Vaccine-induced correlate of protection against fatal COVID-19 in older and frail adults during waves of neutralization-resistant variants of concern: an observational study.

Background: To inform future preventive measures including repeated vaccinations, we have searched for a clinically useful immune correlate of protection against fatal COVID-19 among nursing homes residents. Methods: We performed repeated capillary blood sampling with analysis of S-binding IgG in an open cohort of nursing home residents in Sweden. We analyzed immunological and registry data from 16 September 2021 to 31 August 2022 with follow-up of deaths to 30 September 2022. The study period included implementation of the 3rd and 4th mRNA monovalent vaccine doses and Omicron virus waves. Findings: A total of 3012 nursing home residents with median age 86 were enrolled. The 3rd mRNA dose elicited a 99-fold relative increase of S-binding IgG in blood and corresponding increase of neutralizing antibodies. The 4th mRNA vaccine dose boosted levels 3.8-fold. Half-life of S-binding IgG was 72 days. A total 528 residents acquired their first SARS-CoV-2 infection after the 3rd or the 4th vaccine dose and the associated 30-day mortality was 9.1%. We found no indication that levels of vaccine-induced antibodies protected against infection with Omicron VOCs. In contrast, the risk of death was inversely correlated to levels of S-directed IgG below the 20th percentile. The death risk plateaued at population average above the lower 35th percentile of S-binding IgG. Interpretation: In the absence of neutralizing antibodies that protect from infection, quantification of S-binding IgG post vaccination may be useful to identify the most vulnerable for fatal COVID-19 among the oldest and frailest. This information is of importance for future strategies to protect vulnerable populations against neutralization resistant variants of concern. Funding: Swedish Research Council, SciLifeLab via Knut and Alice Wallenberg Foundation, VINNOVA. Swedish Healthcare Regions, and Erling Persson Foundation.

Tissue factor activity of small and large extracellular vesicles in different diseases

Background Circulating procoagulant extracellular vesicles (EVs) are increased in diseases, such as cancer, sepsis and coronavirus 2019 (COVID-19). EV tissue factor (TF) activity is associated with disseminated intravascular coagulation in sepsis and venous thrombosis in patients with pancreatic cancer and COVID-19. EVs are commonly isolated by centrifugation at ∼20,000 g. In this study, we analyzed TF activity of two EV populations enriched for large and small EVs in patients with either sepsis, pancreatic cancer or COVID-19. Methods EVs were isolated from plasma by sequential centrifugation at 20,000 g (large EVs, LEVs) and then 100,000 g (small EVs, SEVs). We analyzed EVs from plasma prepared from whole blood samples from healthy individuals with or without lipopolysaccharide (LPS) stimulation as well as EVs from plasma samples from patients with either sepsis, pancreatic cancer or COVID-19. TF-dependent (EV-TF activity) and TF-independent factor Xa (FXa) generation of the EVs was measured. Results LPS increased EV-TF activity in LEVs but not SEVs. Similarly, in two patients with sepsis that had EV-TF activity above the background of the assay we observed EV-TF activity in LEVs but not SEVs. Patients with pancreatic cancer or COVID-19 had circulating EV-TF activity in both LEVs and SEVs. Conclusion We recommend that EVs are isolated from plasma from patients by centrifugation at 100,000 g rather than 20,000 g to obtain a more accurate measure of levels of circulating EV-TF activity.

Tissue factor activity of small and large extracellular vesicles in different diseases.

Background: Circulating procoagulant extracellular vesicles (EVs) are increased in diseases, such as cancer, sepsis, and COVID-19. EV tissue factor (TF) activity is associated with disseminated intravascular coagulation in sepsis and venous thrombosis in patients with pancreatic cancer and COVID-19. EVs are commonly isolated by centrifugation at ∼20,000 g. Objectives: In this study, we analyzed the TF activity of 2 EV populations enriched for large and small EVs in patients with either sepsis, pancreatic cancer, or COVID-19. Methods: EVs were isolated from plasma by sequential centrifugation at 20,000 g (large EVs, LEVs) and then 100,000 g (small EVs, SEVs). We analyzed EVs from plasma prepared from whole blood samples from healthy individuals with or without lipopolysaccharide (LPS) stimulation as well as EVs from plasma samples from patients with either sepsis, pancreatic cancer, or COVID-19. TF-dependent (EV-TF activity) and TF-independent factor Xa (FXa) generation of the EVs was measured. Results: LPS increased EV-TF activity in LEVs but not SEVs. Similarly, in 2 patients with sepsis who had EV-TF activity above the background of the assay we observed EV-TF activity in LEVs but not SEVs. Patients with pancreatic cancer or COVID-19 had circulating EV-TF activity in both LEVs and SEVs. Conclusion: We recommend that EVs are isolated from plasma from patients by centrifugation at 100,000 g rather than 20,000 g to obtain a more accurate measure of levels of circulating EV-TF activity.

