Measuring Variant-Specific Neutralizing Antibody Profiles after Bivalent SARS-CoV-2 Vaccinations Using a Multivariant Surrogate Virus Neutralization Microarray.

The capability of antibodies to neutralize different SARS-CoV-2 variants varies among individuals depending on the previous exposure to wild-type or Omicron-specific immunogens by mono- or bivalent vaccinations or infections. Such profiles of neutralizing antibodies (nAbs) usually have to be assessed via laborious live-virus neutralization tests (NTs). We therefore analyzed whether a novel multivariant surrogate-virus neutralization test (sVNT) (adapted from a commercial microarray) that quantifies the antibody-mediated inhibition between the receptor angiotensin-converting enzyme 2 (ACE2) and variant-specific receptor-binding domains (RBDs) can assess the neutralizing activity against the SARS-CoV-2 wild-type, and Delta Omicron BA.1, BA.2, and BA.5 subvariants after a booster with Omicron-adapted bivalent vaccines in a manner similar to live-virus NTs. Indeed, by using the live-virus NTs as a reference, we found a significant correlation between the variant-specific NT titers and levels of ACE2-RBD binding inhibition (p < 0.0001, r ≤ 0.78 respectively). Furthermore, the sVNTs identified higher inhibition values against BA.5 and BA.1 in individuals vaccinated with Omicron-adapted vaccines than in those with monovalent wild-type vaccines. Our data thus demonstrate the ability of sVNTs to detect variant-specific nAbs following a booster with bivalent vaccines.

Effect of previous heterologous flavivirus vaccinations on human antibody responses in tick-borne encephalitis and dengue virus infections.

Arthropod-borne flaviviruses include a number of medically relevant human pathogens such as the mosquito-borne dengue (DEN), Zika, and yellow fever (YF) viruses as well as tick-borne encephalitis virus (TBEV). All flaviviruses are antigenically related and anamnestic responses due to prior immunity can modulate antibody specificities in subsequent infections or vaccinations. In our study, we analyzed the induction of broadly flavivirus cross-reactive antibodies in tick-borne encephalitis (TBE) and DEN patients without or with prior flavivirus exposure through TBE and/or YF vaccination, and determined the contribution of these antibodies to TBE and dengue virus (DENV) neutralization. In addition, we investigated the formation of cross-reactive antibodies in TBE-vaccination breakthroughs (VBTs). A TBEV infection without prior YF or TBE vaccination induced predominantly type-specific antibodies. In contrast, high levels of broadly cross-reactive antibodies were found in samples from TBE patients prevaccinated against YF as well as in DEN patients prevaccinated against TBE and/or YF. While these cross-reactive antibodies did not neutralize TBEV, they were effective in neutralizing DENV. This discrepancy points to structural differences between the two viruses and indicates that broadly cross-reactive epitopes are less accessible in TBEV than in DENV. In TBE VBT infections, type-specific antibodies dominated the antibody response, thus revealing no difference from that of unvaccinated TBE patients. Our results emphasize significant differences in the structural properties of different flaviviruses that have an impact on the induction of broadly cross-reactive antibodies and their functional activities in virus neutralization.

Interrogating ligand-receptor interactions using highly sensitive cellular biosensors.

Interactions of membrane-resident proteins are important targets for therapeutic interventions but most methods to study them are either costly, laborious or fail to reflect the physiologic interaction of membrane resident proteins in trans. Here we describe highly sensitive cellular biosensors as a tool to study receptor-ligand pairs. They consist of fluorescent reporter cells that express chimeric receptors harboring ectodomains of cell surface molecules and intracellular signaling domains. We show that a broad range of molecules can be integrated into this platform and we demonstrate its applicability to highly relevant research areas, including the characterization of immune checkpoints and the probing of cells for the presence of receptors or ligands. The platform is suitable to evaluate the interactions of viral proteins with host receptors and to test for neutralization capability of drugs or biological samples. Our results indicate that cellular biosensors have broad utility as a tool to study protein-interactions.

Monitoring of Sotrovimab-Levels as Pre-Exposure Prophylaxis in Kidney Transplant Recipients Not Responding to SARS-CoV-2 Vaccines.

