CLEVELAND, Ohio The state did not release the number of new COVID-19 cases early Thursday afternoon as usual due to a technical issue, a spokesperson said.
New weekly case numbers are normally released at 2 p.m.
Last week, the number of new COVID-19 cases in Ohio stayed steady at 7,199, only two cases up from the previous week.
The slight increase ended a five-week run of falling weekly case numbers.
As recently as early January, weekly case numbers hit 15,046.
The total COVID-19 case count since early 2020 in Ohio has reached at least 3,712,548.
Previously: Nov. 16 Ohio COVID-19 update
Feb. 22 recap
* Total reported cases: 3,705,349, up 7,197.
* Total individuals with updated vaccine: 1,275,978, up 11,638.
* Total reported deaths: 43,608, up 91.
* Total reported hospitalizations: 149,643, up 236.
* Total reported ICU admissions: 15,722, up 12.
Julie Washington covers healthcare for cleveland.com. Read previous stories at this link.
MONDAY, Feb. 26, 2024 (HealthDay News) -- The Adaptive COVID-19 Treatment Trial (ACTT) risk profile identifies hospitalized COVID-19 patients who benefit most from baricitinib treatment, according to a study published online Feb. 27 in the Annals of Internal Medicine.
Noting that the ACTT risk profile previously demonstrated that hospitalized patients in the high-risk quartile benefit most from remdesivir, Catharine I. Paules, M.D., from the Penn State Health Milton S. Hershey Medical Center, and colleagues examined potential baricitinib-related treatment effects by risk quartile in a post hoc analysis of the ACTT-2 trial, conducted in 999 adults hospitalized with COVID-19 at 67 trial sites in eight countries. Participants received baricitinib plus remdesivir or placebo plus remdesivir.
The researchers found that baricitinib plus remdesivir was associated with a reduced risk for death, reduced progression to invasive mechanical ventilation or death, and improved recovery rate compared with placebo plus remdesivir in the high-risk quartile (hazard ratios, 0.38, 0.57, and 1.53, respectively). Compared with control participants, those receiving baricitinib plus remdesivir had significantly larger increases in absolute lymphocyte count and significantly larger decreases in absolute neutrophil count after five days, with the largest effects seen in the high-risk quartile.
"To our knowledge, no other clinical trials have assessed clinical benefit from an immunomodulator with relation to dynamics in hematologic parameters, and these data suggest the relevance of these measurements in predicting treatment response," the authors write.
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On a ride to high school one morning, Shelley Marshall asked her daughter how things were going with her field hockey team.
At least, that's what she intended to say. The words came out so garbled that her daughter said, "Mom, what is going on? Are you having a stroke or something? Look at me."
Marshall looked fine. Although slurred speech is a classic stroke symptom, she didn't have a droopy face or arm weakness. In a clear voice, she told her daughter not to worry.
Marshall, though, was concerned.
Two days earlier, she noticed that she'd slurred her own name. Her blood pressure had recently been slightly elevated. And she was still recovering from a serious bout of COVID-19, her third. All of this was unusual for Marshall, then 47 and in excellent health, thanks in part to running nearly every day.
Marshall called her boyfriend, Lyle Sarver, to tell him she was on her way to the emergency room at the hospital in Harrisburg, Pennsylvania, where they both worked in administration.
He met her there. By then, she felt totally fine.
A brain scan revealed otherwise.
The carotid arteries in the neck are major blood vessels for the brain. One of Marshall's was almost completely blocked in two places. She also had a carotid artery dissection, which is a tear of the inner layer of the wall of a carotid artery.
Despite those problems, Marshall's symptoms were still somewhat minimal. Doctors wanted to gather more information via an angiogram, a scan that shows blood flow through vessels.
While waiting for it, the symptoms began to build.
Marshall garbled her speech more often. She noticed she could no longer say certain words, especially "perfectly," which she tried over and over.
She had a headache that kept getting worse and some paralysis on her right side.
By now, her daughter, Kennley McCown, was there. Marshall was in so much pain she feared she would die. Just saying "I love you" to her daughter took all the strength she had.
Sarver feared that Marshall might have lasting deficiencies.
The angiogram was done the next morning. That afternoon, Marshall underwent a procedure to clear the blockages in her carotid artery. Doctors placed three stents to improve blood flow. The surgery was expected to last three hours; it took six because her problems turned out to be more complex.