Correlates of protection and viral load trajectories in omicron breakthrough infections in triple vaccinated healthcare workers.

Vaccination offers protection against severe COVID-19 caused by SARS-CoV-2 omicron but is less effective against infection. Characteristics such as serum antibody titer correlation to protection, viral abundance and clearance of omicron infection in vaccinated individuals are scarce. We present a 4-week twice-weekly SARS-CoV-2 qPCR screening in 368 triple vaccinated healthcare workers. Spike-specific IgG levels, neutralization titers and mucosal spike-specific IgA-levels were determined at study start and qPCR-positive participants were sampled repeatedly for two weeks. 81 (cumulative incidence 22%) BA.1, BA.1.1 and BA.2 infections were detected. High serum antibody titers are shown to be protective against infection (p < 0.01), linked to reduced viral load (p < 0.01) and time to viral clearance (p < 0.05). Pre-omicron SARS-CoV-2 infection is independently associated to increased protection against omicron, largely mediated by mucosal spike specific IgA responses (nested models lr test p = 0.02 and 0.008). Only 10% of infected participants remain asymptomatic through the course of their infection. We demonstrate that high levels of vaccine-induced spike-specific WT antibodies are linked to increased protection against infection and to reduced viral load if infected, and suggest that the additional protection offered by pre-omicron SARS-CoV-2 infection largely is mediated by mucosal spike-specific IgA.

Impact of monocyte-related modulators and kidney function on mortality in hospitalized patients with COVID-19.

Patients with chronic kidney disease (CKD) are at high risk of severe complications from COVID-19 and functional monocyte disturbances have been implicated to play a role. Our objective was to analyse the association between kidney function and monocyte modulatory factors, with risk of mortality in patients with COVID-19. Hospitalized patients with COVID-19 (n = 110) were included and in-hospital mortality was analysed with unadjusted and adjusted multiple logistic regression analysis. Plasma levels of monocyte chemoattractant factors (MIP-1α, MCP-1, IL-6) and a monocyte immune modulator (sCD14) were analysed and correlated to kidney function and risk of mortality. Monocyte modulatory factors were also determined in CKD patients without infection (disease controls) and in healthy subjects. Patients who died in hospital were more often in CKD stages 3-5, with lower estimated glomerular filtration rate (eGFR) and had significantly higher MIP-1α and IL-6 levels than survivors. In multiple regression analyses adjusted for age, sex and eGFR, both high MCP-1 and high MIP-1α were significantly associated with risk of in-hospital mortality. Apart from impaired kidney function, also the concentrations of MCP-1 and MIP-1α add important prognostic information in hospitalized patients with COVID-19. These data provide an increased understanding of the impact of monocyte modulators in patients with COVID-19 and normal or impaired kidney function, and warrant consideration in the pursuit of new effective therapies.

"Role of kidney function and concentrations of BAFF, sPD-L1 and sCD25 on mortality in hospitalized patients with COVID-19".

BACKGROUND: Chronic kidney disease (CKD) is a recognized risk factor for severe complications in COVID-19. Our objective was to analyze the association between kidney function / T and B lymphocyte modulatory factors and risk of mortality in COVID-19 patients. METHODS: In-hospital and 30-day mortality were analyzed in COVID-19 patients (n = 110). Plasma levels of selected T and B cell modulators were analyzed and correlated to mortality risk. A subgroup of sex- and eGFR-matched COVID-19 patients was compared to CKD patients without infection and healthy subjects. RESULTS: COVID-19 patients who died in hospital and within 30 days had significantly higher BAFF and sCD25 plasma levels than survivors. In logistic regression models patients with high BAFF, sCD25 and sPD-L1 levels had significantly higher risk of both in-hospital and 30-day mortality while there was no association to eGFR. In the subgroup analysis, a higher level of BAFF, IFN-α, sCD25, sPD-L1 and a lower level of sCD40L was observed in COVID-19 patients compared to the CKD group with corresponding kidney function. CONCLUSIONS: We demonstrate that kidney function and concentrations of BAFF, sCD25 and PD-L1, independent of previously recognized risk factors; age, male gender, and leukocytosis are associated with risk of in-hospital and 30-day mortality in patients with COVID-19. These data indicate the significance of adaptive immune system modulators in COVID-19 and motivate further analysis to identify new potential prognostic and therapeutic approaches.

