile AMA medium was also included, and no bacterial growth was observed in these wells. The final concentration of each CP-Ser sample was 500 μg mL–1 (21 μM), and previous work has shown that CP-Ser inhibits growth of E. coli and S. aureus at this concentration.600 values, which were measured at several time points (0 to 20 h) by using a plate reader (BioTek). Three independent replicates were conducted on different days. Different E. coli freezer stocks were used for each replicate, and at least two independent medium preparations were employed, and two different preparations of each protein were used over the triplicate. The resulting averages and standard errors of the mean are reported (n = 9). μg mL–1  μM, and Nα-benzoyl-l-arginine ethyl ester HCl  was dissolved to 10 mM in 75 mM HEPES, 100 mM NaCl, pH 7.5, and then diluted to 80 μM in the same buffer with 1.5 mM CaCl2 with or without 30 μM MnCl2. These solutions were dispensed into quartz cuvettes (Starna), 1 mL of solution per cuvette. The reaction was initiated by adding trypsin to 8.4 nM in a cuvette and briefly shaking. The reaction was monitored continuously at 253 nm. An analogous experiment was carried out to assay the activities of chymotrypsin and human neutrophil elastase (HNE). For chymotrypsin, the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Enzo Life Sciences) was dissolved to 40 mM in dimethylformamide. In the experiment, the chymotrypsin was diluted to 2 nM and the substrate was at 0.4 mM. The chymotrypsin reactions were continuously monitored at 410 nm in quartz cuvettes. For HNE, the substrate N-succinyl-Ala-Ala-Val-Ala-p-nitroanilide (Santa Cruz Biotech) was dissolved to 100 mM dimethylsulfoxide. In the assay, HNE was diluted to 10 nM, and the substrate was at 0.5 mM. The HNE reactions were continuously monitored at 410 nm in polystyrene cuvettes.To determine if the metals used in the digestion assays altered protease activity, we carried out activity assays with small molecule substrates. Protease stock solutions were prepared as described above. All experiments were carried out in triplicate and averaged. For trypsin, ii) or Mn(ii), the substrate was diluted to 100 μM. For the assays with Zn(ii), CP-Ser (30 μM) and Zn(ii) (60 μM) were combined in the assay buffer and the GluC substrate was added last. This modification was made because precipitation occurred when Zn(ii) was introduced into solutions containing the substrate. The reactions were initiated by adding GluC to 67 nM. The reaction was monitored with all slits set to 0.8 mm, excitation at 340 nm, and scanning from 400–430 nm at a rate of 0.1 nm s–1. The progress of the reaction was determined by monitoring the fluorescence emission at 410 nm.The activity of glutamyl endopeptidase (GluC) was assayed with carboxybenzyl-Leu-Leu-Glu-β-naphthylamide. A 1 mM stock solution of the substrate was made in dimethylsulfoxide. The reactions were carried out on a 1 mL scale in quartz cuvettes (Starna), and monitored by fluorescence emission. In the assays with Ca(ii) was assayed as previously described except that 1 μM ZP1 and 4 μM protein were employed.ii) required for CP variants to outcompete ZP1 for Mn(ii) were performed as previously described, but the ZP1 and MnCl2 concentrations were 1 μM and 3.5 μM, respectively.n = 3).Competition between CP variants and ZP1 for Mn(2) were made at the same time before beginning data acquisition. Wavelength scans were carried out from 190–260 nm with 1 nm steps .An Aviv Model 202 circular dichroism (CD) spectrometer thermostatted at 25 °C was employed for CD spectroscopy. A 1 mm path-length CD cell (Hellma) was employed for all CD measurements. All protein samples (10 μM protein, 300 μL, 1 mM Tris, 0.5 mM EDTA, pH 8.5, ±2 mM CaClSupplementary informationClick here for additional data file.


Novel tuberculosis vaccines should be safe, immunogenic, and effective in various population groups, including HIV-infected individuals. In this phase II multi-centre, double-blind, placebo-controlled trial, the safety and immunogenicity of the novel H1/IC31 vaccine, a fusion protein of Ag85B-ESAT-6 (H1) formulated with the adjuvant IC31, was evaluated in HIV-infected adults.3 and without evidence of active tuberculosis were enrolled and followed until day 182. H1/IC31 vaccine or placebo was randomly allocated in a 5∶1 ratio. The vaccine was administered intramuscularly at day 0 and 56. Safety assessment was based on medical history, clinical examinations, and blood and urine testing. Immunogenicity was determined by a short-term whole blood intracellular cytokine staining assay.HIV-infected adults with CD4+ T cell counts >350/mm47 of the 48 randomised participants completed both vaccinations. In total, 459 mild or moderate and 2 severe adverse events were reported. There were three serious adverse events in two vaccinees classified as not related to the investigational product. Local injection site reactions were more common in H1/IC31 versus placebo recipients . Solicited systemic and unsolicited adverse events were similar by study arm. The baseline CD4+ T cell count and HIV viral load were similar by study arm and remained constant over time. The H1/IC31 vaccine induced a persistent Th1-immune response with predominately TNF-α and IL-2 co-expressing CD4+ T cells, as well as polyfunctional IFN-γ, TNF-α and IL-2 expressing CD4+ T cells.3. The vaccine did not have an effect on CD4+ T cell count or HIV-1 viral load. H1/IC31 induced a specific and durable Th1 immune response.H1/IC31 was well tolerated and safe in HIV-infected adults with a CD4+ Lymphocyte count greater than 350 cells/mmPACTR201105000289276Pan African Clinical Trials Registry (PACTR)  Mycobacterium tuberculosis (M.tb), which according to the World Health Organization (WHO) led to almost 8.6 million new active TB cases and 1.3 million TB deaths in 2012 Mycobacterium bovis Bacille Calmette-Guérin (BCG), the only currently licensed TB vaccine, is effective in preventing severe progressive disease in children but has limited impact on adult pulmonary TB, the driving force of the TB global pandemic Tuberculosis (TB) remains a global public health problem. One third of humankind is infected with Vaccination campaigns may administer TB vaccines to persons without knowing their HIV status. It is therefore essential to evaluate the safety and immunogenicity of TB vaccines in HIV-infected persons. Furthermore, HIV-infected individuals have an increased risk of developing active TB, which may be decreased by an effective TB vaccine that reduces the risk of reactivation of latent TB infection (LTBI) or prevents TB infection or reinfection.3 and without evidence of active TB disease (PACTR Identifier: PACTR201105000289276).The Statens Serum Institut Hybrid (H1) recombinant fusion protein (antigen (Ag)85B- Early Secretory Antigenic Target (ESAT)-6) TB vaccine, adjuvanted with IC31, has been shown to be safe and immunogenic in BCG unvaccinated, TB uninfected participants and in BCG vaccinated and latently TB infected participants The study was conducted in accordance with the Helsinki Declaration and Good Clinical Practice (GCP) and approved by the following local and national ethics committees and regulatory authorities: Medical Research Coordinating Committee of Tanzania, Institutional Review Board of the Ifakara Health Institute, Tanzanian Food and Drug Authority, South African Medicines Control Council and the Human Research Ethics Committee, University of Witwatersrand.3, antiretroviral therapy naïve, generally healthy, had no evidence of active TB, had no history of receiving immunosuppressive medication, immunoglobulins, blood products or known hypersensitivity to any of the vaccine components. Women of child bearing potential were eligible if pregnancy was excluded and they agreed to use at least two forms of acceptable contraception from 21 days prior to administration of the study vaccine through to the end of the study.This was a phase II, multicentre, double-blind, randomized, placebo-controlled trial. Participants were eligible if they were between 18 and 55 years of age, HIV infected with CD4+ lymphocyte counts greater than 350/mmth of September 2012 (last participant last visit); the first participant last visit was on the 2nd of July 2012. Written informed