sCD4-17b bifunctional protein: Extremely broad and potent neutralization of HIV-1 Env pseudotyped viruses from genetically diverse primary isolates

<p>Abstract</p> <p>Background</p> <p>We previously described a potent recombinant HIV-1 neutralizing protein, sCD4-17b, composed of soluble CD4 attached via a flexible polypeptide linker to an SCFv of the 17b human monoclonal antibody directed against the highly conserved CD4-induced bridging sheet of gp120 involved in coreceptor binding. The sCD4 moiety of the bifunctional protein binds to gp120 on free virions, thereby enabling the 17b SCFv moiety to bind and block the gp120/coreceptor interaction required for entry. The previous studies using the MAGI-CCR5 assay system indicated that sCD4-17b (in concentrated cell culture medium, or partially purified) potently neutralized several genetically diverse HIIV-1 primary isolates; however, at the concentrations tested it was ineffective against several other strains despite the conservation of binding sites for both CD4 and 17b. To address this puzzle, we designed variants of sCD4-17b with different linker lengths, and tested the neutralizing activities of the immunoaffinity purified proteins over a broader concentration range against a large number of genetically diverse HIV-1 primary isolates, using the TZM-bl Env pseudotype assay system. We also examined the sCD4-17b sensitivities of isogenic viruses generated from different producer cell types.</p> <p>Results</p> <p>We observed that immunoaffinity purified sCD4-17b effectively neutralized HIV-1 pseudotypes, including those from HIV-1 isolates previously found to be relatively insensitive in the MAGI-CCR5 assay. The potencies were equivalent for the original construct and a variant with a longer linker, as observed with both pseudotype particles and infectious virions; by contrast, a construct with a linker too short to enable simultaneous binding of the sCD4 and 17b SCFv moieties was much less effective. sCD4-17b displayed potent neutralizing activity against 100% of nearly 4 dozen HIV-1 primary isolates from diverse genetic subtypes (clades A, B, C, D, F, and circulating recombinant forms AE and AG). The neutralization breadth and potency were superior to what have been reported for the broadly neutralizing monoclonal antibodies IgG b12, 2G12, 2F5, and 4E10. The activity of sCD4-17b was found to be similar against isogenic virus particles from infectious molecular clones derived either directly from the transfected producer cell line or after a single passage through PBMCs; this contrasted with the monoclonal antibodies, which were less potent against the PMBC-passaged viruses.</p> <p>Conclusions</p> <p>The results highlight the extremely potent and broad neutralizing activity of sCD4-17b against genetically diverse HIV-1 primary isolates. The bifunctional protein has potential applications for antiviral approaches to combat HIV infection.</p

The effect of termination of membrane phospholipid synthesis on cell-dependent events in caulobacter

Membrane phospholipid synthesis in Caulobacter crescentus has been shown to be related to the expression of specific cell cycle events. DNA synthesis was immediately inhibited if phospholipid synthesis was terminated either by glycerol starvation of a glycerol auxotroph or by treatment of mutant and wildtype cultures with cerulenin. Termination of phospholipid synthesis, by either method, resulted in the inhibition of stalk elongation, flagellum biogenesis and cell division. The inability to form a stalk appears to be directly due to the cessation of phospholipid synthesis, whereas the inhibition of flagella formation and cell division is likely a result of the secondary effect on DNA replication. Two cell cycle events, the ejection of the flagellum and stalk initiation, were shown to be independent of phospholipid synthesis and DNA replication.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23265/1/0000200.pd

Molecular mechanisms separating two axonal pathways during embryonic development of the avian optic tectum

During embryonic development of the avian optic tectum, retinal and tectobulbar axons form an orthogonal array of nerve processes. Growing axons of both tracts are transiently very closely apposed to each other. Despite this spatial proximity, axons from the two pathways do not intermix, but instead restrict their growth to defined areas, thus forming two separate plexiform layers, the stratum opticum and the stratum album centrale. In this study we present experimental evidence indicating that the following three mechanisms might play a role in segregating both axonal populations: Retinal and tectobulbar axons differ in their ability to use the extracellular matrix protein laminin as a substrate for axonal elongation; the environment in the optic tectum is generally permissive for retinal axons, but is specifically nonpermissive for tectobulbar axons, resulting in a strong fasciculation of the latter; and growth cones of temporal retinal axons are reversibly inhibited in their motility by direct contact with the tectobulbar axon's membrane

