Adaptive Immunity Dysregulation Is Associated to the Development of Long Covid

Background: The pathogenetic mechanisms behind the development of long- COVID (LC) are largely unknown. Because both plasma SARS-CoV-2 RNAemia and dysregulated immunity have been correlated with COVID-19 severity, we evaluated whether they are associated with LC. Method(s): We consecutively enrolled unvaccinated hospitalized COVID-19 patients during acute-COVID-19 (T0) in March-October 2020 who either developed LC at a follow-up visit 2-3 months from virologic clearance (T1) or did not. LC was defined as persistence >=2 months after recovery of >=1 symptom: anosmia, dysgeusia, fever, gastrointestinal symptoms, dyspnoea, fatigue, musculoskeletal pain, muscle weakness, brain fog. We measured: SARS-CoV-2 RNAemia (RT-qPCR, log10(copies/mL)), magnitude (ELISA, AUC) and functionality (pseudovirus neutralization, ID50;Fc-mediated functions, %ADCC) of SARS-CoV-2-specific antibodies, SARS-CoV-2-specific B and CD4-T-cells (Immunophenotype, AIM and ICS assays). Result(s): We enrolled 48 COVID-19 individuals, 38/48 (79.2%) developed LC (LC+) and 10 did not (LC-). LC+ and LC- had similar co-morbidities and symptoms in the acute phase (Fig.1A), and the majority showed a radiologically documented SARS-CoV-2 pneumonia. The SARS-CoV-2 RNAemia did not differ between groups at both time points. The levels of RBD-specific Abs, as well as their functionality, appeared to increase over time in the LC- group but not in the LC+ (Fig.1B-D). Similarly, a trend towards increased RBD-specific B-cells was observed over time in the LC- group but not in LC+ (Fig.1E). B-cell immunophenotyping showed a significant increase over time of classical memory B cells (MBCs) at the expenses of activated MBCs (Fig.1F-G) as well as an IgA class-switching in the LC- group compared to LC+ (Fig.1H-I). Furthermore, LC+ showed a faster decline of SARS-CoV-2-specific (CD69+CD137+) CD4- TEMRA and CD4-TEM (Fig.1L-M). Finally, IFN-gamma-producing TREG of LC- individuals increased over time (Fig.1N). Conclusion(s): Acutely ill, hospitalized COVID-19 patients developing LC feature a dysregulated SARS-CoV-2-specific humoral as well as B- and T-cell response, in both magnitude and functionality, suggesting a link between dysregulated SARS-CoV-2-specific adaptive immunity and LC development. The fine understanding of the factors contributing to such dysregulation in LC patients is strongly needed, that might further inform targeted therapeutic interventions. (Figure Presented).

BROAD ANTIBODIES TO FUNCTIONALLY CONSTRAINED REGIONS OF SARS-CoV-2 SPIKE

Background: While remarkable and rapid progress was made in fighting the SARS-CoV-2 pandemic with vaccines and therapeutic antibodies, these approaches were quickly compromised by viral evolution. Therapeutic monoclonal antibodies (mAbs) that were once authorized for clinical use, which all target the receptor binding domain (RBD), are no longer effective against current variants of concern (VOCs) due to mutations in this region of Spike. Thus, to achieve durable protection against SARS-CoV-2, novel mAbs need to show breadth and potency across VOCs and target epitopes that are more constrained. Method(s): mAbs from an individual who had a breakthrough Delta VOC infection after vaccination were isolated from Spike-specific memory B cells. mAbs were assessed for binding affinity and neutralization potency using Spike-pseudotyped lentivirus (PSV) and live SARS-CoV-2 virus neutralization assays. Epitopes were mapped using deep mutational sequencing (DMS) and structural-based methods. Result(s): Three novel mAbs (C68.3, C68.13, C68.59) demonstrated binding breadth to Spikes from various VOCs including Omicron VOCs despite that C68 had not yet been exposed to Omicron. These mAbs potently neutralized the Wuhan-Hu-1 vaccine and Delta strains (IC50 = 9-61ng/mL), and early Omicron strains BA.1, BA.2, BA.5 (IC50 = 12-149 ng/mL). C68.3 and C68.59 retained potency against recent VOCs BQ.1.1 and XBB (IC50 = 121-122 ng/mL and 56-82 ng/mL, respectively) in the PSV assay. Similar neutralization activity was observed in the live virus assay. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs previously authorized for treatment and they showed greater breadth. The mAbs target distinct epitopes on the Spike glycoprotein, two in the RBD (C68.3, C68.13) and one in an invariant region downstream of RBD in subdomain 1 (SD1) (C68.59). Structural analysis of C68.59 Fab binding to Spike trimer revealed significant allosteric changes to regions of Spike outside of the epitope in the S2 unit. Finally, DMS escape pathways showed these mAbs target regions highly conserved across VOCs that are also functionally constrained, suggesting escape could incur a fitness cost. Conclusion(s): Overall, these mAbs are novel in their breadth across VOCs and include a potent mAb targeting a rare epitope outside of the RBD in SD1. These mAbs focus on diverse, functionally constrained regions in Spike making them candidates for development as combination therapeutics with good durability against future VOCs.