consent was obtained from all literate patients. In case of illiteracy, informed oral consent was attested by an impartial witness and documented with the patient’s fingerprint according to ICH GCP guidelines (E6 (R1); 4.8.9), as approved by the above mentioned ethics committees and regulatory authorities. The protocol for this trial and CONSORT checklist are available as supporting information; see The trial took place at two African research facilities, the rural Bagamoyo Site of the Ifakara Health Institute in Tanzania and the urban Tembisa Site of the Aurum Institute in South Africa. It was carried out between 19th of December 2011 (first participant first visit) and 10Participants were randomly allocated in a 5∶1 ratio to receive either H1/IC31 vaccine or placebo according to a computer-generated randomisation list. The pharmacist prepared the vaccination according the pre-prepared study randomisation list. The unequal randomisation was employed to maximise exposure to the H1/IC31 vaccine thereby increasing the chance of detecting a serious adverse event to the vaccine. The study monitors, investigators, and participants were blinded to study product. The study pharmacists prepared the investigational product. Syringes were masked with red tape in order to conceal a slight difference in the appearance of the H1/IC31 and placebo.The Hybrid 1 (H1) vaccine is a recombinant fusion protein of the antigens Ag85B and ESAT-6 (Ag85B–ESAT-6), developed and manufactured by the Statens Serum Institut (Denmark). The adjuvant IC31 was developed by Intercell AG (Austria) and consists the cationic polyaminoacid KLK, which is composed of the amino acids lysine (K) and leucine (L), and ODN1a, a single stranded oligodeoxynucleotide with alternating sequences of the nucleic acids inosine and cytidine. A volume of 0.5 mL was administered providing a dose of 50 µg Ag85B-ESAT6 (antigen) and 500 nmol KLK +20 nmol ODN1a (adjuvant) or 0.5 mL Tris buffer (placebo).The H1/IC31 vaccine or placebo was administered intramuscularly with a 22 to 25-gauge needle at study day 0 and 56, after confirming eligibility criteria. The first injection was given into the right and the second into the left deltoid muscle.All solicited local  and systemic adverse events (AEs)  and unsolicited AEs were coded by the Aurum Institute according to the MedDRA Dictionary Version 15.1. AEs and laboratory measures of safety were graded using the Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events  as mild, moderate, severe or potentially life-threatening and assessed for their relationship to study product. CD4+ T cell count and HIV-1 viral load were measured pre and 14 days post vaccination and at the study end.QuantiFERON-TB Gold In-Tube assay (QFT) (Celestis) was performed at screening and at day 182 to identify whether H1/IC31 induced a false positive result. The assay was carried out according to manufacturer’s instructions with a cut-off value of 0.35 IU IFN-γ/mL to either antigen (ESAT-6/CFP-10).All immunological assays described below were performed by the South African TB Vaccine Initiative (SATVI) laboratory in Cape Town, South Africa.The whole blood intracellular cytokine staining assay was used to quantify antigen specific CD4+ and CD8+ T cell responses (interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), interleukin (IL)-2 and IL-17) after stimulation with peptide pools of Ag85B and ESAT-6 peptides  and recombinant H1 fusion protein . CD4+ and CD8+ T cell expression of IFN-γ, TNF-α and IL-2 was measured to assess the polyfunctional characteristics of H1/IC31 induced T cells. The primary immunogenicity endpoint was assessed at day 70 (14 days post 2nd vaccination).Processing of whole blood started within 75 minutes after phlebotomy. One mL whole blood from each study participant and time point were either left unstimulated (negative control) or stimulated with peptide pools of Ag85B, ESAT-6, the H1 fusion protein and phytohemagglutinin (PHA) (positive controls). The co-stimulatory antibodies anti-CD28 and anti-CD49d  were included in all assay conditions. The whole blood was incubated at 37°C for 7 hours and Brefeldin-A  was added thereafter. The blood sample was transferred to a waterbath at 37°C and incubated for a further 5 hours. Thereafter the waterbath was switched off and allowed to reach room temperature. Within the following 10 hours the blood was harvested with EDTA , red blood cells were lysed and white cells fixed with 9 ml of FACS lysing solution (BD Biosciences). Thereafter, white cells were pelleted and cryopreserved in cryosolution containing 50% RPMI (Lonza), 40% fetal calf serum (BioWest) and 10% DMSO (Sigma-Aldrich). The specimens were shipped to SATVI ensuring that the cold chain was maintained.The subsequent intracellular cytokine staining (ICS) procedure was performed as follows: the stimulated, fixed and frozen white cells from whole blood were thawed in a water bath at 37°C. The thawed cells were transferred to labelled tubes containing phosphate buffered saline  and washed. Next, the cells were permeabilised using Perm/Wash solution (BD Biosciences). Cells were then stained with the following anti-human antibodies: CD3 BV421 , CD4-QDot605 , CD8-Cy5.5PerCP , CCR7-PE , IFN-γ-Alexa 700 , TNF-α-Cy7PE , IL-2-FITC , IL-17-Alexa 647  and CD45RA BV 570 . Results of CCR7 and CD45RA staining are not presented here. Samples were stained, acquired and analysed in batches. For every ICS assay experiment, compensation controls (single stained positive and negative mouse kappa compensation beads) were included. These controls were processed in parallel with the study samples during the staining and acquisition process to allow post acquisition flow cytometry data compensation. After the samples were acquired on the BD LSRII cytometer , flow analysis was performed using FlowJo software (TreeStar version 9.6.2).Anti-Ag85B-ESAT-6 specific IgG antibody assay was used to measure antibodies specific to antigens of H1/IC31 as an indicator of humoral reactivity. Plasma was collected and frozen from ex vivo whole blood.Prior to the start of the study both sites laboratories were trained on specimen processing and storage. Both sites passed their proficiency tests for PBMCs cell viability and number and were IATA certified for shipping specimens. IFN-γ EliSpot assay was not possible to conduct on PBMCs collected from the Tembisa site because the cell recovery upon thaw was too low . The cell recovery and yield was excellent for PBMCs from Bagamoyo. However, on Elispot assay, these PBMCs showed high level of spot forming units in the negative control wells that was indistinguishable from the response detected with the assay’s positive control (stimulation with PHA). Therefore, we could not reliably report the IFN-γ EliSpot assay results.A sample size of 40 vaccine and 8 placebo recipients was adequate to assess the safety and immunogenicity of the vaccine. Assuming an incidence of adverse events in the vaccine and placebo arm of 10 and 15 per 100 person years, the probability of observing at least one adverse event was 0.87 and 0.95, respectively. The sample size for immunogenicity response rates were based on the phase I study results where 100% responded by the 2nd vaccination. The sample size was adequate to show an immune response in 70–90% of vaccine recipients and an absolute difference of 66% in the proportion of vaccine and placebo recipients with an immune response with 90% power.Laboratory and immunogenicity parameters were summarised by study arm and visit. The baseline is defined as the measurement at day 0. Comparing cytokine responses  between study arms was conducted using the Wilcoxon rank-sum test and between time points using the Wilcoxon signed-ranks test. The CD4 and log10 change over follow-up was estimated for each individual using linear regression and the estimated slopes compared by study arm using the Wilcoxon rank-sum test. All AEs were summarised by system organ class and preferred term by treatment group and visit. The statistical analysis was performed using software STATA version 13.0.The study protocol and the CONSORT checklist are available as supporting information.In total, 167 adults were screened and 48 volunteers were enrolled (24 in Tanzania and South Africa