Rapid selection of sotrovimab escape variants in SARS-CoV-2 Omicron infected immunocompromised patients.

BACKGROUND: Monoclonal antibodies (mAb) targeting SARS-CoV-2 are predominantly less effective against Omicron variants. Immunocompromised patients often experience prolonged viral shedding and are therefore at increased risk for viral escape mutations, when mAbs are used as monotherapy. METHODS: In an observational, prospective cohort, 57 patients infected with Omicron variants receiving sotrovimab alone or in combination with remdesivir were followed. The study endpoints were a decrease in SARS-CoV-2-RNA <106 copies/ml in nasopharyngeal swabs at day 21 and the emergence of resistance mutations at days 7, 14, and 21 after sotrovimab administration. All SARS-CoV-2 samples were analyzed by whole-genome sequencing, individual variants within the quasispecies were subsequently quantified and further characterized by a pseudovirus neutralization assay. RESULTS: 47/57 patients (82.5%) were infected with Omicron/BA.1 and 10/57 (17.5%) with Omicron/BA.2. The vast majority of patients (43/57, 75.4%) were immunodeficient, predominantly due to immunosuppression after organ transplantation or hematologic malignancies. 21 days after sotrovimab administration, 12/43 (27.9%) of immunodeficient patients had prolonged viral shedding compared to 1/14 (7.1%) immunocompetent patients (p = 0.011). Longitudinal sequencing revealed that 14/43 (32.6%) immunodeficient patients had in part Omicron-specific viral spike protein mutations (e.g., P337S and/or E340D/V) that substantially reduced susceptibility to sotrovimab in a pseudovirus neutralization assay. Combination therapy with remdesivir significantly reduced the selection of escape variants. CONCLUSIONS: Immunocompromised patients face a considerable risk of prolonged viral shedding and emergence of escape mutations after early therapy with sotrovimab. These findings underscore the importance of careful monitoring and the need to conduct dedicated clinical trials for this patient population.

Integrated genomic surveillance enables tracing of person-to-person SARS-CoV-2 transmission chains during community transmission and reveals extensive onward transmission of travel-imported infections, Germany, June to July 2021.

BackgroundTracking person-to-person SARS-CoV-2 transmission in the population is important to understand the epidemiology of community transmission and may contribute to the containment of SARS-CoV-2. Neither contact tracing nor genomic surveillance alone, however, are typically sufficient to achieve this objective.AimWe demonstrate the successful application of the integrated genomic surveillance (IGS) system of the German city of Düsseldorf for tracing SARS-CoV-2 transmission chains in the population as well as detecting and investigating travel-associated SARS-CoV-2 infection clusters.MethodsGenomic surveillance, phylogenetic analysis, and structured case interviews were integrated to elucidate two genetically defined clusters of SARS-CoV-2 isolates detected by IGS in Düsseldorf in July 2021.ResultsCluster 1 (n = 67 Düsseldorf cases) and Cluster 2 (n = 36) were detected in a surveillance dataset of 518 high-quality SARS-CoV-2 genomes from Düsseldorf (53% of total cases, sampled mid-June to July 2021). Cluster 1 could be traced back to a complex pattern of transmission in nightlife venues following a putative importation by a SARS-CoV-2-infected return traveller (IP) in late June; 28 SARS-CoV-2 cases could be epidemiologically directly linked to IP. Supported by viral genome data from Spain, Cluster 2 was shown to represent multiple independent introduction events of a viral strain circulating in Catalonia and other European countries, followed by diffuse community transmission in Düsseldorf.ConclusionIGS enabled high-resolution tracing of SARS-CoV-2 transmission in an internationally connected city during community transmission and provided infection chain-level evidence of the downstream propagation of travel-imported SARS-CoV-2 cases.

Adjusted COVID-19 booster schedules balance age-dependent differences in antibody titers benefitting risk populations

We provide follow-up data on the humoral immune response after COVID-19 vaccinations of two distinct cohorts aged below 60 and over 80 years to screen for age-related differences in the longevity and magnitude of the induction of the antibody responses post booster-vaccinations. While anti-SARS-CoV-2 spike-specific IgG and neutralization capacity waned rapidly after the initial vaccination schedule, additional boosters highly benefitted the humoral immune responses especially in the elderly cohort, including the neutralization of Omikron variants. Thus, adjusted COVID-19 booster vaccination schedules are an appropriate tool to overcome limitations in the success of vaccinations.