Alternative mechanism for bacteriophage adsorption to the motile bacterium Caulobacter crescentus

2D and 3D cryo-electron microscopy, together with adsorption kinetics assays of ϕCb13 and ϕCbK phage-infected Caulobacter crescentus, provides insight into the mechanisms of infection. ϕCb13 and ϕCbK actively interact with the flagellum and subsequently attach to receptors on the cell pole. We present evidence that the first interaction of the phage with the bacterial flagellum takes place through a filament on the phage head. This contact with the flagellum facilitates concentration of phage particles around the receptor (i.e., the pilus portals) on the bacterial cell surface, thereby increasing the likelihood of infection. Phage head filaments have not been well characterized and their function is described here. Phage head filaments may systematically underlie the initial interactions of phages with their hosts in other systems and possibly represent a widespread mechanism of efficient phage propagation

Expression of the Axonal Membrane Glycoprotein M6a Is Regulated by Chronic Stress

It has been repeatedly shown that chronic stress changes dendrites, spines and modulates expression of synaptic molecules. These effects all may impair information transfer between neurons. The present study shows that chronic stress also regulates expression of M6a, a glycoprotein which is localised in axonal membranes. We have previously demonstrated that M6a is a component of glutamatergic axons. The present data reveal that it is the splice variant M6a-Ib, not M6a-Ia, which is strongly expressed in the brain. Chronic stress in male rats (3 weeks daily restraint) has regional effects: quantitative in situ hybridization demonstrated that M6a-Ib mRNA in dentate gyrus granule neurons and in CA3 pyramidal neurons is downregulated, whereas M6a-Ib mRNA in the medial prefrontal cortex is upregulated by chronic stress. This is the first study showing that expression of an axonal membrane molecule is differentially affected by stress in a region-dependent manner. Therefore, one may speculate that diminished expression of the glycoprotein in the hippocampus leads to altered output in the corresponding cortical projection areas. Enhanced M6a-Ib expression in the medial prefrontal cortex (in areas prelimbic and infralimbic cortex) might be interpreted as a compensatory mechanism in response to changes in axonal projections from the hippocampus. Our findings provide evidence that in addition to alterations in dendrites and spines chronic stress also changes the integrity of axons and may thus impair information transfer even between distant brain regions

Comparison of the pathogenesis of the highly passaged MCMV Smith strain with that of the low passaged MCMV HaNa1 isolate in BALB/c mice upon oronasal inoculation

Murine cytomegalovirus (MCMV) Smith strain is widely used in mouse models to study HCMV infections. Due to high serial passages, MCMV Smith has acquired genetic and biological changes. Therefore, a low passaged strain would be more relevant to develop mouse models. Here, the pathogenesis of an infection with MCMV Smith was compared with that of an infection with a low passaged Belgian MCMV isolate HaNa1 in BALB/c adult mice following oronasal inoculation with either a low (10(4) TCID50/mouse) or high (10(6) TCID50/mouse) inoculation dose. Both strains were mainly replicating in nasal mucosa and submandibular glands for one to two months. In nasal mucosa, MCMV was detected earlier and longer (1-49 days post inoculation (dpi)) and reached higher titers with the high inoculation dose compared to the low inoculation dose (14-35 dpi). In submandibular glands, a similar finding was observed (high dose: 7-49 dpi; low dose: 14-42 dpi). In lungs, both strains showed a restricted replication. In spleen, liver and kidneys, only the Smith strain established a productive infection. The infected cells were identified as olfactory neurons and sustentacular cells in olfactory epithelium, macrophages and dendritic cells in NALT, acinar cells in submandibular glands, and macrophages and epithelial cells in lungs for both strains. Antibody analysis demonstrated for both strains that IgG(2a) was the main detectable antibody subclass. Overall, our results show that significant phenotypic differences exist between the two strains. MCMV HaNa1 has been shown to be interesting for use in mouse models in order to get better insights for HCMV infections in immunocompetent humans