Absence of detectable SARS-CoV-2 replication in ex vivo cultured cornea and cornea-derived epithelial cells.

PURPOSE: To study the possibility of SARS-CoV-2 to infect human corneal cells and tissues under standard corneal culture conditions using explants of COVID-19 donors and primary cornea-derived epithelial cells. METHODS: Cornea isolated from deceased COVID-19 donors was cultured for 4 weeks, and SARS-CoV-2 replication was monitored by qRT-PCR. Furthermore, primary corneal epithelial cells from healthy donors were cultured ex vivo and infected with SARS-CoV-2 and human cytomegalovirus (HCMV) as a control. Infection status was assessed by western blotting and reporter gene expression using green fluorescent protein-expressing viral strains. ACE2 and TMPRSS2 receptor expression levels in cornea and epithelial cells were assessed by qRT-PCR. RESULTS: We did not detect SARS-CoV-2 replication in 10 corneas isolated from deceased COVID-19 patients and cultured for 4 weeks, indicating absence of infection under natural conditions. Furthermore, high-titer SARS-CoV-2 infection of ex vivo cultured cornea-derived epithelial cells did not result in productive virus replication. In contrast, the same cells were highly permissive for HCMV. This phenotype could potentially be explained by low ACE2 and TMPRSS2 transcriptional activity in cornea and cornea-derived epithelial cells. CONCLUSIONS: Our data suggest that cornea and limbal epithelial cells are refractory to productive SARS-CoV-2 infection. This could be due to the absence of robust receptor expression levels necessary for viral entry. This study adds further evidence to support the very low possibility of transmission of SARS-CoV-2 from an infected corneal transplant donor to a recipient in corneal organ cultures.

Novel ACE2 fusion protein with adapting activity against SARS-CoV-2 variants in vitro.

Despite the successful development of vaccines and neutralizing antibodies to limit the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerging variants prolong the pandemic and emphasize the persistent need to develop effective antiviral treatment regimens. Recombinant antibodies directed to the original SARS-CoV-2 have been successfully used to treat established viral disease. However, emerging viral variants escape the recognition by those antibodies. Here we report the engineering of an optimized ACE2 fusion protein, designated ACE2-M, which comprises a human IgG1 Fc domain with abrogated Fc-receptor binding linked to a catalytically-inactive ACE2 extracellular domain that displays increased apparent affinity to the B.1 spike protein. The affinity and neutralization capacity of ACE2-M is unaffected or even enhanced by mutations present in the spike protein of viral variants. In contrast, a recombinant neutralizing reference antibody, as well as antibodies present in the sera of vaccinated individuals, lose activity against such variants. With its potential to resist viral immune escape ACE2-M appears to be particularly valuable in the context of pandemic preparedness towards newly emerging coronaviruses.

Pulmonary Surfactant Proteins are Inhibited by IgA Autoantibodies in Severe COVID-19.

RATIONALE: Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distress syndrome with fatal outcomes. Evidence suggests that dysregulated immune responses, including autoimmunity, are key pathogenic factors. OBJECTIVES: To assess whether IgA autoantibodies target lung-specific proteins and contribute to disease severity. METHODS: We collected 147 blood, 9 lung tissue, and 36 bronchoalveolar lavage fluid samples from three tertiary hospitals in Switzerland and one in Germany. Severe COVID-19 was defined by the need to administer oxygen. We investigated the presence of IgA autoantibodies and their effects on pulmonary surfactant in COVID-19 using the following methods: immunofluorescence on tissue samples, immunoprecipitations followed by mass spectrometry on bronchoalveolar lavage fluid samples, enzyme-linked immunosorbent assays on blood samples, and surface tension measurements with medical surfactant. MEASUREMENTS AND MAIN RESULTS: IgA autoantibodies targeting pulmonary surfactant proteins B and C were elevated in patients with severe COVID-19, but not in patients with influenza or bacterial pneumonia. Notably, pulmonary surfactant failed to reduce surface tension after incubation with either plasma or purified IgA from patients with severe COVID-19. CONCLUSIONS: Our data suggest that patients with severe COVID-19 harbor IgA against pulmonary surfactant proteins B and C and that these antibodies block the function of lung surfactant, potentially contributing to alveolar collapse and poor oxygenation. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Inactivation of aerosolized SARS-CoV-2 by 254 nm UV-C irradiation.