respectively); 47 participants completed both vaccinations. The CONSORT diagram is shown in The safety analysis included data from both study sites. In total 461 AEs were reported .H1/IC31 versus placebo recipients were more likely to have one or more local injection site AE (H1/IC31∶26/40 (65.0%), placebo: 1/8 (12.5%), p = 0.015). The local AEs in the H1/IC31 arm were graded as mild  or moderate  and were either not related , possibly , probably  or  definitely related. In the H1/IC31 arm, the most frequent solicited local AEs were pain  and induration . The one local AE in the placebo arm was assessed as mild and probably related.29/40 (72.5%) of H1/IC31 participants experienced a total of 84 solicited systemic AEs, which were graded as mild  or moderate  and 51.2% (43/84) and 22.6% (19/84) were assessed as possibly or probably related respectively. In the placebo arm, 7/8 (87.5%) participants experienced a total of 17 systemic AEs. These were graded as mild  and either unrelated  or possibly related . In the H1/IC31 and placebo arms, headache  and fatigue  were the most common solicited systemic AEs. The percentage of patients with at least one solicited systemic AE was similar by study arm .All participants reported one or more unsolicited systemic AE . The 40 H1/IC31 recipients experienced 251 unsolicited AEs, which were graded as either mild , moderate  of severe , and one AE was ungraded (pregnancy). Of the 251 unsolicited AEs among H1/IC31 recipients, 152 (60.6%) were not related, 96 (38.3%) were possibly related and 3 (1.2%) were probably related. 46 unsolicited AEs were reported in the placebo group, all considered unrelated  or possibly related  and graded as either mild  or moderate . There were three serious individual case safety reports from two H1/IC31 participants, all classified as unrelated to the investigational product. One participant had two separate hospitalisations for malaria and drainage of a perianal abscess. The second participant delivered a premature baby that died as a result of respiratory failure. The participant had a history of one miscarriage in the last trimester and one delivery of another child, who died soon after birth. In the H1/IC31 and placebo arms, unsolicited systemic AE were most frequently categorised as blood and lymphatic disorders  or infections and infestations .The frequencies of one or more local and systemic solicited AE per participant was similar after the first and the second vaccination with H1/IC31 , while unsolicited AEs were less frequent after the second H1/IC31 vaccination .QFT positive, versus negative, H1/IC31 recipients had more solicited local reactions , but not solicited systemic  or unsolicited AEs (100% vs 100%).−4 and −0.54×10−4 copies/ml in the H1/IC31 arm and placebo arms, respectively) was also similar by study arm (p = 0.93).There were no significant differences in haematology, biochemistry or urinalysis test results at any visit by study arm. The median CD4 slope (per day) over the 182 day follow-up period was −0.86 and −0.59 cells per µl in the H1/IC31 and placebo arms, respectively, indicating no difference by study arm (p = 0.85). The median log10  slope per day  of the H1/IC31 and 3/8 (37.5%) of the placebo participants and participants had a positive QFT result. Conversion from QFT negative to QFT positive was observed in 8 of the 23 (34.8%) H1/IC31 recipients, and in none of the placebo recipients. Two of the 17 QFT positive H1/IC31 participants (11.8%) converted to QFT negative by day 182. One initially QFT positive placebo recipient (33.3%) was QFT negative after 182 days. 10 QFT results from 9 H1/IC31 and 1 placebo recipient were missing at day 182 .Only samples from the 24 participants from the Bagamoyo Site were used for the immunogenicity analysis. None of the whole blood intracellular cytokine assay specimens from the Tembisa site were useable, as they thawed as a result of the liquid nitrogen tank in which they were stored running dry. The gating strategy for flow cytometry analysis is shown is shown in Among H1/IC31 recipients, the median magnitude of CD4+ T cells expressing one or more cytokines (IFN-γ and/or IL-2 and/or TNF-α) in response to Ag85B, ESAT-6 and H1 were greatest two weeks after the second vaccination (day 70), and remained elevated compared with baseline up to day 182 . The greAmong H1/IC31 recipients, the median CD4+ T cell IFN-γ and/or IL-2 and/or TNF-α responses to H1 two weeks post second vaccination (day 70) did not differ by baseline CD4 count , or by QFT status  and 182 (data not shown).3. Key findings are: (i) H1/IC31 was safe and well tolerated; (ii) H1/IC31 did not have a negative impact on CD4+ T cell count or on HIV-1 viral load. (iii) H1/IC31 induced a strong, specific and long lasting, CD4+ T cell response; (iv) H1/IC31 vaccination induced primarily triple  or double (TNF-α and IL-2) positive CD4+ T cells.This is the first report on safety and immunogenicity of the H1/IC31 subunit TB vaccine administered to HIV-infected adults with CD4+ lymphocyte counts >350 cells/mmM. vaccaeM.tb, fragmented) of HIV-infected adults was associated with an increased risk of local reactions, including nodules and sterile abscesses There is a concern that in HIV-infected individuals, TB vaccines may promote immune activation leading to increased adverse events and progression of HIV. Vaccination with Modified Vaccinia Ankara virus expressing antigen 85A (MVA85A) 3) induced a specific Th1-type response to the H1 antigen, and its Ag85B and ESAT-6 subunits. We observed some CD4+ T cell IFN-γ responses to the vaccine antigen H1 at enrolment - prior to vaccination. The responses were most prominent in the QFT positive individuals, and probably due to latent TB infections or possibly cross reacting environmental mycobacterial infections. Despite these baseline responses, H1/IC31 vaccination induced significantly higher CD4+ T cell responses that were durable. These results are encouraging and suggest that latent TB infection will not mask an H1/IC31 specific immune response. The magnitude and durability of the Th1 responses in HIV-infected participants, and the lack of humoral response, has similarly been observed in HIV-uninfected participants TB vaccines in HIV-infected individuals potentially may be less immunogenic due to HIV-associated immunosuppression. H1/IC31 vaccination of HIV-infected participants with a high CD4 count  or an inadequate limit of detection H1/IC31 vaccine induced poor CD8+ T cell responses, which has likewise been described for other vaccine candidates As formerly reported, H1/IC31 vaccination can lead to conversion of the QFT result because of its ESAT-6 component New subunit TB vaccines should boost the efficacy of BCG and prevent TB infection and prevent latent TB infection from progressing to active disease. Both mechanisms are essential for the control of the TB epidemic. In order to achieve a better post-exposure protection, the Statens Serum Institut has added a latency-associated antigen (Rv2660c) to the H1/IC31 backbone, to create a multistage vaccine, H56 (Ag85B-ESAT-6-Rv2660c) 3 was necessary because of safety concerns, but limits the generalizability of the results.The present study had a number of limitations. The study was not powered to show difference between subgroups, particularly by baseline QFT status. The whole blood intracellular cytokine staining data from one site and IFN-γ EliSpot assay data from both sites did not meet the internal quality control standards, which limits comparability to other TB vaccine trials. The restriction to HIV-infected individuals with CD4+ lymphocyte counts greater than 350 cells/mm3, from TB endemic areas.In conclusion, the intramuscular injection of the adjuvanted TB subunit vaccine H1/IC31 was safe and immunogenic in HIV-infected adults, with CD4+ lymphocyte counts greater than 350 cells/mmS1 Protocol3.’Study protocol of the study ‘Phase II Double-Blind, Randomized, Placebo-Controlled Study to Evaluate the Safety and Immunogenicity of H1/IC31, an adjuvanted TB Subunit Vaccine, in HIV-Infected Adults with CD4+ Lymphocyte Counts Greater than 350 cells/mm(PDF)Click here for additional data file.S1 ChecklistConsolidated Standards of Reporting Trials (CONSORT) checklist.(DOC)Click here for additional data file.