As soon as her medication wore off, Sarver asked Marshall if she knew who he was and where she was.
"Lyle," she answered. "The hospital."
Kennley also tested her for several days.
"Say 'perfectly,' Mom," she'd ask.
Each time Marshall's pronunciation was perfect.
They all felt better knowing she avoided any major cognitive deficiencies. Since Marshall's stroke in March 2023, her memory is slightly fuzzier, but nothing significant, she said.
While she was in the hospital, doctors made sure she had no other issues. They also sought a reason for her stroke. The lack of other reasons along with the emerging link between COVID-19 and an increased risk of heart attack and stroke led her doctors to believe her severe case of COVID-19 may have contributed to her stroke.
Marshall took two months off work to heal and regain her strength. She and Kennley went on a long-planned trip to the beach in Florida, but only after she got her doctor's clearance to fly and had researched specialists at her destination, just in case.
"I feel tremendously lucky, but I'm also still a little scared, especially about COVID," she said. "I do what I can to prevent it."
Stories From the Heart chronicles the inspiring journeys of heart disease and stroke survivors, caregivers and advocates.
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Children born in October are both more likely to be vaccinated against influenza and least likely to be diagnosed with influenza compared with children born in other months, finds a US study published by The BMJ.
The results suggest that birth month is associated with both timing of flu vaccination and the likelihood of a flu diagnosisand that October is the optimal time for young children to have a flu shot, in line with current recommendations.
Annual influenza vaccination is particularly important for young children, who are at higher risk of flu and severe infection requiring admission to hospital. Vaccination is recommended during September or October to maximize immunity during the peak flu season.
Among young children in the United States, preventive care visits tend to occur during birth months and are a convenient time to receive the influenza vaccine, but large-scale studies of the optimal timing of vaccination are unavailable.
To address this, researchers set out to assess the optimal timing of influenza vaccination in young children.
Using health insurance claims data, they identified over 800,000 children aged 25 years who received an influenza vaccination between 1 August and 31 January during 201118. They then analyzed rates of diagnosed influenza among these children by birth month.
After accounting for a range of potentially influential factors such as age, sex, existing conditions, health care use and family size, the results show that October was the most common month for children to be vaccinated.
Children born in October also had the lowest rate of influenza diagnosis. For example, among children born in August, the average rate of influenza diagnosis across flu seasons studied was 3% compared with 2.7% for children born in October and 2.9% for those born in December.
This is an observational study and the authors acknowledge that their findings are limited to insured children who received medical care. Nor can they rule out the possibility that other unmeasured factors may have influenced their results.
Nevertheless, results were similar after additional analyses to evaluate whether the relation between birth month and influenza risk was due to chance, providing greater confidence in their conclusions.
"Our findings suggest that US public health interventions focused on vaccination of young children in October may yield the best protection in typical flu seasons," they say.
"The study's findings are consistent with current recommendations promoting October vaccination," they add.
More information: Optimal timing of influenza vaccination in young children: population based cohort study, The BMJ (2024). DOI: 10.1136/bmj-2023-077076 www.bmj.com/content/384/bmj-2023-077076
Journal information: British Medical Journal (BMJ)
Moderna (MRNA) shares surged in early trading on Thursday after posting better-than-expected fourth-quarter results. The biotech company reported a surprise profit in the quarter, with CEO Stphane Bancel telling Yahoo Finance Live that "last year was a transition year" for the company.
Oppenheimer & Co. Biotechnology Analyst Hartaj Singh argues that investors should focus less on the Covid-19 business and more on the company's pipeline, given that this should be "a trough year" for Covid-19 vaccine revenues. Singh believes that over the next 6 to 9 months, there will be "a relentless discussion" around the company's pipeline, which he says is "great" given that he thinks "this should be a five-product company in 2026."
Watch the video above to hear why Singh says Moderna is the "kind of stock you want to own."
For more expert insight and the latest market action, click here to watch this full episode of Yahoo Finance Live.
Editor's note: This article was written by Stephanie Mikulich.
- Shares of Moderna up just about 6.5% this morning after reporting better than expected sales when it comes to the estimates that we got here from the street. Now, all of this coming despite the fact that sales for its COVID-19 vaccine pulled back just about 43% from a year ago.
