Multiplex Serology for Sensitive and Specific Flavivirus IgG Detection: Addition of Envelope Protein Domain III to NS1 Increases Sensitivity for Tick-Borne Encephalitis Virus IgG Detection.

Tick-borne encephalitis is a vaccine-preventable disease of concern for public health in large parts of Europe, with EU notification rates increasing since 2018. It is caused by the orthoflavivirus tick-borne encephalitis virus (TBEV) and a diagnosis of infection is mainly based on serology due to its short viremic phase, often before symptom onset. The interpretation of TBEV serology is hampered by a history of orthoflavivirus vaccination and by previous infections with related orthoflaviviruses. Here, we sought to improve TBEV sero-diagnostics using an antigen combination of in-house expressed NS1 and EDIII in a multiplex, low-specimen-volume set-up for the detection of immune responses to TBEV and other clinically important orthoflaviviruses (i.e., West Nile virus, dengue virus, Japanese encephalitis virus, Usutu virus and Zika virus). We show that the combined use of NS1 and EDIII results in both a specific and sensitive test for the detection of TBEV IgG for patient diagnostics, vaccination responses and in seroprevalence studies. This novel approach potentially allows for a low volume-based, simultaneous analysis of IgG responses to a range of orthoflaviviruses with overlapping geographic circulations and clinical manifestations.

Comparative study between virus neutralisation testing and other serological methods detecting anti-SARS-CoV-2 antibodies in Europe, 2021.

One consequence of the ongoing coronavirus disease pandemic was the rapid development of both in-house and commercial serological assays detecting anti-SARS-CoV-2 antibodies, in an effort to reliably detect acute and past SARS-CoV-2 infections. It is crucial to evaluate the quality of these serological tests and consequently the sero-epidemiological studies that are performed with the respective tests. Here, we describe the set-up and results of a comparative study, in which a laboratory contracted by the European Centre for Disease Prevention and Control offered a centralised service to EU/EEA Member and pre-accession Member States to test representative serum specimens with known serological results, with the gold standard technique (virus neutralisation tests) to determine the presence of neutralising antibodies. Laboratories from 12 European countries shared 719 serum specimens with the contractor laboratory. We found that in-house serological tests detecting neutralising antibodies showed the highest percent agreement, both positive and negative, with the virus neutralisation test results. Despite extensive differences in virus neutralisation protocols neutralisation titres showed a strong correlation. From the commercial assays, the best positive percent agreement was found for SARS-CoV-2 IgG (sCOVG) (Siemens - Atellica IM Analyzer). Despite lower positive percent agreement of LIAISON SARS-CoV-2 TrimericS IgG kit (Diasorin Inc.), the obtained results showed relatively good correlation with neutralisation titres. The set-up of this study allowed for high comparability between laboratories and enabled laboratories that do not have the capacity or capability to perform VNTs themselves. Given the variety of in-house protocols detecting SARS-CoV-2 specific neutralising antibodies, including the virus strain, it could be of interest to select reference isolates for SARS-CoV-2 diagnostic to be made available for interested EU Member States and pre-accession countries.

External quality assessment of orthohantavirus and lymphocytic choriomeningitis virus molecular detection and serology in Europe, 2021.

BackgroundRodent-borne viruses such as orthohantaviruses and arenaviruses cause considerable disease burden with regional and temporal differences in incidence and clinical awareness. Therefore, it is important to regularly evaluate laboratory diagnostic capabilities, e.g. by external quality assessments (EQA).AimWe wished to evaluate the performance and diagnostic capability of European expert laboratories to detect orthohantaviruses and lymphocytic choriomeningitis virus (LCMV) and human antibody response towards orthohantaviruses.MethodsWe conducted an EQA in 2021; molecular panels consisted of 12 samples, including different orthohantaviruses (Seoul, Dobrava-Belgrade (DOBV), Puumala (PUUV) and Hantaan orthohantavirus), LCMV and negative controls. Serological panels consisted of six human serum samples reactive to PUUV, DOBV or negative to orthohantaviruses. The EQA was sent to 25 laboratories in 20 countries.ResultsThe accuracy of molecular detection of orthohantaviruses varied (50‒67%, average 62%) among 16 participating laboratories, while LCMV samples were successfully detected in all 11 participating laboratories (91-100%, average 96%). The accuracy of serological diagnosis of acute and past orthohantavirus infections was on average 95% among 20 participating laboratories and 82% in 19 laboratories, respectively. A variety of methods was used, with predominance of in-house assays for molecular tests, and commercial assays for serological ones.ConclusionSerology, the most common tool to diagnose acute orthohantavirus infections, had a high accuracy in this EQA. The molecular detection of orthohantaviruses needs improvement while LCMV detection (performed in fewer laboratories) had 95% accuracy. Further EQAs are recommended to be performed periodically to monitor improvements and challenges in the diagnostics of rodent-borne diseases.

