Characteristics Associated with COVID-19 Breakthrough Infections after Booster Vaccinations in Healthcare Workers: Insights from the TüSeRe:exact Study.

Background: The prevalence of COVID-19 breakthrough infections in healthcare workers (HCWs) remains an issue of concern. This study examines the different characteristics associated with breakthrough infections in HCWs. Methods: From the total participants in the TüSeRe:exact study (n = 1046), we specifically included study participants who had received three vaccinations and were not infected prior to the third vaccination. Participants were invited to complete an online questionnaire, which included inquiries about any breakthrough infections they might have experienced. Univariate Cox regression analysis was used to investigate the association between participant characteristics and breakthrough infections. Results: Among 629 HCWs (497 female and 132 male), 241 (38%) experienced breakthrough infections during the follow-up period. The frequency of breakthrough infections was 39.2% (195/497) among female participants and 34.8% (46/132) among male participants (p = 0.357). The Cox regression model adjusted for age and sex showed that participants with cardiovascular disease (hazard ratio (95%CI) = 0.621 (0.392-0.985); p = 0.043) and those taking antihypertensives (hazard ratio (95%CI) = 0.551 (0.331-0.915); p = 0.021) had a significantly lower hazard ratio for breakthrough infections. The use of analgesics after the first vaccine (hazard ratio (95%CI) = 1.343 (1.025-1.759); p = 0.032) was associated with an increased risk of breakthrough infections. Conclusions: These findings can inform targeted preventive measures and risk management strategies to protect frontline workers and maintain a resilient healthcare system during the ongoing pandemic.

Development and validation of a respiratory syncytial virus multiplex immunoassay.

PURPOSE: Respiratory syncytial virus (RSV) is one of the leading causes of severe respiratory disease in infants and adults. While vaccines and monoclonal therapeutic antibodies either are or will shortly become available, correlates of protection remain unclear. For this purpose, we developed an RSV multiplex immunoassay that analyses antibody titers toward the post-F, Nucleoprotein, and a diverse mix of G proteins. METHODS: A bead-based multiplex RSV immunoassay was developed, technically validated to standard FDA bioanalytical guidelines, and clinically validated using samples from human challenge studies. RSV antibody titers were then investigated in children aged under 2 and a population-based cohort. RESULTS: Technical and clinical validation showed outstanding performance, while methodological developments enabled identification of the subtype of previous infections through use of the diverse G proteins for approximately 50% of samples. As a proof of concept to show the suitability of the assay in serosurveillance studies, we then evaluated titer decay and age-dependent antibody responses within population cohorts. CONCLUSION: Overall, the developed assay shows robust performance, is scalable, provides additional information on infection subtype, and is therefore ideally suited to be used in future population cohort studies.

Long-term efficacy of the peptide-based COVID-19 T cell activator CoVac-1 in healthy adults.

OBJECTIVES: T cell immunity is key for the control of viral infections including SARS-CoV-2, in particular with regard to immune memory and protection against arising genetic variants. METHODS: We recently evaluated a peptide-based SARS-CoV-2 T cell activator termed CoVac-1 in a first-in-human trial in healthy adults. Here, we report on long-term safety and efficacy data of CoVac-1 until month 12. RESULTS: CoVac-1 is well tolerated without long-term immune-related side effects and induces long-lasting anti-viral T cell responses in 100% of study participants, with potent expandability of clusters of differentiation (CD4+) and CD8+ T cells targeting multiple different CoVac-1 T cell epitopes. T cell responses were associated with stronger injection site reaction. Beyond induction of T cell immunity, 89% of subjects developed CoVac-1-specific immunoglobulin G antibodies which associated with the intensity of the T cell response, indicating that CoVac-1-specific CD4+ T cells support the induction of B-cell responses. Vaccination with approved COVID-19 vaccines boosted CoVac-1-specific T cell responses. Overall, a low SARS-CoV-2 infection rate (8.3%) was observed. CONCLUSION: Together, a single application of CoVac-1 elicits long-lived and broad SARS-CoV-2-specific T cell immunity, which further supports the current evaluation of our T cell activator in patients with congenital or acquired B-cell defects.