Multi-robot Sanitization of Railway Stations Based on Deep Q-Learning

Sanitizing railway stations is a relevant issue especially due to the recent evolution of the Covid-19 pandemic. In this work, we propose a multi-robot approach to sanitize railway stations based on a distributed Deep Q-Learning technique. The framework relies on anonymous information from existing WiFi networks to localize passengers inside the station and to develop a map of possible risky areas to be sanitized. Starting from this map, a swarm of cleaning robots, each one endowed with a robot-specific convolutional neural network, learns how to on-line cooperate inside the station in order to maximize the sanitized area depending on the presence of the passengers. © 2021 Copyright for this paper by its authors.

Response of lymphopenic apheresis platelet donors to COVID-19 vaccination

Background: Recent studies have shown that several frequent apheresis platelet donors (more than 20 donations/year for several years) have CD4+ T cell counts below the normal limit (<400 cells/μl) while the levels of other blood cell types (including B cells) remain normal. This lymphopenia does not appear to be associated with an increased susceptibility to infections and cancers, suggesting that it does not affect immune function of the platelet donors with low CD4+ T cell counts. Aims: To assess the immune capacity of apheresis platelet donors with lymphopenia by taking advantage of the recent SARS-CoV-2 vaccination campaign. Methods: Forty-three apheresis platelet donors who donate at least five times per year were recruited. At least half of these participants were frequent donors and were thus more likely to have low CD4+ T cell counts. Their baseline CD4+ T cell count was determined by flow cytometry. The levels of antibodies targeting the SARS-CoV-2 Spike receptor binding domain (RBD) were measured by ELISA before and after vaccination. The level of antibodies against native full-length Spike was determined by flow cytometry using transfected cells expressing different Spike variants of concern (D614G, Delta, Omicron). The avidity of RBD antibodies produced after the first and second dose of vaccine was determined using a modified ELISA in which a chaotropic agent (urea 8 M) was added. The functional properties of vaccine-elicited SARSCoV- 2 antibodies were measured using ADCC and neutralization assays. Results: Of the 43 participants, 27 had pre-vaccination CD4+ T cell counts below 400 cells/μl (low CD4 group) and 16 participants had CD4+ T cell counts in the normal range (400-1600 CD4/μl;normal CD4 group). The levels of RBD-binding antibody did not significantly differ between the low and normal CD4 groups for all three isotypes (IgG, IgA and IgM) after the first and second doses of vaccine. As expected given the maturation of immune response, the avidity of RBD-binding antibodies present in the plasma collected after the second dose of vaccine was significantly higher than that measured in plasma recovered after the first dose. However, the increase was of similar magnitude in both CD4 groups. Recognition of the three Spike variants, as measured by flow cytometry, did not differ significantly between the low and normal CD4 groups. Finally, the antibody(Fc)- mediated effector function (measured by ADCC using cells expressing the wild type Spike) and neutralization capacity of the three variants of concern were comparable for the two groups, after receiving two vaccine doses. Summary/Conclusions: Our data show that low CD4+ T cell counts in apheresis platelet donors do not impair their response to antigenic challenge such as COVID-19 vaccination. This finding is consistent with the previously lack of increased susceptibility of platelet donors with lymphopenia to infections and cancers. Work remains to be done to understand the physiological mechanism behind the low number of CD4+ T cells in the peripheral blood of several plateletpheresis donors.