Background Sotrovimab, a monoclonal antibody against SARS-CoV-2, is used as a pre-exposition prophylaxis (PrEP) against COVID-19, but monitoring strategies using routine test systems have not been defined. Methods Twenty kidney transplant recipients without antibodies after vaccination received 500 mg Sotrovimab. Antibody levels were quantified over eight weeks using live-virus neutralization (BA1 and BA2), antibody binding assays (TrimericS, Elecsys, QuantiVAC) and surrogate virus neutralization tests (sVNTs; TECOmedical, cPass and NeutraLISA). Results Sotrovimab neutralized both Omicron subvariants (BA1 NT titer 90 (+-50) > BA2 NT titer 33 (+-15) one hour post infusion). Sotrovimab was measurable on all used immunoassays, although a prior 1:100 dilution was necessary for Elecsys due to a presumed prozone effect. The best correlation with live-virus neutralization titers was found for QuantiVAC and TrimericS, with a respective R2 of 0.65/0.59 and 0.76/0.57 against BA1/BA2. Elecsys showed an R2 of 0.56/0.54 for BA1/BA2, respectively. sVNT values increased after infusion but had only a poor correlation with live-virus neutralization titers (TECOmedical and cPass) or did not reach positivity thresholds (NeutraLISA). Conclusion Antibody measurements by the used immunoassays showed differences in antibody levels and only a limited correlation with neutralization capacity. We do not recommend sVNTs for monitoring SARS-CoV-2 neutralization by Sotrovimab.

Bivalent COVID-19 mRNA booster vaccination (BA.1 or BA.4/BA.5) increases neutralization of matched Omicron variants.

We report SARS-CoV-2 neutralizing antibody titers in sera of triple-vaccinated individuals who received a booster dose of an original monovalent or a bivalent BA.1- or BA.4/BA.5-adapted vaccine or had a breakthrough infection with Omicron variants BA.1, BA.2 or BA.4/BA.5. A bivalent BA.4/BA.5 booster or Omicron-breakthrough infection induced increased Omicron-neutralization titers compared with the monovalent booster. The XBB.1.5 variant effectively evaded neutralizing-antibody responses elicited by current vaccines and/or infection with previous variants.

A Multivariant Surrogate Neutralization Assay Identifies Variant-Specific Neutralizing Antibody Profiles in Primary SARS-CoV-2 Omicron Infection.

Primary infection with the Omicron variant of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) can be serologically identified with distinct profiles of neutralizing antibodies (nAbs), as indicated by high titers against the Omicron variant and low titers against the ancestral wild-type (WT). Here, we evaluated whether a novel surrogate virus neutralization assay (sVNT) that simultaneously quantifies the binding inhibition of angiotensin-converting enzyme 2 (ACE2) to the proteins of the WT- and Omicron-specific receptor-binding domains (RBDs) can identify nAb profiles after primary Omicron infection with accuracy similar to that of variant-specific live-virus neutralization tests (NTs). Therefore, we comparatively tested 205 samples from individuals after primary infection with the Omicron variant and the WT, and vaccinated subjects with or without Omicron breakthrough infections. Indeed, variant-specific RBD-ACE2 binding inhibition levels significantly correlated with respective NT titers (p < 0.0001, Spearman's r = 0.92 and r = 0.80 for WT and Omicron, respectively). In addition, samples from individuals after primary Omicron infection were securely identified with the sVNT according to their distinctive nAb profiles (area under the curve = 0.99; sensitivity: 97.2%; specificity: 97.84%). Thus, when laborious live-virus NTs are not feasible, the novel sVNT we evaluated in this study may serve as an acceptable substitute for the serological identification of individuals with primary Omicron infection.

Serological screening for tick-borne encephalitis virus in eight Norwegian herds of semi-domesticated reindeer (Rangifer tarandus tarandus).