The envelope (E) of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. After biosynthesis E protein forms a heterodimer with precursor membrane (prM) protein. Recent reports of infection enhancement by anti-prM monoclonal antibodies (mAbs) suggest anti-prM responses could be potentially harmful. Previously, we studied a series of C-terminal truncation constructs expressing DENV type 4 prM/E or E proteins and found the ectodomain of E protein alone could be recognized by all 12 mAbs tested, suggesting E protein ectodomain as a potential subunit immunogen without inducing anti-prM response. The characteristics of DENV E protein ectodomain in the absence of prM protein remains largely unknown.In this study, we investigated the expression, membrane association, glycosylation pattern, secretion and particle formation of E protein ectodomain of DENV4 in the presence or absence of prM protein. E protein ectodomain associated with membrane in or beyond trans-Golgi and contained primarily complex glycans, whereas full-length E protein associated with ER membrane and contained high mannose glycans. In the absence of prM protein, E protein ectodomain can secrete as well as form particles of approximately 49 nm in diameter, as revealed by sucrose gradient ultracentrifugation with or without detergent and electron microscopy. Mutational analysis revealed that the secretion of E protein ectodomain was affected by N-linked glycosylation and could be restored by treatment with ammonia chloride.Considering the enhancement of DENV infectivity by anti-prM antibodies, our findings provide new insights into the expression and secretion of E protein ectodomain in the absence of prM protein and contribute to future subunit vaccine design. Flavivirus in the family Flaviviridae, cause the most common and important arboviral diseases in humans in the tropical and subtropical areas The four serotypes of dengue virus , belonging to the genus DENV enters the cell through receptor-mediated endocytosis The E protein plays an important role in virus entry, and is the major target of neutralizing antibodies and vaccine development After biosynthesis in the rough ER, prM and E proteins form a heterodimer, which was reported to be important for assembly of VLPs Previously, we studied a series of C-terminal truncation constructs expressing DENV4 prM/E or E proteins, and found that the ectodomain of E protein alone can be recognized by all 12 mouse and human mAbs tested The prM/E expression construct of DENV4 , prMEd395 and Ed395 constructs were described previously 5 cells per dish one day before were transfected with 10 µg of plasmid DNA by calcium phosphate method 4Cl at 18 h post-transfection. Culture supernatants were collected at 48 h post-transfection (see below); cells were washed with PBS and treated with 1% NP40 lysis buffer, followed by centrifugation at 20,000×g at 4°C for 30 min to obtain cell lystates 293T cells prepared in a 10 cm-culture dish at 5×10Protein A sepharose beads  was incubated with anti-CD4 mAb (SK3), anti-E mouse mAbs  and human serum at 4°C for 6 h and then incubated with cell lysates or culture supernatants at 4°C overnight. After washing with 1% NP40 buffer 4 times, the beads were mixed with 15 µl 2X sample buffer Plasmid DNA was transfected to 293T cells prepared in a 10-cm culture dish by calcium phosphate method. At 48 h post-transfection, cells were washed, resuspended in PBS, treated with 1% Triton X-100 on ice for 60 min, and then loaded into a 5 to 20% (wt/wt) sucrose gradient made with gradient buffer . The gradient was ultracentrifuged at 247,606×g  at 15°C for 22 h, and each of the 13 fractions was collected and subjected to Western blot analysis 5 cells) prepared in 10 cm dish were transfected with 10 µg DNA by calcium phosphate method. The transfected cells were washed with PBS, lysed in 1 ml of hypotonic buffer  and passed through a 25-gauge needle 20 times. After centrifugation at 1,000×g at 4°C for 5 min, 0.8 ml postnuclear supernatants were mixed with 3 ml of 72% sucrose in low-salt buffer , overlaid with 4 ml of 55% sucrose in LSB and then 1.5 ml of 10% sucrose in LSB, followed by ultracentrifugation at 247,606×g  at 4°C for 14 h 293T cells  at 4°C for 16 h. A total of 9 fractions was collected; each fraction was subjected to Western blot analysis. The density of each fraction was determined by a refractometer.An aliquot of VLPs was absorbed to a glow-discharged 300-mesh formvar/carbon film, stained with 2% phophotungstic acid (PTA) (Electron Microscopy Sciences), and examined by transmission electron microscopy . For immunogold labeling, the pellets overlaid with 5–20% sucrose gradient in PBS, followed by ultracentrifugation at 247,606×g  and 4°C for 90 min. Each fraction was first diluted three folds in PBS, then ultracentrifuged at 65,000×g  and 4°C for 5 h and resuspended in PBS Flat-bottom 96 well plate was coated with mouse mAb (1H10-6-7) against DENV4 E protein at 4°C overnight, followed by blocking with 1% BSA in PBS for 1 h and adding culture supernatants or recombinant DENV4 E protein, anti-E human mAbs and anti-human IgG conjugated with HRP each at 37°C for 1 h, and adding TMB substrate and stop solution To investigate the expression, membrane association and glycosylation pattern of the ectodomain of DENV4 E protein in the presence or absence of prM protein in comparison with those of the full-length E protein, we generated four constructs encoding full-length E protein and E protein ectodomain with or without prM protein . After tWe next analyzed the oligomeric status of E protein ectodomain, E and prM proteins by a sucrose gradient sedimentation analysis under non-denaturing condition To rule out the formation of E aggregates in high density fractions (of mutants prMEd395 and Ed395), we repeated the sucrose gradient sedimentation analysis with 14 fractions, recovered the E protein from the highest fraction (fraction 14) and middle fraction , and conducted enzyme digestion with endo H and PNGaseF. As shown in In the presence of transmembrane domains, flaviviral E protein has been shown to associate with membranes in cells To further study membrane association and intracellular localization, 293T cells transfected with each of the four constructs were subjected to subcellular fractionation to separate the membrane-bound and soluble fractions. In agreement with our previous report To further investigate whether E protein ectodomain can produce extracellular particles, pellets derived from culture supernatants, which contain VLPs We next performed a quantitative ELISA  to measu3  Known concentration of recombinant DENV4 E protein  were added to 96-well plate pre-coated with a human dengue-immune serum, followed by adding mouse anti-E mAb (1H10-6-7) and secondary antibody to generate a standard curve. (B) The OD450 of recombinant DENV4 E protein (left) and E protein in culture supernatants, including total E protein in supernatants , E protein in pellets containing VLPs (pellet-VLPs) and soluble E protein in supernatants post-ultracentrifugation (post-pellet) were read. The [E] was determined by interpolating the OD value to the standard curve. Data are means with standard deviation of duplicates from one representative experiment of two.(TIF)Click here for additional data file.Figure S2Detergent sensitivity and buoyant density of particles derived from culture supernatants of 293T cells transfected with prME, prMEd395 or Ed395 construct. Pellets derived from culture supernatants of 293T cells transfected with prME, prMEd395 or Ed395 were treated with or without 1% Triton X-100, followed by 15 to 55% (wt/wt) sucrose gradient ultracentrifugation; each fraction was subjected to Western blot analysis using a dengue-immune serum (TIF)Click here for additional data file.Figure S3Electron micrographs of purified VLPs from sucrose gradient. Pellets derived from 293T cells transfected with prME or Ed395 were subjected to a 5 to 20% (wt/wt) sucrose gradient ultracentrifugation as in (TIF)Click here for additional data file.Figure S4Recognition of secreted E protein ectodomain by mAbs and human serum using immunoprecipitation assay and capture-ELISA. (A) Culture supernatants derived from 293T cells transfected with prME, prMEd395, E or Ed395 construct and recombinant DENV4 E protein ectodomain were immunoprecipitated using two mouse anti-E mAbs (4G2 and 1H10-6-7) and a confirmed dengue-immune serum (#13), and subjected to Western blot analysis. Anti-CD4 mAb is a negative control. (B) Culture supernatants derived from 293T cells transfected with prMEd395 or Ed395 construct and recombinant DENV4 E protein ectodomain were subjected to capture-ELISA using 9 human mAbs.(TIF)Click here for additional data file.Table S1Summary of the E domains of epitope residues recognized by mAbs in this study.(DOC)Click here for additional data file.