SARS-CoV-2 VARIANTS INCREASE KINETIC STABILITY of OPEN SPIKE CONFORMATIONS

Background: SARS-CoV-2 variants of concern harbor mutations in the Spike (S) glycoprotein that confer more efficient transmission and dampen the efficacy of COVID-19 vaccines and antibody therapies. S mediates virus entry and is the primary target for antibody responses, with structural studies of soluble S variants revealing an increased propensity towards conformations accessible to the human Angiotensin-Converting Enzyme 2 (hACE2) receptor. However, real-time observations of conformational dynamics that govern the structural equilibriums of the S variants have been lacking. Methods: Here, we report single-molecule Förster Resonance Energy Transfer (smFRET) studies of S variants of concern containing critical mutations, including D614G and E484K, in the context of virus particles. Results: Investigated variants were shown by smFRET to predominantly occupy more open hACE2-accessible conformations, agreeing with predictions from structures of soluble trimers. Additionally, S variants exhibited decelerated transitions from hACE2-accessible/bound states. Conclusion: Here, we provide the real-time dimension to distinct structures of Spikes in the context of virus particles and present the first experimental evidence of increased stability of Spike variants. Our finding of increased S kinetic stability in the open conformation provides a new perspective on SARS-CoV-2 adaptation to the human population.

A LONG-INTERVAL VACCINE REGIMEN LEADS to STRONG HUMORAL RESPONSES AGAINST SARS-CoV-2

Background: While the standard regimen of the BNT162b2 mRNA vaccine includes two doses administered three weeks apart, some public health authorities decided to space them in a context of vaccine scarcity. This decision raised concerns about vaccine efficacy, notably against the many circulating variants. In this study, we analyzed the longitudinal humoral responses from before the first dose to 4 months after the second dose in a cohort of SARS-CoV-2 naïve and previously infected (PI) individuals, with an interval of sixteen weeks between the two doses. We compared these responses to those elicited in individuals receiving the three weeks dose interval. Methods: We measured the level of antibodies recognizing SARS-CoV-2 Spike or its receptor-binding domain, and the capacity of these antibodies to neutralize several variants of concern (VOCs) and other human coronaviruses. We also measured B cell responses and Fc-mediated effector functions (ADCC) elicited by vaccination. Results: We observed that in PI individuals, the first dose led to strong humoral responses that could not be significantly improved further upon administration of a second dose. In the naïve individual's group, the first dose induced weak neutralizing activity but strong Fc-mediated functions and the administration of the second dose 16 weeks after led to a significant increase of humoral responses, achieving similar levels to those measured in PI individuals. In both groups, we observed that plasmas were able to recognize and neutralize the Spike of different VOCs but also SARS-CoV-1. Conclusion: Our results show that individuals that received the extended BNT162b2 vaccine interval developed strong humoral responses. For the naïve donors, these responses were superior to those elicited by the three-week dose interval and comparable to the PI responses after one or two doses.

SARS-CoV-2 PLASMA VIREMIA and DYSREGULATED IMMUNITY in SEVERE COVID-19

Background: Critical COVID-19 occurs ca. 7d from symptoms onset, and is associated to immune dysregulation as well as SARS-CoV-2 detection in plasma (i.e. viremia). We hereby sought to detail the association between SARS-CoV-2 viremia measured at the end of the first week of disease and immune phenotypes/function in COVID-19 patients. Methods: We consecutively enrolled patients hospitalized in the acute phase of ascertained SARS-CoV-2 pneumonia. In this disease stage, we studied SARS-CoV-2 viremia (RT-PCR) and cytokines (MACSPlex), HLA-DR+CD38+ activated, GRZB+PRF+ pro-cytolitic T-cells, intracellular cytokine production (IL-2, IFNγ, TNFα, IL-4, IL-17A) after SARS-CoV-2 challenge (S-N-M-peptide pool). Simultaneous Th1-cytokines production (polyfunctionality) and amount (iMFI) was assessed. Humoral response: anti-S1/S2 IgG, anti-RBD total-Ig, IgM, IgA, IgG1 and IgG3 (ELISA), pseudoviruses neutralization (ID50) and Fc-mediated functions (%ADCC). Results: Out of 54 patients, 27 had detectable viremia (V+). Albeit comparable age and co-morbidities, V+ patients more frequently required non-invasive/invasive ventilatory support (p=0.035), with a trend to higher death (p=0.099) vs patients with undetectable viremia (V-)(Fig.1A). V+ displayed higher circulating IFN-α (p=0.002) and IL-6 (0.003), lower activated HLA-DR+CD38+CD4 (p=0.01) and CD8 (p=0.02), with no differences in GRZB+PRF+ T-cells. V+ featured reduced SARS-CoV-2-specific cytokine-producing T-cells, reaching significance for IFNγ+CD4 (p=0.02), TNFα+CD8, IL-4+CD8 (p=0.04) (Fig.1B-C), with lower bi-and tri-functional SARS-CoV-2-specific CD4 Th1, reaching significance for IL-2+TNFα+CD4 (p=0.03) (Fig.1D). A trend towards lower cytokines iMFI in bi-and tri-functional SARS-CoV-2-specific CD4 Th1 was observed in V+, reaching significance for IL-2+TNFα+CD4, p=0.004. V+ displayed lower anti-S IgG, anti-RBD total-Ig, IgM, IgG1 and IgG3 (Fig.1E), with lower ID50 and %ADCC vs V-(Fig.1F-G). Conclusion: Hospitalized COVID-19 patients with detectable plasma SARS-CoV-2 RNA in the acute phase of disease present worse outcome, higher inflammatory cytokines, fewer activated and SARS-CoV-2-specific polyfunctional T-cells, suggesting a link between SARS-CoV-2 viremia at the end of the first stage of disease and immune dysregulation. Whether high ab initium viral burden and/or intrinsic host factors contribute to a delayed and/or exhausted immune response in severe COVID-19 remains to be elucidated, to further inform strategies of targeted therapeutic interventions.