Differential abundance of IgG antibodies against the spike protein of SARS-CoV-2 and seasonal coronaviruses in patients with fatal COVID-19.

Infection with the novel pandemic SARS-CoV-2 virus has been shown to elicit a cross-reactive immune response that could lead to a back-boost of memory recall to previously encountered seasonal (endemic) coronaviruses (eCoVs). Whether this response is associated with a fatal clinical outcome in patients with severe COVID-19 remains unclear. In a cohort of hospitalized patients, we have previously shown that heterologous immune responses to eCoVs can be detected in severe COVID-19. Here, we report that COVID-19 patients with fatal disease have decreased SARS-CoV-2 neutralizing antibody titers at hospital admission, which correlated with lower SARS-CoV-2 spike-specific IgG and was paralleled by a relative abundance of IgG against spike protein of eCoVs of the genus Betacoronavirus. Additional research is needed to assess if eCoV-specific back-boosted IgG is a bystander phenomenon in severe COVID-19, or a factor that influences the development of an efficient anti-viral immune response.

Impaired SARS-CoV-2 specific T-cell response in patients with severe COVID-19.

Cellular immune responses are of pivotal importance to understand SARS-CoV-2 pathogenicity. Using an enzyme-linked immunosorbent spot (ELISpot) interferon-γ release assay with wild-type spike, membrane and nucleocapsid peptide pools, we longitudinally characterized functional SARS-CoV-2 specific T-cell responses in a cohort of patients with mild, moderate and severe COVID-19. All patients were included before emergence of the Omicron (B.1.1.529) variant. Our most important finding was an impaired development of early IFN-γ-secreting virus-specific T-cells in severe patients compared to patients with moderate disease, indicating that absence of virus-specific cellular responses in the acute phase may act as a prognostic factor for severe disease. Remarkably, in addition to reactivity against the spike protein, a substantial proportion of the SARS-CoV-2 specific T-cell response was directed against the conserved membrane protein. This may be relevant for diagnostics and vaccine design, especially considering new variants with heavily mutated spike proteins. Our data further strengthen the hypothesis that dysregulated adaptive immunity plays a central role in COVID-19 immunopathogenesis.

Rescue and in vitro characterization of a divergent TBEV-Eu strain from the Netherlands.

Tick-borne encephalitis virus (TBEV) may cause tick-borne encephalitis (TBE), a potential life-threatening infection of the central nervous system in humans. Phylogenetically, TBEVs can be subdivided into three main subtypes, which differ in endemic region and pathogenic potential. In 2016, TBEV was first detected in the Netherlands. One of two detected strains, referred to as Salland, belonged to the TBEV-Eu subtype, yet diverged ≥ 2% on amino acid level from other members of this subtype. Here, we report the successful rescue of this strain using infectious subgenomic amplicons and its subsequent in vitro characterization by comparison to two well-characterized TBEV-Eu strains; Neudoerfl and Hypr. In the human alveolar epithelial cell line A549, growth kinetics of Salland were comparable to the high pathogenicity TBEV-Eu strain Hypr, and both strains grew considerably faster than the mildly pathogenic strain Neudoerfl. In the human neuroblastoma cell line SK-N-SH, Salland replicated faster and to higher infectious titers than both reference strains. All three TBEV strains infected primary human monocyte-derived dendritic cells to a similar extent and interacted with the type I interferon system in a similar manner. The current study serves as the first in vitro characterization of the novel, divergent TBEV-Eu strain Salland.

A Comprehensive Sampling Study on SARS-CoV-2 Contamination of Air and Surfaces in a Large Meat Processing Plant Experiencing COVID-19 Clusters in June 2020.