Here we suggest a potential role of Zscan10 in controlling progenitor cell populations in vivo. Mice homozygous for a Zscan10 mutation exhibit reduced weight, mild hypoplasia in the spleen, heart and long bones and phenocopy an eye malformation previously described for Sox2 hypomorphs. Phenotypic abnormalities are supported by the nature of Zscan10 expression in midgestation embryos and adults suggesting a role for Zscan10 in either maintaining progenitor cell subpopulation or impacting on fate choice decisions thereof.The transcription factor Zscan10 had been attributed a role as a pluripotency factor in embryonic stem cells based on its interaction with Oct4 and Sox2 in  Expression remained in the otic vesicle, the branchial arches and the hindgut between E9.0–9.5 and became barely detectable at E10.5 Zscan10 indicated expression in the ectoderm and mesoderm but not endoderm of E7.5 embryos and low level expression in all germ layers from E9.5 to E12.5. Northern blot analysis performed with a 60 bp oligonucleotide located in the last exon of Zscan10 revealed strongest expression in embryos at E4.5–E5.5 and E10.5–E13.5, while in the adult expression was limited to the testis et al. indicated upregulation of Zscan10 expression in E9.5 embryos as well as lymph nodes, the spinal cord, testis and epididymis as well as femur and calvarial bones Zscan10, only exon 4 is shared amongst all six known alternative splice variants Zscan10 (previously known as Zfp206) is a 14 zinc finger transcription factor (ZF-TF) of the CDuring post-fertilization pre-implantation stages in mouse, a totipotent zygote develops into a pluripotent blastocyst by E3.5 with only the cells of the ICM retaining the pluripotent state. Pluripotent ESC can be derived from the ICM of blastocysts Zscan10 homozygous mutant mice derived from clone #285B6 (CMMR) of a gene trap located 5′ to the ATG start codon of Zscan10, which should render null all known protein isoforms of Zscan10, yet allow residual 3′ RNA expression to persist. Typically affected organs did retain residual Zscan10 RNA expression in distinct cells during midgestation development and even in the adult and were generally determined to be hypoplastic in the Zscan10 homozygous mutants.Here we propose a possible role for Zscan10 in the maintenance of multipotent progenitor cells based on the pleiotropic phenotype revealed by an exhaustive screen of Zscan10 mutant mice were generated by injecting the R1 mouse ESC clone #285B6, obtained from the Canadian Mouse Mutant Repository (CMMR) , into C57BL/6 2C–8C stage embryos as described in Kraus et al. Zscan10 as confirmed by 3′RACE performed by CMMR. For a schematic of the gene trap allele see ZP3-Cre deleter strain 5′-CGGGTCAAATTACGAGGTGCT-3′) and wild type allele (285B6-wt-F: 5′-CTGGCAAGTCCTCACCTAGAAATT-3′/ 285B6-wt-R: 5′-CTGCAGCAGATGCTCGTGAGTT-3′), generating a 300 bp wild type allele and a 1100 bp and 500 bp mutant allele before and after Cre recombination, respectively.All animal procedures were performed according to the Singapore A*STAR Biopolis Biological Resource Center (BRC) Institutional Animal Care and Use Committee (IACUC) guidelines which are set by the National Advisory Committee for Laboratory Animal Research (NACLAR). The IACUC protocols employed were reviewed and approved by the aforementioned committee before any animal procedures were undertaken for this study described here (IACUC Protocol No: 110689 and 110648). The mouse strains used in this study were housed, maintained and provided by the A*STAR Biopolis Biological Resource Center (BRC) (Singapore) & the German Mouse Clinic (GMC)  and maintained in IVC cages according to BRC and GMC housing conditions and Singaporean and German laws. All tests performed at the GMC and described here were approved for the ethical treatment of animals by the responsible authority of the Regierung von Oberbayern (Government of Upper Bavaria). The lines described here will be made available to the research community upon acceptance of the manuscript.in situ hybridization on 10 um paraffin sections (SISH) was carried out, essentially as described Zscan10 probes have been used for SISH, emphasized here is the 270 bp probe template that spans exons 3 to 5 of the Zscan10 cDNA, precisely from base pair position 667 to 937 of the reference sequence NM_001033425 and the 1355 bp probe template spanning base positions 433 to 1788. PCR was carried out following standard PCR procedures using GoTaq (Promega) at 1.0 mM MgCl2 on a Nexus Eppendorf thermocycler with a 40 s annealing step at 65°C and 2.0 mM MgCl2 at 60°C, respectively, for 35 cycles with primers including the sequence for the T3 and T7 RNA polymerase promoters, to generate a DIG labeled sense and antisense probe, respectively, using the DIG RNA labeling mix (Roche):RNA 5'-CGCGCGTTAATTGGGAGTGATTTCCCCCTCAGAAGAGATTCCAGCCC-3', Zfp206_T7: 5'-GCGCGCGTAATACGACTCACTATAGGGCGCCAAGCTCTCTTCTCTGAGGT-3', Zscan10_433_T3: 5′-CGCGCGCAATTAACCCTCACTAAAGGGCTGGAGCAGTTCCTGAGTGTC-3′, Zscan10_1788_T7:5′-GCGCGCGTAATACGACTCACTATAGGGCTAGTTGCTCGCTTTGTCGGAA-3′. Likewise a ∼1 kb probe for Sox2 was generated using the primers: T7_Sox2_S: 5′- GCGCGCGTAATACGACTCACTATAGGGCTTTGTCCGAGACCGAGAAGC-3′, T3_Sox2_AS: 5′- CGCGCGCAATTAACCCTCACTAAAGGGGAAGCGCCTAACGTACCACT-3′. The ORF Hes5 probe was a generous gift from Urban Lendahl. The probe template for the pUPA reporter gene EGFP was amplified using the primers EGFP_T7: 5′-CGCGCGCAATTAACCCTCACTAAAGGGCCTACGGCGTGCAGTGCTTCAGC-3′ and EGFP_T3: 5:-CGCGCGGTAATACGACTCACTATAGGGCCGGCGAGCTGCACGCTGCCGTCC-3′.Zfp206_T3: Zscan10+/− x Zscan10+/− matings. The homozygous (−/−) line L2 was maintained on gelatinized plates in the absence (GEL) or presence (MEF) of mouse embryonic fibroblast feeder cells all maintained in ES medium+LIF.Western blot was essentially performed as described www.mouseclinic.de) comprises exhaustive, standardized phenotyping in the realms of dysmorphology, cardiovascular health, energy metabolism, clinical chemistry, eye, lung function, molecular phenotyping, behavior, neurology, nociception, immunology, steroid metabolism and pathology. Phenotypic screens at the GMC were performed according to standardized methods The GMC screen . Growth weight and body size was determined. Mice were subjected to morphological observation and hearing abilities were analyzed following the protocol described previously Morphological alterations of the eye such as anterior segment abnormalities were assessed by Scheimpflug imaging, posterior segment abnormalities, eye fundus and retina were analyzed by optical coherence tomography. The axial eye length was measured by laser interference biometry For the assessment of basic neurological functions, muscle function as well as motor coordination and balance, a modified SHIRPA protocol was applied and grip strength and Rota rod performance were measured as described previously Blood samples were collected at 16 weeks of age by retrobulbar puncture under isoflurane anesthesia. Determination of clinical-chemical parameters in heparinized plasma samples was done using an AU480  and reagents kits provided by Beckman-Coulter, as described previously Awake echo was used for the assessment of cardio-vascular functions 2 consumption, CO2 production, respiratory exchange ratio and body composition was assessed by indirect calorimetry and qNMR . The steroid hormones testosterone, corticosterone and androstendione were analyzed out of 50 ul plasma by liquid chromatography tandem mass spectrometry (LC-MS/MS) according to Rathkolb et al Measurement of body mass, body temperature, locomotor activity, OZscan10 mutants were viable and fertile, genotyping offspring (n = 153) from heterozygous x heterozygous matings at weaning age  revealed a slight deviation from the expected Mendelian ratio, with the number of homozygous females being 30% lower than that of wild type females  reduced  (data not shown). Zscan10 mutant mice presented with moderately decreased chloride and mildly reduced albumin concentration in plasma, while creatinine and urea levels were increased in these mice compared to controls  and increased locomotor speed (p<0.05) in the open field and entered the center more frequently while male mutant mice showed the opposite effect (interactions genotype x sex: p<0.05) see . Male muSHIRPA is a standardized observation protocol for assessment of genetically modified mice. During this test increased occurrence of urination was detected for two thirds of the mutant cohort, compared to only one third of the wild type mice . Measuring grip strength for the evaluation of muscle function revealed reduced force in 2- and 4-paw