Mapping the SARS-CoV-2 spike glycoprotein-derived peptidome presented by HLA class II on dendritic cells

Understanding and eliciting protective immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an urgent priority. To facilitate these objectives, we have profiled the repertoire of human leukocyte antigen class II (HLA-II)-bound peptides presented by HLA-DR diverse monocyte-derived dendritic cells pulsed with SARS-CoV-2 spike (S) protein. We identify 209 unique HLA-II-bound peptide sequences, many forming nested sets, which map to sites throughout S including glycosylated regions. Comparison of the glycosylation profile of the S protein to that of the HLA-II-bound S peptides revealed substantial trimming of glycan residues on the latter, likely introduced during antigen processing. Our data also highlight the receptor-binding motif in S1 as a HLA-DR-binding peptide-rich region. Results from this study have application in vaccine design, and will aid analysis of CD4+ T cell responses in infected individuals and vaccine recipients.

Organisms causing secondary pneumonias in COVID-19 patients at 5 UK ICUs as detected with the FilmArray test

IntroductionSeveral viral respiratory infections - notably influenza - are associated with secondary bacterial infection and additional pathology. The extent to which this applies for COVID-19 is unknown. Accordingly, we aimed to define the bacteria causing secondary pneumonias in COVID-19 ICU patients using the FilmArray Pneumonia Panel, and to determine this tests potential in COVID-19 management. MethodsCOVID-19 ICU patients with clinically-suspected secondary infection at 5 UK hospitals were tested with the FilmArray at point of care. We collected patient demographic data and compared FilmArray results with routine culture. ResultsWe report results of 110 FilmArray tests on 94 patients (16 had 2 tests): 69 patients (73%) were male, the median age was 59 yrs; 92 were ventilated. Median hospital stay before testing was 14 days (range 1-38). Fifty-nine (54%) tests were positive, with 141 bacteria detected. Most were Enterobacterales (n=55, including Klebsiella spp. [n= 35]) or Staphylococcus aureus (n=13), as is typical of hospital and ventilator pneumonia. Community pathogens, including Haemophilus influenzae (n=8) and Streptococcus pneumoniae (n=1), were rarer. FilmArray detected one additional virus (Rhinovirus/Enterovirus) and no atypical bacteria. Fewer samples (28 % vs. 54%) were positive by routine culture, and fewer species were reported per sample; Klebsiella species remained the most prevalent pathogens. ConclusionFilmArray had a higher diagnostic yield than culture for ICU COVID-19 patients with suspected secondary pneumonias. The bacteria found mostly were Enterobacterales, S. aureus and P. aeruginosa, as in typical HAP/VAP, but with Klebsiella spp. more prominent. We found almost no viral co-infection. Turnaround from sample to results is around 1h 15 min compared with the usual 72h for culture, giving prescribers earlier data to inform antimicrobial decisions.

Calreticulin in cardiac development and pathology.

Calreticulin is a Ca(2+) binding/storage chaperone resident in the lumen of endoplasmic reticulum (ER). The protein is an important component of the calreticulin/calnexin cycle and the quality control pathways in the ER. In mice, calreticulin deficiency is lethal due to impaired cardiac development. This is not surprising because the protein is expressed at high level at early stages of cardiac development. Overexpression of the protein in developing and postnatal heart leads to bradycardia, complete heart block and sudden death. Recent studies on calreticulin-deficient and transgenic mice revealed that the protein is a key upstream regulator of calcineurin-dependent pathways during cardiac development. Calreticulin and ER may play important role in cardiac development and postnatal pathologies.

Ca2+ signaling and calcium binding chaperones of the endoplasmic reticulum.

The endoplasmic reticulum is a centrally located organelle which affects virtually every cellular function. Its unique luminal environment consists of Ca(2+) binding chaperones, which are involved in protein folding, post-translational modification, Ca(2+) storage and release, and lipid synthesis and metabolism. The environment within the lumen of the endoplasmic reticulum has profound effects on endoplasmic reticulum function and signaling, including apoptosis, stress responses, organogenesis, and transcriptional activity. Calreticulin, a major Ca(2+) binding (storage) chaperone in the endoplasmic reticulum, is a key component of the calreticulin/calnexin cycle which is responsible for the folding of newly synthesized proteins and glycoproteins and for quality control pathways in the endoplasmic reticulum. The function of calreticulin, calnexin and other endoplasmic reticulum proteins is affected by continuous fluctuations in the concentration of Ca(2+) in the endoplasmic reticulum. Thus, changes in Ca(2+) concentration may play a signaling role in the lumen of the endoplasmic reticulum as well as in the cytosol. Recent studies on calreticulin-deficient and transgenic mice have revealed that calreticulin and the endoplasmic reticulum may be upstream regulators in the Ca(2+)-dependent pathways that control cellular differentiation and/or organ development.