Recombinant HIV envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex

Broadly neutralizing antibodies (bnAbs) targeting the trimer apex of HIV envelope are favored candidates for vaccine design and immunotherapy because of their great neutralization breadth and potency. However, methods of isolating bnAbs against this site have been limited by the quaternary nature of the epitope region. Here we report the use of a recombinant HIV envelope trimer, BG505 SOSIP.664 gp140, as an affinity reagent to isolate quaternary-dependent bnAbs from the peripheral blood mononuclear cells of a chronically infected donor. The newly isolated bnAbs, named “PGDM1400–1412,” show a wide range of neutralization breadth and potency. One of these variants, PGDM1400, is exceptionally broad and potent with cross-clade neutralization coverage of 83% at a median IC₅₀ of 0.003 µg/mL. Overall, our results highlight the utility of BG505 SOSIP.664 gp140 as a tool for the isolation of quaternary-dependent antibodies and reveal a mosaic of antibody responses against the trimer apex within a clonal family.Multiple methods have been developed to isolate HIV broadly neutralizing antibodies (bnAbs) (1–12). Hybridoma and phage display techniques were used to isolate the first generation of bnAbs including b12, 2F5, 2G12, 4E10, and Z13 (13–20). These antibodies exhibit a range of neutralization breadth against primary isolates from 30 to 90% but have moderate neutralization potency (median IC₅₀ of ∼2–4 µg/mL). Access to infected donors who have high serum titers of bnAbs (21, 22) and the availability of newer approaches for isolating human mAbs have recently enabled the discovery of a new generation of more potent bnAbs (1–4, 6–8).One of the newer approaches involves the sorting and activation of large numbers of memory B cells using cytokine-secreting feeder cells and the subsequent high-throughput screening of supernatants for neutralization. This method led to the identification and characterization of the first of the new generation of bnAbs, PG9 and PG16 (1), and since then has revealed several sites of vulnerability to bnAb recognition on HIV envelope (Env) (1–4, 6, 7). An alternative method of bnAb isolation involves the use of soluble Env molecules or scaffold proteins as baits to select single IgG⁺ memory B cells of interest by cell sorting (6, 8, 9, 23, 24). However, soluble baits have not been successful in isolating antibody responses targeting quaternary epitopes, including the trimer-apex site surrounding the N160 glycan, because the protein constructs used to date have not properly mimicked native Env trimers. To address this problem, GFP-labeled 293T cells that express cell-surface Env, called “GFP-293T^BaL cells,” were used recently to isolate antibodies 3BC176 and 3BC315 (10, 25). These antibodies do not bind soluble monomeric gp120 but do bind Env trimer, demonstrating the utility of the approach, but the method was reported to be less efficient than the use of soluble protein baits (10, 25).The favorable antigenic profile of the soluble BG505 SOSIP.664 gp140 trimer opens the possibility of its use for isolating quaternary-specific antibodies by single-cell sorting (26). To this end, we used BG505 SOSIP.664 gp140 to select for memory B cells from a donor from whom we previously had isolated the trimer-specific bnAbs PGT141–145 (3, 21). (For naming of PGT and PGDM bnAbs, please see SI Materials and Methods, Antibody Nomenclature.) We describe the isolation of previously unidentified somatic variants that are highly divergent from PGT145 and display a range of neutralization breadth and potency, with some being broader and more potent than the previously described PGT145 family members. Overall, the results reveal a mosaic of antibody responses against the trimer-apex site of vulnerability that have important implications for immunogen design in general and for the future optimization of BG505 SOSIP.664 and related native-like trimers as vaccine candidates.     

Spatiotemporal control of epithelial remodeling by regulated myosin phosphorylation