Tick-borne encephalitis virus (TBEV) is found in Ixodes ricinus ticks throughout the area where viable tick populations exist. In Norway, TBEV is found in I. ricinus from the south coast until Brønnøy municipality in Nordland County and the range of the vector is expanding due to changes in climate, vegetation, host animals and environmental conditions. TBEV might thus have the potential to establish in new areas when I. ricinus expand its geographical distribution. At present, there is little knowledge on the status of the virus in high-altitude areas of inland regions in Norway. It has previously been indicated that reindeer may be an important sentinel species and indicator of the spread of ticks and TBEV in high-altitude regions. In this study, 408 semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus) from eight herds, from Tana in Troms and Finnmark County in northern Norway to Filefjell in Innlandet and Viken Counties in southern Norway, were screened for TBEV antibodies using a commercial enzyme-linked immunosorbent assay (ELISA). We found 16 TBEV reactive reindeer samples by ELISA; however, these results could not be confirmed by the serum neutralization test (SNT). This could indicate that a flavivirusand not necessarily TBEV, may be circulating among Norwegian semi-domesticated reindeer. The results also indicate that TBEV was not enzootic in Norwegian semi-domesticated reindeer in 2013-2015. This knowledge is important as an information base for future TBEV and flavivirus surveillance in Norway.

Three rounds of a national external quality assessment reveal a link between disharmonic anti-SARS-CoV-2 antibody quantifications and the infection stage.

OBJECTIVES: The WHO's standardized measuring unit, "binding antibody units per milliliter (BAU/mL)," should allow the harmonization of quantitative results by different commercial Anti-SARS-CoV-2 immunoassays. However, multiple studies demonstrate inter-assay discrepancies. The antigenic changes of the Omicron variant affect the performance of Spike-specific immunoassays. This study evaluated the variation of quantitative Anti-SARS-CoV-2-Spike antibody measurements among 46, 50, and 44 laboratories in three rounds of a national external quality assessment (EQA) prior to and after the emergence of the Omicron variant in a diagnostic near-to-real-life setting. METHODS: We analyzed results reported by the EQA participant laboratories from single and sequential samples from SARS-CoV-2 convalescent, acutely infected, and vaccinated individuals, including samples obtained after primary and breakthrough infections with the Omicron variant. RESULTS: The three immunoassays most commonly used by the participants displayed a low intra-assay and inter-laboratory variation with excellent reproducibility using identical samples sent to the participants in duplicates. In contrast, the inter-assay variation was very high with all samples. Notably, the ratios of BAU/mL levels quantified by different immunoassays were not equal among all samples but differed between vaccination, past, and acute infection, including primary infection with the Omicron variant. The antibody kinetics measured in vaccinated individuals strongly depended on the applied immunoassay. CONCLUSIONS: Measured BAU/mL levels are only inter-changeable among different laboratories when the same assay was used for their assessment. Highly variable ratios of BAU/mL quantifications among different immunoassays and infection stages argue against the usage of universal inter-assay conversion factors.

The accelerated waning of immunity and reduced effect of booster in patients treated with bDMARD and tsDMARD after SARS-CoV-2 mRNA vaccination.

