Single Prime hAd5 Spike (S) + Nucleocapsid (N) Dual Antigen Vaccination of Healthy Volunteers Induces a Ten-Fold Increase in Mean S- and N- T-Cell Responses Equivalent to T-Cell Responses from Patients Previously Infected with SARS-CoV-2

In response to the need for a safe, efficacious vaccine that elicits vigorous T cell as well as humoral protection against SARS-CoV-2 infection, we have developed a dual-antigen COVID-19 vaccine comprising both the viral spike (S) protein modified to increase cell-surface expression (S-Fusion) and nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to enhance MHC class I and II presentation and T-cell responses. The antigens are delivered using a human adenovirus serotype 5 (hAd5) platform with E1, E2b, and E3 regions deleted that has been shown previously in cancer vaccine studies to be safe and effective in the presence of pre-existing hAd5 immunity. The findings reported here are focused on human T-cell responses due to the likelihood that such responses will sustain efficacy against emerging variants, a hypothesis supported by our in silico prediction of T-cell epitope HLA binding for both the first-wave SARS-CoV-2 A strain and the B.1.351 strain K417N, E484K, and N501Y spike and T201I N variants. We demonstrate the hAd5 S-Fusion + N-ETSD vaccine antigens expressed by previously SARS-CoV-2-infected patient dendritic cells elicit Th1 dominant activation of autologous patient T cells, indicating the vaccine antigens have the potential to elicit immune responses in previously infected patients. For participants in our open-label Phase 1b study of the vaccine (NCT04591717; https://clinicaltrials.gov/ct2/show/NCT04591717), the magnitude of Th-1 dominant S- and N-specific T-cell responses after a single prime subcutaneous injection were comparable to T-cell responses from previously infected patients. Furthermore, vaccinated participant T-cell responses to S were similar for A strain S and a series of spike variant peptides, including S variants in the B.1.1.7 and B.1.351 strains. The findings that this dual-antigen vaccine elicits SARS-CoV-2-relevant T-cell responses and that such cell-mediated protection is likely to be sustained against emerging variants supports the testing of this vaccine as a universal booster that would enhance and broaden existing immune protection conferred by currently approved S-based vaccines.

An apical MRCK-driven morphogenetic pathway controls epithelial polarity.

Polarized epithelia develop distinct cell surface domains, with the apical membrane acquiring characteristic morphological features such as microvilli. Cell polarization is driven by polarity determinants including the evolutionarily conserved partitioning-defective (PAR) proteins that are separated into distinct cortical domains. PAR protein segregation is thought to be a consequence of asymmetric actomyosin contractions. The mechanism of activation of apically polarized actomyosin contractility is unknown. Here we show that the Cdc42 effector MRCK activates myosin-II at the apical pole to segregate aPKC-Par6 from junctional Par3, defining the apical domain. Apically polarized MRCK-activated actomyosin contractility is reinforced by cooperation with aPKC-Par6 downregulating antagonistic RhoA-driven junctional actomyosin contractility, and drives polarization of cytosolic brush border determinants and apical morphogenesis. MRCK-activated polarized actomyosin contractility is required for apical differentiation and morphogenesis in vertebrate epithelia and Drosophila photoreceptors. Our results identify an apical origin of actomyosin-driven morphogenesis that couples cytoskeletal reorganization to PAR polarity signalling.

Anticholinergic burden in older women: not seeing the wood for the trees?

OBJECTIVES: To identify medicines contributing to and describe predictors of anticholinergic burden among community-dwelling older Australian women. DESIGN, SETTING AND PARTICIPANTS: Retrospective longitudinal analysis of data from the Australian Longitudinal Study on Women's Health linked to Pharmaceutical Benefits Scheme medicines data from 1 January 2008 to 30 December 2010; for 3694 women born in 1921-1926. MAIN OUTCOME MEASURES: Anticholinergic burden calculated from Anticholinergic Drug Scale (ADS) scores derived from ADS levels (0 to 3) for all medicines used by each woman, summed over each 6-month period (semester), medicines commonly used by women with high semester ADS scores (defined as 75th percentile of scores). RESULTS: 1126 women (59.9%) used at least one medicine with anticholinergic properties. The median ADS score was 4 or 5 across all semesters. Most anticholinergic medicines used by women who had a high anticholinergic burden (ADS score, ≥ 9) had a low anticholinergic potency (ADS level 1). Increasing age, cardiovascular disease, and number of other medicines used were predictive of a higher anticholinergic burden. CONCLUSIONS: A high anticholinergic medicines burden in this group was driven by the use of multiple medicines with lower anticholinergic potency rather than the use of medicines with higher potency. This is a novel and important finding for clinical practice as doctors would readily identify the risk of a high anticholinergic burden for patients using high potency medicines, but may be less likely to identify this risk for users of multiple medicines with low anticholinergic potency.

Silencing the Metallothionein-2A gene induces entosis in adherent MCF-7 breast cancer cells.

The presence of a live cell cohabiting within another cell has fascinated scientists for many decades. Far from being a spurious event, many have attempted to uncover the molecular mechanism underlying this phenomenon. In this study, we observed anchorage-dependent MCF-7 cells internalizing neighboring epithelial cells (entosis) after siRNA-mediated silencing of the Metallothionein-2A (MT-2A) gene. MTs belong to a family of low-molecular weight proteins, which bind metal ions endogenously and its over-expression has been reported in a variety of cancers that include breast, prostate, and colon. We provide microscopic evidence at light and ultrastructural levels of the occurrence of entosis after altering MT expression in a subpopulation of MCF-7 breast cancer cells by silencing the MT-2A gene. Our results demonstrate that adheren junctions may play important roles in the formation of cell-in-cell cytostructure after MT-2A gene downregulation and the entotic process does not appear to involve genes associated with autophagy. Interiorized cells often underwent lysosomal degradation within the cytoplasmic body of the engulfing cell. It would appear that a subset of breast cancer cells could die via entosis after MT-2A gene silencing.