IL-6 signalling biomarkers in hospitalised patients with moderate to severe SARS-CoV-2 infection in a single centre study in Sweden.

BACKGROUND: COVID-19 disease severity and need for intensive care has been associated with profound immune disturbances in which interleukin 6 (IL-6) is central. IL-6 signals through two pathways: classical IL-6 signalling with C-reactive protein (CRP) as a product is pivotal in the acute immune response against pathogens while IL-6 trans-signalling is involved in prolonged inflammation. We measured biomarkers of the IL-6 classical and trans-signalling pathways in patients with moderate or severe COVID-19 in the first wave of the COVID-19 pandemic. METHOD: In a longitudinal cohort study including patients admitted to Danderyd hospital, Stockholm, Sweden, with COVID-19 (n = 112), plasma IL-6 mirroring activity in both pathways, CRP as marker of classical signalling and the soluble IL-6 receptor (sIL-6R) and soluble glycoprotein 130 (sgp130) as markers of trans-signalling were analysed at baseline. Potential differences in biomarker levels between groups of moderate and severe COVID-19 defined by care level, level of respiratory support and one-month mortality was analysed, as was correlations between biomarkers. In addition, levels 4 months after hospital admission were compared to those at baseline. RESULTS: Levels of IL-6 and CRP were increased in severe COVID-19 whereas IL-6 trans-signalling markers (sIL-6R, sgp130) did not differ between the groups. CRP correlated positively with IL-6 in all patients while correlation with IL-6 could not be demonstrated for sIL-6R and sgp130 in either group. Levels of IL-6, CRP and sIL-6R were significantly decreased after 4 months whereas sgp130 levels increased. CONCLUSION: Classical signalling is the dominating IL-6 pathway in moderate-severe COVID-19.

Infection Rate of SARS-CoV-2 in Asymptomatic Healthcare Workers, Sweden, June 2022.

Given the recent surge in SARS-CoV-2 Omicron infections, we performed a quantitative PCR screening survey during June 28-29, 2022, in Stockholm, Sweden, to investigate SARS-CoV-2 point prevalence in a group with high exposure risk. Results showed SARS-CoV-2 infection in 2.3% of healthcare workers who were asymptomatic at time of sampling.

Clinical grade ACE2 as a universal agent to block SARS-CoV-2 variants.

The recent emergence of multiple SARS-CoV-2 variants has caused considerable concern due to both reduced vaccine efficacy and escape from neutralizing antibody therapeutics. It is, therefore, paramount to develop therapeutic strategies that inhibit all known and future SARS-CoV-2 variants. Here, we report that all SARS-CoV-2 variants analyzed, including variants of concern (VOC) Alpha, Beta, Gamma, Delta, and Omicron, exhibit enhanced binding affinity to clinical grade and phase 2 tested recombinant human soluble ACE2 (APN01). Importantly, soluble ACE2 neutralized infection of VeroE6 cells and human lung epithelial cells by all current VOC strains with markedly enhanced potency when compared to reference SARS-CoV-2 isolates. Effective inhibition of infections with SARS-CoV-2 variants was validated and confirmed in two independent laboratories. These data show that SARS-CoV-2 variants that have emerged around the world, including current VOC and several variants of interest, can be inhibited by soluble ACE2, providing proof of principle of a pan-SARS-CoV-2 therapeutic.

Long-term SARS-CoV-2-specific and cross-reactive cellular immune responses correlate with humoral responses, disease severity, and symptomatology.

BACKGROUND: Cellular immune memory responses post coronavirus disease 2019 (COVID-19) have been difficult to assess due to the risks of contaminating the immune response readout with memory responses stemming from previous exposure to endemic coronaviruses. The work herein presents a large-scale long-term follow-up study investigating the correlation between symptomology and cellular immune responses four to five months post seroconversion based on a unique severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific peptide pool that contains no overlapping peptides with endemic human coronaviruses. METHODS: Peptide stimulated memory T cell responses were assessed with dual interferon-gamma (IFNγ) and interleukin (IL)-2 Fluorospot. Serological analyses were performed using a multiplex antigen bead array. RESULTS: Our work demonstrates that long-term SARS-CoV-2-specific memory T cell responses feature dual IFNγ and IL-2 responses, whereas cross-reactive memory T cell responses primarily generate IFNγ in response to SARS-CoV-2 peptide stimulation. T cell responses correlated to long-term humoral immune responses. Disease severity as well as specific COVID-19 symptoms correlated with the magnitude of the SARS-CoV-2-specific memory T cell response four to five months post seroconversion. CONCLUSION: Using a large cohort and a SARS-CoV-2-specific peptide pool we were able to substantiate that initial disease severity and symptoms correlate with the magnitude of the SARS-CoV-2-specific memory T cell responses.