Bridging the gap - estimation of 2022/2023 SARS-CoV-2 healthcare burden in Germany based on multidimensional data from a rapid epidemic panel.

OBJECTIVES: Throughout the SARS-CoV-2 pandemic, Germany like other countries lacked adaptive population-based panels to monitor the spread of epidemic diseases. METHODS: To fill a gap in population-based estimates needed for winter 2022/23 we resampled in the German SARS-CoV-2 cohort study MuSPAD in mid-2022, including characterization of systemic cellular and humoral immune responses by interferon-γ-release assay (IGRA) and CLIA/IVN assay. We were able to confirm categorization of our study population into four groups with differing protection levels against severe COVID-19 courses based on literature synthesis. Using these estimates, we assessed potential healthcare burden for winter 2022/23 in different scenarios with varying assumptions on transmissibility, pathogenicity, new variants, and vaccine booster campaigns in ordinary differential equation models. RESULTS: We included 9921 participants from eight German regions. While 85% of individuals were located in one of the two highest protection categories, hospitalization estimates from scenario modeling were highly dependent on viral variant characteristics ranging from 30-300% compared to the 02/2021 peak. Our results were openly communicated and published to an epidemic panel network and a newly established modeling network. CONCLUSIONS: We demonstrate feasibility of a rapid epidemic panel to provide complex immune protection levels for inclusion in dynamic disease burden modeling scenarios.

SARS-CoV-2 specific sIgA in saliva increases after disease-related video stimulation.

Secretory immunoglobulin A (sIgA) in saliva is the most important immunoglobulin fighting pathogens in the respiratory tract and may thus play a role in preventing SARS-CoV-2 infections. To gain a better understanding of the plasticity in the mucosal antibody, we investigated the proactive change in secretion of salivary SARS-CoV-2-specific sIgA in 45 vaccinated and/or previously infected, generally healthy persons (18 to 35 years, 22 women). Participants were exposed to a disease video displaying humans with several respiratory symptoms typical for COVID-19 in realistic situations of increased contagion risk. The disease video triggered an increase in spike-specific sIgA, which was absent after a similar control video with healthy people. The increase further correlated inversely with revulsion and aversive feelings while watching sick people. In contrast, the receptor binding domain-specific sIgA did not increase after the disease video. This may indicate differential roles of the two salivary antibodies in response to predictors of airborne contagion. The observed plasticity of spike-specific salivary antibody release after visual simulation of enhanced contagion risk suggests a role in immune exclusion.

Humoral immune response to SARS-CoV-2 and endemic coronaviruses in urban and indigenous children in Colombia.

BACKGROUND: Although anti-SARS-CoV-2 humoral immune responses and epidemiology have been extensively studied, data gaps remain for certain populations such as indigenous people or children especially in low- and middle-income countries. To address this gap, we evaluated SARS-CoV-2 seroprevalence and humoral immunity towards the parental B.1 strain, local SARS-CoV-2 variants, and endemic coronaviruses in children from Colombia from March to April 2021. METHODS: We performed a cross-sectional seroprevalence study with 80 children from Bogotá and expanded our analysis by comparing results with an independent observational study of 82 children from the Wiwa community living in the north-eastern Colombian territories. Antibody IgG titers towards SARS-CoV-2 and the endemic coronaviruses as well as ACE2 binding inhibition as a proxy for neutralization towards several SARS-CoV-2 variants were analyzed using two multiplex-based immunoassays. RESULTS: While we find seroprevalence estimates of 21.3% in children from Bogotá, seroprevalence is higher with 34.1% in Wiwa children. We observe a robust induction of antibodies towards the surface-exposed spike protein, its S1-, S2- and receptor-binding-subdomains in all SARS-CoV-2 seropositive children. Only nucleocapsid-specific IgG is significantly lower in the indigenous participants. ACE2 binding inhibition is low for all SARS-CoV-2 variants examined. We observe a dominance of NL63 S1 IgG levels in urban and indigenous children which suggests an early exposure to this respiratory virus independent of living conditions and geographic location. SARS-CoV-2 seropositivity does not correlate with antibody levels towards any of the four endemic coronaviruses indicating the absence of cross-protective immunity. CONCLUSIONS: Overall, antibody titers, but in particular ACE2 binding inhibition are low within Colombian samples, requiring further investigation to determine any potential clinical significance.

