While five arenaviruses cause human hemorrhagic fevers in the Western Hemisphere, only Junin virus (JUNV) has a vaccine. The GP1 subunit of their envelope glycoprotein binds transferrin receptor 1 (TfR1) using a surface that substantially varies in sequence among the viruses. As such, receptor-mimicking antibodies described to date are type-specific and lack the usual breadth associated with this mode of neutralization. Here we isolate, from the blood of a recipient of the live attenuated JUNV vaccine, two antibodies that cross-neutralize Machupo virus with varying efficiency. Structures of GP1–Fab complexes explain the basis for efficient cross-neutralization, which involves avoiding receptor mimicry and targeting a conserved epitope within the receptor-binding site (RBS). The viral RBS, despite its extensive sequence diversity, is therefore a target for cross-reactive antibodies with activity against New World arenaviruses of public health concern

Clark, Lars E.

Mahmutovic, Selma

Raymond, Donald D.

Dilanyan, Taleen

Koma, Takaaki

Manning, John T.

Shankar, Sundaresh

Levis, Silvana C.

Briggiler, Ana M.

Enria, Delia A.

Wucherpfennig, Kai W.

Paessler, Slobodan

Abraham, Jonathan

Gomilanje građevinskog otpada je veliki problem današnjice. Uslijed neadekvatnog skladištenja otpada, te sve veće potrebe za odlagalištima, otpad se nastoji ponovno iskoristiti za izradu novog betona. U ovom radu opisan je utjecaj punila od mljevene ispitne betonske kocke frakcije 0.125-0.25 i 0-0.5 mm na svojstva betona u svježem i očvrslom stanju. Ispitana je temperatura i veličina slijeganja svježeg, te dinamički modul elastičnosti i tlačna čvrstoća očvrslog betona.The buildup of construction waste is a huge problem nowdays. Due to inadequate storage of construction waste and the increasing need for landfills, waste is being sought to be reused to make new concrete. This study describes the effect of fillers from the ground test concrete cube fraction 0.125-0.25 and 0-0.5 mm on the properties of concrete in fresh and hardened state. Temperature and size of settling were examined for concrete in fresh state, dynamic modulus of elasticity and compressive strenght were examined for concrete in hardened state

Cakalin, Iva

FCEAG Repository

The Use of Ground Test Concrete Cube Fraction 0.125-0.2 and 0-0.5 mm as  Filler in Concrete : Undergraduate Thesis

