Durability of Cross-Neutralizing Antibodies 5.5 Months After Bivalent Coronavirus Disease 2019 Vaccine Booster.

We analyzed neutralizing antibodies in samples from ancestral + BA.1 and ancestral + BA.4/5 boosted individuals, collected around 5.5 months after booster. Titers of neutralizing antibodies generally decreased compared to a time point early after the bivalent booster immunization. This was more pronounced for individuals without infection history and for recently emerged Omicron variants.

Expression of Tissue Factor and Platelet/Leukocyte Markers on Extracellular Vesicles Reflect Platelet-Leukocyte Interaction in Severe COVID-19.

Severe COVID-19 is frequently associated with thromboembolic complications. Increased platelet activation and platelet-leukocyte aggregate formation can amplify thrombotic responses by inducing tissue factor (TF) expression on leukocytes. Here, we characterized TF-positive extracellular vesicles (EVs) and their cellular origin in 12 patients suffering from severe COVID-19 (time course, 134 samples overall) and 25 healthy controls. EVs exposing phosphatidylserine (PS) were characterized by flow cytometry. Their cellular origin was determined by staining with anti-CD41, anti-CD45, anti-CD235a, and anti-CD105 as platelet, leukocyte, red blood cell, and endothelial markers. We further investigated the association of EVs with TF, platelet factor 4 (PF4), C-reactive protein (CRP), and high mobility group box-1 protein (HMGB-1). COVID-19 patients showed higher levels of PS-exposing EVs compared to controls. The majority of these EVs originated from platelets. A higher amount of EVs in patient samples was associated with CRP, HMGB-1, PF4, and TF as compared to EVs from healthy donors. In COVID-19 samples, 16.5% of all CD41+ EVs displayed the leukocyte marker CD45, and 55.5% of all EV aggregates (CD41+CD45+) co-expressed TF, which reflects the interaction of platelets and leukocytes in COVID-19 on an EV level.

Xanthoepocin, a photolabile antibiotic of Penicillium ochrochloron CBS 123823 with high activity against multiresistant gram-positive bacteria.

BACKGROUND: With the steady increase of antibiotic resistance, several strategies have been proposed in the scientific community to overcome the crisis. One of many successful strategies is the re-evaluation of known compounds, which have been early discarded out of the pipeline, with state-of-the-art know-how. Xanthoepocin, a polyketide widespread among the genus Penicillium with an interesting bioactivity spectrum against gram-positive bacteria, is such a discarded antibiotic. The purpose of this work was to (i) isolate larger quantities of this metabolite and chemically re-evaluate it with modern technology, (ii) to explore which factors lead to xanthoepocin biosynthesis in P. ochrochloron, and (iii) to test if it is beside its known activity against methicillin-resistant Staphylococcus aureus (MRSA), also active against linezolid and vancomycin-resistant Enterococcus faecium (LVRE)-a very problematic resistant bacterium which is currently on the rise. RESULTS: In this work, we developed several new protocols to isolate, extract, and quantify xanthoepocin out of bioreactor batch and petri dish-grown mycelium of P. ochrochloron. The (photo)chemical re-evaluation with state-of-the-art techniques revealed that xanthoepocin is a photolabile molecule, which produces singlet oxygen under blue light irradiation. The intracellular xanthoepocin content, which was highest under ammonium-limited conditions, varied considerably with the applied irradiation conditions in petri dish and bioreactor batch cultures. Using light-protecting measures, we achieved MIC values against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), which were up to 5 times lower than previously published. In addition, xanthoepocin was highly active against a clinical isolate of linezolid and vancomycin-resistant Enterococcus faecium (LVRE). CONCLUSIONS: This interdisciplinary work underlines that the re-evaluation of known compounds with state-of-the-art techniques is an important strategy in the combat against multiresistant bacteria and that light is a crucial factor on many levels that needs to receive more attention. With appropriate light protecting measures in the susceptibility tests, xanthoepocin proved to be a powerful antibiotic against MRSA and LVRE. Exploring the light response of other polyketides may be pivotal for re-introducing previously discarded metabolites into the antibiotic pipeline and to identify photosensitizers which might be used for (antimicrobial) photodynamic therapies.