Yahoo Finance spoke with Moderna CEO, Stphane Bancel, about the decline in COVID vaccines. And here's what he had to say.
STPHANE BANCEL: I think we can do much better as a company and also working with public health leaders to increase vaccination rates. Americans need to know, if you are 65 and above, you have five times more chance to get hospitalized because of COVID than because of flu. And as you know, many more people take a flu shot than a COVID shot, so we have to do better there.
- All right, for more on these results, we want to bring in Hartaj Singh, he's Oppenheimer's biotechnology analyst. Hartaj, it's great to see you here. So I'd love to get your reaction to what we just heard from Stphane Bancel and also just putting this in perspective for us. Because over the last couple of quarters, we've talked about the fact, the risk of the decline in the uptake here of COVID-19 vaccines, what that risk or challenge them poses here to Moderna. Are we starting to see that narrative shift just a bit as we get more positive results on RSV? And also some of the excitement surrounding its cancer immunotherapy.
Story continues
HARTAJ SINGH: No, thank you, Seana. And I really appreciate you all having me as always. You know, look, here's the thing, this has been a story around COVID-19 vaccines, the sales, and the underlying trends. When we upgraded earlier this year, we basically said that, look, this is a story that this year will change from COVID-19 vaccine trends and COVID-19 vaccine revenues to basically a pipeline story and with the company being potentially a five product company in 2026.
And I think today what you saw was they reported COVID-19 vaccine revenues that were in line with what we were expecting. This probably should be the trough year. On the call, the company reiterated their guidance for the full year of 2024. And especially I think in January and February, it didn't seem like there was any big change to vaccination trends one way or another. That should give us pause that this should be a trough year for COVID-19 vaccine or COVID vaccine revenues.
The second part to your point that more and more, the conversation is coming up on their pipeline. On conference calls, investor calls, 70%, 80%, 90% of the questions used to be on COVID-19 vaccine revenues. Now, people are talking about flu. Phase two will read out this year. The combination flu and COVID-19. A phase three will read out later this year. CMV cytomegalovirus, this is for pregnant women. That will hopefully read out later this year. And cancer vaccines, rare diseases, those are trials that are initiating.
So you can already see suddenly the change happening where we're going from discussing COVID-19 revenues and vaccination trends relentlessly like Stphane was talking about to now really talking about the pipeline.
- Well, the shares right now down by about 41%, investors waiting for that pipeline story to really ring through to some of the financial results. So where are we in the mRNA platform, messenger RNA as a platform and iterating on top of what they already have to really bring some of these new solutions, treatments to market?
HARTAJ SINGH: Yeah, but so the mRNA platform is a really good question, and it matters in a way. So as a biotech analyst, what does a platform mean to me?
A platform means that the company has an approach with either a type of a molecule. For Regeneron with antibodies. Gilead, infectious diseases. Vertex, they're agnostic but they go after understanding disease pathology. With Moderna, it's the mRNA. And what does that mean? What that means is you should be able to shorten development times, increase the probability of success for your development candidates that follow the first approval, in which in this case, it was the COVID-19 vaccine revenues. Sorry, vaccines.
If you go back to the R&D slides from last year, you'll see that that's exactly what they're doing. As they're developing products in RSV, in flu, in combo vaccines, in cancer vaccines, in rare diseases, they're shortening the development times and they're increasing their probability of success.
So we're already seeing that this is a company that's beating historical estimates. And that's the kind of stock you want to own if you're a kind of a mid to long-term holder because that means that every product will just get on market faster with a higher degree of certainty.
- Right. Cutting down some of the R&D costs as well along the way here. So for investors that are looking around that time horizon, how far out should they be looking?
HARTAJ SINGH: Look, the first six to nine months here is really just going to be all about the pipeline. We're not going to see COVID-19 sales until-- the insights there until about the second quarter call in late July or even the third quarter call in late October.
So really, the discussion about next six to nine months are going to be all centered around the pipeline, which I think is great because people are now going to start getting comfortable with the idea that they should have a flu vaccines approved next year, RSV approved this year, a combination vaccine flu and COVID-19 approved next year. Cytomegalovirus vaccine possibly approved next year. The readout is good later this year.
So what we're going to see now is a relentless discussion next six to nine months around the pipeline and these projects. So we should start seeing it very quickly. And our upgrade stated in January, a couple of months ago, that this should be a five-product company in 2026. And the update today underlined our conviction there.