Croatian Digital Thesis Repository

University of Split Repository

Tokushima University Institutional Repository

Supplementary Information  Vaccine-elicited receptor-binding site antibodies neutralize two New World hemorrhagic fever arenaviruses  Lars E. Clark1#, Selma Mahmutovic2#, Donald D. Raymond2, Takaaki Koma3, John T. Manning3, Taleen Dilanyan1, Sundaresh Shankar1, Silvana C. Levis4, Ana M. Briggiler4, Delia A. Enria4, Kai W. Wucherpfennig1,5,6, Slobodan Paessler3, Jonathan Abraham1,2,7*   1Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA; 2Laboratory of Molecular Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; 3Department of Pathology, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA; 4Instituto Nacional de Enfermedades Virales Humanas “Dr. Julio I Maiztegui”, Monteagudo 251 Pergamino, Buenos Aires 2700, Argentina; 5Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02115, USA; 6Program in Immunology, Harvard Medical School, Boston, MA 02115, USA; 7Department of Medicine, Division of Infectious Diseases, Brigham and Women’s Hospital, Boston, MA 02115, USA.     Supplementary Figure 1. Gating scheme for single B cell sorting. Gating scheme for isolation of antigen-specific memory B cells (CD19+, CD27+, IgM-, JUNV GP1+). Asterisk indicates the exclusion channel; in Sort 2 (Fig. 1c), the exclusion channel also included cells that stained positive for JUNV GP1mut-PerCP.         Supplementary Figure 2. Analysis of CR1-06 and CR1-09 binding to JUNV GP1. (a) ELISA of CR1-06 IgG binding to immobilized JUNV GP1. LUJV GP1 is a control. Error bars, which indicate standard deviation, are smaller than symbols. (b) Sensorgrams for binding of CR1-09 Fab to immobilized JUNV GP1 as measured by surface plasmon resonance. CR1-09 Fab was passed over the surface at concentrations of 400 nM, 200 nM, 100 nM, 50 nM, and 25 nM. The recorded sensorgrams (one of duplicates) are shown. The fast off rate prevented accurate fitting of the data to estimate binding kinetics.   Supplementary Figure 3. CR1-07 and CR1-28 compete with GD01 for binding to JUNV GP1. Competitor IgG (GD01 or 17b) was added at increasing concentrations to plates coated with JUNV GP1. After an incubation step, CR1-10 IgG (left panel), CR1-07 IgG (middle panel), or CR1-28 IgG (right panel) were added at fixed concentrations. After several wash steps, CR1-07, CR1-10, or CR1-28 IgG that bound to the plate was detected using an anti-human IgG secondary antibody. The experiment was performed twice in duplicate and representative data are shown. Error bars indicate standard deviation (S.D.).   Supplementary Figure 4. Electron density for the CR1-10 Fab/JUNV GP1/CR1-28 Fab complex. (a) Part of the CR1-10 Fab/JUNV GP1 interface is shown as sticks with the refined 2Fo-Fc electron density at 1 σ. (b) Part of the CR1-28 Fab/JUNV GP1 interface is shown as sticks with the refined 2Fo-Fc electron density at 1 σ. (c) 2Fo-Fc electron density at 1 σ for a N-linked glycan (shown as green sticks) attached at JUNV GP1 Asn178.        Supplemental Figure 5. CR1-10 binds cell-surface expressed JUNV GPC trimer. We transfected HEK293T cells with JUNV GPC and measured binding of CR1-28 IgG, CR1-10 IgG, or 17b IgG (control) by FACS 48 hr after transfection. Mean fluorescence intensity (MFI) values for antibody-binding to transfected cells were corrected by subtraction of MFI obtained at the same antibody concentration with control, untransfected HEK293T cells. The EC50 for CR1-28 and CR1-10 binding to cells transfected with JUNV GPC was measured as 0.32 µg ml-1 and 1.9 µg ml-1, respectively. Error bars indicate S.D. For some data points, error bars are smaller than symbols. The experiment was performed twice in duplicate and representative data are shown.  Supplementary Figure 6. CR1-07 Fab structure and electron density for MACV GP1/CR1-07 Fab complex. (a) Ribbon diagram of the unliganded CR1-07 Fab (heavy chain in gray, light chain in black). HC: heavy chain; LC: light chain. (b) Part of the MACV GP1/CR1-07 Fab interface is shown as sticks with the refined 2Fo-Fc electron density at 1 σ. (c) One of the four Fab copies (representative) found in the asymmetric unit of the MACV GP1/CR1-07 Fab complex was overlaid onto the unliganded CR1-07 Fab. The tip CR1-07 CDR L1 shifts by about 6 Å to accommodate MACV GP1 in the complex.  