Our knowledge of SARS-CoV-2, the virus causing COVID-19 remains incomplete for certain populations including indigenous people and younger age groups. Here, we aim to understand the extent to which children from urban and indigenous populations of Colombia were previously infected with SARS-CoV-2 and the related common cold coronaviruses. By measuring antibodies, protective proteins produced by the immune system, we find higher levels of previous SARS-CoV-2 infections in indigenous children of the Wiwa community (34.1%) compared to children from urbanized Bogotá (21.3%). Antibody levels towards the common cold coronaviruses were similar in SARS-CoV-2 infected and uninfected children suggesting immune responses to one coronavirus do not automatically protect against closely-related viruses. Further, we find low levels of protective immunity against SARS-CoV-2 in both populations. This finding warrants further investigation as it relates to reinfection risk and future vaccination strategies in these populations.

Differential levels of anti-Mycobacterium tuberculosis-specific IgAs in saliva of household contacts with latent tuberculosis infection.

Introduction: Mucosal immunity is strongly elicited in early stages of many respiratory and enteric infections; however, its role in tuberculosis pathogenesis has been scarcely explored. We aimed to investigate Mycobacterium tuberculosis (Mtb) specific IgA levels in saliva in different stages of latent Tuberculosis Infection (TBI). Methodology: A multiplex bead-based Luminex immunoassay was developed to detect specific IgA against 12 highly immunogenic Mtb antigens. A prospective cohort of household contacts (>14 years) of pulmonary TB cases was established in Santiago, Chile. Contacts were classified as Mtb-infected or not depending on serial interferon-γ release assay results. Saliva samples were collected and tested at baseline and at a 12-week follow-up. Results: Mtb-specific IgA was detectable at all visits in all participants (n = 168), including the "non-Mtb infected" (n = 64). Significantly higher median levels of IgA were found in the "Mtb infected" compared to the uninfected for anti-lipoarabinomannan (LAM) (110 vs. 84.8 arbitrary units (AU), p < 0.001), anti-PstS1 (117 vs. 83 AU, p < 0.001), anti-Cell Membrane Fraction (CMF) (140 vs. 103 AU, p < 0.001) and anti-Culture Filtrate Proteins (CFP) (median 125 vs. 96 AU, p < 0.001), respectively. Nonetheless, the discriminatory performance of these specific mucosal IgA for TBI diagnosis was low. Conclusion: Saliva holds Mtb-specific IgA against several antigens with increased levels for anti-LAM, anti-PstS1, anti-CMF and anti-CFP found in household contacts with an established TBI. The role of these mucosal antibodies in TB pathogenesis, and their kinetics in different stages of Mtb infection merits further exploring.

STAR SIGN study: Evaluation of COVID-19 vaccine efficacy against the SARS-CoV-2 variants BQ.1.1 and XBB.1.5 in patients with inflammatory bowel disease.

BACKGROUND: Vaccine-elicited immune responses are impaired in patients with inflammatory bowel disease (IBD) treated with anti-TNF biologics. AIMS: To assess vaccination efficacy against the novel omicron sublineages BQ.1.1 and XBB.1.5 in immunosuppressed patients with IBD. METHODS: This prospective multicentre case-control study included 98 biologic-treated patients with IBD and 48 healthy controls. Anti-spike IgG concentrations and surrogate neutralisation against SARS-CoV-2 wild-type, BA.1, BA.5, BQ.1.1, and XBB.1.5 were measured at two different time points (2-16 weeks and 22-40 weeks) following third dose vaccination. Surrogate neutralisation was based on antibody-mediated blockage of ACE2-spike protein-protein interaction. Primary outcome was surrogate neutralisation against tested SARS-CoV-2 sublineages. Secondary outcomes were proportions of participants with insufficient surrogate neutralisation, impact of breakthrough infection, and correlation of surrogate neutralisation with anti-spike IgG concentration. RESULTS: Surrogate neutralisation against all tested sublineages was reduced in patients with IBD who were treated with anti-TNF biologics compared to patients treated with non-anti-TNF biologics and healthy controls (each p ≤ 0.001) at visit 1. Anti-TNF therapy (odds ratio 0.29 [95% CI 0.19-0.46]) and time since vaccination (0.85 [0.72-1.00]) were associated with low, and mRNA-1273 vaccination (1.86 [1.12-3.08]) with high wild-type surrogate neutralisation in a ß-regression model. Accordingly, higher proportions of patients treated with anti-TNF biologics had insufficient surrogate neutralisation against omicron sublineages at visit 1 compared to patients treated with non-anti-TNF biologics and healthy controls (each p ≤ 0.015). Surrogate neutralisation against all tested sublineages decreased over time but was increased by breakthrough infection. Anti-spike IgG concentrations correlated with surrogate neutralisation. CONCLUSIONS: Patients with IBD who are treated with anti-TNF biologics show impaired neutralisation against novel omicron sublineages BQ.1.1 and XBB.1.5 and may benefit from prioritisation for future variant-adapted vaccines.