- Oppenheimer analyst, Hartaj Singh. Hartaj, thanks so much for taking the time here today to break down the Moderna results. Certainly appreciate it.
HARTAJ SINGH: Thank you for having me.
Key points:
Moderna reported a surprise profit in the fourth quarter of 2024, with earnings of 55 cents a share, a decrease from $3.61 a share in the same quarter a year ago. The company's net income exceeded expectations, despite a decline in sales of its Covid-19 vaccine, Spikevax. Revenue for the quarter was $2.81 billion, down from $5.08 billion in the year-ago quarter, but above projections for $2.51 billion.
The company's Covid-19 vaccine netted $800 million in U.S. sales and another $2 billion in international markets. The quarter also included the recognition of $600 million in deferred revenue related to Gavi, the Vaccine Alliance, an international organization created to improve vaccine access for children in poor countries.
Moderna's cost-cutting efforts and deferred payments contributed to the surprise profit. The company also set out a commercial roadmap for its vaccines in Europe and its experimental respiratory syncytial virus (RSV) shot. Moderna expects a U.S. approval decision for its RSV vaccine by May 12 and plans to launch the RSV shot in Germany and Australia this year.
Data posted earlier this month showed the RSV vaccine was 63% effective at preventing RSV-related respiratory symptoms after 8.6 months, down from 84% at 3.3 months. Moderna also expects data from late-stage trials of its next generation COVID shot and its cytomegalovirus and COVID-flu combination vaccines this year.
Despite the decrease in earnings and revenue, Moderna reaffirmed its 2024 forecast of $4 billion in sales, the lowest figure for annual revenue since its COVID vaccine got U.S. emergency authorization in late 2020.
Population and clinical data
Inclusion criteria were: having received at least one dose of a COVID-19 vaccine that was being administered in Sweden during the study period (Comirnaty, Spikevax, Vaxzevria), being diagnosed with a condition or event that was attributed to the vaccine, being 18 years or older at time of recruitment, and having the capability to provide informed consent. Causality assessment for AEFIs were performed according to WHO criteria, as described previously11. The vaccines are detailed as follows: Comirnaty is the proprietary name for the Pfizer-BioNTech mRNA vaccine, also known as BNT162b2. Spikevax is the proprietary name for the Moderna mRNA vaccine, also known as mRNA-1273. Vaxzevria is the proprietary name for the Oxford-AstraZeneca adenoviral-vector vaccine, also known as Covishield, ChAdOx1 nCoV-19, and AZD1222. Anonymous blood donor (BD) samples had been collected for research use before the COVID-19 pandemic at Uppsala University Hospital. APS1 patient samples were collected as part of an ongoing registry (Swedish Addisons Disease Registry; ethics approval number: 2008/296-31/2).
Basic demographic data, elapsed time from vaccination until AEFI onset, and other clinical characteristics were collected from medical records and standardized questionnaires. Patients were classified according to AEFI diagnoses into the following groups: coagulation, neurological, allergic, cardiac, major adverse cardiac events (MACE), cytopenia, systemic disease, infection, vascular, and other. Exact diagnoses included in each group are detailed in Supplementary Table 1.
Venous blood samples of patients with AEFIs (n=290) were collected into EDTA-containing tubes. All samples were centrifuged to obtain plasma (1500g, 10min, 4C), aliquoted, and sent for storage at 70C. De-identified samples were received at or transferred to the Medical Biochemistry and Microbiology Department of Uppsala University for analyses.
The screening of type I IFN autoantibodies was performed via an established bead-based anti-IgG assay that has been used previously with demonstration of reproducible results13. The large multiplex assay analysis plan was created to examine autoantibodies against 96 designated bead IDs (antigens, including technical controls) in a grand total of 2112 samples. Antibodies against IFN- (IFNA1, 2, 4, 5, 6, 7, 8, 10, 14, 16, 17 and 21), IFN- (IFNB), IFN- (IFNE), IFN- (IFNK), and IFN- (IFNW) were measured in the study population for the specific purpose of the presented hypothesis. As part of the large multiplex assay, all samples underwent measurement of anti-human immunoglobulin G and antibodies against the primary proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including Spike (S protein), receptor binding domain (RBD), and nucleocapsid (N protein). Antibody levels against the Epstein-Barr virus nuclear antigen 1 (EBNA1) were also measured to assess the detection reliability and reproducibility of measurements.
