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.

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.

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.

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.

Seroprevalence of Hepatitis E virus in children and adolescents living in urban Bogotá: An explorative cross-sectional study.

The majority of Hepatitis E Virus (HEV)-related studies are carried out in adults whereas information about HEV seroprevalence, clinical disease manifestation, molecular epidemiology, and transmission patterns in children is limited. To estimate HEV seroprevalence among scholar children living in an urban setting and to analyze risk factors for an infection, we invited children aged 5-18 years from Bogotá (Colombia) for a cross-sectional survey. We collected self-reported data on demographics, social, clinical, and exposure variables in a structured interview. Venous blood samples were analyzed with two commercially available ELISAs for HEV-specific IgG antibodies. Among the 263 participants, we found three HEV IgG-reactive samples (1.1%) using both assays. We additionally characterized the samples for HEV IgM using a commercially available IgM ELISA and for HEV RNA. Here, we found one IgM-reactive sample, which was also reactive for IgG. In contrast, none of the IgM- and IgG-reactive sera samples showed detectable RNA levels indicating HEV exposure had not been recently. All participants reported access to drinking water and sanitary systems in their households and frequent hand washing routines (76-88%). Eighty percent of children reported no direct contact with pigs, but occasional pork consumption was common (90%). In contrast to the majority of studies performed in Colombian adults, we found a low unadjusted HEV seroprevalence of 1.1% (95% CI: 0.3-3.6%) for both HEV IgG ELISAs in our study population. While the majority of participants reported pork consumption, we speculate in the absence of viral RNA for genotyping in the affected individuals, that existing access to drinking water and sanitary systems within our study group contribute to the low HEV seroprevalence.

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.

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.

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.

Human serum from SARS-CoV-2-vaccinated and COVID-19 patients shows reduced binding to the RBD of SARS-CoV-2 Omicron variant.

BACKGROUND: The COVID-19 pandemic is caused by the betacoronavirus SARS-CoV-2. In November 2021, the Omicron variant was discovered and immediately classified as a variant of concern (VOC), since it shows substantially more mutations in the spike protein than any previous variant, especially in the receptor-binding domain (RBD). We analyzed the binding of the Omicron RBD to the human angiotensin-converting enzyme-2 receptor (ACE2) and the ability of human sera from COVID-19 patients or vaccinees in comparison to Wuhan, Beta, or Delta RBD variants. METHODS: All RBDs were produced in insect cells. RBD binding to ACE2 was analyzed by ELISA and microscale thermophoresis (MST). Similarly, sera from 27 COVID-19 patients, 81 vaccinated individuals, and 34 booster recipients were titrated by ELISA on RBDs from the original Wuhan strain, Beta, Delta, and Omicron VOCs. In addition, the neutralization efficacy of authentic SARS-CoV-2 wild type (D614G), Delta, and Omicron by sera from 2× or 3× BNT162b2-vaccinated persons was analyzed. RESULTS: Surprisingly, the Omicron RBD showed a somewhat weaker binding to ACE2 compared to Beta and Delta, arguing that improved ACE2 binding is not a likely driver of Omicron evolution. Serum antibody titers were significantly lower against Omicron RBD compared to the original Wuhan strain. A 2.6× reduction in Omicron RBD binding was observed for serum of 2× BNT162b2-vaccinated persons. Neutralization of Omicron SARS-CoV-2 was completely diminished in our setup. CONCLUSION: These results indicate an immune escape focused on neutralizing antibodies. Nevertheless, a boost vaccination increased the level of anti-RBD antibodies against Omicron, and neutralization of authentic Omicron SARS-CoV-2 was at least partially restored. This study adds evidence that current vaccination protocols may be less efficient against the Omicron variant.

Comparative Magnitude and Persistence of Humoral SARS-CoV-2 Vaccination Responses in the Adult Population in Germany.