ALTERED FEATURES of B and T CELL RESPONSES to BNT162 VACCINE in HEMODIALYSIS PATIENTS

Background: People receiving hemodialysis (HD) are highly vulnerable to SARS-CoV-2 infection and develop lower antibody responses to SARS-CoV-2 mRNA vaccines. However, the underlying immune defects are poorly understood. We compared immune responses of 27 HD patients with 22 health care workers (HCW) before and up to 4 months after 2 doses of BNT162 SARS-CoV-2 vaccination. All participants were confirmed to be SARS-CoV-2 naïve. Methods: We profiled B cells, CD4 T cells, CD8 T cells and humoral responses and examined associations between these arms of immunity. We used high-parameter flow cytometry to study: i) receptor binding domain (RBD)-specific B cells;ii) the phenotype of Spike (S)-specific CD4 and CD8 T cells identified by an activation-induced marker (AIM) assay;iii) effector functions of S-specific CD4 and CD8 T cells by intracellular staining (ICS). We measured humoral responses by ELISA RBD. Results: In each cohort, two vaccine doses enhanced RBD-specific B cell responses, with a significantly greater increase after the second dose (V2 than after the first dose (V1). Their magnitude was significantly lower in HD than in HCW at V1 (p=0.002) and V2 (p=0.002), which was consistent with the detection of lower anti-RBD IgG antibody levels at the same time points (V1: p<0.001;V2: p<0.001). The subsequent rates of B cell decline were similar in HD and HCW. As CD4 help is critical for B cell and CD8 T cell immunity, we compared Spike (S)-specific T cells responses between cohorts. While we observed no significant quantitative difference in the magnitude of vaccine-specific CD4 T cells between HD and HCW at V2, phenotypic and functional Thelper profiles differed significantly. The frequency of vaccine-specific CXCR3+ Th1 CD4 T cells was significantly increased in HD compared to HCW (p=0.008), and TNFα+ CD4 T cell responses were elevated in HD (p=0.01). In contrast to CD4 T cells, S-specific CD8 T cell responses were quantitatively reduced in HD compared to HCW after each dose (V1: p<0.001;V2: p<0.001). Conclusion: People on HD develop poor B cell and CD8 T cell responses after SARS-CoV-2 mRNA vaccination. These defects are associated with a skewed differentiation of vaccine-specific CD4 T cells toward CXCR3+ and TNFα+ Th1-like profiles, and probable altered crosstalk between Thelper and B cells. Further study is needed to determine if impaired B and T cell vaccine immunity in addition to defective antibody responses increases vulnerability of HD patients to breakthrough COVID-19 infection.