Quantitative analysis of different respiratory specimens on two automated test systems for detection of SARS-CoV-2 RNA.

Molecular testing of SARS-CoV-2 RNA is essential during the pandemic. Here, we compared the results of different respiratory specimens including anterior nasal swabs, pharyngeal swabs, saliva swabs, and gargle lavage samples to nasopharyngeal swabs on two automated SARS-CoV-2 test systems. Samples were collected and tested simultaneously from a total of 36 hospitalized symptomatic COVID-19 patients. Detection and quantification of SARS-CoV-2 was performed on cobas®6800 (Roche) and NeuMoDx™ (Qiagen) systems. Both assays showed reliable detection and quantification of SARS-CoV-2 RNA, with nasopharyngeal swabs showing the highest sensitivity. SARS-CoV-2 RNA concentrations in other respiratory specimens were lower (mean 2.5 log10 copies/ml) or even undetectable in up to 20%. These data clearly indicate that not all respiratory materials are equally suitable for the management of hospitalized patients, especially, in the late phase of COVID-19, when the viral phase subsides and inflammation becomes the predominant factor, making detection of even lower viral loads increasingly important.

Characterization of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection Clusters Based on Integrated Genomic Surveillance, Outbreak Analysis and Contact Tracing in an Urban Setting.

BACKGROUND: Tracing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission chains is still a major challenge for public health authorities, when incidental contacts are not recalled or are not perceived as potential risk contacts. Viral sequencing can address key questions about SARS-CoV-2 evolution and may support reconstruction of viral transmission networks by integration of molecular epidemiology into classical contact tracing. METHODS: In collaboration with local public health authorities, we set up an integrated system of genomic surveillance in an urban setting, combining a) viral surveillance sequencing, b) genetically based identification of infection clusters in the population, c) integration of public health authority contact tracing data, and d) a user-friendly dashboard application as a central data analysis platform. RESULTS: Application of the integrated system from August to December 2020 enabled a characterization of viral population structure, analysis of 4 outbreaks at a maximum care hospital, and genetically based identification of 5 putative population infection clusters, all of which were confirmed by contact tracing. The system contributed to the development of improved hospital infection control and prevention measures and enabled the identification of previously unrecognized transmission chains, involving a martial arts gym and establishing a link between the hospital to the local population. CONCLUSIONS: Integrated systems of genomic surveillance could contribute to the monitoring and, potentially, improved management of SARS-CoV-2 transmission in the population.

Age-dependent Immune Response to the Biontech/Pfizer BNT162b2 Coronavirus Disease 2019 Vaccination.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has led to the development of various vaccines. Real-life data on immune responses elicited in the most vulnerable group of vaccinees older than age 80 years old are still underrepresented despite the prioritization of the elderly in vaccination campaigns. METHODS: We conducted a cohort study with 2 age groups, young vaccinees below the age of 60 years and elderly vaccinees over the age of 80 years, to compare their antibody responses to the first and second dose of the BNT162b2 coronavirus disease 2019 vaccination. RESULTS: Although the majority of participants in both groups produced specific immunoglobulin G antibody titers against SARS-CoV-2 spike protein, titers were significantly lower in elderly participants. Although the increment of antibody levels after the second immunization was higher in elderly participants, the absolute mean titer of this group remained lower than the <60 years of age group. After the second vaccination, 31.3% of the elderly had no detectable neutralizing antibodies in contrast to the younger group, in which only 2.2% had no detectable neutralizing antibodies. CONCLUSIONS: Our data showed differences between the antibody responses raised after the first and second BNT162b2 vaccination, in particular lower frequencies of neutralizing antibodies in the elderly group. This suggests that this population needs to be closely monitored and may require earlier revaccination and/or an increased vaccine dose to ensure stronger long-lasting immunity and protection against infection.

SARS-CoV-2 Infection in Fully Vaccinated Individuals of Old Age Strongly Boosts the Humoral Immune Response.