Recent increases in SARS-CoV-2 infections have led to questions about duration and quality of vaccine-induced immune protection. While numerous studies have been published on immune responses triggered by vaccination, these often focus on studying the impact of one or two immunisation schemes within subpopulations such as immunocompromised individuals or healthcare workers. To provide information on the duration and quality of vaccine-induced immune responses against SARS-CoV-2, we analyzed antibody titres against various SARS-CoV-2 antigens and ACE2 binding inhibition against SARS-CoV-2 wild-type and variants of concern in samples from a large German population-based seroprevalence study (MuSPAD) who had received all currently available immunisation schemes. We found that homologous mRNA-based or heterologous prime-boost vaccination produced significantly higher antibody responses than vector-based homologous vaccination. Ad26.CoV2S.2 performance was particularly concerning with reduced titres and 91.7% of samples classified as non-responsive for ACE2 binding inhibition, suggesting that recipients require a booster mRNA vaccination. While mRNA vaccination induced a higher ratio of RBD- and S1-targeting antibodies, vector-based vaccines resulted in an increased proportion of S2-targeting antibodies. Given the role of RBD- and S1-specific antibodies in neutralizing SARS-CoV-2, their relative over-representation after mRNA vaccination may explain why these vaccines have increased efficacy compared to vector-based formulations. Previously infected individuals had a robust immune response once vaccinated, regardless of which vaccine they received, which could aid future dose allocation should shortages arise for certain manufacturers. Overall, both titres and ACE2 binding inhibition peaked approximately 28 days post-second vaccination and then decreased.

Diminishing Immune Responses against Variants of Concern in Dialysis Patients 4 Months after SARS-CoV-2 mRNA Vaccination.

Patients undergoing chronic hemodialysis were among the first to receive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccinations because of their increased risk for severe coronavirus disease and high case-fatality rates. By using a previously reported cohort from Germany of at-risk hemodialysis patients and healthy donors, where antibody responses were examined 3 weeks after the second vaccination, we assessed systemic cellular and humoral immune responses in serum and saliva 4 months after vaccination with the Pfizer-BioNTech BNT162b2 vaccine using an interferon-γ release assay and multiplex-based IgG measurements. We further compared neutralization capacity of vaccination-induced IgG against 4 SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, and Delta) by angiotensin-converting enzyme 2 receptor-binding domain competition assay. Sixteen weeks after second vaccination, compared with 3 weeks after, cellular and humoral responses against the original SARS-CoV-2 isolate and variants of concern were substantially reduced. Some dialysis patients even had no detectable B- or T-cell responses.

Direct and Indirect Proof of SARS-CoV-2 Infections in Indigenous Wiwa Communities in North-Eastern Colombia-A Cross-Sectional Assessment Providing Preliminary Surveillance Data.

To provide initial data on local SARS-CoV-2 epidemiology and spread in indigenous communities in north-eastern Colombia, respiratory swabs and serum samples from volunteers of indigenous communities were examined in March and April 2021. Samples from non-indigenous Colombians from the same villages were included as well. While previous exposure to SARS-CoV-2 was assessed by analysing serum samples for IgG and IgM with a rapid antibody point-of-care-test (POCT), screening for active infections was carried out with an antigen POCT test and real-time PCR from nasal swabs. In 380 indigenous and 72 non-indigenous volunteers, 61 (13.5%) active infections and an additional 113 (25%) previous infections were identified using diagnostic serology and molecular assays. Previous infections were more frequent in non-indigenous volunteers, and relevant associations of clinical features with active or previous SARS-CoV-2 infections were not observed. Symptoms reported were mild to moderate. SARS-CoV-2 was frequent in the assessed Colombian indigenous communities, as 38.5% of the study participants showed signs of exposure to SARS-CoV-2, which confirms the need to include these indigenous communities in screening and vaccination programs.

Cellular and humoral immunogenicity of a SARS-CoV-2 mRNA vaccine in patients on haemodialysis.