TEMPORAL ASSOCIATIONS of B and T CELL IMMUNITY in A 16-WEEK INTERVAL BNT162b2 REGIMEN

Background: Spacing of the BNT162b2 mRNA doses beyond the standard 3-week interval raised concerns about vaccine efficacy. We longitudinally analyzed B cell, T cell and humoral responses to two BNT162b2 mRNA doses administered 16 weeks apart in 43 SARS-CoV-2 naïve and previously-infected (PI) donors. We examined blood samples at five time points from baseline to 4 months post second dose. Methods: We used high-parameter flow cytometry to study: i) receptor binding domain (RBD)-specific B cells;ii) Spike (S)-specific CD4 and CD8 T cells by activation-induced marker (AIM) assay;iii) S-specific CD4 and CD8 T cells by intracellular staining (ICS) assay. We measured humoral responses by ELISA, neutralization and ADCC assays. We did supervised and unsupervised (FlowSOM) analyses of B and T cell subsets, and temporal association analyses. Results: We observed partial attrition of B and T cell responses between doses at a memory time point 12 weeks post first dose. RBD-specific B cell kinetics differed between cohorts: the first dose led to their robust increase in PI but small magnitude in naïve. The second dose had little effect in PI but briskly expanded RBD-specific B cells in naïve, leading to convergence between cohorts. Robust T cell responses, with a dominance of CD4 over CD8 responses, were universally induced and did not significantly differ in magnitude after either dose, although there was a trend for a gain in CD8 responses after the second dose in naïve. Unsupervised and supervised analyses of S-specific CD4 T cells showed that the first dose was sufficient to generate highly diverse CD4 subsets, including robust populations of follicular T helper cells. The second dose did not elicit new subsets but lead to convergent phenotypic and functional profiles between PI and naïve with qualitative shifts. Integrated analyses of antigen-specific responses showed immune component-specific associations over-time, with early CD4 responses post-first dose (but not at late time points) strongly correlating with B cell responses after the second dose. In contrast, CD8 responses post second dose correlated with CD4 responses at the same time point. Conclusion: The 16-week interval schedule is associated with robust, multi-faceted recall cellular responses after the second dose, consistent with highly functional immune memory. The early induction of robust CD4 responses and their associations with longer-term B cell and humoral immunity support their central role in the efficacy of this vaccine regimen.

SARS-CoV-2 SPIKE EXPRESSION at the SURFACE of INFECTED HUMAN AIRWAY EPITHELIAL CELLS

Background: Emerging evidence points out to potential benefits from Fc-mediated effector functions in SARS-CoV-2 infection. Some Fc-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) require recognition of the antigen at the surface of infected cells. Methods: To evaluate the expression levels of SARS-CoV-2 Spike at the surface of infected airway epithelial cells, we developed an intracellular staining against SARS-CoV-2 nucleocapsid (N). This assay allows the distinction between infected versus uninfected cells. Human primary airway epithelial cells (pAECs) were infected with authentic SARS-CoV-2 D614G or Alpha variants. Infected cells were identified with an anti-N antibody and cell surface expression of Spike measured with the conformational-independent anti-S2 CV3-25 antibody. Results: We found robust SARS-CoV-2 Spike expression at the cell surface of pAECs. Infected cells were readily recognized with plasma from convalescent and vaccinated individuals. Importantly, recognition of SARS-CoV-2 infected cells strongly correlated with Fc-mediated effector functions measured in a cohort of vaccinated naïve and previously-infected individuals. Conclusion: Altogether, our findings further support the importance of measuring Fc-mediated effector function in infection and vaccination settings for SARS-CoV-2.