Surface residing SARS-CoV-2 is efficiently inactivated by UV-C irradiation. This raises the question whether UV-C-based technologies are also suitable to decontaminate SARS-CoV-2- containing aerosols and which doses are needed to achieve inactivation. Here, we designed a test bench to generate aerosolized SARS-CoV-2 and exposed the aerosols to a defined UV-C dose. Our results demonstrate that the exposure of aerosolized SARS-CoV-2 with a low average dose in the order of 0.42-0.51 mJ/cm2 UV-C at 254 nm resulted in more than 99.9% reduction in viral titers. Altogether, UV-C-based decontamination of aerosols seems highly effective to achieve a significant reduction in SARS-CoV-2 infectivity.

COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants.

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.

Biparatopic nanobodies protect mice from lethal challenge with SARS-CoV-2 variants of concern.

The ongoing COVID-19 pandemic and the emergence of new SARS-CoV-2 variants of concern (VOCs) requires continued development of effective therapeutics. Recently, we identified high-affinity neutralizing nanobodies (Nbs) specific for the receptor-binding domain (RBD) of SARS-CoV-2. Taking advantage of detailed epitope mapping, we generate two biparatopic Nbs (bipNbs) targeting a conserved epitope outside and two different epitopes inside the RBD:ACE2 interface. Both bipNbs bind all currently circulating VOCs with high affinities and are capable to neutralize cellular infection with VOC B.1.351 (Beta) and B.1.617.2 (Delta) in vitro. To assess if the bipNbs NM1267 and NM1268 confer protection against SARS-CoV-2 infection in vivo, human ACE2 transgenic mice are treated intranasally before infection with a lethal dose of SARS-CoV-2 B.1, B.1.351 (Beta) or B.1.617.2 (Delta). Nb-treated mice show significantly reduced disease progression and increased survival rates. Histopathological analyses further reveal a drastically reduced viral load and inflammatory response in lungs. These data suggest that both bipNbs are broadly active against a variety of emerging SARS-CoV-2 VOCs and represent easily applicable drug candidates.

Iota-Carrageenan Inhibits Replication of SARS-CoV-2 and the Respective Variants of Concern Alpha, Beta, Gamma and Delta.

The COVID-19 pandemic continues to spread around the world and remains a major public health threat. Vaccine inefficiency, vaccination breakthroughs and lack of supply, especially in developing countries, as well as the fact that a non-negligible part of the population either refuse vaccination or cannot be vaccinated due to age, pre-existing illness or non-response to existing vaccines intensify this issue. This might also contribute to the emergence of new variants, being more efficiently transmitted, more virulent and more capable of escaping naturally acquired and vaccine-induced immunity. Hence, the need of effective and viable prevention options to reduce viral transmission is of outmost importance. In this study, we investigated the antiviral effect of iota-, lambda- and kappa-carrageenan, sulfated polysaccharides extracted from red seaweed, on SARS-CoV-2 Wuhan type and the spreading variants of concern (VOCs) Alpha, Beta, Gamma and Delta. Carrageenans as part of broadly used nasal and mouth sprays as well as lozenges have the potential of first line defense to inhibit the infection and transmission of SARS-CoV-2. Here, we demonstrate by using a SARS-CoV-2 spike pseudotyped lentivirus particles (SSPL) system and patient-isolated SARS-CoV-2 VOCs to infect transgenic A549ACE2/TMPRSS2 and Calu-3 human lung cells that all three carrageenan types exert antiviral activity. Iota-carrageenan exhibits antiviral activity with comparable IC50 values against the SARS-CoV-2 Wuhan type and the VOCs. Altogether, these results indicate that iota-carrageenan might be effective for prophylaxis and treatment of SARS-CoV-2 infections independent of the present and potentially future variants.

Long-Term Humoral Immune Response against SARS-CoV-2 after Natural Infection and Subsequent Vaccination According to WHO International Binding Antibody Units (BAU/mL).