OBJECTIVE: We aimed to assess SARS-CoV-2 contamination of air and surfaces to gain insight into potential occupational exposure in a large meat processing plant experiencing COVID-19 clusters. Methods: Oro-nasopharyngeal SARS-CoV-2 screening was performed in 76 workers. Environmental samples ( n = 275) including air, ventilation systems, sewage, and swabs of high-touch surfaces and workers' hands were tested for SARS-CoV-2 RNA by real-time quantitative polymerase chain reaction. Results: Twenty-seven (35.5%) of the (predominantly asymptomatic) workers tested positive with modest to low viral loads (cycle threshold ≥ 29.7). Six of 203 surface swabs, 1 of 12 personal air samples, and one of four sewage samples tested positive; other samples tested negative. Conclusions: Although one third of workers tested positive, environmental contamination was limited. Widespread SARS-CoV-2 transmission via air and surfaces was considered unlikely within this plant at the time of investigation while strict COVID-19 control measures were already implemented.

Elevated risk of infection with SARS-CoV-2 Beta, Gamma, and Delta variants compared with Alpha variant in vaccinated individuals.

The extent to which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) break through infection- or vaccine-induced immunity is not well understood. We analyzed 28,578 sequenced SARS-CoV-2 samples from individuals with known immune status obtained through national community testing in the Netherlands from March to August 2021. We found evidence of an increased risk of infection by the Beta (B.1.351), Gamma (P.1), or Delta (B.1.617.2) variants compared with the Alpha (B.1.1.7) variant after vaccination. No clear differences were found between vaccines. However, the effect was larger in the first 14 to 59 days after complete vaccination compared with ≥60 days. In contrast to vaccine-induced immunity, there was no increased risk for reinfection with Beta, Gamma, or Delta variants relative to the Alpha variant in individuals with infection-induced immunity.

Evaluation of the use of alternative sample types for mosquito-borne flavivirus surveillance: Using Usutu virus as a model.

Wild birds are reservoirs of several zoonotic arboviruses including West Nile virus (WNV) and Usutu virus (USUV), and are often monitored as indicators for virus introduction and spread. To optimize the bird surveillance for arboviruses in the Netherlands and to explore the possibilities for citizen science in surveillance, we investigated the suitability of using alternative sample types from live and dead birds. The sensitivity of molecular detection via RT-PCR of viral RNA in feather, heart, lung, throat and cloaca swabs from dead birds, and serum, dried blood spots (DBS) and throat and cloaca swabs from live birds were compared. IgY antibody detection was also assessed from DBS relative to serum on protein-microarray and virus neutralization test. Feathers showed a high detection sensitivity for USUV RNA in both live and dead birds, and no significant decrease was observed in the RNA loads in the feathers after being stored dry at room temperature for 43 days. Additionally, viral RNAs extracted from feathers of day 0 and 43 were successfully sequenced. The results indicated no statistical significant difference in sensitivity and viral loads detection in heart, spleen, and lung relative to corresponding brain samples in dead birds. In live birds, viral RNA loads did not differ between throat and cloaca swabs. This study identified less-invasive sample types that allows involvement of citizens in collecting samples from wild birds for arbovirus surveillance. Sensitivity and specificity of DBS-based antibody detections were significantly lower and therefore need optimization.

Sensitivity of Detection and Variant Typing of SARS-CoV-2 in European Laboratories.

The molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is key for clinical management and surveillance. Funded by the European Centre for Disease Prevention and Control, we conducted an external quality assessment (EQA) on the molecular detection and variant typing of SARS-CoV-2 that included 59 European laboratories in 34 countries. The EQA panel consisted of 12 lyophilized inactivated samples, 10 of which were SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, Epsilon, Eta, parental B.1 strain) ranging from 2.5 to 290.0 copies/µL or pooled respiratory viruses (adenovirus, enterovirus, influenza virus A, respiratory syncytial virus, or human coronaviruses 229E and OC43). Of all participants, 72.9% identified the presence of SARS-CoV-2 RNA correctly. In samples containing 25.0 or more genome copies/µL, SARS-CoV-2 was detected by 98.3% of the participating laboratories. Laboratories applying commercial tests scored significantly better (P < 0.0001, Kruskal-Wallis test) than those using in-house assays. Both the molecular detection and the typing of the SARS-CoV-2 variants were associated with the RNA concentrations (P < 0.0001, Kruskal-Wallis test). On average, only 5 out of the 10 samples containing different SARS-CoV-2 variants at different concentrations were correctly typed. The identification of SARS-CoV-2 variants was significantly more successful among EQA participants who combined real-time reverse transcription polymerase chain reaction (RT-PCR)-based assays for mutation detection and high-throughput genomic sequencing than among those who used a single methodological approach (P = 0.0345, Kruskal-Wallis test). Our data highlight the high sensitivity of SARS-CoV-2 detection in expert laboratories as well as the importance of continuous assay development and the benefits of combining different methodologies for accurate SARS-CoV-2 variant typing.

