Gut as an Alternative Entry Route for SARS-CoV-2: Current Evidence and Uncertainties of Productive Enteric Infection in COVID-19.

The gut has been proposed as a potential alternative entry route for SARS-CoV-2. This was mainly based on the high levels of SARS-CoV-2 receptor expressed in the gastrointestinal (GI) tract, the observations of GI disorders (such as diarrhea) in some COVID-19 patients and the detection of SARS-CoV-2 RNA in feces. However, the underlying mechanisms remain poorly understood. It has been proposed that SARS-CoV-2 can productively infect enterocytes, damaging the intestinal barrier and contributing to inflammatory response, which might lead to GI manifestations, including diarrhea. Here, we report a methodological approach to assess the evidence supporting the sequence of events driving SARS-CoV-2 enteric infection up to gut adverse outcomes. Exploring evidence permits to highlight knowledge gaps and current inconsistencies in the literature and to guide further research. Based on the current insights on SARS-CoV-2 intestinal infection and transmission, we then discuss the potential implication on clinical practice, including on long COVID. A better understanding of the GI implication in COVID-19 is still needed to improve disease management and could help identify innovative therapies or preventive actions targeting the GI tract.

Factors Modulating COVID-19: A Mechanistic Understanding Based on the Adverse Outcome Pathway Framework.

Addressing factors modulating COVID-19 is crucial since abundant clinical evidence shows that outcomes are markedly heterogeneous between patients. This requires identifying the factors and understanding how they mechanistically influence COVID-19. Here, we describe how eleven selected factors (age, sex, genetic factors, lipid disorders, heart failure, gut dysbiosis, diet, vitamin D deficiency, air pollution and exposure to chemicals) influence COVID-19 by applying the Adverse Outcome Pathway (AOP), which is well-established in regulatory toxicology. This framework aims to model the sequence of events leading to an adverse health outcome. Several linear AOPs depicting pathways from the binding of the virus to ACE2 up to clinical outcomes observed in COVID-19 have been developed and integrated into a network offering a unique overview of the mechanisms underlying the disease. As SARS-CoV-2 infectibility and ACE2 activity are the major starting points and inflammatory response is central in the development of COVID-19, we evaluated how those eleven intrinsic and extrinsic factors modulate those processes impacting clinical outcomes. Applying this AOP-aligned approach enables the identification of current knowledge gaps orientating for further research and allows to propose biomarkers to identify of high-risk patients. This approach also facilitates expertise synergy from different disciplines to address public health issues.

COVID-19 mRNA vaccine and antibody response in lactating women: a prospective cohort study.

BACKGROUND: Immunological protection via breastfeeding is well known. The immunological profile of human milk changes during lactation. No clinical trials have been conducted in lactating women with the newest mRNA vaccines against SARS- CoV-2. A Few studies have shown the presence of antibodies in breastmilk after vaccination. The aim of this work is to study possible antibodies transfer via breastmilk and also the immunological characteristics of lactating women compared to non-lactating women, after using the BNT162b2 Pfizer vaccine. METHODS: This is a prospective cohort study with a convenience homogenous sample of 24 healthcare workers (14 lactating and 10 non-lactating women) enrolled at the time of COVID-19 vaccination. Clinical data was registered in a questionnaire. Titers of SARS-CoV-2 spike IgG, IgA and IgM were quantified in post vaccination blood and human milk. Antibody quantification was performed by an in-house ELISA to SARS-CoV-2 trimeric spike protein. RESULTS: All women showed immunity after vaccination with positive antibodies for IgM, IgA and IgG antibodies. The dominant serum antibody response was IgG. Modest levels of antibodies in breastmilk of lactating mothers were observed in this study, especially IgG in 42.9%. There was a moderate association between higher titers of IgG and a longer duration of breastfeeding (R= 0.55, p=0.041). CONCLUSIONS: Evidence of antibody transfer in human milk after COVID-19 vaccination is scarce. The presence of antibodies in human milk is reported, but immunization through breastfeeding is still to be established.

Effects of spike protein and toxin-like peptides found in COVID-19 patients on human 3D neuronal/glial model undergoing differentiation: Possible implications for SARS-CoV-2 impact on brain development.

The possible neurodevelopmental consequences of SARS-CoV-2 infection are presently unknown. In utero exposure to SARS-CoV-2 has been hypothesized to affect the developing brain, possibly disrupting neurodevelopment of children. Spike protein interactors, such as ACE2, have been found expressed in the fetal brain, and could play a role in potential SARS-CoV-2 fetal brain pathogenesis. Apart from the possible direct involvement of SARS-CoV-2 or its specific viral components in the occurrence of neurological and neurodevelopmental manifestations, we recently reported the presence of toxin-like peptides in plasma, urine and fecal samples specifically from COVID-19 patients. In this study, we investigated the possible neurotoxic effects elicited upon 72-hour exposure to human relevant levels of recombinant spike protein, toxin-like peptides found in COVID-19 patients, as well as a combination of both in 3D human iPSC-derived neural stem cells differentiated for either 2 weeks (short-term) or 8 weeks (long-term, 2 weeks in suspension + 6 weeks on MEA) towards neurons/glia. Whole transcriptome and qPCR analysis revealed that spike protein and toxin-like peptides at non-cytotoxic concentrations differentially perturb the expression of SPHK1, ELN, GASK1B, HEY1, UTS2, ACE2 and some neuronal-, glia- and NSC-related genes critical during brain development. Additionally, exposure to spike protein caused a decrease of spontaneous electrical activity after two days in long-term differentiated cultures. The perturbations of these neurodevelopmental endpoints are discussed in the context of recent knowledge about the key events described in Adverse Outcome Pathways relevant to COVID-19, gathered in the context of the CIAO project (https://www.ciao-covid.net/).