Supplementary Figure 7. CR1-07 contact residues in MACV GP1. (a) Ribbon diagram of MACV GP1 with residues that are contacted by CR1-07 shown as sticks and colored red. Asparagines that are potential sites of N-linked glycan attachment are shown as sticks and indicated with an asterisk. (b) List of CR1-07 contact residues in MACV GP1 and residues at the analogous positions in the GP1s of other New World arenaviruses (using MACV GP1 numbering). Residues highlighted in grey are identical between MACV and the indicated GP1.    Supplementary Figure 8. Model of neutralizing antibody binding to the pre-fusion arenavirus GPC ectodomain. (a) The MACV GP1/TfR1 complex (PDB: 3KAS)1 was overlaid onto the structure of the prefusion LASV GPC ectodomain (PDB: 5VK2)2 by aligning the GP1 proteins, which have a similar fold. One of the three GP1:GP2 protomers is colored for GPC. (b) Left: Structure of the JUNV GP1/GD01 Fab complex (PDB: 5EN2)3 overlaid onto the prefusion LASV GPC ectodomain structure as in a. Right: Structure of the JUNV GP1/CR1-28 Fab subcomplex overlaid onto the LASV GPC ectodomain structure as in a. GD01 and CR1-28 are receptor-mimicking antibodies. (c) Structure of the MACV GP1/CR1-07 Fab complex overlaid onto the structure of the LASV GPC as in a. CR1-07 is a JUNV/MACV cross-neutralizing antibody. (d) The JUNV GP1/CR1-10 Fab complex was overlaid onto LASV GPC as in a, with two of the protomers in trimeric GPC shown as transparent to allow for better visualization of the binding site. CR1-10 is a non-neutralizing antibody. For all panels, the New World arenavirus GP1 protein used for structural alignment is omitted for clarity.   Supplementary Figure 9. Sample electron density for the reported X-ray crystal structures. (a) Sample electron density for the unliganded CR1-07 Fab crystal structure. The volume shown in stereoview is from the 2Fo-Fc electron density map contoured at 1.5 σ in the region corresponding to light chain residues 39-47. (b) Sample electron density for the CR1-10 Fab/JUNV GP1/CR1-28 Fab crystal structure. The volume shown in stereoview is from the 2Fo-Fc electron density map contoured at 1.0 σ in the region corresponding to residues 32-39 of the CR1-28 heavy chain (chain T in the asymmetric unit). (c) Sample electron density for the MACV GP1/CR1-07 Fab crystal structure. The volume shown in stereoview is from the 2Fo-Fc electron density map contoured at 1.0 σ in the region corresponding to CR1-07 Fab heavy chain residues 32-39 (chain F in the asymmetric unit).  Supplementary Table 1. Properties of GP1-directed antibodies.  Antibody VH D JH CDR3 Length Mutations % Mutation CR1-06 3-7 3-10 4/5 AKVGGHSAFYKTGRVGFDS 19 39 13.4% CR1-07 3-30 3-16/3-9 4 AKDLSPPYSYAWDIFQY  17 39 13.2% CR1-09 4-4 2-15 4 ARYCSGSACVPFDF 14 27 9.2% CR1-10 4-31 2-15 4 ARLTSDCSGGNCYQAFDY 18 33 11.1% CR1-28 3-33 6-13 4 ATDKTYVSGYTSTWYYFNY 19 11 3.7% *GD01 1-4/1-7 1-1 4 ARRRVYYGSNYIYALDY 17 38 13.0%           Vk Jk CDR3 Length Mutations % Mutation  CR1-06 1-12/1D-12 2 QQGNSLPYT 9 39 13.70%  CR1-07 4-1 4 QQYYSSPPT 9 14 6.6%  CR1-09 3-20 1 LQYGTRPRT 9 21 7.3%  CR1-10 3-20 2 QQYGRSGYT 9 9 3.2%  CR1-28 1-5 1 QHRT 4 4 1.5%  *GD01 6-13 5 QQYSSYPLA 9 6 2.1%   *For GD01, murine antibody gene usage patterns are shown.           Supplementary Table 2. Binding rate constants of surface plasmon resonance analysis of antibody Fabs binding to immobilized GP1.   Analyte Ligand ka (1/Ms) kd (1/s) KD (M) CR1-07 Fab JUNV GP1 8.52E+03 7.34E-04 8.62E-08 CR1-10 Fab JUNV GP1 8.10E+05 3.65E-05 4.50E-11 CR1-28 Fab JUNV GP1 1.05E+05 5.54E-04 5.30E-09 CR1-07 Fab MACV GP1 4.13E+04 6.76E-04 1.60E-08    Supplementary Table 3. Data collection and refinement statistics.    