Objectives: This study aimed to assess the duration of humoral responses after two doses of SARS-CoV-2 mRNA vaccines in patients with inflammatory joint diseases and IBD and booster vaccination compared with healthy controls. It also aimed to analyze factors influencing the quantity and quality of the immune response. Methods: We enrolled 41 patients with rheumatoid arthritis (RA), 35 with seronegative spondyloarthritis (SpA), and 41 suffering from inflammatory bowel disease (IBD), excluding those receiving B-cell-depleting therapies. We assessed total anti-SARS-CoV-2 spike antibodies (Abs) and neutralizing Ab titers 6 months after two and then after three doses of mRNA vaccines compared with healthy controls. We analyzed the influence of therapies on the humoral response. Results: Patients receiving biological or targeted synthetic disease-modifying antirheumatic drugs (b/tsDMARDs) showed reduced anti-SARS-CoV-2 S Abs and neutralizing Ab titers compared with HC or patients receiving conventional synthetic (cs)DMARDs 6 months after the first two vaccination doses. Anti-SARS-CoV-2 S titers of patients with b/tsDMARDs declined more rapidly, leading to a significant reduction in the duration of vaccination-induced immunity after two doses of SARS-CoV-2 mRNA vaccines. While 23% of HC and 19% of patients receiving csDMARDs were without detectable neutralizing Abs 6 months after the first two vaccination doses, this number was 62% in patients receiving b/tsDMARDs and 52% in patients receiving a combination of csDMARDs and b/tsDMARDs. Booster vaccination led to increased anti-SARS-CoV-2 S Abs in all HC and patients. However, anti-SARS-CoV-2 S Abs after booster vaccination was diminished in patients receiving b/tsDMARDs, either alone or in combination with csDMARDs compared to HC. Conclusion: Patients receiving b/tsDMARDs have significantly reduced Abs and neutralizing Ab titers 6 months after mRNA vaccination against SARS-CoV-2. This was due to a faster decline in Ab levels, indicating a significantly reduced duration of vaccination-induced immunity compared with HC or patients receiving csDMARDs. In addition, they display a reduced response to a booster vaccination, warranting earlier booster vaccination strategies in patients under b/tsDMARD therapy, according to their specific Ab levels.

Comprehensive Comparison of Seven SARS-CoV-2-Specific Surrogate Virus Neutralization and Anti-Spike IgG Antibody Assays Using a Live-Virus Neutralization Assay as a Reference.

Neutralizing antibodies (nAbs) are considered a valuable marker for measuring humoral immunity against SARS-CoV-2. However, live-virus neutralization tests (NTs) require high-biosafety-level laboratories and are time-consuming. Therefore, surrogate virus neutralization tests (sVNTs) have been widely applied, but unlike most anti-spike (S) antibody assays, NTs and sVNTs are not harmonized, requiring further evaluation and comparative analyses. This study compared seven commercial sVNTs and anti-S-antibody assays with a live-virus NT as a reference, using a panel of 720 single and longitudinal serum samples from 666 convalescent patients after SARS-CoV-2 infection. The sensitivity of these assays for detecting antibodies ranged from 48 to 94% after PCR-confirmed infection and from 56% to 100% relative to positivity in the in-house live-virus NT. Furthermore, we performed receiver operating characteristic (ROC) curve analyses to determine which immunoassays were most suitable for assessing nAb titers exceeding a specific cutoff (NT titer, ≥80) and found that the NeutraLISA and the cPass assays reached the highest area under the curve (AUC), exceeding 0.91. In addition, when the assays were compared for their correlation with nAb kinetics over time in a set of longitudinal samples, the extent of the measured decrease of nAbs after infection varied widely among the evaluated immunoassays. Finally, in vaccinated convalescent patients, high titers of nAbs exceeded the upper limit of the evaluated assays' quantification ranges. Based on data from this study, we conclude that commercial immunoassays are acceptable substitutes for live-virus NTs, particularly when additional adapted cutoffs are employed to detect nAbs beyond a specific threshold titer. IMPORTANCE While the measurement of neutralizing antibodies is considered a valuable tool in assessing protection against SARS-CoV-2, neutralization tests employ live-virus isolates and cell culture, requiring advanced laboratory biosafety levels. Including a large sample panel (over 700 samples), this study provides adapted cutoff values calculated for seven commercial immunoassays (including four surrogate neutralization assays and a protein-based microarray) that robustly correlate with specific titers of neutralizing antibodies.

Limited role of children in transmission of SARS-CoV-2 virus in households-Immunological analysis of 26 familial clusters.