A high leukocyte count and administration of hydrocortisone hamper PCR-based diagnostics for bloodstream infections.

Bloodstream infections (BSIs) require an accurate and fast identification of causative pathogens. Molecular diagnostics, in particular polymerase chain reaction (PCR)-based approaches for BSI diagnostics directly from whole blood, suffer from limitations such as inhibition leading to invalid results. In this retrospective study, we analyzed 23 parameters for their potential interference with LightCycler SeptiFast PCR tests (n = 2167) routinely performed at our institution. The overall inhibition rate was 9.1%. Test date, type of ward, procalcitonin levels, high leukocyte counts, and absolute neutrophil count were significantly associated with inhibition. For a subset (n = 448), cut-off values for leukocyte counts of < 5700 cells/µL and ≥ 26,900 cells/µL were significantly associated with a low (5%) and high (67%) inhibition risk. For patients with a moderate to high leukocyte count (5700-26,900 cells/µL), the additional administration of hydrocortisone significantly increased the inhibition risk. Furthermore, freezing of blood samples prior to DNA extraction and SF testing appeared to neutralize inhibitory factors. It remains to be investigated whether other molecular diagnostic tests are susceptible to similar inhibiting parameters.

Estimating seroprevalence of SARS-CoV-2 antibodies using three self-reported symptoms: development of a prediction model based on data from Ischgl, Austria.

We report the development of a regression model to predict the prevalence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) antibodies on a population level based on self-reported symptoms. We assessed participant-reported symptoms in the past 12 weeks, as well as the presence of SARS-CoV-2 antibodies during a study conducted in April 2020 in Ischgl, Austria. We conducted multivariate binary logistic regression to predict seroprevalence in the sample. Participants (n = 451) were on average 47.4 years old (s.d. 16.8) and 52.5% female. SARS-CoV-2 antibodies were found in n = 197 (43.7%) participants. In the multivariate analysis, three significant predictors were included and the odds ratios (OR) for the most predictive categories were cough (OR 3.34, CI 1.70-6.58), gustatory/olfactory alterations (OR 13.78, CI 5.90-32.17) and limb pain (OR 2.55, CI 1.20-6.50). The area under the receiver operating characteristic curve was 0.773 (95% CI 0.727-0.820). Our regression model may be used to estimate the seroprevalence on a population level and a web application is being developed to facilitate the use of the model.

Lipophilic Arginine Esters: The Gateway to Preservatives without Side Effects.

This study hypothesized that long carbon chain cationic arginine (Arg) esters can be considered as toxicologically harmless preservatives. Arg-esters with C18 and C24 carbon chains, namely, arginine-oleate (Arg-OL) and arginine-decyltetradecanoate (Arg-DT), were synthesized. Structures were confirmed by FT-IR, 1H NMR, and mass spectroscopy. Both Arg-esters were tested regarding hydrophobicity in terms of log Poctanol/water, critical micelle concentration (CMC), biodegradability, cytotoxicity, hemolysis, and antimicrobial activity against Escherichiacoli (E. coli), Staphylococcusaureus (S. aureus), Bacillussubtilis (B. subtilis), and Enterococcusfaecalis (E. faecalis). Log Poctanol/water of arginine was raised from -1.9 to 0.3 and 0.6 due to the attachment of C18 and C24 carbon chains, respectively. The critical micelle concentration of Arg-OL and Arg-DT was 0.52 and 0.013 mM, respectively. Both Arg-esters were biodegradable by porcine pancreatic lipase. In comparison to the well-established antimicrobials, benzalkonium chloride (BAC) and cetrimide, Arg-esters showed significantly less cytotoxic and hemolytic activity. Both esters exhibited pronounced antimicrobial properties against Gram-positive and Gram-negative bacteria comparable to that of BAC and cetrimide. The minimum inhibitory concentration (MIC) of Arg-esters was <50 µg mL-1 against all tested microbes. Overall, results showed a high potential of Arg-esters with long carbon chains as toxicologically harmless novel preservatives.

