Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models.

The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains. We found that a single intranasal (i.n.) dose of serotype-5 adenoviral vectors expressing either the receptor binding domain (Ad5-RBD) or the complete ectodomain (Ad5-S) of the SARS-CoV-2 spike protein was effective in inducing i) serum and bronchoalveolar lavage (BAL) anti-spike IgA and IgG, ii) robust SARS-CoV-2-neutralizing activity in the serum and BAL, iii) rigorous spike-directed T helper 1 cell/cytotoxic T cell immunity, and iv) protection of mice from a challenge with the SARS-CoV-2 beta variant. Intramuscular (i.m.) Ad5-RBD or Ad5-S administration did not induce serum or BAL IgA, and resulted in lower neutralizing titers in the serum. Moreover, prior immunity induced by an intramuscular mRNA vaccine could be potently enhanced and modulated towards a mucosal IgA response by an i.n. Ad5-S booster. Notably, Ad5 DNA was found in the liver or spleen after i.m. but not i.n. administration, indicating a lack of systemic spread of the vaccine vector, which has been associated with a risk of thrombotic thrombocytopenia. Unlike in otherwise genetically identical HLA-DQ6 mice, in HLA-DQ8 mice Ad5-RBD vaccine was inferior to Ad5-S, suggesting that the RBD fragment does not contain a sufficient collection of helper-T cell epitopes to constitute an optimal vaccine antigen. Our data add to previous promising preclinical results on intranasal SARS-CoV-2 vaccination and support the potential of this approach to elicit mucosal immunity for preventing transmission of SARS-CoV-2.

Intranasal trimeric sherpabody inhibits SARS-CoV-2 including recent immunoevasive Omicron subvariants.

The emergence of increasingly immunoevasive SARS-CoV-2 variants emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Intranasal administration of neutralizing antibodies has shown encouraging protective potential but there remains a need for SARS-CoV-2 blocking agents that are less vulnerable to mutational viral variation and more economical to produce in large scale. Here we describe TriSb92, a highly manufacturable and stable trimeric antibody-mimetic sherpabody targeted against a conserved region of the viral spike glycoprotein. TriSb92 potently neutralizes SARS-CoV-2, including the latest Omicron variants like BF.7, XBB, and BQ.1.1. In female Balb/c mice intranasal administration of just 5 or 50 micrograms of TriSb92 as early as 8 h before but also 4 h after SARS-CoV-2 challenge can protect from infection. Cryo-EM and biochemical studies reveal triggering of a conformational shift in the spike trimer as the inhibitory mechanism of TriSb92. The potency and robust biochemical properties of TriSb92 together with its resistance against viral sequence evolution suggest that TriSb92 could be useful as a nasal spray for protecting susceptible individuals from SARS-CoV-2 infection.

Clinical and Serological Findings of COVID-19 Participants in the Region of Makkah, Saudi Arabia.

Makkah in Saudi Arabia hosts the largest annual religious event in the world. Despite the many strict rules enacted, including Hajj cancellation, city lockdowns, and social distancing, the region has the second highest number of new COVID-19 cases in Saudi Arabia. Public health interventions that identify, isolate, and manage new cases could slow the infection rate. While RT-PCR is the current gold standard in SARS-CoV-2 identification, it yields false positive and negative results, which mandates the use of complementary serological tests. Here, we report the utility of serological assays during the acute phase of individuals with moderate and severe clinical manifestations of SARS-CoV-2 (COVID19). Fifty participants with positive RT-PCR results for SARS-CoV-2 were enrolled in this study. Following RT-PCR diagnosis, serum samples from the same participants were analyzed using in-house ELISA (IgM, IgA, and IgG) and microneutralization test (MNT) for the presence of antibodies. Of the 50 individuals analyzed, 43 (86%) showed a neutralizing antibody titer of ≥20. Univariate analysis with neutralizing antibodies as a dependent variable and the degree of disease severity and underlying medical conditions as fixed factors revealed that patients with no previous history of non-communicable diseases and moderate clinical manifestation had the strongest neutralizing antibody response "Mean: 561.11". Participants with severe symptoms and other underlying disorders, including deceased individuals, demonstrated the lowest neutralizing antibody response. Anti-spike protein antibody responses, as measured by ELISA, showed a statistically significant correlation with neutralizing antibodies. This reinforces the speculation that serological assays complement molecular testing for diagnostics; however, patients' previous medical history (anamnesis) should be considered in interpreting serological results.

