Nanobodies against SARS-CoV-2 non-structural protein Nsp9 inhibit viral replication by targeting innate immunity (preprint)

Nanobodies are emerging as critical tools for drug design. Several have been recently created to serve as inhibitors of SARS-Cov-2 entry in the host cell by targeting surface-exposed Spike protein. However, due to the high frequency of mutations that affect Spike, these nanobodies may not target it to their full potential and as a consequence, inhibition of viral entry may not be efficient. Here we have established a pipeline that instead targets highly conserved viral proteins that are made only after viral entry into the host cell when the SARS-Cov-2 RNA-based genome is translated. As proof of principle, we designed nanobodies against the SARS-CoV-2 non-structural protein Nsp9, required for viral genome replication. To find out if this strategy efficiently blocks viral replication, one of these anti-Nsp9 nanobodies, 2NSP23, previously characterized using immunoassays and NMR spectroscopy for epitope mapping, was encapsulated into lipid nanoparticles (LNP) as mRNA. We show that this nanobody, hereby referred to as LNP-mRNA-2NSP23, is internalized and translated in HEK293 cells. We next infected HEK293-ACE2 cells with multiple SARS-CoV-2 variants and subjected them to LNP-mRNA-2NSP23 treatment. Analysis of total RNA isolated from infected cells treated or untreated with LNP-mRNA-2NSP23 using qPCR and RNA deep sequencing shows that the LNP-mRNA-2NSP23 nanobody protects HEK293-ACE2 cells and suppresses replication of several SARS-CoV-2 variants. These observations indicate that following translation, the nanobody 2NSP23 inhibits viral replication by targeting Nsp9 in living cells. We speculate that LNP-mRNA-2NSP23 may be translated into an innovative technology to generate novel antiviral drugs highly efficient across coronaviruses.

"Photolangage©" Psychoterapy Group Held in Person during Pandemic in a Psychiatric Day-Care Centre

Introduction: The Sars-Covid-19 pandemic situation has placed services dedicated to the care and rehabilitation of patients with psychotic disorders in the face of the choice of which therapeutic resources should be provided "in-person" and which "remotely". The choice of the two authors was to maintain most of the group activities "in-person", binding the participants to all the restrictions set by the norms, and to translate "remotely" mainly the individual activities. This choice was the result of a reflection of the working group, stemming from concern that a practice carried out "remotely" and "in group" in an already extremely compromised group of patients might further impair the transformative effect of the treatment itself. The underlying hypothesis was that the lack of a real physical presence and the loss of sensory input that allow one to "listen to the patient with all the senses" (Bastianini, 2019) might affect the possibility to access pre-symbolic experiences, experiences that cannot be evoked and expressed in words and that usually emerge not through verbal and symbolic communication but through nonverbal one. Method(s): In particular, this consideration has been applied from the beginning to the Photolangage © group, in which participants are already stimulated by a verbal content and asked to find answers without the use of words, through the physical choice of some photos after a "silent observation" of a selection of images taken by the conductors from some dossier;just at a subsequent, participants are encouraged to translate their choice into word, and then to share all their reflections and feelings with the rest of the group. All these steps - seen as key elements of this kind of group setting - were considered "untranslatable" as a step of a "remote" setting for this kind of groupusers, while in other contexts (e.g., didactic field) they were adopted effectively by the two authors. Thus, the hypothesis is that it was the pathology and level of functioning of the targeted treatment subjects that was mandatory for the choice rather than administration procedure of the treatment itself. Result(s): Finally, despite the limitations given by the pandemic (e.g., use of devices before and during the group session, distancing, reduction of the number of participants), group members have expressed their gratitude for the guarantee of effective treatment, experiencing a continuity in the sense of being "taken care of" and "thought about" by services. "In-person" treatment also provided the fostering and maintaining moments of "good sociability" (Corbella, 2021) among group members, who often lack it due to the consequences of their pathology and at a time when the pandemic situation affected actually that dimension.