Influence of antibiotic treatment on the detection of S. aureus in whole blood following pathogen enrichment.

BACKGROUND: Early pathogen detection and identification are crucial for an effective and targeted antibiotic therapy in patients suffering from blood stream infection. Molecular diagnostic methods can accelerate pathogen identification as compared to blood culture, but frequently suffer from the inhibition of polymerase chain reation (PCR) by sample matrix components, such as host DNA, anticoagulants, or plasma proteins. To overcome this limitation, molecular diagnostic methods commonly rely on pathogen enrichment by selective lysis of blood cells and pelleting of intact pathogens prior to analysis. RESULTS: Here, we investigated the impact of antibiotic treatment on the recovery of pathogen DNA using an established pathogen enrichment protocol. Based on the hypothesis that induction of bacterial cell wall disintegration following antibiotic administration leads to incomplete pelleting of pathogen DNA, S. aureus was grown in human whole blood with or without addition of cell wall active (vancomycin, piperacillin) or non cell wall active (ciprofloxacin, clindamycin) antibiotics at clinically relevant concentrations. Pathogen detection remained unaffected by non cell wall active antibiotics or even increased in the presence of cell wall active antibiotics, indicating improved accessibility of pathogen DNA. Likewise, mechanical lysis of S. aureus prior to pathogen enrichment resulted in increased recovery of pathogen DNA. Quantification of pathogen and human DNA after selective lysis of blood cells and pathogen enrichment confirmed partial depletion of human DNA, leading to a net enrichment of pathogen DNA over human DNA. CONCLUSION: Concurrent antibiotic administration does not reduce the recovery of pathogen DNA during pathogen enrichment by selective lysis and centrifugation. Leads to a 10-fold human DNA depletion as compared to pathogen DNA. Moreover, we confirm that the recovery of pathogen DNA after pathogen enrichment is not negatively influenced by concurrent antibiotic administration.

Shiga toxin 2a binds antithrombin and heparin, but does not directly activate platelets.

Escherichia coli-induced hemolytic uremic syndrome (eHUS) is a life-threatening complication of infection with Shiga toxin (Stx), in particular Stx2a-producing Escherichia coli. Enhanced coagulation activation with formation of microthrombi seems to be a key event in development of eHUS. Platelet activation has been postulated as a possible, but controversially debated mechanism. The present study investigated the effect of Stx2a on plasmatic coagulation and platelets. Binding studies were initially performed with ELISA and co-immunoprecipitation and supported by quartz crystal microbalance with dissipation monitoring (QCM-D). Antithrombin (AT) activity was measured using the automated BCS XP® system. ROTEM® was used for functional coagulation testing. Platelet binding and activation was studied with FACS and light-transmission aggregometry. We found binding of Stx2a to AT, an important inhibitor of blood coagulation, but only a mild albeit significant reduction of AT activity against FXa in the presence of Stx2a. QCM-D analysis also showed binding of Stx2a to heparin and an impaired binding of AT to Stx2a-bound heparin. ROTEM® using Stx2a-treated platelet-poor plasma revealed a significant, but only moderate shortening of clotting time. Neither binding nor activation of platelets by Stx2a could be demonstrated. In summary, data of this study suggest that Stx2a binds to AT, but does not induce major effects on plasmatic coagulation. In addition, no interaction with platelets occurred. The well-known non-beneficial administration of heparin in eHUS patients could be explained by the interaction of Stx2a with heparin.

Blood transcriptomics analysis offers insights into variant-specific immune response to SARS-CoV-2.