Experimental Infection of Mink with SARS-COV-2 Omicron Variant and Subsequent Clinical Disease.

We report an experimental infection of American mink with SARS-CoV-2 Omicron variant and show that mink remain positive for viral RNA for days, experience clinical signs and histopathologic changes, and transmit the virus to uninfected recipients. Preparedness is crucial to avoid spread among mink and spillover to human populations.

Inhibition of SARS-CoV-2 Alpha Variant and Murine Noroviruses on Copper-Silver Nanocomposite Surfaces.

With the continued scenario of the COVID-19 pandemic, the world is still seeking out-of-the-box solutions to break its transmission cycle and contain the pandemic. There are different transmission routes for viruses, including indirect transmission via surfaces. To this end, we used two relevant viruses in our study. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the pandemic and human norovirus (HuNV), both known to be transmitted via surfaces. Several nanoformulations have shown attempts to inhibit SARS-CoV-2 and other viruses. However, a rigorous, similar inactivation scheme to inactivate the cords of two tedious viruses (SARS-CoV-2 Alpha variant and HuNV) is lacking. The present study demonstrates the inactivation of the SARS-CoV-2 Alpha variant and the decrease in the murine norovirus (MNV, a surrogate to HuNV) load after only one minute of contact to surfaces including copper-silver (Cu-Ag) nanocomposites. We thoroughly examined the physicochemical characteristics of such plated surfaces using diverse microscopy tools and found that Cu was the dominanting element in the tested three different surfaces (~56, ~59, and ~48 wt%, respectively), hence likely playing the major role of Alpha and MNV inactivation followed by the Ag content (~28, ~13, and ~11 wt%, respectively). These findings suggest that the administration of such surfaces within highly congested places (e.g., schools, public transportations, public toilets, and hospital and live-stock reservoirs) could break the SARS-CoV-2 and HuNV transmission. We suggest such an administration after an in-depth examination of the in vitro (especially on skin cells) and in vivo toxicity of the nanocomposite formulations and surfaces while also standardizing the physicochemical parameters, testing protocols, and animal models.

Common Laboratory Mice Are Susceptible to Infection with the SARS-CoV-2 Beta Variant.

Small animal models are of crucial importance for assessing COVID-19 countermeasures. Common laboratory mice would be well-suited for this purpose but are not susceptible to infection with wild-type SARS-CoV-2. However, the development of mouse-adapted virus strains has revealed key mutations in the SARS-CoV-2 spike protein that increase infectivity, and interestingly, many of these mutations are also present in naturally occurring SARS-CoV-2 variants of concern. This suggests that these variants might have the ability to infect common laboratory mice. Herein we show that the SARS-CoV-2 beta variant attains infectibility to BALB/c mice and causes pulmonary changes within 2-3 days post infection, consistent with results seen in other murine models of COVID-19, at a reasonable virus dose (2 × 105 PFU). The findings suggest that common laboratory mice can serve as the animal model of choice for testing the effectiveness of antiviral drugs and vaccines against SARS-CoV-2.