Maternal transfer of IgA and IgG SARS-CoV-2 specific antibodies transplacentally and via breast milk feeding.

BACKGROUND: Although there have been many studies on antibody responses to SARS-CoV-2 in breast milk, very few have looked at the fate of these in the infant, and whether they are delivered to immunologically relevant sites in infants. METHODS: Mother/infant pairs (mothers who breast milk fed and who were SARS-CoV-2 vaccinated before or after delivery) were recruited for this cross-sectional study. Mother blood, mother breast milk, infant blood, infant nasal specimen, and infant stool was tested for IgA and IgG antibodies against SARS-CoV-2 spike trimer. RESULTS: Thirty-one mother/infant pairs were recruited. Breast milk fed infants acquired systemic anti-spike IgG antibodies only if their mothers were vaccinated antepartum (100% Antepartum; 0% Postpartum; P<0.0001). Breast milk fed infants acquired mucosal anti-spike IgG antibodies (in the nose) only if their mothers were vaccinated antepartum (89% Antepartum; 0% Postpartum; P<0.0001). None of the infants in either group had anti-spike IgA in the blood. Surprisingly, 33% of the infants whose mothers were vaccinated antepartum had high titer anti-spike IgA in the nose (33% Antepartum; 0% Postpartum; P = 0.03). Half-life of maternally transferred plasma IgG antibodies in the Antepartum infant cohort was ~70 days. CONCLUSION: Vaccination antepartum followed by breast milk feeding appears to be the best way to provide systemic and local anti-SARS-CoV-2 antibodies for infants. The presence of high titer SARS-CoV-2-specific IgA in the nose of infants points to the potential importance of breast milk feeding early in life for maternal transfer of mucosal IgA antibodies. Expectant mothers should consider becoming vaccinated antepartum and consider breast milk feeding for optimal transfer of systemic and mucosal antibodies to their infants.

Histopathology and SARS-CoV-2 Cellular Localization in Eye Tissues of COVID-19 Autopsies.

Ophthalmic manifestations and tissue tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported in association with coronavirus disease 2019 (COVID-19), but the pathology and cellular localization of SARS-CoV-2 are not well characterized. The objective of this study was to evaluate macroscopic and microscopic changes and investigate cellular localization of SARS-CoV-2 across ocular tissues at autopsy. Ocular tissues were obtained from 25 patients with COVID-19 at autopsy. SARS-CoV-2 nucleocapsid gene RNA was previously quantified by droplet digital PCR from one eye. Herein, contralateral eyes from 21 patients were fixed in formalin and subject to histopathologic examination. Sections of the droplet digital PCR-positive eyes from four other patients were evaluated by in situ hybridization to determine the cellular localization of SARS-CoV-2 spike gene RNA. Histopathologic abnormalities, including cytoid bodies, vascular changes, and retinal edema, with minimal or no inflammation in ocular tissues were observed in all 21 cases evaluated. In situ hybridization localized SARS-CoV-2 RNA to neuronal cells of the retinal inner and outer layers, ganglion cells, corneal epithelia, scleral fibroblasts, and oligodendrocytes of the optic nerve. In conclusion, a range of common histopathologic alterations were identified within ocular tissue, and SARS-CoV-2 RNA was localized to multiple cell types. Further studies will be required to determine whether the alterations observed were caused by SARS-CoV-2 infection, the host immune response, and/or preexisting comorbidities.