Secretory IgA and T cells targeting SARS-CoV-2 spike protein are transferred to the breastmilk upon mRNA vaccination.

In view of the scarcity of data to guide decision making, we evaluated how BNT162b2 and mRNA-1273 vaccines affect the immune response in lactating women and the protective profile of breastmilk. Compared with controls, lactating women had a higher frequency of circulating RBD memory B cells and higher anti-RBD antibody titers but similar neutralizing capacity. We show that upon vaccination, immune transfer to breastmilk occurs through a combination of anti-spike secretory IgA (SIgA) antibodies and spike-reactive T cells. Although we found that the concentration of anti-spike IgA in breastmilk might not be sufficient to directly neutralize SARS-CoV-2, our data suggest that cumulative transfer of IgA might provide the infant with effective neutralization capacity. Our findings put forward the possibility that breastmilk might convey both immediate (through anti-spike SIgA) and long-lived (via spike-reactive T cells) immune protection to the infant. Further studies are needed to address this possibility and to determine the functional profile of spike T cells.

Evaluating SARS-CoV-2 Seroconversion Following Relieve of Confinement Measures.

Seroprevalence studies are crucial both for estimating the prevalence of SARS-CoV-2 exposure and to provide a measure for the efficiency of the confinement measures. Portuguese universities were closed on March 16th 2020, when Portugal only registered 62 SARS-CoV-2 infection cases per million. We have validated a SARS-CoV-2 ELISA assay to a stabilized full-length spike protein using 216 pre-pandemic and 19 molecularly diagnosed SARS-CoV-2 positive individual's samples. At NOVA University of Lisbon, presential work was partially resumed on May 25th with staggered schedules. From June 15th to 30th, 3-4 weeks after the easing of confinement measures, we screened 1,636 collaborators of NOVA university of Lisbon for the presence of SARS-CoV-2 spike specific IgA and IgG antibodies. We found that spike-specific IgG in 50 of 1,636 participants (3.0%), none of which had anti-spike IgA antibodies. As participants self-reported as asymptomatic or paucisymptomatic, our study also provides a measurement of the prevalence of asymptomatic/paucisymptomatic SARS-CoV-2 infections. Our study suggests that essential workers have a 2-fold increase in viral exposure, when compared to non-essential workers that observed confinement. Additional serological surveys in different population subgroups will paint a broader picture of the effect of the confinement measures in the broader community.

Signatures in SARS-CoV-2 spike protein conferring escape to neutralizing antibodies.

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population. Determining signatures in SARS-CoV-2 that render the virus resistant to neutralizing antibodies is critical. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations in the spike protein, including all defining mutations in the variants of concern, to identify the effect of single and synergic amino acid substitutions in promoting immune escape. We confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and with mutations able to increase binding to ACE2, such as N501Y. Our analysis of synergic mutations provides a signature for hotspots for immune evasion and for targets of therapies, vaccines and diagnostics.

Staff SARS-CoV-2 Seroprevalence and Mental Health as Key Factors in University Response to COVID-19 Pandemic.

Background: In response to rapid global spread of the newly emerged coronavirus disease 2019 (COVID-19), universities transitioned to online learning and telework to decrease risks of inter-person contact. To help administrators respond to the COVID-19 pandemic and better understand its impacts, we surveyed SARS-CoV-2 seroprevalence among NOVA University employees and assessed community mental health. Methods: Data were collected from voluntary participants at six NOVA University locations, in the Lisbon metropolitan area, from June 15-30, 2020. All subjects provided written informed consent. Of 1,627 recruited participants (mean age 42.0 ± 12.3 years), 1,624 were tested. Prior to blood collection, participants completed a questionnaire that assessed: COVID-19 symptoms during the previous 14 days, chronic non-communicable diseases, chronic medication, anxiety, and depression symptoms. SARS-CoV-2 serology tests were then performed, and results communicated approximately 4 days after blood draw. Participants with positive serology tests were contacted to assess COVID-19 symptoms since February. Results: Estimated prevalence of SARS-CoV-2 IgG antibodies was 3.1% (n = 50), of which 43.5% reported symptoms in the previous 4 months. The Medical School had the highest seroprevalence (6.2%). Participants reported having at least one chronic disease (63.7%), depression-like symptoms (2.1%), and anxiety symptoms (8.1%). Rates of depression and anxiety symptoms were significantly higher in women, with sleep hours and occasional alcohol consumption negatively associated with depression. Male gender, older age, and sleep hours negatively associated with anxiety symptoms. School of employment and presence of comorbidities positively associated with anxiety. Conclusion: By measuring seroprevalence of SARS-CoV-2 antibodies among NOVA employees and assessing subjects' mental health, we aim to help administrators at European public universities in urban areas, such as Lisbon, Portugal, better understand the needs of their communities. This study resulted in implementation of a stricter contingency plan in the Medical School, while other schools continued to follow Government mitigation guidelines. These findings may also guide the development of tailored strategies to ensure physical and mental health of the academic community during this pandemic crisis. We conclude that, together with COVID-19 contingency plans, psychological support services and facilities to help people effectively face pandemic-associated challenges and minimise anxiety and depression should be implemented.