Prophylactic vaccination against SARS-CoV-2 is one of the most important measures to contain the COVID-19 pandemic. Recently, break-through infections following vaccination against this virus have been reported. Here, we describe the humoral immune response of break-through infections in fully vaccinated individuals of old age from an outbreak in a nursing home. In cooperation with the local health authority, blood samples from fully vaccinated and infected as well as fully vaccinated and uninfected residents of the nursing home were collected 4 weeks after the onset of the outbreak. The humoral immune response was determined in a neutralisation assay with replication-competent virus isolates and by a quantitative ELISA. In this outbreak a total of 23 residents and four health care workers were tested positive for SARS-CoV-2. Four residents were unvaccinated, including one with a severe course of disease who later severe disease course who later succumbed to infection. Despite their old age, all vaccinated residents showed no or only mild disease. Comparison of the humoral immune response revealed significantly higher antibody levels in fully vaccinated infected individuals compared to fully vaccinated uninfected individuals (p < 0.001). Notably, although only a minority of the vaccinated uninfected group showed neutralisation capacity against SARS-CoV-2, all vaccinated and infected individuals showed high-titre neutralisation of SARS-CoV-2 including the alpha and beta variant. Large SARS-CoV-2 outbreaks can occur in fully vaccinated populations, but seem to associate with mild disease. SARS-CoV-2 infection in fully vaccinated individuals is a strong booster of the humoral immune response providing enhanced neutralisation capacity against immune evasion variants.

Students in Dormitories Were Not Major Drivers of the Pandemic during Winter Term 2020/2021: A Cohort Study with RT-PCR and Antibody Surveillance in a German University City

The role of educational facilities, including schools and universities, in the SARS-CoV-2 pandemic is heavily debated. Specifically, the risk of infection in student dormitories has not been studied. This cohort study monitored students living in dormitories in Bochum, Germany, throughout the winter term of 2020/2021. Over the course of four months, participants were tested repeatedly for SARS-CoV-2 infections using RT-PCR from gargle samples and serological testing. An online questionnaire identified individual risk factors. A total of 810 (46.5% female) students participated. Of these, 590 (72.8%) students participated in the final visit. The cross-sectional antibody prevalence was n = 23 (2.8%) in November 2020 and n = 29 (4.9%) in February 2021. Of 2513 gargle samples analyzed, 19 (0.8%) tested positive for SARS-CoV-2, corresponding to 14 (2.4%) infections detected within the study period. Gargle samples available of cases with confirmed present infection were always positive. The person-time incidence rate was 112.7 (95% CI: 54.11–207.2) per 100,000 person weeks. The standardized incidence ratio was 0.9 (95% CI 0.51–1.46, p = 0.69). In conclusion, students living in student dormitories do not appear to be major drivers of SARS-CoV-2 infections. RT-PCR from gargle samples is a patient-friendly and scalable surveillance tool for detection of SARS-CoV-2 infections.

Case Report: Convalescent Plasma Achieves SARS-CoV-2 Viral Clearance in a Patient With Persistently High Viral Replication Over 8 Weeks Due to Severe Combined Immunodeficiency (SCID) and Graft Failure.

We describe the unique disease course and cure of SARS-CoV-2 infection in a patient with SCID and graft failure. In absence of a humoral immune response, viral clearance was only achieved after transfusion of convalescent plasma. This observation underscores the necessity of the humoral immune response for SARS-CoV-2 clearance.

Emergence of the E484K mutation in SARS-COV-2-infected immunocompromised patients treated with bamlanivimab in Germany.

BACKGROUND: Monoclonal antibodies (mAb) have been introduced as a promising new therapeutic approach against SARS-CoV-2. At present, there is little experience regarding their clinical effects in patient populations underrepresented in clinical trials, e.g. immunocompromised patients. Additionally, it is not well known to what extent SARS-CoV-2 treatment with monoclonal antibodies could trigger the selection of immune escape viral variants. METHODS: After identifying immunocompromised patients with viral rebound under treatment with bamlanivimab, we characterized the SARS-CoV-2-isolates by whole genome sequencing. Viral load measurements and sequence analysis were performed consecutively before and after bamlanivimab administration. FINDINGS: After initial decrease of viral load, viral clearance was not achieved in five of six immunocompromised patients treated with bamlanivimab. Instead, viral replication increased again over the course of the following one to two weeks. In these five patients, the E484K substitution - known to confer immune escape - was detected at the time of viral rebound but not before bamlanivimab treatment. INTERPRETATION: Treatment of SARS-CoV-2 with bamlanivimab in immunocompromised patients results in the rapid development of immune escape variants in a significant proportion of cases. Given that the E484K mutation can hamper natural immunity, the effectiveness of vaccination as well as antibody-based therapies, these findings may have important implications not only for individual treatment decisions but may also pose a risk to general prevention and treatment strategies. FUNDING: All authors are employed and all expenses covered by governmental, federal state, or other publicly funded institutions.