The new WHO reference standard allows for the definition of serum antibodies against various SARS-CoV-2 antigens in terms of binding antibody units (BAU/mL) and thus to compare the results of different ELISA systems. In this study, the concentration of antibodies (ABs) against both the S- and the N-protein of SARS-CoV-2 as well as serum neutralization activity were evaluated in three patients after a mild course of COVID-19. Serum samples were collected frequently during a period of over one year. Furthermore, in two individuals, the effects of an additional vaccination with a mRNA vaccine containing the S1-RBD sequence on these antibodies were examined. After natural infection, the antibodies (IgA, IgG) against the S1-protein remained elevated above the established cut-off to positivity (S-IgA 60 BAU/mL and S-IgG 50 BAU/mL, respectively) for over a year in all patients, while this was not the case for ABs against the N-protein (cut-off N-IgG 40 BAU/mL, N-IgA 256 BAU/mL). Sera from all patients retained the ability to neutralize SARS-CoV-2 for more than a year. Vaccination resulted in a rapid boost of antibodies to S1-protein but, as expected, not to the N-protein. Most likely, the wide use of the WHO reference preparation will be very useful in determining the individual immune status of patients after an infection with SARS-CoV-2 or after vaccination.

Rapid, dose-dependent and efficient inactivation of surface dried SARS-CoV-2 by 254 nm UV-C irradiation.

BACKGROUND: The COVID-19 pandemic urges for cheap, reliable, and rapid technologies for disinfection and decontamination. One frequently proposed method is ultraviolet (UV)-C irradiation. UV-C doses necessary to achieve inactivation of high-titre SARS-CoV-2 are poorly defined. AIM: We investigated whether short exposure of SARS-CoV-2 to UV-C irradiation sufficiently reduces viral infectivity and doses necessary to achieve an at least 6-log reduction in viral titres. METHODS: Using 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 high-titre SARS-CoV-2. RESULTS: Drying for 2 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 for at least a certain amount of time. Short exposure of high titre 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 titres, whereas 1.75 mJ/cm2 lowered infectivity only by one order of magnitude. CONCLUSIONS: SARS-CoV-2 is rapidly inactivated by relatively low doses of UV-C irradiation and the relationship between UV-C dose and log-viral titre reduction of surface residing SARS-CoV-2 is nonlinear. Our findings emphasize that it is necessary 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 option to decontaminate surfaces from high-titre infectious SARS-CoV-2.

Lectin from Triticum vulgaris (WGA) Inhibits Infection with SARS-CoV-2 and Its Variants of Concern Alpha and Beta.

Even in the face of global vaccination campaigns, there is still an urgent need for effective antivirals against SARS-CoV-2 and its rapidly spreading variants. Several natural compounds show potential as antiviral substances and have the advantages of broad availabilities and large therapeutic windows. Here, we report that lectin from Triticum vulgaris (Wheat Germ Agglutinin) displays antiviral activity against SARS-CoV-2 and its major Variants of Concern (VoC), Alpha and Beta. In Vero B4 cells, WGA potently inhibits SARS-CoV-2 infection with an IC50 of <10 ng/mL. WGA is effective upon preincubation with the virus or when added during infection. Pull-down assays demonstrate direct binding of WGA to SARS-CoV-2, further strengthening the hypothesis that inhibition of viral entry by neutralizing free virions might be the mode of action behind its antiviral effect. Furthermore, WGA exhibits antiviral activity against human coronavirus OC43, but not against other non-coronaviruses causing respiratory tract infections. Finally, WGA inhibits infection of the lung cell line Calu-3 with wild type and VoC viruses with comparable IC50 values. Altogether, our data indicate that topical administration of WGA might be effective for prophylaxis or treatment of SARS-CoV-2 infections.

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.

[Biology and pathology of coronaviruses]. / Biologie und Pathologie von Coronaviren.

The worldwide novel coronavirus SARS-CoV­2 pandemic is ongoing. SARS-CoV­2 belongs to the coronavirus family, the first representatives of which have been known since the 1960s. Coronaviruses are present in animals and humans and show similarities as well as differences in their biology and pathology regarding each genus. Besides mild flu-like and gastroenterological symptoms, SARS-CoV­2 can lead to dysfunctions of the lungs and other organs including the heart as already observed during SARS and MERS infections.