External quality assessment of SARS-CoV-2 serology in European expert laboratories, April 2021.

BackgroundCountries worldwide are focusing to mitigate the ongoing SARS-CoV-2 pandemic by employing public health measures. Laboratories have a key role in the control of SARS-CoV-2 transmission. Serology for SARS-CoV-2 is of critical importance to support diagnosis, define the epidemiological framework and evaluate immune responses to natural infection and vaccine administration.AimThe aim of this study was the assessment of the actual capability among laboratories involved in sero-epidemiological studies on COVID-19 in EU/EEA and EU enlargement countries to detect SARS-CoV-2 antibodies through an external quality assessment (EQA) based on proficiency testing.MethodsThe EQA panels were composed of eight different, pooled human serum samples (all collected in 2020 before the vaccine roll-out), addressing sensitivity and specificity of detection. The panels and two EU human SARS-CoV-2 serological standards were sent to 56 laboratories in 30 countries.ResultsThe overall performance of laboratories within this EQA indicated a robust ability to establish past SARS-CoV-2 infections via detection of anti-SARS-CoV-2 antibodies, with 53 of 55 laboratories using at least one test that characterised all EQA samples correctly. IgM-specific test methods provided most incorrect sample characterisations (24/208), while test methods detecting total immunoglobulin (0/119) and neutralising antibodies (2/230) performed the best. The semiquantitative assays used by the EQA participants also showed a robust performance in relation to the standards.ConclusionOur EQA showed a high capability across European reference laboratories for reliable diagnostics for SARS-CoV-2 antibody responses. Serological tests that provide robust and reliable detection of anti-SARS-CoV-2 antibodies are available.

Longitudinal Household Assessment of Respiratory Illness in Children and Parents During the COVID-19 Pandemic.