Successful lung transplantation using an allograft from a COVID-19–recovered donor: a potential role for subgenomic RNA to guide organ utilization

Although the risk of SARS-CoV-2 transmission through lung transplantation from acutely infected donors is high, the risks of virus transmission and long-term lung allograft outcomes are not as well described when using pulmonary organs from COVID-19–recovered donors. We describe successful lung transplantation for a COVID-19–related lung injury using lungs from a COVID-19–recovered donor who was retrospectively found to have detectable genomic SARS-CoV-2 RNA in the lung tissue by multiple highly sensitive assays. However, SARS-CoV-2 subgenomic RNA (sgRNA), a marker of viral replication, was not detectable in the donor respiratory tissues. One year after lung transplantation, the recipient has a good functional status, walking 1 mile several times per week without the need for supplemental oxygen and without any evidence of donor-derived SARS-CoV-2 transmission. Our findings highlight the limitations of current clinical laboratory diagnostic assays in detecting the persistence of SARS-CoV-2 RNA in the lung tissue. The persistence of SARS-CoV-2 RNA in the donor tissue did not appear to represent active viral replication via sgRNA testing and, most importantly, did not negatively impact the allograft outcome in the first year after lung transplantation. sgRNA is easily performed and may be a useful assay for assessing viral infectivity in organs from donors with a recent infection.

SARS-CoV-2 infection and persistence in the human body and brain at autopsy.

Coronavirus disease 2019 (COVID-19) is known to cause multi-organ dysfunction1-3 during acute infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with some patients experiencing prolonged symptoms, termed post-acute sequelae of SARS-CoV-2 (refs. 4,5). However, the burden of infection outside the respiratory tract and time to viral clearance are not well characterized, particularly in the brain3,6-14. Here we carried out complete autopsies on 44 patients who died with COVID-19, with extensive sampling of the central nervous system in 11 of these patients, to map and quantify the distribution, replication and cell-type specificity of SARS-CoV-2 across the human body, including the brain, from acute infection to more than seven months following symptom onset. We show that SARS-CoV-2 is widely distributed, predominantly among patients who died with severe COVID-19, and that virus replication is present in multiple respiratory and non-respiratory tissues, including the brain, early in infection. Further, we detected persistent SARS-CoV-2 RNA in multiple anatomic sites, including throughout the brain, as late as 230 days following symptom onset in one case. Despite extensive distribution of SARS-CoV-2 RNA throughout the body, we observed little evidence of inflammation or direct viral cytopathology outside the respiratory tract. Our data indicate that in some patients SARS-CoV-2 can cause systemic infection and persist in the body for months.

SARS-CoV-2 RNA in dental biofilms: Supragingival and subgingival findings from inpatients in a COVID-19 intensive care unit.

BACKGROUND: Saliva, salivary glands, gingival crevicular fluid, and supragingival biofilms may harbor SARS-CoV-2 RNA. This observational study aimed to investigate the presence and load of SARS-CoV-2 RNA in supragingival, and subgingival biofilms obtained from intensive care unit (ICU) patients. METHODS: A convenience sample, composed of 52 COVID-19+ participants (48.6 ± 14.8 years, 26.9% females), were evaluated for pre-existing comorbidities, number of teeth, and periodontal data [visible plaque (VPI), bleeding on probing (BOP), periodontal probing depth (PPD), and attachment loss (AL)]. Supragingival and subgingival samples (SubDeep: four sites with the deepest PPD; SubRemain: remaining shallower sites) were analyzed by RT-qPCR with corresponding cycle quantification (Cq). Statistical analyses considered the individual (P = 5%). RESULTS: Twenty-six participants tested positive for dental biofilms (Biofilm+) with 96.2% of them being positive for subgingival samples. Pre-existing comorbidities, number of teeth examined, VPI, PPD, AL, and BOP were similar between Biofilm+ and Biofilm-. SubDeep PPD (3.72 ± 0.86), AL (4.34 ± 1.33), and % of BOP (66.0 ± 31.1) values were significantly greater compared to SubRemain values (2.84 ± 0.48, 3.37 ± 0.34, and 20.4 ± 24.1, respectively). Biofilm+ Cqs showed no association with the periodontal condition. Cqs from Nasopharynx/Oropharynx (Naso/Oro; n = 36) were similar between Biofilm+ and Biofilm- participants. Length of time since ICU intake, last Naso/Oro RT-qPCR readings, onset of COVID-19 symptoms, and biofilm samplings were greater for Biofilm-. CONCLUSIONS: ICU patients harbored SARS-CoV-2 RNA in supragingival and subgingival biofilms, irrespective of the periodontal condition, and systemic viral load. The high number of positive patients highlights the need to better understand this habit to provide adequate oral care.