IN VIVO EFFICACY of ENGINEERED ACE2-Fc in PREVENTING LETHAL SARS-CoV-2 INFECTION

Background: Soluble Angiotensin Converting Enzyme 2 (ACE2) constitutes an attractive therapeutic candidate with natural resistance to viral escape. To date, ACE2-Fcs, dimeric forms of soluble ACE2, were mostly tested as robust SARS-CoV-2 neutralizers but their potential as antiviral agents capable of Fc-effector functions is largely unknown and has not been tested for effectiveness in vivo, in any model of SARS-CoV2 infection. Methods: We used structure-guided design to select ACE2 mutations that improve SARS-CoV-2 spike (S) affinity and remove angiotensin enzymatic activity. ACE2-Fc variants were engineered into a human IgG1 or IgG3 backbone and produced in mammalian HEK293 cells. S binding was tested by ELISA and surface plasmon resonance (SPR). Mutational effects were validated by X-Ray crystallography. Neutralization activities were measured against SARS-CoV-2 variants of concern (VOCs) using an in vitro pseudovirus (PsV) assay and dynamic bioluminescence imaging (BLI). Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) were also quantified using established methods (1, 2). A K18-hACE2 transgenic mouse model challenged by lethal SARS-CoV-2 nLuc infection (3) was used for in vivo evaluation of prophylactic and therapeutic administration of engineered ACE2-Fcs, as monitored by dynamic BLI. Results: Our lead variant, ACE2740 LFMYQY2HA-Fc GASDALIE, increased RBD binding by ∼7-13 fold as compared to wild type, cross-neutralized SARS-CoV-2 VOCs with an IC50 range of 0.23-2.06 nM and mediated robust ADCC and ADCP in vitro. When tested in humanized K18-hACE2 mice, in either a prophylatic or a multi-dosage therapeutic setting, our lead ACE2-Fc variant provided protection from lethal SARS-CoV-2 infection. Our studies in K18-hACE2 mouse model revealed that efficient in vivo efficacy of ACE2-Fcs under prophylaxis or therapeutic settings required Fc-effector functions in addition to neutralization. Conclusion: Our data confirm the utility of engineered ACE2-Fcs as valuable SARS-CoV-2 antivirals and demonstrate that the efficient ACE2-Fc therapeutic activity required both neutralization and Fc-effector functions.

ANTIGENICITY of the MU (B.1.621) and A.2.5 SARS-CoV-2 SPIKES

Background: The rapid emergence of SARS-CoV-2 variants is fueling the recent waves of the COVID-19 pandemic. Recently identified Mu (B.1.621) and A.2.5 variants carry some mutations shared by other variants of concerns (VOCs). For example, N501Y and E484K mutations in the receptor-binding domain (RBD) domain detected in B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) are now present within the Mu variant. Similarly, the L452R mutation of B.1.617.2 (Delta) variant is now present in A.2.5. Here, we evaluated the capacity of Mu and A.2.5 Spikes to interact with angiotensin-converting enzyme 2 (ACE2) and performed binding and neutralization assays with plasma from vaccinated individuals. In addition, to better understand their antigenic properties, we compared both Mu and A.2.5 with Alpha, Beta, Gamma and Delta VOCs Spikes. Methods: Cells expressing the different Spikes were interrogated for their capacity to interact with the ACE2 receptor using a recombinant ACE2-Fc recombinant protein. We also evaluated their recognition by plasma from BNT162b2 vaccinated individuals. Biolayer interferometry (BLI) was used to measure the binding kinetics of selected RBD mutants to soluble ACE2 (sACE2). Finally, we evaluated the susceptibility of pseudoviral particles bearing the different Spikes to neutralization by plasma from vaccinated individuals. Results: All SARS-CoV-2 S-glycoprotein variants were recognized less efficiently by plasma from vaccinated SARS-CoV-2 naïve and previously-infected individuals compared to D614G Spike with the exception of B.1.1.7 S-glycoprotein. Enhanced ACE2 interaction by the Spikes tested was associated with a decrease in the off-rate of the ACE2-RBD interaction. Pseudoviral particles bearing the Spike of Mu variant were similarly neutralized by plasma from vaccinated individuals than those carrying the Beta and Delta Spikes. Conclusion: Plasma from vaccinated SARS-CoV-2 naïve and previously-infected individuals efficiently recognized all the Spikes tested. The decreased neutralization susceptibility of pseudoviral particles expressing the Mu Spike was similar to Beta and Delta, thus underscoring the importance of functionally tracking emerging variants. In summary, our results highlight the importance of measuring critical parameters such as ACE2 interaction, plasma recognition and neutralization from each emerging variant.