BACKGROUND: The impact of children on the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains uncertain. This study provides an insight into distinct patterns of SARS-CoV-2 household transmission in case of pediatric and adult index cases as well as age-dependent susceptibility to SARS-CoV-2 infection. METHODS: Immune analysis, medical interviewing, and contact tracing of 26 families with confirmed SARS-CoV-2 infection cases have been conducted. Blood samples were analyzed serologically with the use of a SARS-CoV-2-specific IgG assay and virus neutralization test (VNT). Uni- and multivariable linear regression and mixed effect logistic regression models were used to describe potential risk factors for higher contagiousness and susceptibility to SARS-CoV-2 infection. RESULTS: SARS-CoV-2 infection could be confirmed in 67 of 124 family members. Fourteen children and 11 adults could be defined as index cases in their households. Forty of 82 exposed family members were defined as secondarily infected. The mean secondary attack rate in households was 0.48 and was significantly higher in households with adult than with pediatric index cases (0.85 vs 0.19; p < 0.0001). The age (grouped into child and adult) of index case, severity of disease, and occurrence of lower respiratory symptoms in index cases were significantly associated with secondary transmission rates in households. Children seem to be equally susceptible to acquire a SARS-CoV-2 infection as adults, but they suffer milder courses of the disease or remain asymptomatic. CONCLUSION: SARS-CoV-2 transmission from infected children to other household members occurred rarely in the first wave of the pandemic, despite close physical contact and the lack of hygienic measures.

Tick-Borne Encephalitis in Vaccinated Patients: A Retrospective Case-Control Study and Analysis of Vaccination Field Effectiveness in Austria From 2000 to 2018.

BACKGROUND: There are discrepant observations on the severity of tick-borne encephalitis (TBE) in vaccinated persons. We, therefore, analyzed the occurrence of severe and mild disease in hospitalized vaccinated and nonvaccinated patients with TBE and determined the field effectiveness (FE) of vaccination against these forms of disease. METHODS: The study covered all patients hospitalized with TBE in Austria from 2000 to 2018. Clinical diagnoses in vaccinated and age- and sex-matched nonvaccinated patients were compared in a nested case-control study. FE was calculated based on vaccination coverage and incidences in the nonvaccinated and vaccinated population. RESULTS: Of 1545 patients hospitalized with TBE, 206 were vaccinated. In those, a higher proportion of severe TBE was observed, especially in children. FE was high in all age groups and against all forms of disease. The higher proportion of severe TBE can be explained by a lower FE against severe than against mild disease, a difference especially pronounced in children (FE, 82.7% for severe vs 94.7% for mild disease). CONCLUSIONS: The FE of TBE vaccination is excellent. The observed higher proportion of severe disease in vaccinated persons with TBE does not reflect a higher risk associated with vaccination but is rather due to a somewhat lower FE against severe TBE. Because this effect was more pronounced in children, we recommend adapting the immunization schedule.

Safety and immunogenicity of a third COVID-19 vaccination in patients with immune-mediated inflammatory diseases compared with healthy controls.

OBJECTIVES: A third COVID-19 vaccination is recommended for immunosuppressed patients. However, data on immunogenicity and safety of a third COVID-19 vaccination in patients with immune-mediated inflammatory diseases (IMIDs) are sparse and therefore addressed within this clinical trial. METHODS: 60 immunosuppressed patients and 48 healthy controls (HCs) received a third vaccination with an mRNA vaccine. The primary endpoint was defined as the presence of antibody levels against the receptor-binding domain (RBD)>1500 BAU/mL in patients with IMIDs versus HCs. Further endpoints included differences in neutralising antibodies and cellular immune responses after the third vaccination. Reactogenicity was recorded for 7 days, and safety was evaluated until week 4. RESULTS: Rate of individuals with anti-RBD antibodies>1500 BAU/mL was not significantly different after the third vaccination between patients with IMIDs and HCs (91% vs 100% p=0.101). Anti-RBD and neutralising antibody levels were significantly lower in patients with IMIDs after the third vaccination than in HCs (p=0.002 and p=0.016, respectively). In contrast, fold increase in antibody levels between week 0 and 4 was higher in patients with IMIDs. Treatment with biological (b) disease-modifying anti-rheumatic drugs (DMARD) or combination of bDMARDs and conventional synthetic DMARDs was associated with reduced antibody levels. Enhanced cellular immune response to wild type and Omicron peptide stimulation was observed after the third vaccination. No serious adverse event was attributed to the third vaccination. CONCLUSION: Our clinical trial data support the immunogenicity and safety of a third COVID-19 vaccination in patients with IMIDs. However, effects of DMARD therapy on immunogenicity should be considered. TRIAL REGISTRATION NUMBER: EudraCT No: 2021-002693-10.