Bulk RNA sequencing (RNA-seq) of blood is typically used for gene expression analysis in biomedical research but is still rarely used in clinical practice. In this study, we propose that RNA-seq should be considered a diagnostic tool, as it offers not only insights into aberrant gene expression and splicing but also delivers additional readouts on immune cell type composition as well as B-cell and T-cell receptor (BCR/TCR) repertoires. We demonstrate that RNA-seq offers insights into a patient's immune status via integrative analysis of RNA-seq data from patients infected with various SARS-CoV-2 variants (in total 196 samples with up to 200 million reads sequencing depth). We compare the results of computational cell-type deconvolution methods (e.g., MCP-counter, xCell, EPIC, quanTIseq) to complete blood count data, the current gold standard in clinical practice. We observe varying levels of lymphocyte depletion and significant differences in neutrophil levels between SARS-CoV-2 variants. Additionally, we identify B and T cell receptor (BCR/TCR) sequences using the tools MiXCR and TRUST4 to show that-combined with sequence alignments and BLASTp-they could be used to classify a patient's disease. Finally, we investigated the sequencing depth required for such analyses and concluded that 10 million reads per sample is sufficient. In conclusion, our study reveals that computational cell-type deconvolution and BCR/TCR methods using bulk RNA-seq analyses can supplement missing CBC data and offer insights into immune responses, disease severity, and pathogen-specific immunity, all achievable with a sequencing depth of 10 million reads per sample.

Machine Learning to Identify Critical Biomarker Profiles in New SARS-CoV-2 Variants.

The global dissemination of SARS-CoV-2 resulted in the emergence of several variants, including Alpha, Alpha + E484K, Beta, and Omicron. Our research integrated the study of eukaryotic translation factors and fundamental components in general protein synthesis with the analysis of SARS-CoV-2 variants and vaccination status. Utilizing statistical methods, we successfully differentiated between variants in infected individuals and, to a lesser extent, between vaccinated and non-vaccinated infected individuals, relying on the expression profiles of translation factors. Additionally, our investigation identified common causal relationships among the translation factors, shedding light on the interplay between SARS-CoV-2 variants and the host's translation machinery.

Direct comparison of SARS-CoV-2 variant specific neutralizing antibodies in human and hamster sera.

Antigenic characterization of newly emerging SARS-CoV-2 variants is important to assess their immune escape and judge the need for future vaccine updates. To bridge data obtained from animal sera with human sera, we analyzed neutralizing antibody titers in human and hamster single infection sera in a highly controlled setting using the same authentic virus neutralization assay performed in one laboratory. Using a Bayesian framework, we found that titer fold changes in hamster sera corresponded well to human sera and that hamster sera generally exhibited higher reactivity.

High prevalence group testing in epidemiology with geometrically inspired algorithms.

Demand for mass surveillance during peak times of the SARS-CoV-2 pandemic caused high workload for clinical laboratories. Efficient and cost conserving testing designs by means of group testing can substantially reduce resources during possible future emergency situations. The novel hypercube algorithm proposed by Mutesa et al. 2021 published in Nature provides methodological proof of concept and points out the applicability to epidemiological testing. In this work, the algorithm is explored and expanded for settings with high group prevalence. Numerical studies investigate the limits of the adapted hypercube methodology, allowing to optimize pooling designs for specific requirements (i.e. number of samples and group prevalence). Hyperparameter optimization is performed to maximize test-reduction. Standard deviation is examined to investigate resilience and precision. Moreover, empirical validation was performed by elaborately pooling SARS-CoV-2 virus samples according to numerically optimized pooling designs. Laboratory experiments with SARS-CoV-2 sample groups, ranging from 50 to 200 items, characterized by group prevalence up to 10%, are successfully processed and analysed. Test-reductions from 50 to 72.5% were achieved in the experimental setups when compared to individual testing. Higher theoretical test-reduction is possible, depending on the number of samples and the group prevalence, indicated by simulation results.

Characterizing SARS-CoV-2 neutralization profiles after bivalent boosting using antigenic cartography.