DIMENSIONALITY REDUCTION for STEPWISE ANALYSIS of IMMUNE RESPONSE DYNAMICS in COVID19

Background: COVID-19 is highly heterogeneous in clinical severity and outcome. Considerable advances have uncovered biomolecular traits associated with fatal outcome. However, novel analytical tools are needed to rapidly and accurately delineate patient subgroups with various immunovirological profiles, analyze diverging disease trajectories and prioritize in-depth molecular studies. Methods: To find how immunovirological features are interrelated, we profiled 12 plasma analytes (SARS-CoV-2 vRNA, SARS-CoV-2-specifc antibodies, cytokine and tissue injury markers) in 500 acute longitudinal plasma samples collected from 214 hospitalized COVID-19 patients. We analyzed them simultaneously using PHATE algorithm (potential of heat diffusion for affinity-based transition embedding, Moon et al, Nature Biotech 2019), which can reduce multiple input variables to two salient features for visualization. We performed whole blood transcriptomic analyses to identify molecular signatures associated with survival vs death in a patient cluster identified as being at extreme mortality risk. Results: PHATE analysis of samples collected 11 days after symptom onset (DSO11) revealed four distinct k-means clusters of patients, which aligned with disease severity and outcome. Two groups were highly enriched in critical patients requiring mechanical ventilation: a high-fatality critical cluster 1 accounted for 59% of fatal outcomes (16/27) by DSO60, while critical cluster 2 had good prognosis. Clusters 3 and 4 consisted almost entirely of non-critical survivors delineated respectively by low and high antibody responses. Averaged trajectories between DSO3 to DSO30 diverged between clusters. All patients of the high-fatality cluster had detectable plasma vRNA, which lingered unlike the critical survivor cluster. Their antibody response had a 4-day delay, while their cytokine profile diverged from the other clusters by DSO8, remaining distinct until DSO22. Transcriptome profiles differed between deceased and survivors of the high-fatality cluster 1, with differential expression of GO terms associated with metabolic processes, protein regulation, cell signaling and immune pathways. Conclusion: This unbiased approach gives an integrated view of dysregulated immune response components in fatal COVID-19, which may be explained through differences in molecular pathways. This approach allows to efficiently target detailed investigations on very high-risk patient subgroups who may most likely benefit from new therapeutic interventions.

A FC-ENHANCED NON-NEUTRALIZING ANTIBODY DELAYS SARS-CoV-2-INDUCED DEATH in MICE

Background: Both neutralizing activity and Fc-mediated effector functions of antibodies are believed to contribute to protection against SARS-CoV-2. However, it is unclear if antibody effector functions alone could protect against SARS-CoV-2 infection. Methods: We isolated CV3-13 from a convalescent individual with potent Fc-mediated effector functions. Neutralization capacity of this antibody was measured by both a pseudovirus neutralization assay and an authentic virus microneutralization assay. We mutated the Fc-portion of CV3-13 to enhance (GASDALIE) or reduce (LALA) its capacity to mediate antibody dependant cellular cytotoxicity (ADCC). Structural analysis of CV3-13 was done by cryo-EM to characterize its epitope and its angle of approach. Finally, CV3-13 and CV3-13 GASDALIE were used in vivo in a K18-hACE2 transgenic mouse model challenged with SARS-CoV-2-nLuc to see if they altered viral replication and/or contributed to protection against SARS-CoV-2. Results: While CV3-13 did not neutralize SARS-CoV-2, it demonstrated nanomolar affinity towards the SARS-CoV-2 Spike and mediated strong ADCC. The cryo-EM structure of CV3-13 in complex with the SARS-CoV-2 Spike revealed that the antibody bound to a novel NTD epitope that partially overlapped with a frequently mutated NTD supersite in SARS-CoV-2 variants. Interestingly, this angle of approach was not observed for previously described NTD-directed antibodies. While CV3-13 did not alter the replication dynamics of SARS-CoV-2 in a K18-hACE2 transgenic mouse model, a Fc-enhanced CV3-13 significantly delayed neuroinvasion and death in prophylactic settings. Conclusion: CV3-13 represents a new class of non-neutralizing NTD-directed mAbs that can mediate Fc-effector functions both in vitro and in vivo. While effector functions alone did not protect K18-hACE2 mice from SARS-CoV-2-nLuc challenge, our data indicate that along with neutralization, additional antibody properties including Fc-mediated effector functions contribute to limiting viral spread and aid in fighting SARS-CoV-2 infection.

Pediatric inflammatory multisystemic syndrome temporally associated with COVID 19 (PIMS-TS): characteristics of the Sainte Justine Hospital cohort