Samples (1 ul) were diluted with a 2-step process: 1:25 in phosphate-buffered saline (PBS) and then a further 1:10 in a solution containing 0.05% Tween-20, 3% BSA and 5% non-fat milk in PBS. Magnetic beads (MagPlex, Luminex Corp.) were coupled with commercially-available type I IFNs (and other proteins examined) at a concentration of 3 ug per 1.5 10^6 beads. For coupling, the AnteoTech Activation Kit for Multiplex Microspheres was used (Catalog code: A-LMPAKMM-10). The diluted samples (250 ul total volume) were incubated with 5l of the bead solution for two hours at room temperature with slight agitation achieved by a shaker set to 650rpm. Following the incubation, beads were magnetized before washing with 0.05% Tween-20 in PBS (3X), and then resuspended in 50 microliters of 0.2% paraformaldehyde for 10min. After another 3X wash process, a 30-min incubation with the secondary antibody (Invitrogen, H10104 lot#2384336) was performed. Measurement was carried out with a FlexMap 3D instrument (Luminex Corp) and results were recorded in AUs.
The initial multiplex screening (bead-based assay, Luminex) results were confirmed via optimized ELISA methods for several IFNs that were selected due to the presence of at least one sample with elevated response in the bead-based screening. That is, ELISA re-analysis was performed for a certain IFN if at least one elevated response had been observed in either the AEFI or the BD group for said IFN (defined as >1500 AUs, based on Bastard et al.14). According to this criterion, we performed confirmatory testing for IFNA2 (number of samples with elevated response = 1), IFNA6 (n=7), IFNA8 (n=2), and IFNK (n=1). For each antigen, the highest 8 samples (including those with elevated response) were included in the analyses (total n=32). Starting sample dilution was 1:10 and was increased based on optimization goals described in the Supplementary Methods (1:20, 1:40, 1:80, 1:100, 1:160, 1:320, 1:1000, 1:2000, 1:5000, 1:10000, 1:20000, 1:25000, 1:50000, and 1:100000). In addition to the tested samples, we included three patients with APS1 as positive controls, one sample known to have high cross-reactivity (or non-specific binding), and three known-negative BDs during the course of each ELISA optimization (Supplementary Fig. 1).
The neutralization properties of equivocal responses (n=4) detected in the multiplex autoantibody assay were analyzed via cell culture experiments modified from previously-reported methods23. The experimental design involved (i) cell plating, (ii) co-transfection with Firefly (type I IFN-stimulable) and Renilla luciferase (constitutive expression) genes, (iii) stimulation with IFNA2 & addition of samples, and (iv) detection via a dual luciferase reporter assay. On day 1, HEK293T cells were seeded at 45000 cells/well in a 96-well plate (clear, flat-bottom, cell culture) with a final volume of 90 ul growth media per well (Gibco DMEM GlutaMAX + 10% fetal bovine serum + 100 units of penicillin-streptomycin). Transfection was performed with the Firefly pGL4.45[luc2P/ISRE/Hygro] and Renilla pRL-SV40 internal control luciferase vectors (Promega; #E414A and #E2231, respectively). The transfection solution was created in OptiMEM media with a 3:1 (ul:ug) ratio between the X-tremeGENE9 transfection reagent (Sigma-Aldrich; 6365787001) and total DNA (inter-vector ratio: 2:1 between Firefly and Renilla). The solution was incubated for 15min and added to the wells (10l). On day 2, following overnight incubation, stimulation was performed with a final concentration of 10ng/ml IFNA2 in wells (MedChemExpress; HY-P7022), except for non-stimulation controls. Immediately after stimulation, plasma samples were added into the wells to create a final plasma dilution of 1:10 in media, except for non-plasma controls. The plasma samples tested for neutralization included APS1 samples, AEFI samples with equivocal positivity (n=4), and BD samples. On day 3, following 24h of incubation, the Dual-Luciferase Reporter Assay System (Promega; #E1960) was used for analysis as described by the manufacturer (cell lysis, transfer of lysates to white opaque plates, and measurement with sequential addition of substrate and inhibitory/activating solutions). To perform quantification, we employed a plate reader that had luminescence quantification capabilities with magenta (Firefly) and green (Renilla) filters (Tecan, Magellan). The Firefly:Renilla ratio was used to assess neutralization. Technical controls confirmed experimental success, APS1 samples showed strong neutralization (ratios of <0.050), and BDs showed similar results to non-plasma controlsindicating non-neutralization (Supplementary Fig. 1).