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test.

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different antibody isotypes because they appear at different stages of the host immune response. With Lyme borreliosis, the pathogenic agent can be one of the multiple members of the Borrelia species. Therefore, correct sample classification requires evaluating the immunoreactivity against different antigens of different Borrelia species. Additionally, anti-pathogen IgG and IgM responses can have different elicitation time courses during disease progression. Here we demonstrate the development of a two-reporter multiplex immunoassay that has utility in identifying Borrelia-specific immune response in human serum samples by simultaneously evaluating both IgG and IgM immunoreactivity against different bacterial antigens in the same reaction well. This dual-reporter approach retains the analytical performance of single-reporter methods while conserving time and resources and reducing sample size requirements. This assay allows essentially double the serological information to be generated from a blood sample in half the time.

Correlation between Clinical Characteristics and Antibody Levels in COVID-19 Convalescent Plasma Donor Candidates.

COVID-19 convalescent plasma (CCP) with high neutralizing antibodies has been suggested in preventing disease progression in COVID-19. In this study, we investigated the relationship between clinical donor characteristics and neutralizing anti-SARS-CoV-2 antibodies in CCP donors. COVID-19 convalescent plasma donors were included into the study. Clinical parameters were recorded and anti-SARS-CoV-2 antibody levels (Spike Trimer, Receptor Binding Domain (RBD), S1, S2 and nucleocapsid protein) as well as ACE2 binding inhibition were measured. An ACE2 binding inhibition < 20% was defined as an inadequate neutralization capacity. Univariate and multivariable logistic regression analysis was used to detect the predictors of inadequate neutralization capacity. Ninety-one CCP donors (56 female; 61%) were analyzed. A robust correlation between all SARS-CoV-2 IgG antibodies and ACE2 binding inhibition, as well as a positive correlation between donor age, body mass index, and a negative correlation between time since symptom onset and antibody levels were found. We identified time since symptom onset, normal body mass index (BMI), and the absence of high fever as independent predictors of inadequate neutralization capacity. Gender, duration of symptoms, and number of symptoms were not associated with SARS-CoV-2 IgG antibody levels or neutralization. Neutralizing capacity was correlated with SARS-CoV-2 IgG antibodies and associated with time since symptom onset, BMI, and fever. These clinical parameters can be easily incorporated into the preselection of CCP donors.

Inflammatory CSF profiles and longitudinal development of cognitive decline in sporadic and GBA-associated PD.

Inflammation modifies the incidence and progression of Parkinson's disease (PD). By using 30 inflammatory markers in CSF in 498 people with PD and 67 people with dementia with Lewy bodies (DLB) we show that: (1) levels of ICAM-1, Interleukin-8, MCP-1, MIP-1 beta, SCF and VEGF were associated with clinical scores and neurodegenerative CSF biomarkers (Aß1-42, t-Tau, p181-Tau, NFL and α-synuclein). (2) PD patients with GBA mutations show similar levels of inflammatory markers compared to PD patients without GBA mutations, even when stratified by mutation severity. (3) PD patients who longitudinally developed cognitive impairment during the study had higher levels of TNF-alpha at baseline compared to patients without the development of cognitive impairment. (4) Higher levels of VEGF and MIP-1 beta were associated with a longer duration until the development of cognitive impairment. We conclude that the majority of inflammatory markers is limited in robustly predicting longitudinal trajectories of developing cognitive impairment.