Multi-Faceted Approach to COVID-19 Surveillance at a Large University Campus in the Southeastern United States.

The coronavirus disease 2019 (COVID-19) pandemic continues to present unique public health challenges both within the United States and across the globe. Institutions of higher learning are tasked with preventing and responding to COVID-19 on campus while also considering implications for the surrounding communities. The process of re-opening campus, whether at full or partial capacity, has tasked these institutions with overcoming complex challenges associated with balancing the resumption of campus operations while simultaneously protecting university affiliates and surrounding community members from COVID-19 through robust surveillance, contact tracing, and testing efforts. Here, we provide a concise outline related to the development and implementation of the comprehensive and sustainable COVID-19 surveillance program at the University of Florida. We also critically discuss the successes and pitfalls of this program while also providing recommendations for the development of similar programs in the future.

SARS-CoV-2 mRNA vaccine induced higher antibody affinity and IgG titers against variants of concern in post-partum vs non-post-partum women.

BACKGROUND: Limited knowledge exists in post-partum women regarding durability of SARS-CoV-2 vaccine-induced antibody responses and their neutralising ability against SARS-CoV-2 variants of concern (VOC). METHODS: We elucidated longitudinal mRNA vaccination-induced antibody profiles of 13 post-partum and 13 non-post-partum women (control). FINDINGS: The antibody neutralisation titres against SARS-CoV-2 WA-1 strain were comparable between post-partum and non-post-partum women and these levels were sustained up to four months post-second vaccination in both groups. However, neutralisation titers declined against several VOCs, including Beta and Delta. Higher antibody binding was observed against SARS-CoV-2 receptor-binding domain (RBD) mutants with key VOC amino acids when tested with post-second vaccination plasma from post-partum women compared with controls. Importantly, post-vaccination plasma antibody affinity against VOCs RBDs was significantly higher in post-partum women compared with controls. INTERPRETATION: This study demonstrates that there is a differential vaccination-induced immune responses in post-partum women compared with non-post-partum women, which could help inform future vaccination strategies for these groups. FUNDING: The antibody characterisation work described in this manuscript was supported by FDA's Medical Countermeasures Initiative (MCMi) grant #OCET 2021-1565 to S.K and intramural FDA-CBER COVID-19 supplemental funds.

SARS-CoV-2 mRNA vaccine induced higher antibody affinity and IgG titers against variants of concern in post-partum vs non-post-partum women

Summary Background Limited knowledge exists in post-partum women regarding durability of SARS-CoV-2 vaccine-induced antibody responses and their neutralising ability against SARS-CoV-2 variants of concern (VOC). Methods We elucidated longitudinal mRNA vaccination-induced antibody profiles of 13 post-partum and 13 non-post-partum women (control). Findings The antibody neutralisation titres against SARS-CoV-2 WA-1 strain were comparable between post-partum and non-post-partum women and these levels were sustained up to four months post-second vaccination in both groups. However, neutralisation titers declined against several VOCs, including Beta and Delta. Higher antibody binding was observed against SARS-CoV-2 receptor-binding domain (RBD) mutants with key VOC amino acids when tested with post-second vaccination plasma from post-partum women compared with controls. Importantly, post-vaccination plasma antibody affinity against VOCs RBDs was significantly higher in post-partum women compared with controls. Interpretation This study demonstrates that there is a differential vaccination-induced immune responses in post-partum women compared with non-post-partum women, which could help inform future vaccination strategies for these groups.