measurement. However, the mutant mice were also significantly lighter see  and inclZscan10 mutants were detected, present already at midgestation  and a decreased respiration rate mainly in male mutants. Further, Zscan10 mutants showed a decreased heart rate when compared with control mice (data not shown). Thus, only mild genotype-related differences without relevance for the cardiovascular system were found. Lung function screening showed a slight yet significant but functionally rather irrelevant decreased dynamic compliance (p = 0.026) and tidal volume (p = 0.004) (data not shown). Body temperature showed a tendency to be slightly lower in male mutants (data not shown).Awake echocardiography of In homozygous male mutant mice an increase in liver weight normalized to body weight was determined see . A slighZscan10 expression. Since expression pattern analysis was previously established by WISH www.repeatmasker.org), the Zscan10 probes were free of repetitive elements and specific to Zscan10. In several independent experiments (n = 6) we observed a punctate expression pattern (a positive cell surrounded by numerous non-expressing cells) in E12.5 and E15.5 embryos, namely in kidneys, heart, liver, muscle and developing bones of the forelimb, but in a similar matter in neural tissue like the neural tube, the brain and in dorsal root ganglia and sensory organs like the eye  in Zscan10 homozygous male mutant mice, and a trend in the opposite direction in female mutants. Significantly increased urination was observed in Zscan10 mutants, female Zscan10 mutants were slightly more active in the OFT, including more entries into and more distance travelled in the “anxiogenic” center zone of the OFT. It could be hypothesized based on the expression of Zscan10 in the embryonic brain that an impact on a pluripotent progenitor cell population for example in the limbic system could influence anxiety related behavior along the way of observations made in Dlx1 loss-of function mutants Dlx1 sense Zscan10 homozygous mutants also show an increased acoustic startle response, which is not consistent with the notion of reduced anxiety, and demonstrated a functional visual impairment in the optokinetic drum test. It cannot be excluded that this visual impairment might have played a role in the observed OFT behavior of female Zscan10 homozygous mutants, since it could affect the perception of the arena and its center zone as anxiogenic. The increased urination observed in the neurology screen could however also be due to a renal dysfunction as indicated by the results from the clinical chemistry screen which revealed increased urea and creatinine levels as well was a slight hypocalcemia in males that among other causes can also be attributed to renal dysfunction.The Open Field Test was originally described to study emotionality in rats Zscan10 homozygous mutant behavior.Taken together, further neurological examinations and immunohistochemistry would be required to identify the exact nature of Zcan10 gene expression patterns established by SISH. Previous WISH expression patterns in midgestation embryos could have misinterpreted the punctate expression pattern of Zscan10 as background staining. We propose based on a more refined expression pattern by SISH, the known interaction partners of Zscan10 Zscan10 mutants, that Zscan10 could play a role in regulating the availability of progenitor cells during mouse embryonic development and adulthood. In the case of Zscan10 hypomorphic or loss-of-function mutations the number of progenitors might be reduced, either by a decrease in progenitor cell division or by premature terminal differentiation causing an overall reduction in cell mass of a variety of target tissues namely eye, heart, long bones and spleen. Identified grip strength, immunological and blood chemistry abnormalities could be downstream effects of the impact on progenitor cell populations in the affected organs. The pluripotency marker Sox2 is coexpressed in many but not all of these organs, yet generally in fewer cells compared to Zscan10. This is not surprising as progenitor cells might have acquired a certain maturity and as such are no longer of pluripotent nature, yet can act as multipotent progenitors for cells at an advanced differentiation level. The exact mechanism by which Zscan10 acts in a given progenitor cell population in vivo remains to be determined.All phenotypic findings were coherent with the embryonic and adult Zscan10 mutant mice supports previously proposed in vivo interactions between Sox2 and Zscan10 based on earlier in vitro assays. The nature of the cellular expression pattern of Zscan10 in midgestation embryos and adult organs and the changes in absolute weight of several organs as well as the composition of several cell populations in the homozygous mutants suggests a role for Zscan10 in maintaining a multipotent progenitor cell population or impacting on fate choice decisions thereof.In summary, the observed eye phenotype in homozygous Figure S1Gene trap strategy. Illustrated is the pUPA-TRAP vector  as used by CMMR that was integrated into intron 1 upstream of the ATG start codon. Exons of the currently known five Zscan10 transcripts are drawn to scale as rectangular boxes, yellow and white representing coding and untranslated regions, respectively. The 270 bp SISH probe (purple lines) is covering most of exon 3, all of exon 4 and part of exon 5 and is drawn to scale for transcript 1. Transcripts after insertion of the pUPA-TRAP vector before and after Cre-recombination are indicated. Generation of Zscan10 protein after Cre-recombination is highly unlikely based on the design of the pUPA-TRAP vector. Transcription start site , translation start (green arrow heads), translation stop (red arrow heads), splice acceptor site (SA), internal ribosome entry site (IRES), polyadenylation site (pA), RNA polymerase II promoter (RNAPII), recognition site for Cre recombinase (loxP), splice donor site (SD). Integration site of pUPA-TRAP vector is indicated by the blue arrow.(EPS)Click here for additional data file.Figure S2Results of the immunological screen. (A) Frequencies of leukocyte subpopulations during flow cytometry in peripheral blood after erythrocyte lysis . Means, standard deviation and p-values calculated by a linear model. (B) Levels of immunoglobulins (ug/ml) in blood plasma. Medians, first and third quartile and p-values calculated by Wilcoxon rank-sum test. Missing measurements caused by Ig levels above and below measurability were replaced by 0.9*min/1.1*max of respective Ig measurement (n = 57). (C) Total IgE in plasma were in normal range. Medians, first and third quartile and p-values calculated by a Wilcoxon rank-sum test.(TIF)Click here for additional data file.Figure S3Open Field Testing. Means, standard deviation and p-values calculated by a linear model (n = 60).(TIF)Click here for additional data file.Figure S4Organ weight. Medians, first and third quartile and p-values calculated by a Wilcoxon rank-sum test (n = 57).(TIF)Click here for additional data file.Figure S5Western blot of the homozygous Zscan10 mutant (−/−) ESC line L2 in comparison to wild type (+/+) and heterozygous (+/−) ESC. Using 15 ug each with the GIS206-Ab (EPS)Click here for additional data file.Figure S6Sox2, Hes5 and EGFP expression in comparison to Zscan10. Sections through E15.5 (A-X) and E12.5 (Y-P′) Zscan10−/−  and Zscan10+/−  mouse embryos showing expression of the transcription factors Hes5 (A-L), Zscan10 (S-D′) and Sox2 (E′-P′) as well as the pUPA reporter EGFP (M-R). Organs expressing the Notch pathway effector Hes5 did not show any obvious difference in Hes5 expression between Zscan10−/− and Zscan10+/− embryos as shown for the brain  neural tube (nt)  gut  and liver  with exception of the retinal layer , where the Hes5 signal appeared less intense, either due to reduced expression levels or fewer cells expressing Hes5 in the −/−Zscan10 eye (I). Ectopic upregulation of Hes5 in chondrogenic tissue was not observed . EGFP expression was similar to Zscan10 expression in all organs assayed for namely: Adrenal , kidney , heart , lung  vertebrae body and forming intervertebral disc  and dorsal root ganglia (drg) . Sox2 was not detected in kidney and adrenal , only faintly expressed in nt and drg  but found coexpressed with Zscan10 in the heart , the apical ectodermal ridge (aer)  and the retina .(TIF)Click here for additional data file.Table S1Genotyping results from offspring at weaning age (n = 153). P-value is calculated as one tailed probability values with n = 2 degrees of freedom.(EPS)Click here for additional data file.


