Reduced serum neutralization capacity against SARS-CoV-2 variants in a multiplex ACE2 RBD competition assay

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

A broadly neutralizing biparatopic Nanobody protects mice from lethal challenge with SARS-CoV-2 variants of concern

The ongoing COVID-19 pandemic and the frequent emergence of new SARS-CoV-2 variants of concern (VOCs), requires continued development of fast and effective therapeutics. Recently, we identified high-affinity neutralizing nanobodies (Nb) specific for the receptor-binding domain (RBD) of SARS-CoV-2, which are now being used as biparatopic Nbs (bipNbs) to investigate their potential as future drug candidates. Following detailed in vitro characterization, we chose NM1267 as the most promising candidate showing high affinity binding to several recently described SARS-CoV-2 VOCs and strong neutralizing capacity against a patient isolate of B.1.351 (Beta). To assess if bipNb NM1267 confers protection against SARS-CoV-2 infection in vivo, human ACE2 transgenic mice were treated by intranasal route before infection with a lethal dose of SARS-CoV-2. NM1267-treated mice showed significantly reduced disease progression, increased survival rates and secreted less infectious virus via their nostrils. Histopathological analyses and in situ hybridization further revealed a drastically reduced viral load and inflammatory response in lungs of NM1267-treated mice. These data suggest, that bipNb NM1267 is a broadly active and easily applicable drug candidate against a variety of emerging SARS-CoV-2 VOCs.

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

The SARS-CoV-2 pandemic virus is consistently evolving with mutations within the receptor binding domain (RBD)1 being of particular concern2-4. To date, there is little research into protection offered following vaccination or infection against RBD mutants in emerging variants of concern (UK3, South African5, Mink6 and Southern California7). To investigate this, serum and saliva samples were obtained from groups of vaccinated (Pfizer BNT-162b28), infected and uninfected individuals. Antibody responses among groups, including salivary antibody response and antibody binding to RBD mutant strains were examined. The neutralization capacity of the antibody response against a patient-isolated South African variant was tested by viral neutralization tests and further verified by an ACE2 competition assay. We found that humoral responses in vaccinated individuals showed a robust response after the second dose. Interestingly, IgG antibodies were detected in large titers in the saliva of vaccinated subjects. Antibody responses showed considerable differences in binding to RBD mutants in emerging variants of concern. A substantial reduction in RBD binding and neutralization was detected for the South African variant. Taken together our data reinforces the importance of administering the second dose of Pfizer BNT-162b2 to acquire high levels of neutralizing antibodies. High antibody titers in saliva suggest that vaccinated individuals may have reduced transmission potential. Substantially reduced neutralization for the South African variant highlights importance of surveillance strategies to detect new variants and targeting these in future vaccines.

Rapid and efficient inactivation of surface dried SARS-CoV-2 by UV-C irradiation

BackgroundThe SARS-CoV-2 pandemic urges for cheap, reliable, and rapid technologies for disinfection and decontamination. One frequently proposed method is UV-C irradiation. However, UV-C doses necessary to achieve inactivation of high-titer SARS-CoV-2 are poorly defined. MethodsUsing a box and two handheld systems designed to decontaminate objects and surfaces we evaluated the efficacy of 254 nm UV-C treatment to inactivate surface dried SARS-CoV-2. ResultsDrying for two hours did not have a major impact on the infectivity of SARS-CoV-2, indicating that exhaled virus in droplets or aerosols stays infectious on surfaces at least for a certain amount of time. Short exposure of high titer surface dried virus (3-5*10^6 IU/ml) with UV-C light (16 mJ/cm2) resulted in a total inactivation of SARS-CoV-2. Dose-dependency experiments revealed that 3.5 mJ/cm2 were still effective to achieve a > 6-log reduction in viral titers whereas 1.75 mJ/cm2 lowered infectivity only by one order of magnitude. ConclusionsOur results demonstrate that SARS-CoV-2 is rapidly inactivated by relatively low doses of UV-C irradiation. Furthermore, the data reveal that the relationship between UV-C dose and log-viral titer reduction of surface residing SARS-CoV-2 is non-linear. In the context of UV-C-based technologies used to disinfect surfaces, our findings emphasize the necessity to assure sufficient and complete exposure of all relevant areas by integrated UV-C doses of at least 3.5 mJ/cm2 at 254 nm. Altogether, UV-C treatment is an effective non-chemical possibility to decontaminate surfaces from high-titer infectious SARS-CoV-2.