BACKGROUND: Patients with chronic renal insufficiency on maintenance haemodialysis face an increased risk of COVID-19 induced mortality and impaired vaccine responses. To date, only a few studies have addressed SARS-CoV-2 vaccine elicited immunity in this immunocompromised population. METHODS: We assessed immunogenicity of the mRNA vaccine BNT162b2 in at-risk dialysis patients and characterised systemic cellular and humoral immune responses in serum and saliva using interferon γ release assay and multiplex-based cytokine and immunoglobulin measurements. We further compared binding capacity and neutralization efficacy of vaccination-induced immunoglobulins against emerging SARS-CoV-2 variants Alpha, Beta, Epsilon and Cluster 5 by ACE2-RBD competition assay. FINDINGS: Patients on maintenance haemodialysis exhibit detectable but variable cellular and humoral immune responses against SARS-CoV-2 and variants of concern after a two-dose regimen of BNT162b2. Although vaccination-induced immunoglobulins were detectable in saliva and plasma, both anti-SARS-CoV-2 IgG and neutralization efficacy was reduced compared to a vaccinated non-dialysed control population. Similarly, T-cell mediated interferon γ release after stimulation with SARS-CoV-2 spike peptides was significantly diminished. INTERPRETATION: Quantifiable humoral and cellular immune responses after BNT162b2 vaccination in individuals on maintenance haemodialysis are encouraging, but urge for longitudinal follow-up to assess longevity of immunity. Diminished virus neutralization and interferon γ responses in the face of emerging variants of concern may favour this at-risk population for re-vaccination using modified vaccines at the earliest opportunity. FUNDING: Initiative and Networking Fund of the Helmholtz Association of German Research Centres, EU Horizon 2020 research and innovation program, State Ministry of Baden-Württemberg for Economic Affairs, Labour and Tourism.

Immune response to SARS-CoV-2 variants of concern in vaccinated individuals.

SARS-CoV-2 is evolving with mutations in the receptor binding domain (RBD) being of particular concern. It is important to know how much cross-protection is offered between strains following vaccination or infection. Here, we obtain serum and saliva samples from groups of vaccinated (Pfizer BNT-162b2), infected and uninfected individuals and characterize the antibody response to RBD mutant strains. Vaccinated individuals have a robust humoral response after the second dose and have high IgG antibody titers in the saliva. Antibody responses however show considerable differences in binding to RBD mutants of emerging variants of concern and substantial reduction in RBD binding and neutralization is observed against a patient-isolated South African variant. Taken together our data reinforce the importance of the second dose of Pfizer BNT-162b2 to acquire high levels of neutralizing antibodies and high antibody titers in saliva suggest that vaccinated individuals may have reduced transmission potential. Substantially reduced neutralization for the South African variant further highlights the importance of surveillance strategies to detect new variants and targeting these in future vaccines.

NeutrobodyPlex-monitoring SARS-CoV-2 neutralizing immune responses using nanobodies.

In light of the COVID-19 pandemic, there is an ongoing need for diagnostic tools to monitor the immune status of large patient cohorts and the effectiveness of vaccination campaigns. Here, we present 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD), of which 8 Nbs potently inhibit the interaction of RBD with angiotensin-converting enzyme 2 (ACE2) as the major viral docking site. Following detailed epitope mapping and structural analysis, we select two inhibitory Nbs, one of which binds an epitope inside and one of which binds an epitope outside the RBD:ACE2 interface. Based on these, we generate a biparatopic nanobody (bipNb) with viral neutralization efficacy in the picomolar range. Using bipNb as a surrogate, we establish a competitive multiplex binding assay ("NeutrobodyPlex") for detailed analysis of the presence and performance of neutralizing RBD-binding antibodies in serum of convalescent or vaccinated patients. We demonstrate that NeutrobodyPlex enables high-throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, to monitor immune status or to guide vaccine design.

Exploring beyond clinical routine SARS-CoV-2 serology using MultiCoV-Ab to evaluate endemic coronavirus cross-reactivity.

The humoral immune response to SARS-CoV-2 is a benchmark for immunity and detailed analysis is required to understand the manifestation and progression of COVID-19, monitor seroconversion within the general population, and support vaccine development. The majority of currently available commercial serological assays only quantify the SARS-CoV-2 antibody response against individual antigens, limiting our understanding of the immune response. To overcome this, we have developed a multiplex immunoassay (MultiCoV-Ab) including spike and nucleocapsid proteins of SARS-CoV-2 and the endemic human coronaviruses. Compared to three broadly used commercial in vitro diagnostic tests, our MultiCoV-Ab achieves a higher sensitivity and specificity when analyzing a well-characterized sample set of SARS-CoV-2 infected and uninfected individuals. We find a high response against endemic coronaviruses in our sample set, but no consistent cross-reactive IgG response patterns against SARS-CoV-2. Here we show a robust, high-content-enabled, antigen-saving multiplex assay suited to both monitoring vaccination studies and facilitating epidemiologic screenings for humoral immunity towards pandemic and endemic coronaviruses.