Copper-Silver Nanohybrids: SARS-CoV-2 Inhibitory Surfaces.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a severe health threat. The COVID-19 infections occurring in humans and animals render human-animal interfaces hot spots for spreading the pandemic. Lessons from the past point towards the antiviral properties of copper formulations; however, data showing the "contact-time limit" surface inhibitory efficacy of copper formulations to contain SARS-CoV-2 are limited. Here, we show the rapid inhibition of SARS-CoV-2 after only 1 and 5 min on two different surfaces containing copper-silver (Cu-Ag) nanohybrids. We characterized the nanohybrids' powder and surfaces using a series of sophisticated microscopy tools, including transmission and scanning electron microscopes (TEM and SEM) and energy-dispersive X-ray spectroscopy (EDX). We used culturing methods to demonstrate that Cu-Ag nanohybrids with high amounts of Cu (~65 and 78 wt%) and lower amounts of Ag (~7 and 9 wt%) inhibited SARS-CoV-2 efficiently. Collectively, the present work reveals the rapid SARS-CoV-2 surface inhibition and the promising application of such surfaces to break the SARS-CoV-2 transmission chain. For example, such applications could be invaluable within a hospital or live-stock settings, or any public place with surfaces that people frequently touch (i.e., public transportation, shopping malls, elevators, and door handles) after the precise control of different parameters and toxicity evaluations.

A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection-SARS-CoV-2 as a Proof of Concept.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has seen an unprecedented increase in the demand for rapid and reliable diagnostic tools, leaving many laboratories scrambling for resources. We present a fast and simple assay principle for antigen detection and demonstrate its functionality by detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in nasopharyngeal swabs. The method is based on the detection of SARS-CoV-2 nucleoprotein (NP) and S protein (SP) via time-resolved Förster resonance energy transfer (TR-FRET) with donor- and acceptor-labeled polyclonal anti-NP and -SP antibodies. Using recombinant proteins and cell culture-grown SARS-CoV-2, the limits of detection were established as 25 pg of NP or 20 infectious units (IU) and 875 pg of SP or 625 IU. Testing reverse transcription-PCR (RT-PCR)-positive (n = 48, with cycle threshold [CT ] values from 11 to 30) or -negative (n = 96) nasopharyngeal swabs demonstrated that the assay yielded positive results for all samples with CT values of <25 and for a single RT-PCR-negative sample. Virus isolation from the RT-PCR-positive nasopharyngeal swabs showed a strong association between the presence of infectious virus and a positive antigen test result. The NP-based assay showed 97.4% (37/38) sensitivity and 100% (10/10) specificity in comparison with virus isolation and 77.1% (37/48) sensitivity and 99.0% (95/96) specificity in comparison with SARS-CoV-2 RT-PCR. The assay is performed in a buffer that neutralizes SARS-CoV-2 infectivity, and the assay is relatively simple to set up as an "in-house" test. Here, SARS-CoV-2 served as the model pathogen, but the assay principle is applicable to other viral infections, and the test format could easily be adapted to high-throughput testing.IMPORTANCE PCR is currently the gold standard for the diagnosis of many acute infections. While PCR and its variants are highly sensitive and specific, the time from sampling to results is measured in hours at best. Antigen tests directly detect parts of the infectious agent, which may enable faster diagnosis but often at lower sensitivity and specificity. Here, we describe a technique for rapid antigen detection and demonstrate the test format's potential using SARS-CoV-2 as the model pathogen. The 10-min test, performed in a buffer that readily inactivates SARS-CoV-2, from nasopharyngeal samples identified 97.4% (37/38) of the samples from which we could isolate the virus. This suggests that the test performs well in identifying patients potentially shedding the virus. Although SARS-CoV-2 served as the model pathogen to demonstrate proof of concept, the test principle itself would be applicable to a wide variety of infectious and perhaps also noninfectious diseases.

A 10-Minute "Mix and Read" Antibody Assay for SARS-CoV-2.