CR1-07 Fab  CR1-10 Fab/JUNV GP1/CR1-28 Fab  MACV GP1/CR1-07 Fab Data collection       Space group  C2  P21  P42212 Cell dimensions           a, b, c  89.8 137.4 78.7  154.8 132.2 167.4  206.9 206.9 238.23 Resolution (Å)  2.00 (2.07-2.00)a  3.99 (4.23-3.99)  3.91 (4.15-3.91) Rmerge  0.12 (1.02)  0.72 (1.58)  0.32 (8.38) CC1/2  99.4 (64.6)  79.6 (46.5)  99.5 (21.5) Avg I/σI  9.57 (1.65)  2.34 (1.03)  7.72 (0.52) Completeness (%)  97.9 (96.7)  99.1 (97.6)  98.8 (96.8) Average redundancy  4.5 (4.6)  3.4 (3.4)  8.1 (8.1)        Refinement       Resolution (Å)  44.21-2.00  49.35-3.99  49.12-3.91 No. reflections  32,718  57,194  46,614 Rwork/Rfree  0.23/0.28  0.24/0.29  0.25/0.26 Number of Atoms          Protein  3,378  30,971  18,299    Water  223  -  - R.m.s. deviations           Bond lengths (Å)  0.005  0.008  0.012     Bond angles (°)  0.646  1.15  1.33 Avg B-factor (Å2)           Protein  47.8  69.0  33.5     Water  44.8  -  - One crystal was used to collect each data set.  aValues in parentheses are for highest-resolution shell    Supplementary Table 4. XSCALE4 output summary.  Subset of intensity data with signal to noise >=-3.0 as function of resolution for JUNV GP1 complex  Resolution limit Reflections Observed Reflections Unique Reflections Possible Completeness of data R-factor observed R-factor expected Compared I/SIGMA R-meas CC(1/2) 11.68 7797 2333 2406 97.00% 13.40% 13.80% 7764 7.53 16.00% 98.8* 8.38 13247 3931 3973 98.90% 16.60% 16.90% 13167 6.55 19.80% 98.2* 6.87 17016 5072 5115 99.20% 45.50% 45.20% 16869 2.91 54.10% 90.4* 5.97 19914 5866 5902 99.40% 79.60% 79.30% 19792 1.84 94.60% 73.2* 5.34 23300 6838 6867 99.60% 83.30% 82.20% 23160 1.78 99.00% 72.4* 4.88 25365 7432 7450 99.80% 73.80% 73.40% 25241 1.97 87.80% 77.0* 4.52 27660 8086 8114 99.70% 81.30% 80.50% 27538 1.85 96.60% 73.9* 4.23 29698 8680 8705 99.70% 102.20% 100.70% 29599 1.57 121.60% 64.4* 3.99 30534 8957 9177 97.60% 158.60% 156.70% 30401 1.03 188.60% 46.5* Total 194531 57195 57709 99.10% 72.10% 71.40% 193531 2.34 85.70% 79.6*    Subset of intensity data with signal to noise >=-3.0 as function of resolution for MACV GP1 complex  Resolution limit Reflections Observed Reflections Unique Reflections Possible Completeness of data R-factor observed R-factor expected Compared I/SIGMA R-meas CC(1/2) 11.45 14549 2013 2093 96.20% 2.90% 3.60% 14523 46.89 3.10% 99.9* 8.21 24884 3299 3341 98.70% 4.70% 5.00% 24836 33.4 5.00% 99.9* 6.73 33344 4196 4237 99.00% 17.20% 17.30% 33296 12.92 18.40% 99.3* 5.84 39740 4909 4943 99.30% 42.70% 42.80% 39700 6.31 45.60% 95.7* 5.24 44300 5425 5464 99.30% 65.20% 65.30% 44265 4.42 69.60% 92.2* 4.78 50412 6149 6173 99.60% 87.10% 87.00% 50371 3.49 92.90% 88.1* 4.43 53993 6554 6581 99.60% 153.20% 152.80% 53962 2.15 163.30% 75.1* 4.15 57007 6911 6937 99.60% 262.50% 261.40% 56980 1.25 279.70% 49.5* 3.91 59863 7376 7621 96.80% 838.00% 834.60% 59833 0.52 894.00% 21.5* total 378092 46832 47390 98.80% 32.90% 33.10% 377766 7.72 35.10% 99.5*  Definitions Compared Number of reflections used for calculating R-factor I/SIGMA Mean of intensity/Sigma(I) of unique reflections (after merging symmetry-related observations) Sigma(I) Standard deviation of reflection intensity I estimated from sample statistics R-meas Redundancy independent R-factor (intensities) CC(1/2)  Anomal Corr Percentage of correlation between intensities from random half-datasets. Correlation significant at the 0.1% level is marked by an asterisk. Percentage of correlation between random half-sets of anomalous intensity differences. Correlation significant at the 0.1% level is marked. SigAno Mean anomalous difference in units of its estimated standard deviation (|F(+)-F(-)|/Sigma). F(+), F(-) are structure factor estimates obtained from the merged intensity observations in each parity class. Nano Number of unique reflections used to calculate Anomal_Corr & SigAno. At least two observations for each (+ and -) parity are required.                       Supplementary References  1. Abraham, J. et al. Structural basis for receptor recognition by New World hemorrhagic fever arenaviruses. Nature Structural & Molecular Biology 17, 438–444 (2010). 2. Hastie, K. M. et al. Structural basis for antibody-mediated neutralization of Lassa virus. Science 356, 923–928 (2017). 3. Mahmutovic, S. et al. Molecular Basis for antibody-mediated neutralization of New World hemorrhagic fever mammarenaviruses. Cell Host & Microbe 18, 705–713 (2015). 4. Kabsch, W. XDS. Acta Crystallographica. Section D, Biological Crystallography 66, 125–132 (2010).  