Impact of structural dynamics on biological functions of flaviviruses.

Flaviviruses comprise a number of mosquito- or tick-transmitted human pathogens of global public health importance. Advances in structural biology techniques have contributed substantially to our current understanding of the life cycle of these small enveloped RNA viruses and led to deep insights into details of virus assembly, maturation and cell entry. In addition to large-scale conformational changes and oligomeric rearrangements of envelope proteins during these processes, there is increasing evidence that smaller-scale protein dynamics (referred to as virus "breathing") can confer extra flexibility to these viruses for the fine-tuning of their interactions with the immune system and possibly with cellular factors they encounter in their complex ecological cycles in arthropod and vertebrate hosts. In this review, we discuss how work with tick-borne encephalitis virus has extended our view on flavivirus breathing, leading to the identification of a novel mechanism of antibody-mediated infection enhancement and demonstrating breathing intermediates of the envelope protein in the process of membrane fusion. These data are discussed in the context of other flaviviruses and the perspective of a potential role of virus breathing to cope with the requirements of adaptation and replication in evolutionarily very different hosts.

Reactogenicity and immunogenicity of the second COVID-19 vaccination in patients with inborn errors of immunity or mannan-binding lectin deficiency.

Background: Patients with inborn errors of immunity (IEI) are at increased risk for severe courses of SARS-CoV-2 infection. COVID-19 vaccination provides effective protection in healthy individuals. However, it remains unclear whether vaccination is efficient and safe in patients with constitutional dysfunctions of the immune system. Thus, we analyzed the humoral response, adverse reactions and assessed the disease activity of the underlying disease after COVID-19 vaccination in a cohort of patients suffering from IEIs or mannan-binding lectin deficiency (MBLdef). Methods: Vaccination response was assessed after basic immunization using the Elecsys anti-SARS-CoV-2 S immunoassay and via Vero E6 cell based assay to detect neutralization capabilities. Phenotyping of lymphocytes was performed by flow cytometry. Patient charts were reviewed for disease activity, autoimmune phenomena as well as immunization status and reactogenicity of the vaccination. Activity of the underlying disease was assessed using a patient global numeric rating scale (NRS). Results: Our cohort included 11 individuals with common variable immunodeficiency (CVID), one patient with warts hypogammaglobulinemia immunodeficiency myelokathexis (WHIM) syndrome, two patients with X-linked agammaglobulinemia (XLA), one patient with Muckle Wells syndrome, two patients with cryopyrin-associated periodic syndrome, one patient with Interferon-gamma (IFN-gamma) receptor defect, one patient with selective deficiency in pneumococcal antibody response combined with a low MBL level and seven patients with severe MBL deficiency. COVID-19 vaccination was generally well tolerated with little to no triggering of autoimmune phenomena. 20 out of 26 patients developed an adequate humoral vaccine response. 9 out of 11 patients developed a T cell response comparable to healthy control subjects. Tested immunoglobulin replacement therapy (IgRT) preparations contained Anti-SARS-CoV-2 S antibodies implicating additional protection through IgRT. Summary: In summary the data support the efficacy and safety of a COVID-19 vaccination in patients with IEIs/MBLdef. We recommend evaluation of the humoral immune response and testing for virus neutralization after vaccination in this cohort.

Heterologous vector versus homologous mRNA COVID-19 booster vaccination in non-seroconverted immunosuppressed patients: a randomized controlled trial.

Impaired response to COVID-19 vaccination is of particular concern in immunosuppressed patients. To determine the best vaccination strategy for this vulnerable group we performed a single center, 1:1 randomized blinded clinical trial. Patients who failed to seroconvert upon two mRNA vaccinations (BNT162b2 or mRNA-1273) are randomized to receive either a third dose of the same mRNA or the vector vaccine ChAdOx1 nCoV-19. Primary endpoint is the difference in SARS-CoV-2 spike antibody seroconversion rate between vector and mRNA vaccinated patients four weeks after the third dose. Secondary outcomes include cellular immune responses. Seroconversion rates at week four are significantly higher in the mRNA (homologous vaccination, 15/24, 63%) as compared to the vector vaccine group (heterologous vaccination, 4/22, 18%). SARS-CoV-2-specific T-cell responses are reduced but could be increased after a third dose of either vector or mRNA vaccine. In a multivariable logistic regression analysis, patient age and vaccine type are associated with seroconversion. No serious adverse event is attributed to COVID-19 booster vaccination. Efficacy and safety data underline the importance of a booster vaccination and support the use of a homologous mRNA booster vaccination in immunosuppressed patients.Trial registration: EudraCT No.: 2021-002693-10.

