Textbook of histology

HIV employs multiple means to evade the humoral immune response, particularly the elicitation of and recognition by broadly neutralizing antibodies (bnAbs). Such antibodies can act antivirally against a wide spectrum of viruses by targeting relatively conserved regions on the surface HIV envelope trimer spike. Elicitation of and recognition by bnAbs are hindered by the arrangement of spikes on virions and the relatively difficult access to bnAb epitopes on spikes, including the proximity of variable regions and a high density of glycans. Yet, in a small proportion of HIV-infected individuals, potent bnAb responses do develop, and isolation of the corresponding monoclonal antibodies has been facilitated by identification of favorable donors with potent bnAb sera and by development of improved methods for human antibody generation. Molecular studies of recombinant Env trimers, alone and in interaction with bnAbs, are providing new insights that are fueling the development and testing of promising immunogens...

Broadly Neutralizing Antibodies to HIV and Their Role in Vaccine Design

The envelope glycoprotein trimer (Env) on the surface of HIV-1 recognizes CD4(+) T cells and mediates viral entry. During this process, Env undergoes substantial conformational rearrangements, making it difficult to study in its native state. Soluble stabilized trimers have provided valuable insights into the Env structure, but they lack the hydrophobic membrane proximal external region (MPER, an important target of broadly neutralizing antibodies), the transmembrane domain, and the cytoplasmic tail. Here we present (i) a cryogenic electron microscopy (cryo-EM) structure of a clade B virus Env, which lacks only the cytoplasmic tail and is stabilized by the broadly neutralizing antibody PGT151, at a resolution of 4.2 angstroms and (ii) a reconstruction of this form of Env in complex with PGT151 and MPER-targeting antibody 10E8 at a resolution of 8.8 angstroms. These structures provide new insights into the wild-type Env structure.

https://science.sciencemag.org/content/sci/351/6277/1043.full.pdf

Cryo-EM structure of a native, fully glycosylated, cleaved HIV-1 envelope trimer.

Trimeric HIV-1-Env Structures Define Glycan Shields from Clades A, B and G

Exploitation of glycosylation in enveloped virus pathobiology.

The envelope spike of HIV-1 employs a 'glycan shield' to protect itself from antibody-mediated neutralization. Paradoxically, however, potent broadly neutralizing antibodies (bnAbs) that target this shield have been isolated. The unusually high glycan density on the gp120 subunit limits processing during biosynthesis, leaving a region of under-processed oligomannose-type structures, which is a primary target of these bnAbs. Here we investigate the contribution of individual glycosylation sites in the formation of this so-called intrinsic mannose patch. Deletion of individual sites has a limited effect on the overall size of the intrinsic mannose patch but leads to changes in the processing of neighbouring glycans. These structural changes are largely tolerated by a panel of glycan-dependent bnAbs targeting these regions, indicating a degree of plasticity in their recognition. These results support the intrinsic mannose patch as a stable target for vaccine design.

/pdf/glycan-clustering-stabilizes-the-mannose-patch-of-hiv-1-and-1e1ln4u723.pdf

Glycan clustering stabilizes the mannose patch of HIV-1 and preserves vulnerability to broadly neutralizing antibodies.

There is an urgent need to develop simple and fast antimicrobial susceptibility tests (ASTs) that allow informed prescribing of antibiotics. Here, we describe a label-free AST that can deliver results within an hour, using an actively dividing culture as starting material. The bacteria are incubated in the presence of an antibiotic for 30 min, and then approximately 105 cells are analysed one-by-one with microfluidic impedance cytometry for 2–3 min. The measured electrical characteristics reflect the phenotypic response of the bacteria to the mode of action of a particular antibiotic, in a 30-minute incubation window. The results are consistent with those obtained by classical broth microdilution assays for a range of antibiotics and bacterial species. There is an urgent need to develop simple and fast antimicrobial susceptibility tests. Here, Spencer et al. describe a label-free test that can deliver results within an hour, consisting of a 30-min antibiotic treatment followed by single-cell analysis of phenotypic responses with microfluidic impedance cytometry.

/pdf/a-fast-impedance-based-antimicrobial-susceptibility-test-55znase8t1.pdf

A fast impedance-based antimicrobial susceptibility test.

Single cell impedance cytometry is a label-free electrical analysis method that requires minimal sample preparation and has been used to count and discriminate cells on the basis of their impedance properties. This paper shows experimental and numerically simulated impedance signals for test particles (6 μm diameter polystyrene) flowing through a microfluidic channel. The variation of impedance signal with particle position is mapped using numerical simulation and these results match closely with experimental data. We demonstrate that for a nominal 40 μm × 40 μm channel, the impedance signal is independent of position over the majority of the channel area, but shows large experimentally verifiable variation at extreme positions. The parabolic flow profile in the channel ensures that most of the sample flows through the area of uniform signal. At high flow rates inertial focusing is observed; the particles flow in equal numbers through two equilibrium positions reducing the coefficient of variance (CV) in the impedance signals to negligible values.

Positional dependence of particles in microfludic impedance cytometry

Single-cell impedance cytometry is a label-free analysis technique that is now widely used to measure the electrical properties of a cell and to differentiate different subpopulations. Current techniques are limited to measuring the impedance of a single cell at one or two simultaneous frequencies. Also, there are no methods that extrapolate the intrinsic electrical properties of single cells. We demonstrate a new approach that uses multifrequency impedance measurements to determine the complete intrinsic electrical properties of thousands of single cells at high throughput. The applicability of the method is demonstrated by measuring the properties of red blood cells and red cell ghosts, deriving the unique values of conductivity and permittivity of the membrane and cytoplasm for each individual cell.

/pdf/high-speed-single-cell-dielectric-spectroscopy-43a17jkvgr.pdf

High-Speed Single-Cell Dielectric Spectroscopy.

This paper describes a new design of microfluidic impedance cytometer enabling accurate characterization of particles without the need for focusing. The approach uses multiple pairs of electrodes to measure the transit time of particles through the device in two simultaneous different current measurements, a transverse (top to bottom) current and an oblique current. This gives a new metric that can be used to estimate the vertical position of the particle trajectory through the microchannel. This parameter effectively compensates for the non-uniform electric field in the channel that is an unavoidable consequence of the use of planar parallel facing electrodes. The new technique is explained and validated using numerical modelling. Impedance data for 5, 6 and 7 μm particles are collected and compared with simulations. The method gives excellent coefficient of variation in (electrical) radius of particles of 1% for a sheathless configuration.

/pdf/high-accuracy-particle-analysis-using-sheathless-4ws6msetff.pdf

Daniel Spencer

Papers

Glycan clustering stabilizes the mannose patch of HIV-1 and preserves vulnerability to broadly neutralizing antibodies.

A fast impedance-based antimicrobial susceptibility test.

Positional dependence of particles in microfludic impedance cytometry

High-Speed Single-Cell Dielectric Spectroscopy.

High accuracy particle analysis using sheathless microfluidic impedance cytometry