Since emergence of the initial SARS-CoV-2 BA.1, BA.2 and BA.5 variants, Omicron has diversified substantially. Antigenic characterization of these new variants is important to analyze their potential immune escape from population immunity and implications for future vaccine composition. Here, we describe an antigenic map based on human single-exposure sera and live-virus isolates that includes a broad selection of recently emerged Omicron variants such as BA.2.75, BF.7, BQ, XBB and XBF variants. Recent Omicron variants clustered around BA.1 and BA.5 with some variants further extending the antigenic space. Based on this antigenic map we constructed antibody landscapes to describe neutralization profiles after booster immunization with bivalent mRNA vaccines based on ancestral virus and either BA.1 or BA.4/5. Immune escape of BA.2.75, BQ, XBB and XBF variants was also evident in bivalently boosted individuals, however, cross-neutralization was improved for those with hybrid immunity. Our results indicate that future vaccine updates are needed to induce cross-neutralizing antibodies against currently circulating variants.

Omicron (B.1.1.529) BA.1 or BA.2-related effects on immune responses in previously naïve versus imprinted individuals: immune imprinting as an advantage in the humoral immune response against novel variants.

Background: Immune imprinting is a phenomenon in which a person's immune system develops a specific immunological memory of the pathogen or vaccine due to a previous exposure. This memory basically leads to a faster and stronger immune response in a subsequent contact to the same pathogen or vaccine. However, what happens if the pathogen has changed considerably in the meantime due to mutations in the main target region of antibodies, as in the evolution of SARS-CoV-2 from the ancestral strain to B.1.1.529 (Omicron)? In this case, does immune imprinting also confer an advantage in repeated contact and does it lead to a stronger immune response? Methods: To clarify these questions, we investigated the effects of immune imprinting in the context of SARS-CoV-2 by comparing a group of previously infection-naïve versus imprinted study participants and determined differences in humoral and cellular immune responses during and after infection with strain SARS-CoV-2 B.1.1.529 BA.1 and BA.2, respectively. We used a commercial CLIA, immunoblots, IFN-γ ELISpots and a plaque-reduction neutralization test to generate a clear and comparable picture of the humoral and cellular immune response in the two study groups. Results: Imprinted participants developed significantly higher antibody titers and showed significantly stronger neutralization capacity against the ancestral strain, BA.1 and BA.5. The immune response of naïve study participants was narrower and related mainly to the receptor-binding domain, which resulted in a lower neutralization capacity against other strains including BA.5. Naïve study participants showed a significantly higher cellular immune response than the imprinted study group, indicating a higher antigenic challenge. The cellular immune response was directed against general structures of SARS-CoV-2 and not specifically against the receptor-binding domain. Conclusion: Viral variant infection elicits variant-specific antibodies and prior mRNA vaccination or infection with a previous SARS-CoV-2 variant imprints serological responses toward the ancestral strain rather than variant antigens. On the other hand, our study shows that the initially higher specific antibody titers due to former imprinting via vaccination or prior infection significantly increased the humoral immune response, and therefore outperformed the humoral immune response of naïve study participants.

Blood transcriptomics analysis offers insights into variant-specific immune response to SARS-CoV-2.

Bulk RNA sequencing (RNA-seq) of blood is typically used for gene expression analysis in biomedical research but is still rarely used in clinical practice. In this study, we argue that RNA-seq should be considered a routine diagnostic tool, as it offers not only insights into aberrant gene expression and splicing but also delivers additional readouts on immune cell type composition as well as B-cell and T-cell receptor (BCR/TCR) repertoires. We demonstrate that RNA-seq offers vital insights into a patient's immune status via integrative analysis of RNA-seq data from patients infected with various SARS-CoV-2 variants (in total 240 samples with up to 200 million reads sequencing depth). We compare the results of computational cell-type deconvolution methods (e.g., MCP-counter, xCell, EPIC, quanTIseq) to complete blood count data, the current gold standard in clinical practice. We observe varying levels of lymphocyte depletion and significant differences in neutrophil levels between SARS-CoV-2 variants. Additionally, we identify B and T cell receptor (BCR/TCR) sequences using the tools MiXCR and TRUST4 to show that - combined with sequence alignments and pBLAST - they could be used to classify a patient's disease. Finally, we investigated the sequencing depth required for such analyses and concluded that 10 million reads per sample is sufficient. In conclusion, our study reveals that computational cell-type deconvolution and BCR/TCR methods using bulk RNA-seq analyses can supplement missing CBC data and offer insights into immune responses, disease severity, and pathogen-specific immunity, all achievable with a sequencing depth of 10 million reads per sample.