Sensitivity of anti-SARS-CoV-2 serological assays in a high-prevalence setting.

Evaluation and power of seroprevalence studies depend on the performed serological assays. The aim of this study was to assess four commercial serological tests from EUROIMMUN, DiaSorin, Abbott, and Roche as well as an in-house immunofluorescence and neutralization test for their capability to identify SARS-CoV-2 seropositive individuals in a high-prevalence setting. Therefore, 42 social and working contacts of a German super-spreader were tested. Consistent with a high-prevalence setting, 26 of 42 were SARS-CoV-2 seropositive by neutralization test (NT), and immunofluorescence test (IFT) confirmed 23 of these 26 positive test results (NT 61.9% and IFT 54.8% seroprevalence). Four commercial assays detected anti-SARS-CoV-2 antibodies in 33.3-40.5% individuals. Besides an overall discrepancy between the NT and the commercial assays regarding their sensitivity, this study revealed that commercial SARS-CoV-2 spike-based assays are better to predict the neutralization titer than nucleoprotein-based assays are.

SARS-CoV-2 targets neurons of 3D human brain organoids.

COVID-19 pandemic caused by SARS-CoV-2 infection is a public health emergency. COVID-19 typically exhibits respiratory illness. Unexpectedly, emerging clinical reports indicate that neurological symptoms continue to rise, suggesting detrimental effects of SARS-CoV-2 on the central nervous system (CNS). Here, we show that a Düsseldorf isolate of SARS-CoV-2 enters 3D human brain organoids within 2 days of exposure. We identified that SARS-CoV-2 preferably targets neurons of brain organoids. Imaging neurons of organoids reveal that SARS-CoV-2 exposure is associated with altered distribution of Tau from axons to soma, hyperphosphorylation, and apparent neuronal death. Our studies, therefore, provide initial insights into the potential neurotoxic effect of SARS-CoV-2 and emphasize that brain organoids could model CNS pathologies of COVID-19.

Extraction-free SARS-CoV-2 detection by rapid RT-qPCR universal for all primary respiratory materials.

BACKGROUND: Fast and reliable detection of SARS-CoV-2 is crucial for efficient control of the COVID-19 pandemic. Due to the high demand for SARS-CoV-2 testing there is a worldwide shortage of RNA extraction reagents. Therefore, extraction-free RT-qPCR protocols are urgently needed. OBJECTIVES: To establish a rapid RT-qPCR protocol for the detection of SARS-CoV-2 without the need of RNA extraction suitable for all respiratory materials. MATERIAL AND METHODS: Different SARS-CoV-2 positive respiratory materials from our routine laboratory were used as crude material after heat inactivation in direct RT-qPCR with the PrimeDirect™ Probe RT-qPCR Mix (TaKaRa). SARS-CoV-2 was detected using novel primers targeted to the E-gene. RESULTS: The protocol for the detection of SARS-CoV-2 in crude material used a prepared frozen-PCR mix with optimized primers and 5 µl of fresh, undiluted and pre-analytically heat inactivated respiratory material. For validation, 91 respiratory samples were analyzed in direct comparison to classical RNA-based RT-qPCR. Overall 81.3 % of the samples were detected in both assays with a strong correlation between both Ct values (r = 0.8492, p < 0.0001). The SARS-CoV-2 detection rate by direct RT-qPCR was 95.8 % for Ct values <35. All negative samples were characterized by low viral loads (Ct >35) and/or long storage times before sample processing. CONCLUSION: Direct RT-qPCR is a suitable alternative to classical RNA RT-qPCR, provided that only fresh samples (storage <1 week) are used. RNA extraction should be considered if samples have longer storage times or if PCR inhibition is observed. In summary, this protocol is fast, inexpensive and suitable for all respiratory materials.

Genetic structure of SARS-CoV-2 reflects clonal superspreading and multiple independent introduction events, North-Rhine Westphalia, Germany, February and March 2020.

We whole-genome sequenced 55 SARS-CoV-2 isolates from Germany to investigate SARS-CoV-2 outbreaks in 2020 in the Heinsberg district and Düsseldorf. While the genetic structure of the Heinsberg outbreak indicates a clonal origin, reflecting superspreading dynamics from mid-February during the carnival season, distinct viral strains were circulating in Düsseldorf in March, reflecting the city's international links. Limited detection of Heinsberg strains in the Düsseldorf area despite geographical proximity may reflect efficient containment and contact-tracing efforts.