Severe acute respiratory Syndrome-Coronavirus-2: Can it be detected in the retina?

BACKGROUND/OBJECTIVES: The systemic organ involvement of SARS-CoV-2 needs to be thoroughly investigated including the possibility of an ocular reservoir in humans. To examine retinal tissues and vitreous for histopathology and SARS-CoV-2 presence with regard to possible effects on the human retina and/ or vitreous. We performed histopathological analyses and quantitative (q)RT-PCR-testing for SARS-CoV-2 RNA on retinal tissues and vitreous of COVID-19 postmortem donors. SUBJECTS/METHODS: Included in this study were 10 eyes of 5 deceased COVID-19 patients. The diagnosis of SARS-CoV-2 infection was confirmed via pharyngeal swabs and broncho-alveolar fluids. The highest level of personal protective equipment (PPE) and measures was employed during fluid-tissue procurement and preparation. Histopathological examinations and qRT-PCR-testing were carried out for all retinal tissues and vitreous fluids. RESULTS: The histopathological examinations revealed no signs of morphologically identifiable retinal inflammation or vessel occlusions based on hematoxylin and eosin stains. By qRT-PCRs, we detected no significant level of viral RNA in human retina and vitreous. CONCLUSIONS: In this study, no significant level of SARS-CoV-2-RNA was detected in the human retinal and vitreous fluid samples of deceased COVID-19 patients. Histopathological examinations confirmed no morphological sign of damage to retinal vasculature or tissues. Further studies are needed to confirm or refute the results.

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.

Designing a SARS-CoV-2 T-Cell-Inducing Vaccine for High-Risk Patient Groups.

We describe the results of two vaccinations of a self-experimenting healthy volunteer with SARS-CoV-2-derived peptides performed in March and April 2020, respectively. The first set of peptides contained eight peptides predicted to bind to the individual's HLA molecules. The second set consisted of ten peptides predicted to bind promiscuously to several HLA-DR allotypes. The vaccine formulation contained the new TLR 1/2 agonist XS15 and was administered as an emulsion in Montanide as a single subcutaneous injection. Peripheral blood mononuclear cells isolated from blood drawn before and after vaccinations were assessed using Interferon-γ ELISpot assays and intracellular cytokine staining. We detected vaccine-induced CD4 T cell responses against six out of 11 peptides predicted to bind to HLA-DR after 19 days, following vaccination, for one peptide already at day 12. We used these results to support the design of a T-cell-inducing vaccine for application in high-risk patients, with weakened lymphocyte performance. Meanwhile, an according vaccine, incorporating T cell epitopes predominant in convalescents, is undergoing clinical trial testing.

Inactivation of SARS-CoV-2 through Treatment with the Mouth Rinsing Solutions ViruProX® and BacterX® Pro.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic effects daily dental work. Therefore, infection control measures are necessary to prevent infection of dental personnel during dental treatments. The use of a preprocedural mouth rinse with chlorhexidine (CHX), cetylpyridinium chloride (CPC), or hydrogen peroxide (H2O2) solution for 30-60 s may reduce the viral load and may protect the personnel in a dental practice. In the present study the virucidal effect of the mouth rinsing solutions ViruProX® with 0.05% CPC and 1.5% H2O2 and BacterX® pro containing 0.1% CHX, 0.05% CPC, and 0.005% sodium fluoride (F-) was investigated in vitro. The mouth rinsing solutions successfully inactivated infectious SARS-CoV-2 particles, the causative agent of coronavirus disease 2019 (COVID-19), within 30 s. To determine the effective components, CHX, CPC, H2O2, and a combination of CHX and CPC, were tested against SARS-CoV-2 in addition. While a combination of CPC and CHX as well as CPC alone led to a significant reduction of infectious viral particles, H2O2 and CHX alone had no virucidal effect against SARS-CoV-2. It can be assumed that preprocedural rinsing of the mouth with ViruProX® or BacterX® pro will reduce the viral load in the oral cavity and could thus lower the transmission of SARS-CoV-2 in dental practice.