Analytical performance of 17 commercially available point-of-care tests for CRP to support patient management at lower levels of the health system.

Accurate and precise point-of-care (POC) testing for C-reactive protein (CRP) can help support healthcare providers in the clinical management of patients. Here, we compared the analytical performance of 17 commercially available POC CRP tests to enable more decentralized use of the tool. The following CRP tests were evaluated. Eight quantitative tests: QuikRead go (Aidian), INCLIX (Sugentech), Spinit (Biosurfit), LS4000 (Lansionbio), GS 1200 (Gensure Biotech), Standard F200 (SD Biosensor), Epithod 616 (DxGen), IFP-3000 (Xincheng Biological); and nine semi-quantitative tests: Actim CRP (ACTIM), NADAL Dipstick (nal von minden), NADAL cassette (nal von minden), ALLTEST Dipstick (Hangzhou Alltest Biotech), ALLTEST Cassette cut-off 10-40-80 (Hangzhou Alltest Biotech), ALLTEST Cassette cut-off 10-30 (Hangzhou Alltest Biotech), Biotest (Hangzhou Biotest Biotech), BTNX Quad Line (BTNX), BTNX Tri Line (BTNX). Stored samples (n = 660) had previously been tested for CRP using Cobas 8000 Modular analyzer (Roche Diagnostics International AG, Rotkreuz, Switzerland (reference standards). CRP values represented the clinically relevant range (10-100 mg/L) and were grouped into four categories (<10 mg/L, 10-40 mg/L or 10-30 mg/L, 40-80 mg/L or 30-80 mg/L, and > 80mg/L) for majority of the semi-quantitative tests. Among the eight quantitative POC tests evaluated, QuikRead go and Spinit exhibited better agreement with the reference method, showing slopes of 0.963 and 0.921, respectively. Semi-quantitative tests with the four categories showed a poor percentage agreement for the intermediate categories and higher percentage agreement for the lower and upper limit categories. Analytical performance varied considerably for the semi-quantitative tests, especially among the different categories of CRP values. Our findings suggest that quantitative tests might represent the best choice for a variety of use cases, as they can be used across a broad range of CRP categories.

Antibody Binding and Angiotensin-Converting Enzyme 2 Binding Inhibition Is Significantly Reduced for Both the BA.1 and BA.2 Omicron Variants.

BACKGROUND: The rapid emergence of the Omicron variant and its large number of mutations led to its classification as a variant of concern (VOC) by the World Health Organization. Subsequently, Omicron evolved into distinct sublineages (eg, BA.1 and BA.2), which currently represent the majority of global infections. Initial studies of the neutralizing response toward BA.1 in convalescent and vaccinated individuals showed a substantial reduction. METHODS: We assessed antibody (immunoglobulin G [IgG]) binding, ACE2 (angiotensin-converting enzyme 2) binding inhibition, and IgG binding dynamics for the Omicron BA.1 and BA.2 variants compared to a panel of VOCs/variants of interest, in a large cohort (N = 352) of convalescent, vaccinated, and infected and subsequently vaccinated individuals. RESULTS: While Omicron was capable of efficiently binding to ACE2, antibodies elicited by infection or immunization showed reduced binding capacities and ACE2 binding inhibition compared to wild type. Whereas BA.1 exhibited less IgG binding compared to BA.2, BA.2 showed reduced inhibition of ACE2 binding. Among vaccinated samples, antibody binding to Omicron only improved after administration of a third dose. CONCLUSIONS: Omicron BA.1 and BA.2 can still efficiently bind to ACE2, while vaccine/infection-derived antibodies can bind to Omicron. The extent of the mutations within both variants prevents a strong inhibitory binding response. As a result, both Omicron variants are able to evade control by preexisting antibodies.

Pulmonary Surfactant Proteins Are Inhibited by Immunoglobulin A Autoantibodies in Severe COVID-19.