Evidence of SARS-CoV-2-Specific T-Cell-Mediated Myocarditis in a MIS-A Case.

A 26-year-old otherwise healthy man died of fulminant myocarditis. Nasopharyngeal specimens collected premortem tested negative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Histopathological evaluation of the heart showed myocardial necrosis surrounded by cytotoxic T-cells and tissue-repair macrophages. Myocardial T-cell receptor (TCR) sequencing revealed hyper-dominant clones with highly similar sequences to TCRs that are specific for SARS-CoV-2 epitopes. SARS-CoV-2 RNA was detected in the gut, supporting a diagnosis of multisystem inflammatory syndrome in adults (MIS-A). Molecular targets of MIS-associated inflammation are not known. Our data indicate that SARS-CoV-2 antigens selected high-frequency T-cell clones that mediated fatal myocarditis.

Maternal transfer of IgA and IgG SARS-CoV-2 specific antibodies transplacentally and via breastfeeding (preprint)

Although there have been many studies on antibody responses to SARS-CoV-2 in breastmilk, very few have looked at the fate of these in the baby. We carried out a study in 22 mother/baby pairs (mothers who breastfed and who were SARS-CoV-2 vaccinated before or after delivery) looking at mother blood, mother milk, baby blood, baby nose, and baby stool. Breastfed infants only acquired systemic anti-SARS-CoV-2 IgG antibodies if their mothers were vaccinated antepartum. None of the infants had SARS-CoV-2-specific IgA in the blood, but surprisingly, half of the infants in the Antepartum group had high titer SARS-CoV-2-specific IgA in the nose that exceeded titers found in breastmilk. Vaccination antepartum followed by breastfeeding appears to be the best way to provide systemic and local anti-SARS-CoV-2 antibodies for infants.

A Scalable, Easy-to-Deploy Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material.

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly trained personnel, and large upfront investment. Here, we showcase an orthogonal pipeline we call CREST (Cas13-based, rugged, equitable, scalable testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows a binary interpretation of results. CREST may provide a point-of-care solution to increase the distribution of COVID-19 surveillance.

Comparison of Severe Acute Respiratory Syndrome Coronavirus 2 Screening Using Reverse Transcriptase-Quantitative Polymerase Chain Reaction or CRISPR-Based Assays in Asymptomatic College Students.

Importance: The reopening of colleges and universities in the US during the coronavirus disease 2019 (COVID-19) pandemic is a significant public health challenge. The development of accessible and practical approaches for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in the college population is paramount for deploying recurrent surveillance testing as an essential strategy for virus detection, containment, and mitigation. Objective: To determine the prevalence of SARS-CoV-2 in asymptomatic participants in a university community by using CREST (Cas13-based, rugged, equitable, scalable testing), a CRISPR-based test developed for accessible and large-scale viral screening. Design, Setting, and Participants: For this cohort study, a total of 1808 asymptomatic participants were screened for SARS-CoV-2 using a CRISPR-based assay and a point-of-reference reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) test. Viral prevalence in self-collected oropharyngeal swab samples collected from May 28 to June 11, 2020, and from June 23 to July 2, 2020, was evaluated. Exposures: Testing for SARS-CoV-2. Main Outcomes and Measures: SARS-CoV-2 status, viral load, and demographic information of the study participants were collected. Results: Among the 1808 participants (mean [SD] age, 27.3 [11.0] years; 955 [52.8%] female), 732 underwent testing from May to early June (mean [SD] age, 28.4 [11.7] years; 392 [53.6%] female). All test results in this cohort were negative. In contrast, 1076 participants underwent testing from late June to early July (mean [SD] age, 26.6 [10.5] years; 563 [52.3%] female), with 9 positive results by RT-qPCR. Eight of these positive samples were detected by the CRISPR-based assay and confirmed by Clinical Laboratory Improvement Amendments-certified diagnostic testing. The mean (SD) age of the positive cases was 21.7 (3.3) years; all 8 individuals self-identified as students. These metrics showed that a CRISPR-based assay was effective at capturing positive SARS-CoV-2 cases in this student population. Notably, the viral loads detected in these asymptomatic cases resemble those seen in clinical samples, highlighting the potential of covert viral transmission. The shift in viral prevalence coincided with the relaxation of stay-at-home measures. Conclusions and Relevance: These findings reveal a shift in SARS-CoV-2 prevalence in a young and asymptomatic population and uncover the leading edge of a local outbreak that coincided with rising case counts in the surrounding county and the state of California. The concordance between CRISPR-based and RT-qPCR testing suggests that CRISPR-based assays are reliable and offer alternative options for surveillance testing and detection of SARS-CoV-2 outbreaks, as is required to resume operations in higher-education institutions in the US and abroad.