Accurate and rapid diagnostic tools are needed for management of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Antibody tests enable detection of individuals past the initial phase of infection and help examine vaccine responses. The major targets of human antibody response in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the spike glycoprotein (SP) and nucleocapsid protein (NP). We have developed a rapid homogenous approach for antibody detection termed LFRET (protein L-based time-resolved Förster resonance energy transfer immunoassay). In LFRET, fluorophore-labeled protein L and antigen are brought to close proximity by antigen-specific patient immunoglobulins of any isotype, resulting in TR-FRET signal. We set up LFRET assays for antibodies against SP and NP and evaluated their diagnostic performance using a panel of 77 serum/plasma samples from 44 individuals with COVID-19 and 52 negative controls. Moreover, using a previously described SP and a novel NP construct, we set up enzyme linked immunosorbent assays (ELISAs) for antibodies against SARS-CoV-2 SP and NP. We then compared the LFRET assays with these ELISAs and with a SARS-CoV-2 microneutralization test (MNT). We found the LFRET assays to parallel ELISAs in sensitivity (90-95% vs. 90-100%) and specificity (100% vs. 94-100%). In identifying individuals with or without a detectable neutralizing antibody response, LFRET outperformed ELISA in specificity (91-96% vs. 82-87%), while demonstrating an equal sensitivity (98%). In conclusion, this study demonstrates the applicability of LFRET, a 10-min "mix and read" assay, to detection of SARS-CoV-2 antibodies.

SARS-CoV-2 infections among healthcare workers at Helsinki University Hospital, Finland, spring 2020: Serosurvey, symptoms and risk factors.

BACKGROUND: Exposure, risks and immunity of healthcare workers (HCWs), a vital resource during the SARS-CoV-2 pandemic, warrant special attention. METHODS: HCWs at Helsinki University Hospital, Finland, filled in questionnaires and provided serum samples for SARS-CoV-2-specific antibody screening by Euroimmun IgG assay in March-April 2020. Positive/equivocal findings were confirmed by Abbott and microneutralization tests. Positivity by two of the three assays or RT-PCR indicated a Covid-19 case (CoV+). RESULTS: The rate of CoV(+) was 3.3% (36/1095) and seropositivity 3.0% (33/1095). CoV(+) was associated with contact with a known Covid-19 case, and working on a Covid-19-dedicated ward or one with cases among staff. The rate in the Covid-19-dedicated ICU was negligible. Smoking and age <55 years were associated with decreased risk. CoV(+) was strongly associated with ageusia, anosmia, myalgia, fatigue, fever, and chest pressure. Seropositivity was recorded for 89.3% of those with prior documented RT-PCR-positivity and 2.4% of those RT-PCR-negative. The rate of previously unidentified cases was 0.7% (8/1067) and asymptomatic ones 0% (0/36). CONCLUSION: Undiagnosed and asymptomatic cases among HCWs proved rare. An increased risk was associated with Covid-19-dedicated wards. Particularly high rates were seen for wards with liberal HCW-HCW contacts, highlighting the importance of social distancing also among HCWs.

Longitudinal proteomic profiling reveals increased early inflammation and sustained apoptosis proteins in severe COVID-19.

SARS-CoV-2 infection has a risk to develop into life-threatening COVID-19 disease. Whereas age, hypertension, and chronic inflammatory conditions are risk factors, underlying host factors and markers for disease severity, e.g. requiring intensive care unit (ICU) treatment, remain poorly defined. To this end, we longitudinally profiled blood inflammation markers, antibodies, and 101 plasma proteins of hospitalized COVID-19 patients who did or did not require ICU admission. While essentially all patients displayed SARS-CoV-2-specific antibodies and virus-neutralization capacity within 12-15 days, a rapid, mostly transient upregulation of selective inflammatory markers including IL-6, CXCL10, CXCL11, IFNγ, IL-10, and monocyte-attracting CCL2, CCL7 and CCL8, was particularly evident in ICU patients. In addition, there was consistent and sustained upregulation of apoptosis-associated proteins CASP8, TNFSF14, HGF, and TGFB1, with HGF discriminating between ICU and non-ICU cohorts. Thus, COVID-19 is associated with a selective inflammatory milieu within which the apoptotic pathway is a cardinal feature with potential to aid risk-based patient stratification.