Duration of SARS-CoV-2 Immune Responses Up to Six Months Following Homologous or Heterologous Primary Immunization with ChAdOx1 nCoV-19 and BNT162b2 mRNA Vaccines.

Heterologous primary immunization against SARS-CoV-2 is part of applied recommendations. However, little is known about duration of immune responses after heterologous vaccine regimens. To evaluate duration of immune responses after primary vaccination with homologous adeno-vectored ChAdOx1 nCoV-19 vaccine (ChAd) or heterologous ChAd/BNT162b2 mRNA vaccine (BNT), anti-spike-IgG and SARS-CoV-2 VOC-neutralizing antibody responses were measured in 354 healthcare workers (HCW) at 2 weeks, 3 months, 5 months and 6 months after the second vaccine dose. T-cell responses were investigated using a whole blood interferon gamma (IFN-γ) release assay 2 weeks and 3 months post second vaccine dose. Two hundred and ten HCW immunized with homologous BNT were enrolled for comparison of antibody responses. In study participants naïve to SARS-CoV-2 prior to vaccination, heterologous ChAd/BNT resulted in 6-fold higher peak anti-spike IgG antibody titers compared to homologous ChAd vaccination. The half-life of antibody titers was 3.1 months (95% CI 2.8-3.6) following homologous ChAd vaccination and 1.9 months (95% CI 1.7-2.1) after heterologous vaccination, reducing the GMT difference between the groups to 3-fold 6 months post vaccination. Peak T-cell responses were stronger in ChAd/BNT vaccinees, but no significant difference was observed 3 months post vaccination. SARS-CoV-2 infection prior to vaccination resulted in substantially higher peak GMTs and IFN-γ levels and enhanced SARS-CoV-2 specific antibody and T cell responses over time. Heterologous primary SARS-CoV-2 immunization with ChAd and BNT elicits a stronger initial immune response compared to homologous vaccination with ChAd. However, although the differences in humoral responses remain over 6 months, the difference in SARS-CoV-2 specific T cell responses are no longer significant three months after vaccination.

A Model Predicting Mortality of Hospitalized Covid-19 Patients Four Days After Admission: Development, Internal and Temporal-External Validation.

Objective: To develop and validate a prognostic model for in-hospital mortality after four days based on age, fever at admission and five haematological parameters routinely measured in hospitalized Covid-19 patients during the first four days after admission. Methods: Haematological parameters measured during the first 4 days after admission were subjected to a linear mixed model to obtain patient-specific intercepts and slopes for each parameter. A prediction model was built using logistic regression with variable selection and shrinkage factor estimation supported by bootstrapping. Model development was based on 481 survivors and 97 non-survivors, hospitalized before the occurrence of mutations. Internal validation was done by 10-fold cross-validation. The model was temporally-externally validated in 299 survivors and 42 non-survivors hospitalized when the Alpha variant (B.1.1.7) was prevalent. Results: The final model included age, fever on admission as well as the slope or intercept of lactate dehydrogenase, platelet count, C-reactive protein, and creatinine. Tenfold cross validation resulted in a mean area under the receiver operating characteristic curve (AUROC) of 0.92, a mean calibration slope of 1.0023 and a Brier score of 0.076. At temporal-external validation, application of the previously developed model showed an AUROC of 0.88, a calibration slope of 0.95 and a Brier score of 0.073. Regarding the relative importance of the variables, the (apparent) variation in mortality explained by the six variables deduced from the haematological parameters measured during the first four days is higher (explained variation 0.295) than that of age (0.210). Conclusions: The presented model requires only variables routinely acquired in hospitals, which allows immediate and wide-spread use as a decision support for earlier discharge of low-risk patients to reduce the burden on the health care system. Clinical Trial Registration: Austrian Coronavirus Adaptive Clinical Trial (ACOVACT); ClinicalTrials.gov, identifier NCT04351724.