Importance: In the early COVID-19 pandemic, SARS-CoV-2 testing was only accessible and recommended for symptomatic persons or adults. This restriction hampered assessment of the true incidence of SARS-CoV-2 infection in children as well as detailed characterization of the SARS-CoV-2 disease spectrum and how this spectrum compared with that of other common respiratory illnesses. Objective: To estimate the community incidence of SARS-CoV-2 infection in children and parents and to assess the symptoms and symptom severity of respiratory illness episodes involving SARS-CoV-2-positive test results relative to those with SARS-CoV-2-negative test results. Design, Setting, and Participants: This cohort study randomly selected Dutch households with at least 1 child younger than 18 years. A total of 1209 children and adults from 307 households were prospectively followed up between August 25, 2020, and July 29, 2021, covering the second and third waves of the COVID-19 pandemic. Participation included SARS-CoV-2 screening at 4- to 6-week intervals during the first 23 weeks of participation (core study period; August 25, 2020, to July 29, 2021). Participants in all households finishing the core study before July 1, 2021, were invited to participate in the extended follow-up and to actively report respiratory symptoms using an interactive app until July 1, 2021. At new onset of respiratory symptoms or a SARS-CoV-2 positive test result, a household outbreak study was initiated, which included daily symptom recording, repeated polymerase chain reaction testing (nose-throat swabs and saliva and fecal samples), and SARS-CoV-2 antibody measurement (paired dried blood spots) in all household members. Outbreaks, households, and episodes of respiratory illness were described as positive or negative depending on SARS-CoV-2 test results. Data on participant race and ethnicity were not reported because they were not uniformly collected in the original cohorts and were therefore not representative or informative. Exposures: SARS-CoV-2-positive and SARS-CoV-2-negative respiratory illness episodes. Main Outcomes and Measures: Age-stratified incidence rates, symptoms, and symptom severity for SARS-CoV-2-positive and SARS-CoV-2-negative respiratory illness episodes. Results: Among 307 households including 1209 participants (638 female [52.8%]; 403 [33.3%] aged <12 years, 179 [14.8%] aged 12-17 years, and 627 [51.9%] aged ≥18 years), 183 household outbreaks of respiratory illness were observed during the core study and extended follow-up period, of which 63 (34.4%) were SARS-CoV-2 positive (59 outbreaks [32.2%] during the core study and 4 outbreaks [2.2%] during follow-up). SARS-CoV-2 incidence was similar across all ages (0.24/person-year [PY]; 95% CI, 0.21-0.28/PY). Overall, 33 of 134 confirmed SARS-CoV-2 episodes (24.6%) were asymptomatic. The incidence of SARS-CoV-2-negative respiratory illness episodes was highest in children younger than 12 years (0.94/PY; 95% CI, 0.89-0.97/PY). When comparing SARS-CoV-2-positive vs SARS-CoV-2-negative respiratory illness episodes in children younger than 12 years, no differences were observed in number of symptoms (median [IQR], 2 [2-4] for both groups), symptom severity (median [IQR] maximum symptom severity score, 6 [4-9] vs 7 [6-13]), or symptom duration (median [IQR], 6 [5-12] days vs 8 [4-13] days). However, among adults, SARS-CoV-2-positive episodes had a significantly higher number (median [IQR], 6 [4-8] vs 3 [2-4]), severity (median [IQR] maximum symptom severity score, 15 [9-19] vs 7 [6-11]), and duration (median [IQR] 13 [8-29] days vs 5 [3-11] days; P < .001 for all comparisons) of symptoms vs SARS-CoV-2-negative episodes. Conclusions and Relevance: In this cohort study, during the first pandemic year when mostly partial or full in-person learning occurred, the SARS-CoV-2 incidence rate in children was substantially higher than estimated from routine testing or seroprevalence data and was similar to that of adult household members. Unlike in unvaccinated adults, SARS-CoV-2 symptoms and symptom severity in children were similar to other common respiratory illnesses. These findings may prove useful when developing pediatric COVID-19 vaccine recommendations.

Discriminating cross-reactivity in polyclonal IgG1 responses against SARS-CoV-2 variants of concern.

Existing assays to measure antibody cross-reactivity against different SARS-CoV-2 spike (S) protein variants lack the discriminatory power to provide insights at the level of individual clones. Using a mass spectrometry-based approach we are able to monitor individual donors' IgG1 clonal responses following a SARS-CoV-2 infection. We monitor the plasma clonal IgG1 profiles of 8 donors who had experienced an infection by either the wild type Wuhan Hu-1 virus or one of 3 VOCs (Alpha, Beta and Gamma). In these donors we chart the full plasma IgG1 repertoires as well as the IgG1 repertoires targeting the SARS-CoV-2 spike protein trimer VOC antigens. The plasma of each donor contains numerous anti-spike IgG1 antibodies, accounting for <0.1% up to almost 10% of all IgG1s. Some of these antibodies are VOC-specific whereas others do recognize multiple or even all VOCs. We show that in these polyclonal responses, each clone exhibits a distinct cross-reactivity and also distinct virus neutralization capacity. These observations support the need for a more personalized look at the antibody clonal responses to infectious diseases.

Regional importation and asymmetric within-country spread of SARS-CoV-2 variants of concern in the Netherlands.

Background: Variants of concern (VOCs) of SARS-CoV-2 have caused resurging waves of infections worldwide. In the Netherlands, the Alpha, Beta, Gamma, and Delta VOCs circulated widely between September 2020 and August 2021. We sought to elucidate how various control measures, including targeted flight restrictions, had impacted the introduction and spread of these VOCs in the Netherlands. Methods: We performed phylogenetic analyses on 39,844 SARS-CoV-2 genomes collected under the Dutch national surveillance program. Results: We found that all four VOCs were introduced before targeted flight restrictions were imposed on countries where the VOCs first emerged. Importantly, foreign introductions, predominantly from other European countries, continued during these restrictions. After their respective introductions into the Netherlands, the Alpha and Delta VOCs largely circulated within more populous regions of the country with international connections before asymmetric bidirectional transmissions occurred with the rest of the country and the VOC became the dominant circulating lineage. Conclusions: Our findings show that flight restrictions had limited effectiveness in deterring VOC introductions due to the strength of regional land travel importation risks. As countries consider scaling down SARS-CoV-2 surveillance efforts in the post-crisis phase of the pandemic, our results highlight that robust surveillance in regions of early spread is important for providing timely information for variant detection and outbreak control. Funding: None.