Rationale: Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distress syndrome with fatal outcomes. Evidence suggests that dysregulated immune responses, including autoimmunity, are key pathogenic factors. Objectives: To assess whether IgA autoantibodies target lung-specific proteins and contribute to disease severity. Methods: We collected 147 blood, 9 lung tissue, and 36 BAL fluid samples from three tertiary hospitals in Switzerland and one in Germany. Severe COVID-19 was defined by the need to administer oxygen. We investigated the presence of IgA autoantibodies and their effects on pulmonary surfactant in COVID-19 using the following methods: immunofluorescence on tissue samples, immunoprecipitations followed by mass spectrometry on BAL fluid samples, enzyme-linked immunosorbent assays on blood samples, and surface tension measurements with medical surfactant. Measurements and Main Results: IgA autoantibodies targeting pulmonary surfactant proteins B and C were elevated in patients with severe COVID-19 but not in patients with influenza or bacterial pneumonia. Notably, pulmonary surfactant failed to reduce surface tension after incubation with either plasma or purified IgA from patients with severe COVID-19. Conclusions: Our data suggest that patients with severe COVID-19 harbor IgA autoantibodies against pulmonary surfactant proteins B and C and that these autoantibodies block the function of lung surfactant, potentially contributing to alveolar collapse and poor oxygenation.

Borrelia multiplex: a bead-based multiplex assay for the simultaneous detection of Borrelia specific IgG/IgM class antibodies.

BACKGROUND: Lyme borreliosis (LB) is the most common tick-borne infectious disease in the northern hemisphere. The diagnosis of LB is usually made by clinical symptoms and subsequently supported by serology. In Europe, a two-step testing consisting of an enzyme-linked immunosorbent assay (ELISA) and an immunoblot is recommended. However, due to the low sensitivity of the currently available tests, antibody detection is sometimes inaccurate, especially in the early phase of infection, leading to underdiagnoses. METHODS: To improve upon Borrelia diagnostics, we developed a multiplex Borrelia immunoassay (Borrelia multiplex), which utilizes the new INTELLIFLEX platform, enabling the simultaneous dual detection of IgG and IgM antibodies, saving further time and reducing the biosample material requirement. In order to enable correct classification, the Borrelia multiplex contains eight antigens from the five human pathogenic Borrelia species known in Europe. Six antigens are known to mainly induce an IgG response and two antigens are predominant for an IgM response. RESULTS: To validate the assay, we compared the Borrelia multiplex to a commercial bead-based immunoassay resulting in an overall assay sensitivity of 93.7% (95% CI 84.8-97.5%) and a specificity of 96.5% (95%CI 93.5-98.1%). To confirm the calculated sensitivity and specificity, a comparison with a conventional 2-step diagnostics was performed. With this comparison, we obtained a sensitivity of 95.2% (95% CI 84.2-99.2%) and a specificity of 93.0% (95% CI 90.6-94.7%). CONCLUSION: Borrelia multiplex is a highly reproducible cost- and time-effective assay that enables the profiling of antibodies against several individual antigens simultaneously.

Diminished neutralization responses towards SARS-CoV-2 Omicron VoC after mRNA or vector-based COVID-19 vaccinations.

SARS-CoV-2 variants accumulating immune escape mutations provide a significant risk to vaccine-induced protection against infection. The novel variant of concern (VoC) Omicron BA.1 and its sub-lineages have the largest number of amino acid alterations in its Spike protein to date. Thus, they may efficiently escape recognition by neutralizing antibodies, allowing breakthrough infections in convalescent and vaccinated individuals in particular in those who have only received a primary immunization scheme. We analyzed neutralization activity of sera from individuals after vaccination with all mRNA-, vector- or heterologous immunization schemes currently available in Europe by in vitro neutralization assay at peak response towards SARS-CoV-2 B.1, Omicron sub-lineages BA.1, BA.2, BA.2.12.1, BA.3, BA.4/5, Beta and Delta pseudotypes and also provide longitudinal follow-up data from BNT162b2 vaccinees. All vaccines apart from Ad26.CoV2.S showed high levels of responder rates (96-100%) towards the SARS-CoV-2 B.1 isolate, and minor to moderate reductions in neutralizing Beta and Delta VoC pseudotypes. The novel Omicron variant and its sub-lineages had the biggest impact, both in terms of response rates and neutralization titers. Only mRNA-1273 showed a 100% response rate to Omicron BA.1 and induced the highest level of neutralizing antibody titers, followed by heterologous prime-boost approaches. Homologous BNT162b2 vaccination, vector-based AZD1222 and Ad26.CoV2.S performed less well with peak responder rates of 48%, 56% and 9%, respectively. However, Omicron responder rates in BNT162b2 recipients were maintained in our six month longitudinal follow-up indicating that individuals with cross-protection against Omicron maintain it over time. Overall, our data strongly argue for booster doses in individuals who were previously vaccinated with BNT162b2, or a vector-based primary immunization scheme.