Cellular signaling transduction critically depends on molecular interactions that are in turn governed by dynamic subcellular distributions of the signaling system components. Comprehensive insight into signal transduction requires an understanding of such distributions and cellular structures driving them. To investigate the activation of primary murine T cells by antigen presenting cells (APC) we have imaged more than 60 signaling intermediates during T cell stimulation with microscopy across resolution limits. A substantial number of signaling intermediates associated with a transient, wide, and actin-associated lamellum extending from an interdigitated T cell:APC interface several micrometers into the T cell, as characterized in detail here. By mapping the more than 60 signaling intermediates onto the spatiotemporal features of cell biological structures, the lamellum and other ones previously described, we also define distinct spatial and temporal characteristics of T cell signal initiation, amplification, and core signaling in the activation of primary T cells by APCs. These characteristics differ substantially from ones seen when T cells are activated using common reductionist approaches. T cell activation occurs in cellular interactions between T cells and antigen-presenting cells (APC). During T cell activation signaling intermediates enrich in distinct locations at specific times within the cell –5. Yet, The spatiotemporal organization of T cell activation on APCs is dynamic and complex . AccumulA powerful way to discover organizing principles in signal transduction is to analyze the spatiotemporal organization of the signaling network at the system-scale. Such analyses can elucidate higher-order mechanisms in the formation and resolution of signalling assemblies that are inaccessible to single gene/protein studies. To identify cellular processes controlling signaling organization, we have extended our live primary T cell:APC conjugate imaging data to more than 60 molecules involved in T cell activation and have furthered our understanding of the T cell signaling organization with microscopy across resolution limits. This system-scale imaging analysis of T cell signaling in response to APC stimulation revealed an actin-associated lamellum that organizes a substantial part of the T cell signaling system. By mapping a large and diverse set of signaling intermediates onto the spatiotemporal features of cell biological structures including the lamellum we define distinct spatial and temporal characteristics of T cell signal initiation, amplification, and core signaling in the activation of primary T cells by APCs.In vitro primed primary 5C.C7 TCR transgenic CD4+ T cells were retrovirally transduced to express fluorescently tagged signaling intermediates and sensors (> 60). Time-lapsed fluorescence microscopy was performed with transduced T cells activated by CH27 B cell lymphoma APCs pulsed with 10μM moth cytochrome C (MCC) antigenic peptide. This experimental setup provides an in vitro model for the reactivation of primed T cells, e.g. in the delivery of T cell help. 3D accumulation patterns at the T cell:APC interface (2) sensor pleckstrin homology domain of phospholipase Cδ (PLCδPH), and the Rho family guanine nucleotide exchange factor Vav1   even thoTo determine whether geometrical features of F-actin distributions are comparable to those of the membrane undulations, we further analyzed the fixed 5C.C7:CH27 conjugates stained with phalloidin and imaged by STED microscopy. Discreet regions of F-actin staining were oriented perpendicular to the interface plane early, as opposed to a more diffuse distribution late . The depThus we have characterized that actin and frequent membrane undulations reaching deep into the T cell across the entire T cell:APC interface are most prominent within the first few minutes to T cell activation.2, and Vav . The extent to which pSLP-76 clusters and F-actin extend from the interface into the T cell correlated strongly   . Address0.002) ( 3] ind ind). ThLamellal signaling intermediates represent a third of the T cell signaling network components studied here and substantially overlap with the transient deep actin matrix emanating from the undulating T cell:APC interface. As discussed in more detail below, two mechanistic scenarios are conceivable. Signaling intermediates/complexes could bind directly to lamellal actin or actin could function as a three-dimensional lattice to trap signaling complexes. Either way, restraining signaling intermediates in the lamellal actin matrix should enhance the efficiency of lamellal signaling interactions.1/2 = 0.32±0.04, p<0.0001)  experiments with Themis-GFP or PLCδPH-GFP the average half times of recovery of actin and the lamellal signaling intermediates were indistinguishable ranging from 1.3±0.2s for PLCδPH to 2.1±0.3s for actin (p>0.05) . GFP as <0.0001) . Signali<0.0001)  with sig.0001)  detergeT cells must detect low doses of antigen with only modest distinction from self. Stable central signaling even in response to weak stimuli, as suggested by effective central PKCθ accumulation at limiting peptide concentrations , may proBeyond amplifying signaling by constraining a large part of the T cell signaling system in an actin matrix, the lamellum with its associated membrane undulations provides additional potential mechanisms to shape T cell activation. The more than four-fold increase in T cell:APC contact area resulting from the early membrane undulations should enhance ligand scanning. Altered membrane curvature and the dramatically increased membrane surface to cytoplasm volume ratio in T cell membrane extensions should alter signal progression –33. PlasIn summary, as part of a large-scale investigation of signaling organization in primary T cells as activated by APCs we have characterized a transient F-actin matrix extending from an undulating and highly interdigitated T cell:APC interface several micrometers into the T cell that is associated with a large part of the signaling system at the peak of T cell signaling activity. Similar actin sheets with embedded signaling complexes linked to undulating cellular interfaces should play a comparably important role whenever dynamic cell contacts drive cell fate decisions, such as in development , stem diAntibodies for fixed cell microscopy were Alexa 488-α-SLP76 pY128 (BD Pharmingen) and α-LAT pY191  with Alexa 488-goat α-rabbit IgG (Invitrogen). F-actin was stained with Alexa 633 or 488-Phalloidin (Invitrogen). CFSE and Cell Trace Violet were used as whole cell stains (Invitrogen).In vitro primed primary 5C.C7 T cells were set up from preferentially female 5C.C7 TCR transgenic mice of about 2 months of age, as previously described  among amongA-N(TIF)Click here for additional data file.S1 Tablehttp://www.bristol.ac.uk/cellmolmed/research/infect-immune/wuelfing/spatiotemporal-patterning/.For the signaling intermediates covered in (DOCX)Click here for additional data file.S1 VideoDIC images are shown on the top, with matching top-down, maximum projections of 3D sensor fluorescence data on the bottom. The sensor fluorescence intensity is displayed in a rainbow-like, false-color scale (increasing from blue to red). 