Differential vaccine-induced kinetics of humoral and cellular immune responses in SARS-CoV-2 naive and convalescent health care workers.

Effective vaccination is a key element in the exit strategy from the current severe acute respiratory syndrome-CoV coronavirus-2 (SARS-CoV-2) pandemic, and may also offer protection against severe disease from future variants of concern. Here, we prospectively monitored T-cell responses over time, using ELISpot interferon-γ (INF-y) release assays, and B-cell responses, using serological tests, after vaccination and booster with BioNTech/Pfizer mRNA (Pfizer) and Janssen vector (Janssen/Johnson & Johnson) vaccines in hospital health care workers. Vaccine recipients were divided into seropositive and seronegative individuals at baseline, in order to determine the effect of natural immunity on vaccine-induced immune kinetics. We found that convalescent individuals mounted higher spike-specific INF-y-secreting T-cell responses and B-cell-mediated IgG responses, after receiving the Janssen vaccine or the first dose of the Pfizer vaccine. IgG levels corresponded to the virus neutralization capacity as measured by VNT assay. At 8 months postvaccination, spike-specific cellular immunity waned to low levels in individuals with or without prior natural immunity, whereas waning of humoral immunity occurred predominantly in naive individuals. The booster shot effectively reinduced both cellular and humoral immune responses. To conclude, our data supports the implemented single-dose mRNA booster strategy employed in the Netherlands. Furthermore, the level of pre-existing natural immunity may be factored into determining the optimal time window between future booster vaccines.

Antigenic cartography using sera from sequence-confirmed SARS-CoV-2 variants of concern infections reveals antigenic divergence of Omicron.

Large-scale vaccination campaigns have prevented countless hospitalizations and deaths due to COVID-19. However, the emergence of SARS-CoV-2 variants that escape from immunity challenges the effectiveness of current vaccines. Given this continuing evolution, an important question is when and how to update SARS-CoV-2 vaccines to antigenically match circulating variants, similarly to seasonal influenza viruses where antigenic drift necessitates periodic vaccine updates. Here, we studied SARS-CoV-2 antigenic drift by assessing neutralizing activity against variants of concern (VOCs) in a set of sera from patients infected with viral sequence-confirmed VOCs. Infections with D614G or Alpha strains induced the broadest immunity, whereas individuals infected with other VOCs had more strain-specific responses. Omicron BA.1 and BA.2 were substantially resistant to neutralization by sera elicited by all other variants. Antigenic cartography revealed that Omicron BA.1 and BA.2 were antigenically most distinct from D614G, associated with immune escape, and possibly will require vaccine updates to ensure vaccine effectiveness.

Protection of COVID-19 vaccination and previous infection against Omicron BA.1, BA.2 and Delta SARS-CoV-2 infections.

Given the emergence of the SARS-CoV-2 Omicron BA.1 and BA.2 variants and the roll-out of booster COVID-19 vaccination, evidence is needed on protection conferred by primary vaccination, booster vaccination and previous SARS-CoV-2 infection by variant. We employed a test-negative design on S-gene target failure data from community PCR testing in the Netherlands from 22 November 2021 to 31 March 2022 (n = 671,763). Previous infection, primary vaccination or both protected well against Delta infection. Protection against Omicron BA.1 infection was much lower compared to Delta. Protection was similar against Omicron BA.1 compared to BA.2 infection after previous infection, primary and booster vaccination. Higher protection was observed against all variants in individuals with both vaccination and previous infection compared with either one. Protection against all variants decreased over time since last vaccination or infection. We found that primary vaccination with current COVID-19 vaccines and previous SARS-CoV-2 infections offered low protection against Omicron BA.1 and BA.2 infection. Booster vaccination considerably increased protection against Omicron infection, but decreased rapidly after vaccination.

SARS-CoV-2 RNA and antibody dynamics in a Dutch household study with dense sampling frame.