CRISPR-based and RT-qPCR surveillance of SARS-CoV-2 in asymptomatic individuals uncovers a shift in viral prevalence among a university population (preprint)

Background: The progress of the COVID-19 pandemic profoundly impacts the health of communities around the world, with unique impacts on colleges and universities. Transmission of SARS-CoV-2 by asymptomatic people is thought to be the underlying cause of a large proportion of new infections. However, the local prevalence of asymptomatic and pre-symptomatic carriers of SARS-CoV-2 is influenced by local public health restrictions and the community setting. Objectives: This study has three main objectives. First, we looked to establish the prevalence of asymptomatic SARS-CoV-2 infection on a university campus in California. Second, we sought to assess the changes in viral prevalence associated with the shifting community conditions related to non-pharmaceutical interventions (NPIs). Third, we aimed to compare the performance of CRISPR- and PCR-based assays for large-scale virus surveillance sampling in COVID-19 asymptomatic persons. Methods: We enrolled 1,808 asymptomatic persons for self-collection of oropharyngeal (OP) samples to undergo SARS-CoV-2 testing. We compared viral prevalence in samples obtained in two time periods: May 28th-June 11th; June 23rd-July 2nd. We detected viral genomes in these samples using two assays: CREST, a CRISPR-based method recently developed at UCSB, and the RT-qPCR test recommended by US Centers for Disease Control and Prevention (CDC). Results: Of the 1,808 participants, 1,805 were affiliates of the University of California, Santa Barbara, and 1,306 were students. None of the tests performed on the 732 samples collected between late May to early June were positive. In contrast, tests performed on the 1076 samples collected between late June to early July, revealed nine positive cases. This change in prevalence met statistical significance, p = 0.013. One sample was positive by RT-qPCR at the threshold of detection, but negative by both CREST and CLIA-confirmation testing. With this single exception, there was perfect concordance in both positive and negative results obtained by RT-qPCR and CREST. The estimated prevalence of the virus, calculated using the confirmed cases, was 0.74%. The average age of our sample population was 28.33 (18-75) years, and the average age of the positive cases was 21.7 years (19-30). Conclusions: Our study revealed that there were no COVID-19 cases in our study population in May/June. Using the same methods, we demonstrated a substantial shift in prevalence approximately one month later, which coincided with changes in community restrictions and public interactions. This increase in prevalence, in a young and asymptomatic population which would not have otherwise accessed COVID-19 testing, indicated the leading wave of a local outbreak, and coincided with rising case counts in the surrounding county and the state of California. Our results substantiate that large, population-level asymptomatic screening using self-collection may be a feasible and instructive aspect of the public health approach within large campus communities, and the almost perfect concordance between CRISPR- and PCR-based assays indicate expanded options for surveillance testing

A Scalable, Easy-to-Deploy, Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material (preprint)

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly-trained personnel, and large upfront investment. Here we showcase an orthogonal pipeline we call CREST (Cas13-based, Rugged, Equitable, Scalable Testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows for a binary interpretation of results. CREST may provide a point- of-care solution to increase the distribution of COVID-19 surveillance.