Stabilized recombinant SARS-CoV-2 spike antigen enhances vaccine immunogenicity and protective capacity.

The SARS-CoV-2 spike (S) glycoprotein is synthesized as a large precursor protein and must be activated by proteolytic cleavage into S1 and S2. A recombinant modified vaccinia virus Ankara (MVA) expressing native, full-length S protein (MVA-SARS-2-S) is currently under investigation as a candidate vaccine in phase I clinical studies. Initial results from immunogenicity monitoring revealed induction of S-specific antibodies binding to S2, but low-level antibody responses to the S1 domain. Follow-up investigations of native S antigen synthesis in MVA-SARS-2-S-infected cells revealed limited levels of S1 protein on the cell surface. In contrast, we found superior S1 cell surface presentation upon infection with a recombinant MVA expressing a stabilized version of SARS-CoV-2 S protein with an inactivated S1/S2 cleavage site and K986P and V987P mutations (MVA-SARS-2-ST). When comparing immunogenicity of MVA vector vaccines, mice vaccinated with MVA-SARS-2-ST mounted substantial levels of broadly reactive anti-S antibodies that effectively neutralized different SARS-CoV-2 variants. Importantly, intramuscular MVA-SARS-2-ST immunization of hamsters and mice resulted in potent immune responses upon challenge infection and protected from disease and severe lung pathology. Our results suggest that MVA-SARS-2-ST represents an improved clinical candidate vaccine and that the presence of plasma membrane-bound S1 is highly beneficial to induce protective antibody levels.

Longitudinal cellular and humoral immune responses after triple BNT162b2 and fourth full-dose mRNA-1273 vaccination in haemodialysis patients.

Haemodialysis patients respond poorly to vaccination and continue to be at-risk for severe COVID-19. Therefore, dialysis patients were among the first for which a fourth COVID-19 vaccination was recommended. However, targeted information on how to best maintain immune protection after SARS-CoV-2 vaccinations in at-risk groups for severe COVID-19 remains limited. We provide, to the best of our knowledge, for the first time longitudinal vaccination response data in dialysis patients and controls after a triple BNT162b2 vaccination and in the latter after a subsequent fourth full-dose of mRNA-1273. We analysed systemic and mucosal humoral IgG responses against the receptor-binding domain (RBD) and ACE2-binding inhibition towards variants of concern including Omicron and Delta with multiplex-based immunoassays. In addition, we assessed Spike S1-specific T-cell responses by interferon γ release assay. After triple BNT162b2 vaccination, anti-RBD B.1 IgG and ACE2 binding inhibition reached peak levels in dialysis patients, but remained inferior compared to controls. Whilst we detected B.1-specific ACE2 binding inhibition in 84% of dialysis patients after three BNT162b2 doses, binding inhibition towards the Omicron variant was only detectable in 38% of samples and declining to 16% before the fourth vaccination. By using mRNA-1273 as fourth dose, humoral immunity against all SARS-CoV-2 variants tested was strongly augmented with 80% of dialysis patients having Omicron-specific ACE2 binding inhibition. Modest declines in T-cell responses in dialysis patients and controls after the second vaccination were restored by the third BNT162b2 dose and significantly increased by the fourth vaccination. Our data support current advice for a four-dose COVID-19 immunisation scheme for at-risk individuals such as haemodialysis patients. We conclude that administration of a fourth full-dose of mRNA-1273 as part of a mixed mRNA vaccination scheme to boost immunity and to prevent severe COVID-19 could also be beneficial in other immune impaired individuals. Additionally, strategic application of such mixed vaccine regimens may be an immediate response against SARS-CoV-2 variants with increased immune evasion potential.