Immunogenicity and safety of a fourth COVID-19 vaccination in rituximab-treated patients: an open-label extension study.

OBJECTIVES: Patients under rituximab therapy are at high risk for a severe COVID-19 disease course. Humoral immune responses to SARS-CoV-2 vaccination are vastly diminished in B-cell-depleted patients, even after a third vaccine dose. However, it remains unclear whether these patients benefit from a fourth vaccination and whether continued rituximab therapy affects antibody development. METHODS: In this open-label extension trial, 37 rituximab-treated patients who received a third dose with either a vector or mRNA-based vaccine were vaccinated a fourth time with an mRNA-based vaccine (mRNA-1273 or BNT162b2). Key endpoints included the humoral and cellular immune response as well as safety after a fourth vaccination. RESULTS: The number of patients who seroconverted increased from 12/36 (33%) to 21/36 (58%) following the fourth COVID-19 vaccination. In patients with detectable antibodies to the spike protein's receptor-binding domain (median: 8.0 binding antibody units (BAU)/mL (quartiles: 0.4; 13.8)), elevated levels were observed after the fourth vaccination (134.0 BAU/mL (quartiles: 25.5; 1026.0)). Seroconversion and antibody increase were strongly diminished in patients who received rituximab treatment between the third and the fourth vaccination. The cellular immune response declined 12 weeks after the third vaccination, but could only be slightly enhanced by a fourth vaccination. No unexpected safety signals were detected, one serious adverse event not related to vaccination occurred. CONCLUSIONS: A fourth vaccine dose is immunogenic in a fraction of rituximab-treated patients. Continuation of rituximab treatment reduced humoral immune response, suggesting that rituximab affects a second booster vaccination. It might therefore be considered to postpone rituximab treatment in clinically stable patients. TRIAL REGISTRATION NUMBER: 2021-002348-57.

Different Neutralization Profiles After Primary SARS-CoV-2 Omicron BA.1 and BA.2 Infections.

Background and Methods: The SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Omicron (B.1.1.529) variant is the antigenically most distinct variant to date. As the heavily mutated spike protein enables neutralization escape, we studied serum-neutralizing activities of naïve and vaccinated individuals after Omicron BA.1 or BA.2 sub-lineage infections in live virus neutralization tests with Omicron BA.1, Omicron BA.2, wildtype (WT, B1.1), and Delta (B.1.617.2) strains. Serum samples obtained after WT infections and three-dose mRNA vaccinations with and without prior infection were included as controls. Results: Primary BA.1 infections yielded reduced neutralizing antibody levels against WT, Delta, and Omicron BA.2, while samples from BA.2-infected individuals showed almost no cross-neutralization against the other variants. Serum neutralization of Omicron BA.1 and BA.2 variants was detectable after three-dose mRNA vaccinations, but with reduced titers. Vaccination-breakthrough infections with either Omicron BA.1 or BA.2, however, generated equal cross-neutralizing antibody levels against all SARS-CoV-2 variants tested. Conclusions: Our study demonstrates that although Omicron variants are able to enhance cross-neutralizing antibody levels in pre-immune individuals, primary infections with BA.1 or BA.2 induced mostly variant-specific neutralizing antibodies, emphasizing the differently shaped humoral immunity induced by the two Omicron variants. These data thus contribute substantially to the understanding of antibody responses induced by primary Omicron infections or multiple exposures to different SARS-CoV-2 variants and are of particular importance for developing vaccination strategies in the light of future emerging variants.