Longitudinal analysis of virus load, serum antibody levels and virus neutralizing activity in vitro in cases with less severe COVID-19

SummaryO_ST_ABSBackgroundC_ST_ABSPatients infected with SARS-CoV-2 exhibit a highly variable clinical course, varying from barely discernible signs of disease, to moderate flu-like symptoms and, occasionally, with life-threatening pneumonia and/or cytokine storm. The relationship between the nasopharyngeal virus load, IgA and IgG antibodies to both the S1-RBD-protein and the N-protein as well the neutralizing activity (NAbs) against SARS-CoV-2 in the blood of moderately afflicted COVID-19 patients has not been investigated longitudinally so far. MethodsSeveral new serological methods to examine these parameters were developed and validated for the longitudinal investigation in three patients of a family which underwent a mild course of COVID-19. FindingsWe observed that the virus load had almost completely disappeared after about four weeks, whereas serum antibodies showed a contrasting course. IgA levels to S1-RBD-protein and, to a lesser extent, to the N-protein, peaked about three weeks after clinical disease onset but declined soon thereafter. IgG levels rose continuously, reaching a plateau approximately six weeks after disease onset. NAbs in serum reached a peak about four weeks after disease onset but dropped to a lower level about six weeks later. InterpretationOur data establishes associations of virus neutralization and a serological immune response foremost against Sars-CoV-2 S1-RDB-protein in a longitudinal manner.

Neutralizing antibody response in non-hospitalized SARS-CoV-2 patients

The majority of infections with SARS-CoV-2 are asymptomatic or mild without the necessity of hospitalization. It is of importance to reveal if these patients develop an antibody response against SARS-CoV-2 and to define which antibodies confer virus neutralization. We conducted a comprehensive serological survey of 49 patients with a mild course of disease and quantified neutralizing antibody responses against a clinical SARS-CoV-2 isolate employing human cells as targets. Four patients (8%), even though symptomatic, did not develop antibodies against SARS-CoV-2 and two other patients (4%) were only positive in one of the six serological assays employed. For the remainder, antibody response against the S-protein correlated with serum neutralization whereas antibodies against the nucleocapsid were poor predictors of virus neutralization. Regarding neutralization, only six patients (12%) could be classified as highly neutralizers. Furthermore, sera from several individuals with fairly high antibody levels had only poor neutralizing activity. In addition, employing a novel serological Western blot system to characterize antibody responses against seasonal coronaviruses, we found that antibodies against the seasonal coronavirus 229E might contribute to SARS-CoV-2 neutralization. Altogether, we show that there is a wide breadth of antibody responses against SARS-CoV-2 in patients that differentially correlate with virus neutralization. This highlights the difficulty to define reliable surrogate markers for immunity against SARS-CoV-2.

A throughput serological Western blot system using whole virus lysate for the concomitant detection of antibodies against SARS-CoV-2 and human endemic Coronaviridae

BO_SCPLOWACKGROUNDC_SCPLOWSeroreactivity against human endemic coronaviruses has been linked to disease severity after SARS-CoV-2 infection. Assays that are capable of concomitantly detecting antibodies against endemic coronaviridae such as OC43, 229E, NL63, and SARS-CoV-2 may help to elucidate this question. We set up a platform for serum-screening and developed a bead-based Western blot system, namely DigiWest, capable of running hundreds of assays using microgram amounts of protein prepared directly from different viruses. MO_SCPLOWETHODSC_SCPLOWThe parallelized and miniaturised DigiWest assay was adapted for detecting antibodies using whole protein extract prepared from isolated SARS-CoV-2 virus particles. After characterisation and optimization of the newly established test, whole virus lysates of OC43, 229E, and NL63 were integrated into the system. RO_SCPLOWESULTSC_SCPLOWThe DigiWest-based immunoassay system for detection of SARS-CoV-2 specific antibodies shows a sensitivity of 87.2 % and diagnostic specificity of 100 %. Concordance analysis with the SARS-CoV-2 immunoassays available by Roche, Siemens, and Euroimmun indicates a comparable assay performance (Cohens Kappa ranging from 0.8799-0.9429). In the multiplexed assay, antibodies against the endemic coronaviruses OC43, 229E, and NL63 were detected, displaying a high incidence of seroreactivity against these coronaviruses. CO_SCPLOWONCLUSIONC_SCPLOWThe DigiWest-based immunoassay, which uses authentic antigens from isolated virus particles, is capable of detecting individual serum responses against SARS-CoV-2 with high specificity and sensitivity in one multiplexed assay. It shows high concordance with other commercially available serologic assays. The DigiWest approach enables a concomitant detection of antibodies against different endemic coronaviruses and will help to elucidate the role of these possibly cross-reactive antibodies.