SARS-CoV-2 induces a durable and antigen specific humoral immunity after asymptomatic to mild COVID-19 infection.

Current SARS-CoV-2 serological assays generate discrepant results, and the longitudinal characteristics of antibodies targeting various antigens after asymptomatic to mild COVID-19 are yet to be established. This longitudinal cohort study including 1965 healthcare workers, of which 381 participants exhibited antibodies against the SARS-CoV-2 spike antigen at study inclusion, reveal that these antibodies remain detectable in most participants, 96%, at least four months post infection, despite having had no or mild symptoms. Virus neutralization capacity was confirmed by microneutralization assay in 91% of study participants at least four months post infection. Contrary to antibodies targeting the spike protein, antibodies against the nucleocapsid protein were only detected in 80% of previously anti-nucleocapsid IgG positive healthcare workers. Both anti-spike and anti-nucleocapsid IgG levels were significantly higher in previously hospitalized COVID-19 patients four months post infection than in healthcare workers four months post infection (p = 2*10-23 and 2*10-13 respectively). Although the magnitude of humoral response was associated with disease severity, our findings support a durable and functional humoral response after SARS-CoV-2 infection even after no or mild symptoms. We further demonstrate differences in antibody kinetics depending on the antigen, arguing against the use of the nucleocapsid protein as target antigen in population-based SARS-CoV-2 serological surveys.

A cell-free high throughput assay for assessment of SARS-CoV-2 neutralizing antibodies.

Highly accurate serological tests are key to assessing the prevalence of SARS-CoV-2 antibodies and the level of immunity in the population. This is important to predict the current and future status of the pandemic. With the recent emergence of new and more infectious SARS-CoV-2 variants, assays allowing for high throughput analysis of antibodies able to neutralize SARS-CoV-2 become even more important. Here, we report the development and validation of a robust, high throughput method, which enables the assessment of antibodies inhibiting the binding between the SARS-CoV-2 spike protein and angiotensin converting enzyme 2 (ACE2). The assay uses recombinantly produced spike-f and ACE2 and is performed in a bead array format, which allows analysis of up to 384 samples in parallel per instrument over seven hours, demanding only one hour of manual handling. The method is compared to a microneutralization assay utilising live SARS-CoV-2 and is shown to deliver highly correlating data. Further, a comparison with a serological method that measures all antibodies recognizing the spike protein shows that this type of assessment provides important insights into the neutralizing efficiency of the antibodies, especially for individuals with low antibody levels. This method can be an important and valuable tool for large-scale assessment of antibody-based neutralization, including neutralization of new spike variants that might emerge.

An evaluation of a FluoroSpot assay as a diagnostic tool to determine SARS-CoV-2 specific T cell responses.

Numerous assays evaluating serological and cellular responses have been developed to characterize immune responses against SARS-CoV-2. Serological assays are both cost- and time-effective compared to cellular assays, but cellular immune responses may provide a diagnostic value to determine previous SARS-CoV-2 infection in seronegative individuals. However, potential cross-reactive T cell responses stemming from prior encounters with human coronaviruses (HCoVs) may affect assay specificity. In this study, we evaluated the specificity and sensitivity of a SARS-CoV-2 IFN-γ Release Assay (IGRA) based on the FluoroSpot method employing commercially available SARS-CoV-2-specific peptide pools, as well as an in-house designed SARS-CoV-2 peptide pool restricted to 5 amino acid stretches or less aligning with endemic HCoVs. Blood samples were obtained from healthcare workers (HCW) 5-6 months post SARS-CoV-2 spike (S) IgG and nucleocapsid (N) IgG dual seroconversion (n = 187) and HCW who had been S IgG and N IgG dual seronegative at repeated occasions, including the current sampling time point (n = 102). In addition, samples were obtained 4 to 5 months post infection from 55 polymerase chain reaction (PCR)-confirmed COVID-19 patients. Assay specificity and sensitivity were calculated with serology as a reference standard for HCW. The in-house generated peptide pool displayed a specificity of 96.1%, while the commercially available peptide pools displayed specificities of 80.4% and 85.3%, respectively. Sensitivity was higher in a cohort of previously hospitalized COVID-19 patients (96.4% and 84.0% for the commercially available peptide pools and 92.7% for the in-house generated peptide pool) compared to the HCW cohort (92.0% and 66.8% for the commercially available peptide pools and 76.0% for the in-house generated peptide pool). Based on these findings, the individual diagnostic value of T cell immune responses against SARS-CoV-2 currently appears to be limited but remain an important research tool ahead.