Delta and Omicron: protective measures and SARS-CoV-2 infections in day care centres in Germany in the 4th and 5th wave of the pandemic 2021/2022

Background During the five waves of the SARS-CoV-2 pandemic so far, German early childhood education and care (ECEC) centres implemented various protective measures, such as wearing a face mask, fixed children-staff groups or regular ventilation. In addition, parents and ECEC staff were increasingly vaccinated throughout 2021. During the 4th wave, variant of concern (VOC) Delta-driven transmission indicators reached record values at the end of 2021. Those values were even exceeded in the 5th wave at the beginning of 2022 when Omicron dominated. We examine which factors facilitated or prevented infection with SARS-CoV-2 in ECEC centres, and if these differed between different phases within wave 4 (Delta) and 5 (Omicron). Methods Since August 2020, a weekly online survey among approximately 8000 ECEC managers has been conducted, monitoring both incident SARS-CoV-2 infections and protective measures taken. We included data from calendar week 26/2021 to 05/2022. We estimate the probability of any infections and the number of SARS-CoV-2 infections in children, parents and staff using random-effect-within-between (REWB) panel models for binomial and count data. Results While children, parents and staff of ECEC centres with a high proportion of children from families with low socioeconomic status (SES) have a higher risk of infections in the beginning of wave 4 (OR up to 1.99 [1.56; 2.56]), this effect diminishes for children and parents with rising incidences. Protective measures, such as wearing face masks, tend to have more extensive effects with rising incidences in wave 5 (IRR up to 0.87 [0.8; 0.93]). Further, the protective effect of vaccination against infection among staff is decreasing from wave 4 to wave 5 (OR 0.3 [0.16; 0.55] to OR 0.95, [0.84; 1.07, n.s.]). The degree of transmission from staff to child and from staff to parent is decreasing from wave 4 to wave 5, while transmission from child to staff seems to increase. Conclusion While Omicron seems to affect children and parents from ECEC centres with families with all SES levels more equally than Delta, the protective effect of vaccination against infection is decreasing and the effect of protective measures like face masks becomes increasingly important. In order to prevent massive closures of ECEC centres due to infection of staff, protective measures should be strictly adhered to, especially to protect staff in centres with a high proportion of children from families with low socioeconomic status.Peer Reviewe

Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

BACKGROUND: The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. RESULTS: Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. CONCLUSION: Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo

HIV-1 Nef Employs Two Distinct Mechanisms to Modulate Lck Subcellular Localization and TCR Induced Actin Remodeling

The Nef protein acts as critical factor during HIV pathogenesis by increasing HIV replication in vivo via the modulation of host cell vesicle transport and signal transduction processes. Recent studies suggested that Nef alters formation and function of immunological synapses (IS), thereby modulating exogenous T-cell receptor (TCR) stimulation to balance between partial T cell activation required for HIV-1 spread and prevention of activation induced cell death. Alterations of IS function by Nef include interference with cell spreading and actin polymerization upon TCR engagement, a pronounced intracellular accumulation of the Src kinase Lck and its reduced IS recruitment. Here we use a combination of Nef mutagenesis and pharmacological inhibition to analyze the relative contribution of these effects to Nef mediated alterations of IS organization and function on TCR stimulatory surfaces. Inhibition of actin polymerization and IS recruitment of Lck were governed by identical Nef determinants and correlated well with Nef's association with Pak2 kinase activity. In contrast, Nef mediated Lck endosomal accumulation was separable from these effects, occurred independently of Pak2, required integrity of the microtubule rather than the actin filament system and thus represents a distinct Nef activity. Finally, reduction of TCR signal transmission by Nef was linked to altered actin remodeling and Lck IS recruitment but did not require endosomal Lck rerouting. Thus, Nef affects IS function via multiple independent mechanisms to optimize virus replication in the infected host

Intracellular Lck accumulation is a conserved activity of lentiviral Nef proteins.

<p>(A) Confocal microscopy analysis of Jurkat T lymphocytes transfected with the indicated expression plasmids after staining for endogenous Lck. Depicted is a representative confocal section through the middle of the cell. Asterisks denote GFP positive cells. White bar = 10 µM. (B) Quantification of the experiment shown in A. Values are the arithmetic means of at least three independent experiments+s.d. in which over 100 cells were counted per condition.</p

Sustained Nef induced Lck accumulation requires microtubule integrity.

<p>(A) Confocal microscopy analysis of Jurkat T lymphocytes transfected with GFP or Nef.GFP expression plasmids and stained for endogenous Lck after 30min incubation with the indicated drugs (CytD 5 µM; LatB 0.5 µM; Lck Inh. 3 µM; Noco. 10 µM; control, solvent control). Depicted is a representative confocal section through the middle of the cell. White bar = 10 µM. (B) Quantification of the experiment shown in A. Values are the arithmetic means of at least three independent experiments+s.d. in which over 100 cells were counted per condition.</p

Intracellular Lck accumulation requires membrane attachment and the SH3 binding motif of Nef.

<p>(A) Confocal microscopy analysis of Jurkat T lymphocytes transfected with the indicated expression plasmids after staining for endogenous Lck. Depicted is a representative confocal section through the middle of the cell. White bar = 10 µM. (B) Quantification of the experiment shown in A. Values are the arithmetic means of at least three independent experiments+s.d. in which over 100 cells were counted per condition.</p

Inhibition of TCR induced actin remodeling depends on the protein interaction motif surrounding F195 in Nef.

<p>(A) Confocal microscopy analysis of Jurkat T lymphocytes transfected with the indicated expression plasmids after 5min incubation on anti-CD3 coated coverglasses and subsequent staining for F-actin. Depicted is a representative confocal section near the coverglass. White bar = 10 µM. (B) Quantification of the experiment shown in A. Shown is the percentage of cells exhibiting pronounced actin ring formation relative to the mean value of GFP expressing control cells that was arbitrarily set to 100%. Values are the arithmetic means of at least three independent experiments+s.d. in which over 100 cells were counted per condition.</p

Correlation between Nef mediated inhibition of TCR induced actin dynamics and tyrosine phosphorylation.

<p>Jurkat T cells expressing GFP or the indicated Nef.GFP proteins were incubated on TCR stimulatory surfaces, fixed and analyzed for the subcellular distribution of p-Tyr positive signaling complexes by confocal microscopy. (A) Distribution of p-Tyr and GFP 5min after TCR stimulation. Results depict images representative for at least three independent experiments. White bar = 10 µM. (B) Quantification of overall p-Tyr levels of the transfected cells shown in A. Values represent the mean of three independent experiments+s.e.m. from at least 10 cells representing the phenotypes depicted in (A).</p