To obtain descriptive data and perform statistical analyses, we utilized the SPSS software (version 25.0; IBM, NY, USA). Continuous data were summarized in the form of meanstandard deviation. Categorical data were summarized with absolute (n) and relative frequency (%). Normality of distribution in continuous variables was checked via evaluation of Q-Q plots or histograms. When required, the lack of normal distribution was confirmed via the Shapiro-Wilk or the Kolmogorov-Smirnov (Lilliefors correction) tests. The Kruskal-Wallis test was used to compare continuous variables among diagnosis subgroups (and the BD group), and post hoc adjustments were performed with the Bonferroni correction. In the comparison of groups formed according to the presence/absence of elevated response (>1500 AUs), analyses for continuous data were performed with the MannWhitney U test and we used appropriate Chi-square tests for categorical data. For data visualization in the form of scatterplots and the heatmap, we respectively used the ggplot2 and pheatmap packages installed on RStudio software (Cherry Blossom release, 2023.03.1-Build 446)24. All code used to analyze data are available upon reasonable request from the corresponding authors. Dimensionality reduction was performed via principal component analysis (PCA) with use of the prcomp and factoextra packages in RStudio. The APS1 group was excluded from PCA. All type I IFN values were standardized with the calculation of Z-scores. Antibody levels for IgG, EBNA1 and SARS-CoV-2-related proteins were not included in the PCA in order to be able to detect the potential effects of IFNs since smaller effects (and subgroups) could have been masked by variables with far greater impact (Supplementary Fig. 3).
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Study design and participants
We conducted a prospective cohort study of adults receiving pandemic COVID-19 vaccine (BNT162b2, Pfizer BioNTech) at Eidsvg general practice and Haukeland University Hospital in Bergen, Norway. All participants received the first two doses of BNT162b2 vaccine at a 3-week interval in JanuaryFebruary 2021, and the third dose of BNT162b2 vaccine 1011 months later in NovemberDecember 2021. No vaccinee had tested rt-PCR positive for SARS-CoV-2 or had any COVID-19 symptom before receiving the 1st dose mRNA vaccine.
The COVID-19 patients were participants of a larger case-ascertained study conducted in Bergen, Norway. All patients tested rt-PCR positive for SARS-CoV-2 from nasopharyngeal swabs during March and April 2020. None of the infected patients received any COVID-19 vaccine within 12 months post diagnosis.
The study was approved by the regional ethics committee (Regional Committee for Medical Research Ethics, Western Norway (REK Vest number 118664) and Northern Norway (REK Nord number 218629)) and is registered in the National Institute for Health database Clinical trials.gov (NCT04706390). All participants provided written informed consent before inclusion in the study.
Electronic case report forms (eCRF) were used to collect demographics, comorbidities, infection history (rt-PCR test results and presence of COVID-19 symptoms), vaccination data and side reactions.
The vaccine used in the study was a monovalent COVID-19 mRNA vaccine BNT162b2 embedded in lipid nanoparticles contained 30g of a purified single-stranded, 5-capped messenger RNA (mRNA), encoding the viral spike protein of SARS-CoV-2 from the founder Wuhan-Hu1 strain (pre-alpha). The vaccine was supplied as a multidose vial reconstituted in sodium chloride 9mg/mL (0.9%) containing 0.45ml per dose, 5 doses per vial, and administered by intramuscular injection.
Serum samples were collected pre-, post 1st (day 21) and 2nd doses (2 months), and pre- (9 months) and post 3rd (12 months) vaccination from all COVID-19 vaccinees, and during the acute (08 days post diagnosis), convalescent phase (1676 days post diagnosis) and 12 months (334387 days post diagnosis) after infection from all COVID-19 patients. Sera were separated, aliquoted and stored at 80C until use.
The hCoV-19/Norway/Bergen-01/2020 (GISAID accession ID EPI_ISL_541970, termed as Bergen-1 hereafter) virus was isolated in-house from an rt-PCR-confirmed patient in March 2020 and propagated in Vero cells in a certified Biosafety Level-3 Laboratory.