Spatiotemporally regulated actomyosin contractility generates the forces that drive epithelial cell rearrangements and tissue remodeling. Phosphorylation of the myosin II regulatory light chain (RLC) promotes the assembly of myosin monomers into active contractile filaments and is an essential mechanism regulating the level of myosin activity. However, the effects of phosphorylation on myosin localization, dynamics, and function during epithelial remodeling are not well understood. In Drosophila, planar polarized myosin contractility is required for oriented cell rearrangements during elongation of the body axis. We show that regulated myosin phosphorylation influences spatial and temporal properties of contractile behavior at molecular, cellular, and tissue length scales. Expression of myosin RLC variants that prevent or mimic phosphorylation both disrupt axis elongation, but have distinct effects at the molecular and cellular levels. Unphosphorylatable RLC produces fewer, slower cell rearrangements, whereas phosphomimetic RLC accelerates rearrangement and promotes higher-order cell interactions. Quantitative live imaging and biophysical approaches reveal that both phosphovariants reduce myosin planar polarity and mechanical anisotropy, altering the orientation of cell rearrangements during axis elongation. Moreover, the localized myosin activator Rho-kinase is required for spatially regulated myosin activity, even when the requirement for phosphorylation is bypassed by the expression of phosphomimetic myosin RLC. These results indicate that myosin phosphorylation influences both the level and the spatiotemporal regulation of myosin activity, linking molecular properties of myosin activity to tissue morphogenesis.Contractile assemblies of actin filaments and the nonmuscle myosin II motor protein produce mechanical forces that generate changes in cell shape during cytokinesis, cell movements during cell migration, and the dynamic remodeling of multicellular tissues (1–3). During development, spatiotemporal patterns of actomyosin contractility remodel simple epithelia into functional tissues with complex form and structure. Contractile force generation requires the assembly of inactive myosin monomers into active bipolar filaments (4). Phosphorylation of the myosin II regulatory light chain promotes myosin filament assembly and the movement of the myosin motor along actin filaments in vitro (5, 6) and is necessary for cytokinesis (7–9), oogenesis (7, 10, 11), and tissue morphogenesis (12–15). However, the in vivo effects of regulatory light chain phosphorylation on myosin localization, dynamics, and force generation, and how these properties are integrated to achieve complex changes in the shapes of tissues, are not well understood.Epithelial elongation in the Drosophila embryo is driven by cell rearrangements that are coordinated by spatiotemporal patterns of actomyosin contractility. Myosin contractility is spatially regulated in the plane of the epithelium (termed planar polarity), driving the planar polarized contraction of cell interfaces oriented close to perpendicular to the anterior–posterior axis (AP edges) (16–20). New interfaces preferentially form between dorsal and ventral cells (DV edges), resulting in oriented cell rearrangements that rapidly elongate the body axis of the animal from head to tail (16–18, 21). The myosin II activator Rho-associated protein kinase (Rho-kinase) localizes to regions of cells that display strong actomyosin activity during development (14, 15, 22) and promotes myosin contractility by phosphorylating the myosin regulatory light chain (RLC) and inactivating myosin phosphatase (23). Rho-kinase is required for localized myosin contractility during Drosophila axis elongation (14), neural tube closure in chick (22), and in other epithelia that undergo planar polarized cell behaviors (24, 25). Localized phosphorylation by Rho-kinase could therefore serve as an instructive cue that determines where and when myosin is activated within the cell. However, constitutively active myosin variants that evade this upstream regulation are sufficient to support normal myosin function during cytokinesis (26, 27), oogenesis (7), and wing hair formation (12), indicating that in some contexts regulated myosin phosphorylation is dispensable for morphogenesis. This raises the alternative possibility that the primary requirement for phosphorylation is to activate myosin, and additional mechanisms regulate the spatiotemporal patterns of myosin localization within epithelia.Here we use time-lapse imaging and biophysical approaches to analyze the role of myosin phosphorylation in polarized cell rearrangements during Drosophila axis elongation. Myosin II regulatory light chain variants that prevent or mimic phosphorylation both disrupt axis elongation, but have distinct effects on myosin localization, dynamics, and cell behavior. These results indicate that regulated myosin phosphorylation provides an instructive cue that directs both the magnitude and spatiotemporal pattern of myosin activity during epithelial remodeling, linking molecular-scale myosin activity to cell behavior and tissue morphogenesis.     

Dysfunctional breathing treated with continuous positive airway pressure in newly diagnosed obstructive sleep apnoea: a prospective cohort study

A prospective cohort study investigating patients with obstructive sleep apnoea (OSA) was conducted to determine the prevalence of dysfunctional breathing and if continuous positive airway pressure (CPAP) therapy improves associated symptoms. Almost half of newly diagnosed patients with OSA had dysfunctional breathing and CPAP was not an effective treatment. Dysfunctional breathing is common in patients with OSA.