Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4+ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses

Cross-linking electrospun type II collagen tissue engineering scaffolds with carbodiimide in ethanol

In trying to assess the structural integrity of electrospun type II collagen scaffolds, a modified but new technique for cross-linking collagen has been developed. Carbodiimides have been previously used to cross-link collagen in gels and in lyophilized native tissue specimens but had not been used for electrospun mats until recently. This cross-linking agent, and in particular 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), is of extreme interest, especially for tissue-engineered scaffolds composed specifically of native polymers (e.g., collagen), because it is a zero-length cross-linking agent that has not been shown to cause any cytotoxic reactions. The unique aspect of the cross-linking protocol in this study involves the use of ethanol as the solvent for the cross-linking agent, because the pure collagen electrospun mats immediately disintegrate when placed in an aqueous solution. This study examines 2 concentrations of EDC with and without the addition of N-hydroxysuccinimide to the reaction (which has been shown to result in higher cross-linking yields in aqueous solutions) to test the hypothesis that the use of EDC in a nonaqueous solution will cross-link electrospun type II collagen fibrous matrices in a comparable manner to typical glutaraldehyde fixation protocols. The use of EDC is compared with the cross-linking effects of glutaraldehyde via mechanical testing (uniaxial tensile testing) and biochemical testing (analysis of the percentage of free amino groups). The stress-strain curves of the cross-linked samples demonstrated uniaxial tensile behavior more characteristic of native tissue than do the dry, untreated samples. The heated, 50% glutaraldehyde cross-linking protocol resulted in a mean peak stress of 0.76 MPa, a mean strain at break of 127.30%, and a mean tangential modulus of 0.89 MPa; mean values for the samples treated with the EDC protocols ranged from 0.35 to 0.60 MPa for peak stress, from 111.83 to 159.23% for strain at break, and from 0.57 to 0.92 MPa for tangential modulus. Low and high concentrations (20 mM and 200 mM, respectively) of EDC alone were comparable in extent of cross-linking (29% and 29%, respectively) to the heated 50% glutaraldehyde cross-linking protocol (30% cross-linked). © Mary Ann Liebert, Inc

Interaction of the ^FtDHPS module with Compound 1.

<p>(A) Schematic comparison between the scaffolds of Compound 1 and DHP-PP. Compound 1 comprises a pterin-like core and is missing half of the B-ring as highlighted in orange. (B) Stereo view of Compound 1 (orange) bound within the pterin pocket of the TIM-barrel. Residues that make van der Waals and hydrogen-bond contacts are labeled and shown as pink sticks. The <i>F</i>o-<i>F</i>c simulated-annealing omit electron density for Compound 1 is shown as a blue mesh contoured at 3.5 σ.</p

The overall structure of the HPPK-DHPS bifunctional enzyme from <i>Francisella tularensis</i>.

<p>(A) A stereo view of the overall fold and domain organization showing the secondary structure elements within each module. Each element is labeled with the prefixes ‘H’ and ‘D’ to reflect their locations in the HPPK (blue) and DHPS (purple) domains, respectively. The N- and C-termini and the linker region (green) are labeled. Note that helix Dα8 in the canonical DHPS TIM-barrel is missing. (B) A surface representation of the view shown in (A) that highlights the position of the domain linker and the cleft within the DHPS module corresponding to the missing Dα8 TIM-barrel α-helix.</p

The primary structure of the HPPK-DHPS bifunctional enzyme from <i>Francisella tularensis</i> and its homology to other HPPK and DHPS enzymes.

<p>The organisms shown are <i>Francisella tularensis</i> (Ft), <i>Saccharomyces cerevisiae</i> (Sc), <i>Yersinia pestis</i> (Yp), <i>Escherichia coli</i> (Ec) and <i>Bacillus anthracis</i> (Ba), and numbering is with respect to the Ft enzyme. Secondary structure elements and key structural regions are labeled according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014165#pone-0014165-g003" target="_blank">Fig. 3A</a>. Strictly conserved regions are blocked in red, and conserved regions are boxed. Important loop regions are highlighted and labeled according to their domain association. (A) Multiple sequence alignment of the HPPK module. Residues that contribute to substrate binding are shown as blue triangles. The conserved motif that binds Mg²⁺ is shown as gray circles within blue triangles. (B) Alignment of the DHPS module. The inter-domain linker regions of <i>F. tularensis</i> and <i>S. cerevisiae</i> are highlighted in green and the corresponding β-hairpin of monofunctional DHPS is highlighted in orange. Residues that interact with substrates are indicated as purple triangles. Residues known to contribute to sulfonamide drug resistance are indicated by red circles. The missing Dα8 helix at the C-terminus is highlighted in purple. Sequence alignments were performed using ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014165#pone.0014165-Thompson1" target="_blank">[39]</a> and analyzed using ESPript2.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014165#pone.0014165-Gouet1" target="_blank">[54]</a>.</p

Data Collection and Refinement Statistics.

<p>*Data were collected from a single crystal. Values in parentheses are for the highest-resolution shell.</p>a<p>R_free was calculated using 5% of the reflections.</p

Analytical ultracentrifugation of the HPPK-DHPS bifunctional enzyme from <i>Francisella tularensis</i>.

<p>(A) The sedimentation velocity profiles (fringe displacement) were fitted to a continuous sedimentation coefficient distribution model c(s). The experiment was conducted at a loading protein concentration of 0.69 mg/ml in at 20°C and at a rotor speed of 60,000 rpm. (B) Absorbance scans at 280 nm at equilibrium are plotted <i>versus</i> the distance from the axis of rotation. The protein was centrifuged at 4°C for at least 24 h at each rotor speed of 15 k (red), 22 k (blue) and 27 k (black) rpm. The <i>solid lines</i> represent the global nonlinear least squares best-fit of all the data sets to a monomer-dimer self-association model with a very weak K_D (2.7 mM). The loading protein concentration was 20 µM and the r.m.s. deviation for this fit was 0.0037 absorbance units.</p