SARS-CoV-2 Seroprevalence in Germany.

BACKGROUND: Until now, information on the spread of SARS-CoV-2 infections in Germany has been based mainly on data from the public health offices. It may be assumed that these data do not include many cases of asymptomatic and mild infection. METHODS: We determined seroprevalence over the course of the pandemic in a sequential, multilocal seroprevalence study (MuSPAD). Study participants were recruited at random in seven administrative districts (Kreise) in Germany from July 2020 onward; each participant was tested at two different times 3-5 months apart. Test findings on blood samples were used to determine the missed-case rate of reported infections, the infection fatality rate (IFR), and the association between seropositivity and demographic, socio-economic, and health-related factors, as well as to evaluate the self-reported results of PCR and antigenic tests. The registration number of this study is DRKS00022335. RESULTS: Among non-vaccinated persons, the seroprevalence from July to December 2020 was 1.3-2.8% and rose between February and May 2021 to 4.1-13.1%. In July 2021, 35% of tested persons in Chemnitz were not vaccinated, and the seroprevalence among these persons was 32.4% (07/2021). The surveillance detection ratio (SDR), i.e., the ratio between the true number of infections estimated from seroprevalence and the actual number or reported infections, varied among the districts included in the study from 2.2 to 5.1 up to December 2020 and from 1.3 to 2.9 up to June 2021, and subsequently declined. The IFR was in the range of 0.8% to 2.4% in all regions except Magdeburg, where a value of 0.3% was calculated for November 2020. A lower educational level was associated with a higher seropositivity rate, smoking with a lower seropositivity rate. On average, 1 person was infected for every 8.5 persons in quarantine. CONCLUSION: Seroprevalence was low after the first wave of the pandemic but rose markedly during the second and third waves. The missed-case rate trended downward over the course of the pandemic.

SARS-CoV-2-derived peptides define heterologous and COVID-19-induced T cell recognition.

T cell immunity is central for the control of viral infections. To characterize T cell immunity, but also for the development of vaccines, identification of exact viral T cell epitopes is fundamental. Here we identify and characterize multiple dominant and subdominant SARS-CoV-2 HLA class I and HLA-DR peptides as potential T cell epitopes in COVID-19 convalescent and unexposed individuals. SARS-CoV-2-specific peptides enabled detection of post-infectious T cell immunity, even in seronegative convalescent individuals. Cross-reactive SARS-CoV-2 peptides revealed pre-existing T cell responses in 81% of unexposed individuals and validated similarity with common cold coronaviruses, providing a functional basis for heterologous immunity in SARS-CoV-2 infection. Diversity of SARS-CoV-2 T cell responses was associated with mild symptoms of COVID-19, providing evidence that immunity requires recognition of multiple epitopes. Together, the proposed SARS-CoV-2 T cell epitopes enable identification of heterologous and post-infectious T cell immunity and facilitate development of diagnostic, preventive and therapeutic measures for COVID-19.

RhoA determines disease progression by controlling neutrophil motility and restricting hyperresponsiveness.

Neutrophil responses are central to host protection and inflammation. Neutrophil activation follows a 2-step process in which priming amplifies responses to activating stimuli. Priming is essential for life span extension, chemotaxis, and respiratory burst activity. Here we show that the cytoskeletal organizer RhoA suppresses neutrophil priming via formins. Premature granule exocytosis in Rho-deficient neutrophils activated numerous signaling pathways and amplified superoxide generation. Deletion of Rho altered front-to-back coordination by simultaneously increasing uropod elongation, leading edge formation, and random migration. Concomitant negative and positive regulation of ß2 integrin-independent and ß2 integrin-dependent migration, respectively, reveal Rho as a key decision point in the neutrophil response to discrete chemotactic agents. Although even restricted influx of Rho-deficient hyperactive neutrophils exacerbated lipopolysaccharide-mediated lung injury, deleting Rho in innate immune cells was highly protective in influenza A virus infection. Hence, Rho is a key regulator of disease progression by maintaining neutrophil quiescence and suppressing hyperresponsiveness.