Long COVID symptoms in exposed and infected children, adolescents and their parents one year after SARS-CoV-2 infection: A prospective observational cohort study.

BACKGROUND: Long COVID in children and adolescents remains poorly understood due to a lack of well-controlled studies with long-term follow-up. In particular, the impact of the family context on persistent symptoms following SARS-CoV-2 infection remains unknown. We examined long COVID symptoms in a cohort of infected children, adolescents, and adults and their exposed but non-infected household members approximately 1 year after infection and investigated clustering of persistent symptoms within households. METHODS: 1267 members of 341 households (404 children aged <14 years, 140 adolescents aged 14-18 years and 723 adults) were categorized as having had either a SARS-CoV-2 infection or household exposure to SARS-CoV-2 without infection, based on three serological assays and history of laboratory-confirmed infection. Participants completed questionnaires assessing the presence of long COVID symptoms 11-12 months after infection in the household using online questionnaires. FINDINGS: The prevalence of moderate or severe persistent symptoms was statistically significantly higher in infected than in exposed women (36.4% [95% CI: 30.7-42.4%] vs 14.2% [95% CI: 8.7-21.5%]), infected men (22.9% [95% CI: 17.9-28.5%] vs 10.3% [95% CI: 5.8-16.9%]) and infected adolescent girls (32.1% 95% CI: 17.2-50.5%] vs 8.9% [95%CI: 3.1-19.8%]). However, moderate or severe persistent symptoms were not statistically more common in infected adolescent boys aged 14-18 (9.7% [95% CI: 2.8-23.6%] or in infected children <14 years (girls: 4.3% [95% CI: 1.2-11.0%]; boys: 3.7% [95% CI: 1.1-9.6%]) than in their exposed counterparts (adolescent boys: 0.0% [95% CI: 0.0-6.7%]; girls < 14 years: 2.3% [95% CI: 0·7-6·1%]; boys < 14 years: 0.0% [95% CI: 0.0-2.0%]). The number of persistent symptoms reported by individuals was associated with the number of persistent symptoms reported by their household members (IRR=1·11, p=·005, 95% CI [1.03-1.20]). INTERPRETATION: In this controlled, multi-centre study, infected men, women and adolescent girls were at increased risk of negative outcomes 11-12 months after SARS-CoV-2 infection. Amongst non-infected adults, prevalence of negative outcomes was also high. Prolonged symptoms tended to cluster within families, suggesting family-level interventions for long COVID could prove useful. FUNDING: Ministry of Science, Research and the Arts, Baden-Württemberg, Germany.

COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants.

As global vaccination campaigns against SARS-CoV-2 proceed, there is particular interest in the longevity of immune protection, especially with regard to increasingly infectious virus variants. Neutralizing antibodies (Nabs) targeting the receptor binding domain (RBD) of SARS-CoV-2 are promising correlates of protective immunity and have been successfully used for prevention and therapy. As SARS-CoV-2 variants of concern (VOCs) are known to affect binding to the ACE2 receptor and by extension neutralizing activity, we developed a bead-based multiplex ACE2-RBD inhibition assay (RBDCoV-ACE2) as a highly scalable, time-, cost-, and material-saving alternative to infectious live-virus neutralization tests. By mimicking the interaction between ACE2 and the RBD, this serological multiplex assay allows the simultaneous analysis of ACE2 binding inhibition to the RBDs of all SARS-CoV-2 VOCs and variants of interest (VOIs) in a single well. Following validation against a classical virus neutralization test and comparison of performance against a commercially available assay, we analyzed 266 serum samples from 168 COVID-19 patients of varying severity. ACE2 binding inhibition was reduced for ten out of eleven variants examined compared to wild-type, especially for those displaying the E484K mutation such as VOCs beta and gamma. ACE2 binding inhibition, while highly individualistic, positively correlated with IgG levels. ACE2 binding inhibition also correlated with disease severity up to WHO grade 7, after which it reduced.