20 s intervals in video acquisition are played back as 2 frames per second. The 5C.C7 T cell in S1 Video is transduced with SLP-76-GFP. Cell coupling occurs in frame 5 (2s indicated video time). A rapid transition from central to lamellal accumulation that subsequently fades more slowly is shown.(MOV)Click here for additional data file.S2 VideoThe 5C.C7 T cell in S2 Video is transduced with Vav1-GFP. Cell coupling occurs in frame 4 (1s indicated video time). A transient lamellal accumulation is shown.(MOV)Click here for additional data file.S3 VideoThe 5C.C7 T cell in S3 Video is transduced with F-Tractin-GFP. Cell coupling occurs in frame 6 (2s indicated video time). Accumulation in a mix of peripheral, lamellal, and asymmetric patterns is shown.(MOV)Click here for additional data file.S4 VideoIn S4 Video individual z-slices of the EM tomogram reconstruction with T and B cell membranes marked in red and green are shown.(MOV)Click here for additional data file.S5 Video(MOV)Click here for additional data file.S6 VideoThe 5C.C7 T cell in S6 video is transduced with ADAM10-GFP.(MOV)Click here for additional data file.S7 VideoThe 5C.C7 T cell in S7 video is transduced with GFP-ADAP.(MOV)Click here for additional data file.S8 VideoThe 5C.C7 T cell in S8 video is transduced with Akt-GFP.(MOV)Click here for additional data file.S9 VideoThe 5C.C7 T cell in S9 video is transduced with Chronophin-GFP.(MOV)Click here for additional data file.S10 VideoThe 5C.C7 T cell in S10 video is transduced with tandem C1-GFP, a sensor for DAG.(MOV)Click here for additional data file.S11 VideoThe 5C.C7 T cell in S11 video is transduced with Ezrin-GFP.(MOV)Click here for additional data file.S12 VideoThe 5C.C7 T cell in S12 video is transduced with Grb2-GFP.(MOV)Click here for additional data file.S13 VideoThe 5C.C7 T cell in S13 video is transduced with Itk-GFP.(MOV)Click here for additional data file.S14 VideoThe 5C.C7 T cell in S14 video is transduced with Lck-GFP.(MOV)Click here for additional data file.S15 VideoThe 5C.C7 T cell in S15 video is transduced with Moesin-GFP.(MOV)Click here for additional data file.S16 VideoThe 5C.C7 T cell in S16 video is transduced with MLCK-YFP-CaM-CFP.(MOV)Click here for additional data file.S17 VideoThe 5C.C7 T cell in S17 video is transduced with Myosin 1C-GFP.(MOV)Click here for additional data file.S18 VideoThe 5C.C7 T cell in S18 video is transduced with Nck-GFP.(MOV)Click here for additional data file.S19 VideoThe 5C.C7 T cell in S19 video is transduced with GFP-p65.(MOV)Click here for additional data file.S20 VideoThe 5C.C7 T cell in S20 video is transduced with GFP-α-Pix.(MOV)Click here for additional data file.S21 VideoThe 5C.C7 T cell in S21 video is transduced with PKCζ-GFP.(MOV)Click here for additional data file.S22 VideoThe 5C.C7 T cells in S22 video is transduced with GFP-R-pre, a sensor for negatively charged lipids.(MOV)Click here for additional data file.S23 VideoThe 5C.C7 T cell in S23 video is transduced with SHP-1-GFP.(MOV)Click here for additional data file.S24 VideoThe 5C.C7 T cell in S24 video is transduced with GFP-SLAT.(MOV)Click here for additional data file.S25 VideoThe 5C.C7 T cell in S25 video is transduced with GFP-VASP.(MOV)Click here for additional data file.S26 VideoThe 5C.C7 T cell in S26 video is transduced with GFP-WASH.(MOV)Click here for additional data file.S27 VideoThe 5C.C7 T cell in S27 video is transduced with GFP-WDR34-GFP.(MOV)Click here for additional data file.


It coordinates essential cellular functions with the cell cycle including histone biogenesis, splicing, centrosome duplication and origin firing for DNA replication. The two E-cyclins, E1 and E2, are assumed to act interchangeably in these functions. However recent reports have identified unique functions for cyclins E1 and E2 in different tissues, and particularly in breast cancer.The cyclin E oncogene activates CDK2 to drive cells from GCyclins E1 and E2 localise to distinct foci in breast cancer cells as well as co-localising within the cell. Both E-cyclins are found in complex with CDK2, at centrosomes and with the splicing machinery in nuclear speckles. However cyclin E2 uniquely co-localises with NPAT, the main activator of cell-cycle regulated histone transcription. Increased cyclin E2, but not cyclin E1, expression is associated with high expression of replication-dependent histones in breast cancers.The preferential localisation of cyclin E1 or cyclin E2 to distinct foci indicates that each E-cyclin has unique roles. Cyclin E2 uniquely interacts with NPAT in breast cancer cells, and is associated with higher levels of histones in breast cancer. This could explain the unique correlations of high cyclin E2 expression with poor outcome and genomic instability in breast cancer.The online version of this article (doi:10.1186/s13008-015-0007-9) contains supplementary material, which is available to authorized users. In thisWe previously identified that cyclin E1 and E2 are expressed in different cell line subpopulations due to distinct cell cycle regulation . Close eCyclins E1 and E2 have cytoplasmic, nuclear and chromatin associated functions [Waf1/Cip1 and p27Kip1. Both cyclin E1 and E2 form cyclin/CDK2/CDK inhibitor complexes, although these complexes are mutually exclusive  which localise to histone gene clusters on chromosomes 1 and 6 [Cyclin E directly coordinates histone gene transcription with G 1 and 6 -9. We fo 1 and 6 . Howeverin situ Proximity Ligation Assay (PLA), which detects the co-localisation of two antibodies within 40nm on fixed cells by PCR amplification of a linker probe. PLA analysis identified an average of 22 nuclear NPAT-E2 foci per cell, consistent with the multiple HLBs which are detected in aneuploid cancer cell lines [We confirmed our findings using the ll lines   and HE11 (full-length protein) antibodies which are raised using epitopes that may not effectively discriminate cyclin E1 and cyclin E2 . While cOur observation of a specific NPAT-cyclin E2 interaction in breast cancer cell lines was supported by our findings of high expression of replication-dependent histones in breast cancers that have high expression of cyclin E2. We examined the transcript profiles of breast cancers from The Cancer Genome Atlas (TCGA) for cyclin E and histone expression. In 526 breast cancers, high CCNE2 expression is associated with high levels of replication-dependent histones that are under the control of NPAT  and cultured for <6 months after authentication. Cyclin E1 and E2 siRNA treatment was performed and validated by western blotting as described in .Collection of whole cell lysates , chromatCells were fixed with 4% PFA/PBS for 20 min at room temperature, with or without methanol post-fixation ( -20°C for 20 min). Samples were blocked with 1% BSA/PBS, stained with the indicated antibodies and counterstained with ToPro3/DAPI (Jackson ImmunoResearch Laboratories). Co-localisation was quantitated by detecting overlapping pixels with Imaris v8.0 (Bitplane) and analysed with Pearson’s Correlation Coefficient . For PLACCNE1, CCNE2 and representative replication-dependent and -independent histones (Additional file Expression values in 526 breast cancer samples of