Multiplexed Serum Antibody Screening Platform Using Virus Extracts from Endemic Coronaviridae and SARS-CoV-2.

The presence of antibodies against endemic coronaviruses has been linked to disease severity after SARS-CoV-2 infection. Assays capable of concomitantly detecting antibodies against endemic coronaviridae such as OC43, 229E, NL63, and SARS-CoV-2 may help to elucidate this question. We developed a serum screening platform using a bead-based Western blot system called DigiWest, capable of running hundreds of assays using microgram amounts of protein prepared directly from different viruses. Characterization of the immunoassay for detection of SARS-CoV-2 specific antibodies revealed a sensitivity of 90.3% and a diagnostic specificity of 98.1%. Concordance analysis with the SARS-CoV-2 immunoassays available by Roche, Siemens, and Euroimmun indicates comparable assay performances (Cohen's κ ranging from 0.8874 to 0.9508). Analogous assays for OC43, 229E, and NL63 were established and combined into one multiplex with the SARS-CoV-2 assay. Seroreactivity for different coronaviruses was detected with high incidence, and the multiplex assay was adapted for serum screening.

Antibody Response against SARS-CoV-2 and Seasonal Coronaviruses in Nonhospitalized COVID-19 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 positive in only one of the six serological assays employed. For the remaining 88%, antibody response against the S protein correlated with serum neutralization whereas antibodies against the nucleocapsid were poor predictors of virus neutralization. None of the sera enhanced infection of human cells with SARS-CoV-2 at any dilution, arguing against antibody-dependent enhancement of infection in our system. Regarding neutralization, only six patients (12%) could be classified as high 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.IMPORTANCE There is strong interest in the nature of the neutralizing antibody response against SARS-CoV-2 in infected individuals. For vaccine development, it is especially important which antibodies confer protection against SARS-CoV-2, if there is a phenomenon called antibody-dependent enhancement (ADE) of infection, and if there is cross-protection by antibodies directed against seasonal coronaviruses. We addressed these questions and found in accordance with other studies that neutralization is mediated mainly by antibodies directed against the spike protein of SARS-CoV-2 in general and the receptor binding site in particular. In our test system, utilizing human cells for infection experiments, we did not detect ADE. However, using a novel diagnostic test we found that antibodies against the coronavirus 229E might be involved in cross-protection to SARS-CoV-2.

Absence of Severe Acute Respiratory Syndrome-Coronavirus-2 RNA in Human Corneal Tissues.

PURPOSE: To examine corneal tissue for severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) positivity regarding implications for tissue procurement, processing, corneal transplantation, and ocular surgery on healthy patients. We performed quantitative reverse transcription-polymerase chain reaction qRT-PCR-testing for SARS-CoV-2 RNA on corneal stroma and endothelium, bulbar conjunctiva, conjunctival fluid swabs, anterior chamber fluid, and corneal epithelium of coronavirus disease 2019 (COVID-19) postmortem donors. METHODS: Included in this study were 10 bulbi of 5 COVID-19 patients who died because of respiratory insufficiency. Informed consent and institutional review board approval was obtained before this study (241/2020BO2). SARS-CoV-2 was detected by using a pharyngeal swab and bronchoalveolar lavage. Tissue procurement and tissue preparation were performed with personal protective equipment (PPE) and the necessary protective measures. qRT-PCR-testing was performed for each of the abovementioned tissues and intraocular fluids. RESULTS: The qRT-PCRs yielded no viral RNA in the following ocular tissues and intraocular fluid: corneal stroma and endothelium, bulbar-limbal conjunctiva, conjunctival fluid swabs, anterior chamber fluid, and corneal epithelium. CONCLUSIONS: In this study, no SARS-CoV-2-RNA was detected in conjunctiva, anterior chamber fluid, and corneal tissues (endothelium, stroma, and epithelium) of COVID-19 donors. This implicates that the risk for SARS-CoV-2 infection using corneal or conjunctival tissue is very low. However, further studies on a higher number of COVID-19 patients are necessary to confirm these results. This might be of high importance for donor tissue procurement, processing, and corneal transplantation.

Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.