The human coronavirus (HCoV) strain NL63 (GenBank: AY567487) was obtained from BEI Resources (Cat. NR-470) and propagated in LLC-MK2 cells (ATCC CCL-7) in biosafety level-2 laboratory. The HCoV strain OC43 (GenBank: AY585228) was obtained from BEI Resources (Cat. NR-52725) and propagated in HCT-8 cells (ATCC CCL-244) in biosafety level-2 laboratory.
The full-length spike proteins from SARS-CoV-2 Wuhan-1 isolate (GenBank: QHD43416), HCoV 229E strain (GenBank: A0G74783), NL63 strain (GenBank: AFV53148), HKU1 strain (UniProtKB/Swiss-Prot: Q0ZME7), and OC43 strain (GenBank: AIL49484) were produced in-house in Expi293F cells (Thermo Fisher Scientific) using the constructs provided by Prof. Barney Graham. The S1 and S2 domains of spike protein from SARS-CoV-2 Wuhan-Hu-1 isolate were obtained commercially (Sino Biological Cat. 40591-V08H and 40590-V08B, respectively).
To quantify the SARS-CoV-2 and HCoV spike specific binding IgG, Maxi Sorp 96-well plates (Thermo Fisher) were coated with in-house prepared full-length spike proteins (Wuhan-Hu-1 spike 0.05g/well; 229E, HKU1 and OC43 spikes 0.1g/well; NL63 spike 0.3g/well) or commercial spike proteins (Wuhan-Hu-1 S1 and S2 domains, 0.05g/well) at 4C overnight. Sera were 5-fold serially diluted from 1:100 and tested in duplicates. Biotin labelled anti-human IgG (1:1000, Sigma-Aldrich Cat. B-1140); horseradish peroxidase (HRP) labelled streptavidin (1:1400, Southern Biotech Cat. 7105-05) were added followed by o-Phenylenediamine dihydrochloride (OPD, 0.05mg/well, Sigma-Aldrich Cat. P-8287). The chromogenic reaction was stopped by sulfuric acid. Optical density (OP) values were read at 490nm using a synergy H1 plate reader (BioTek). Immunoglobulin concentrations were interpolated as binding antibody unit (BAU)/ml from the standard curve with purified human IgG (Sigma-Aldrich Cat. I-4506).
To measure serum neutralizing antibody titres, the microneutralization assay was performed in a certified Biosafety Level 3 Laboratory against the infectious hCoV-19/Norway/Bergen-01/2020 (GISAID accession ID. EPI_ISL_541970). Briefly, sera were heat inactivated at 56C for 60min, analysed in serial dilutions (duplicated, starting from 1:20), and mixed with 100 50% tissue culture infectious doses (TCID50) viruses in 96-well plates (ThermoFisher). After one hour incubation, the sera-virus mixtures were added to Vero cells and further incubated at 37C for 24h. Cells were fixed and permeabilized with methanol (Sigma-Aldrich) and 0.6% H2O2 (Sigma-Aldrich) and incubated with rabbit monoclonal IgG against SARS-CoV-2 NP (1:2000, Sino Biological Cat. 40143-R019). Cells were further incubated with biotinylated goat anti-rabbit IgG (H+L) (1:2500, Southern Biotech Cat. 4050-08), and Streptavidin-HRP (1:1400, Southern Biotech Cat. 7105-05). The reactions were developed with OPD (0.05mg/well, Sigma-Aldrich Cat. P-8287). The neutralizing (IC50) titer was determined as the reciprocal of the serum dilution giving 50% inhibition of virus infectivity. Negative samples were assigned a value of 10 for calculation purpose.
A virus neutralizing assay against the HCoV NL63 strain was developed. Serum samples were heat inactivated and 2-fold serially diluted (starting from 1:10) in DMEM supplemented with 2% heat-inactivated fetal bovine serum, 1% non-essential amino acid (Sigma-Aldrich) and 1.5g/L sodium bicarbonate (NaHCO3), then incubated with 100 TCID50 NL63 virus at 3334C for 60min. The mixture was then added into 96-well plates (ThermoFisher) pre-seeded with LLC-MK2 cells (7000 cells/well). The virus neutralization (VN) endpoint titer against NL63 virus was determined as the highest sera dilution giving 100% inhibition of cytopathic effect on LLC-MK2 cells 7 days after infection. Negative samples were assigned a value of 5 for calculation purpose.