Limited cross-variant immune response from SARS-CoV-2 Omicron BA.2 in naïve but not previously infected outpatients.

Omicron is currently the dominant SARS-CoV-2 variant and several sublineages have emerged. Questions remain about the impact of previous SARS-CoV-2 exposure on cross-variant immune responses elicited by BA.2 infection compared to BA.1. Here we show that without previous history of COVID-19, BA.2 infection induces a reduced immune response against all variants of concern (VOC) compared to BA.1 infection. The absence of ACE2 binding in sera of previously naïve BA.1 and BA.2 patients indicates a lack of meaningful neutralization. In contrast, anti-spike antibody levels and neutralizing activity greatly increased in the BA.1 and BA.2 patients with a previous history of COVID-19. Transcriptome analyses of peripheral immune cells showed significant differences in immune response and specific antibody generation between BA.1 and BA.2 patients as well as significant differences in expression of specific immune genes. In summary, prior infection status significantly impacts the innate and adaptive immune response against VOC following BA.2 infection.

Limited cross-variant immune response from SARS-CoV-2 Omicron BA.2 in naïve but not previously infected outpatients.

Omicron is currently the dominant SARS-CoV-2 variant and several sublineages have emerged. Questions remain about the impact of previous SARS-CoV-2 exposure on cross-variant immune responses elicited by the SARS-CoV-2 Omicron sublineage BA.2 compared to BA.1. Here we show that without previous history of COVID-19, BA.2 infection induces a reduced immune response against all variants of concern (VOC) compared to BA.1 infection. The absence of ACE2 binding in sera of previously naïve BA.1 and BA.2 patients indicates a lack of meaningful neutralization. In contrast, anti-spike antibody levels and neutralizing activity greatly increased in the BA.1 and BA.2 patients with a previous history of COVID-19. Transcriptome analyses of peripheral immune cells showed significant differences in immune response and specific antibody generation between BA.1 and BA.2 patients as well as significant differences in the expression of specific immune genes. In summary, prior infection status significantly impacts the innate and adaptive immune response against VOC following BA.2 infection.

BNT162b2 vaccination enhances interferon-JAK-STAT-regulated antiviral programs in COVID-19 patients infected with the SARS-CoV-2 Beta variant.

Background: SARS-CoV-2 infection activates interferon-controlled signaling pathways and elicits a wide spectrum of immune responses and clinical manifestations in human patients. Methods: Here, we investigate the impact of prior vaccination on the innate immune response of hospitalized COVID-19 patients infected with the SARS-CoV-2 Beta variant through RNA sequencing of peripheral blood immune cells. Four patients had received the first dose of BNT162b2 about 11 days prior to the onset of COVID-19 symptoms and five patients were unvaccinated. Patients had received dexamethasone treatment. Immune transcriptomes were obtained at days 7-13, 20-32 and 42-60 after first symptomology. Results: RNA-seq reveals an enhanced JAK-STAT-mediated immune transcriptome response at day 10 in vaccinated patients as compared to unvaccinated ones. This increase subsides by day 35. Expression of the gene encoding the antiviral protein oligoadenylate synthetase (OAS) 1, which is inversely correlated with disease severity, and other key antiviral proteins increases in the vaccinated group. We also investigate the immune transcriptome in naïve individuals receiving their first dose of BNT162b2 and identify a gene signature shared with the vaccinated COVID-19 patients. Conclusions: Our study demonstrates that RNA-seq can be used to monitor molecular immune responses elicited by the BNT162b2 vaccine, both in naïve individuals and in COVID-19 patients, and it provides a biomarker-based approach to systems vaccinology.