This study investigated the dynamics of SARS-CoV-2 infection and diagnostics in 242 household members of different ages and with different symptom severity after SARS-CoV-2 exposure early in the pandemic (March-April 2020). Households with a SARS-CoV-2 confirmed positive case and at least one child in the Netherlands were followed for 6 weeks. Naso (NP)- and oropharyngeal (OP) swabs, oral fluid and feces specimens were analyzed for SARS-CoV-2 RNA and serum for SARS-CoV-2-specific antibodies. The dynamics of the presence of viral RNA and the serological response was modeled to determine the sampling time-frame and sample type with the highest sensitivity to confirm or reject a SARS-CoV-2 diagnosis. In children higher viral loads compared to adults were detected at symptom onset. Early in infection, higher viral loads were detected in NP and OP specimens, while RNA in especially feces were longer detectable. SARS-CoV-2-specific antibodies have 90% probability of detection from 7 days (total Ig) and 18 days (IgG) since symptom onset. For highest probability of detection in SARS-CoV-2 diagnostics early in infection, RT-PCR on NP and OP specimens are more sensitive than on oral fluid and feces. For SARS-CoV-2 diagnostics late after infection, RT-PCR on feces specimens and serology are more valuable.

Frequent Infection of Cats With SARS-CoV-2 Irrespective of Pre-Existing Enzootic Coronavirus Immunity, Brazil 2020.

Carnivores such as cats and minks are highly susceptible to SARS-CoV-2. Brazil is a global COVID-19 hot spot and several cases of human-to-cat transmission have been documented. We investigated the spread of SARS-CoV-2 by testing 547 domestic cats sampled between July-November 2020 from seven states in southern, southeastern, and northeastern Brazil. Moreover, we investigated whether immune responses elicited by enzootic coronaviruses affect SARS-CoV-2 infection in cats. We found infection with significantly higher neutralizing antibody titers against the Gamma variant of concern, endemic in Brazil during 2020, than against an early SARS-CoV-2 B.1 isolate (p<0.0001), validating the use of Gamma for further testing. The overall SARS-CoV-2 seroprevalence in Brazilian cats during late 2020 validated by plaque reduction neutralization test (PRNT90) was 7.3% (95% CI, 5.3-9.8). There was no significant difference in SARS-CoV-2 seroprevalence in cats between Brazilian states, suggesting homogeneous infection levels ranging from 4.6% (95% CI, 2.2-8.4) to 11.4% (95% CI, 6.7-17.4; p=0.4438). Seroprevalence of the prototypic cat coronavirus Feline coronavirus (FCoV) in a PRNT90 was high at 33.3% (95% CI, 24.9-42.5) and seroprevalence of Bovine coronavirus (BCoV) was low at 1.7% (95% CI, 0.2-5.9) in a PRNT90. Neutralizing antibody titers were significantly lower for FCoV than for SARS-CoV-2 (p=0.0001), consistent with relatively more recent infection of cats with SARS-CoV-2. Neither the magnitude of SARS-CoV-2 antibody titers (p=0.6390), nor SARS-CoV-2 infection status were affected by FCoV serostatus (p=0.8863). Our data suggest that pre-existing immunity against enzootic coronaviruses neither prevents, nor enhances SARS-CoV-2 infection in cats. High SARS-CoV-2 seroprevalence already during the first year of the pandemic substantiates frequent infection of domestic cats and raises concerns on potential SARS-CoV-2 mutations escaping human immunity upon spillback.

Heterologous Immune Responses of Serum IgG and Secretory IgA Against the Spike Protein of Endemic Coronaviruses During Severe COVID-19.

Defining immune correlates of disease severity is important to better understand the immunopathogenesis in COVID-19. Here we made use of a protein microarray platform to detect IgG- and IgA-reactive antibodies in sera and saliva respectively, and assess cross-reactivity between SARS-CoV-2 and endemic coronaviruses (eCoVs). IgG responses against the full protein of spike, but not the S1 subunit, were significantly higher in convalescent sera of patients with severe disease compared to mild disease and healthy controls. In addition, we detected reactivity of secretory IgA to eCoVs in saliva of patients with severe disease, not present in patients with moderate disease or seropositive healthy controls. These heterologous immune responses are in line with non-protective cross-reactivity, and support a potential role for immune imprinting in the pathogenesis of severe COVID-19.