When testing for virus neutralizing antibodies against the HCoV OC43 strain, serum samples were heat inactivated and 2-fold serially diluted (starting from 1:10) in RPMI-1640 supplemented with 2% heat-inactivated horse serum, then incubated with 100 TCID50 OC43 virus at 3334C for 60min. The mixture was then added into 96-well plates (ThermoFisher) pre-seeded with HCT-8 cells (15,000 cells/well). After 13 days incubation, 100l/well supernatant were mixed with 50l human O erythrocytes (0.7% v/v). The VN endpoint titer against OC43 virus was determined as the highest sera dilution giving 100% inhibition of hemagglutination. Negative samples were assigned a value of 5 for calculation purpose.
The spike protein amino acid sequences from HCoVs and SARS-CoV-2 used in ELISA and micro-/virus neutralization assays were obtained from NCBI database. Phylogenetic analyses were performed at ngPhylogeny.fr35 using MAFFT (Multiple Alignment using Fast Fourier Transform, default settings), BMGE (Block Mapping and Gathering with Entropy, default settings), and PhyML (Phylogeny software based on the Maximum-likelihood, default settings).
Biological replicates were used in all experiments. Antibody titers and fold-inductions were Ln transformed prior to all statistical analyses. RM one-way or two-way ANOVA with the Geisser-Greenhouse correction and Turkeys multiple comparisons were performed among time points within the same vaccinee or patient group. To compare adult and elderly vaccinees the unpaired t test was performed at each time point. The one-way ANOVA and Bunnetts multiple comparisons were used to compare between COVID-19 patients and vaccinees at different time points. All statistical analyses were performed with GraphPad Prism 9.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Disruptions in the blood-brain barrier along with a hyperactive immune system are the likely mechanisms behind "brain fog" in patients who are experiencing long COVID, an Irish research team reported today in Nature Neuroscience.
Brain fog has been reported during acute COVID infection and has also been reported in nearly 50% of patients who experience long COVID, or symptoms well past the acute phase of COVID-19.
The blood-brain barrier disruption mechanism was suspected before, but to test the connection, the group first analyzed blood samples to look for any biomarker differences between those who did and didn't report brain fog. They examined blood samples from 76 patients who were hospitalized with acute COVID in early 2020, comparing findings with pre-pandemic samples from 25 other patients to look for any differences in coagulation patterns and immune response.
Those who reported brain fog had higher levels of a protein (S100) produced by brain cells not normally found in the blood, which hinted at a "leaky" blood-brain barrier.
For the second part of the study, the researchers conducted brain scans using dynamic contrast-enhanced MRI to examine brain circulation in 11 people who had recovered from COVID and 22 who had long COVID, which included 11 people who reported brain fog. They found that long-COVID patients with brain fog had a leaky blood-brain barrier when compared to other long COVID patients and to others who had recovered.
The group's experiments also found that long-COVID patients with brain fog had increased levels of clotting markers in their blood.
Matthew Campbell, PhD, one of the study coauthors, said in a Trinity College Dublin press release that the findings show for the first time that leaky vessels in the brain along with a hyperactive immune system may be the key drivers of brain fog in people experiencing long COVID.
"This is critically important, as understanding the underlying cause of these conditions will allow us to develop targeted therapies for patients in the future," he said. Campbell is a genetics professor at Trinity College.
The concept that many other viral infections that lead to post-viral syndromes might drive blood vessel leakage in the brain is potentially game changing.
The findings will likely change the understanding and treatment of post-viral neurologic conditions, said Colin Doherty, MD, a study coauthor who is a neurology professor and head of the school of medicine at Trinity College. "It also confirms that the neurological symptoms of Long Covid are measurable with real and demonstrable metabolic and vascular changes in the brain," he added.
Campbell concluded, "The concept that many other viral infections that lead to post-viral syndromes might drive blood vessel leakage in the brain is potentially game changing and is under active investigation by the team."
Two new, but exceptionally rare COVID-19 vaccine side effects, have been found in the largest vaccine safety study ever recorded. Researchers found an uncommon link between recipients of the vaccine and a neurological disorder caused by swelling in the brain and spinal cord. Still, researchers say the benefits of receiving a COVID-19 vaccine far outweigh the risks. Katherine Ward has this story and more in Health Matters for Feb. 23, 2024.
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