Introduction: Since the beginning of SARS-Cov-2 pandemic, children represent a small proportion of patients with an acute disease and present with mild symptoms or are either asymptomatic. However pediatric patients might present with late, post infectious, manifestations of COVID 19, namely Pediatric Inflammatory Multisystemic Syndrome temporally associated with COVID 19 (PIMS-TS). Objectives: We aim to describe the characteristics of pediatric patients with PIMS-TS hospitalised in Sainte Justine Hospital and report their evolution according to their treatment and care. Methods: Patients were recruited prospectively since April 2020. Patient were included if they were less than 18 years old, had at least 2 days of fever, had multisystemic involvement (at least 2 systems) and had a temporal association with COVID 19. This temporal association was defined as: positive reverse transcription (RT)-PCR or antibodies to SARS-Cov2, history of contact with a confirmed or suspected SARS-Cov 2 infected individual or symptom appearance during the pandemic. Patient were excluded if they had an alternative diagnosis that explained the symptoms. Patient were sub categorized into two categories depending on their clinical presentation: 1- Complete or incomplete Kawasaki disease (KD), following the American Heart Association Criteria;or 2- Toxic shock syndrome (TSS). Data were collected during the hospitalization and the follow up. Results: Between April 30th, 2020 and April 30th, 2021, 72 patients were included, 13 (18.1%) with complete KD, 40 (55.6%) with incomplete KD and 19 (26.4%) with TSS. There was more male in the complete KD group (69.2%) and more female in the TSS group (52.6%) but there was no significative difference between the two groups (p = 0.35). Patient with TSS presentation were significatively older than those with KD presentation (10.3 years vs 4.9 years, p < 0.01). Caucasian patients are the most represented ethnic group in the cohort (26.4 %) but Afro-American patient are over-represented in the TSS group (6/19 patients, 31.6%;p= 0.2). Gastro-intestinal symptoms were seen in 50.9% (27/53) and 94.7% (18/19) patients with KD and TSS respectively (p < 0.001). Patients in the TSS group had higher mean value of PCR (239.1 mg/L vs 143.9 mg/L;p< 0.001) and more frequent lymphopenia (89.5% vs 34%;p< 0.001) than those in KD group. Only 40/72 (55.6%) patients in the cohort had either a positive RT-PCR and/or positive antibodies to SARS-Cov-2 and/or a contact with confirmed or suspected infected individual. These proportion increase to 18/19 (94.7%) in the TSS group (p < 0.001). Twenty-three patients (31.9%) required ICU hospitalization including 17 on 19 patients in the TSS group (89.5%;p < 0.001). Cardiac involvement was the most frequent complication either as coronary aneurysm, (15/72 - 20.8%) mainly in patient with KD presentation or as cardiac dysfunction (24/72 - 33.3%), mainly in patient with TSS presentation. Patient received intravenous immunoglobulins (69/72 - 95.8%), steroids (49/72 - 68.1%), sometimes both (48/72 - 66.7%). Five patients (6.9%) required biotherapy: 1 with Enbrel and 4 with Anakinra. Patient received a treatment 6.6 days after the beginning of the symptoms. After 3 months, 4 patients (0.5%) had persistent coronary dilatation and 2 (0.3%) had mitral insufficiency. Conclusion: This cohort study enables a better description of PIMS clinical and biological presentation, which can sometimes be confusing. It also highlights the importance of fast and adequate diagnosis and treatment to avoid the risk of acute and chronic complications, especially cardiac complications.

An ENV-GAG MRNA vaccine protects macaques from heterologous TIER-2 SHIV infection

Background: The development of a preventive vaccine remains a critical priority for ending the HIV/AIDS pandemic. Critical improvements in mRNA technology, as attested by recent successes in preventing COVID-19 disease, led us to develop an mRNA platform for HIV vaccines. Methods: In this regard, we designed an mRNA vaccine with different HIV-1 envelope mRNAs from 3 different clades co-formulated with SIV gag mRNA, which can assemble virus like particles (VLPs) in vivo. Rhesus macaques were primed with a transmitted-founder clade-B Env lacking the 276 N-glycan followed by multiple glycan-repaired autologous and bivalent heterologous (clades A and C) booster immunizations. Results: Immunized animals rapidly developed autologous neutralizing antibodies and eventually, after the second heterologous boost, cross-reactive tier-2 neutralizing antibodies, albeit at low titers. Vaccinated animals were protected from repeated low-dose rectal challenges with a heterologous tier-2 simian-human immunodeficiency virus (AD8). Protection was correlated with the presence of antibodies to the CD4-binding site. Conclusion: Thus, the Gag-Env VLP mRNA platform offers a promising strategy for the development of an HIV-1 vaccine.

Release of ammonia, particulate matter and nitrogen oxides during the Covid-19 quarantine: what is the role of livestock activities?

Several gases contribute to air pollution and most of all to the formation of secondary particulate matter (PM2.5), which is recognized as a source of severe risk to human health. Even if huge steps forward have been done worldwide, traffic, industrial activities, and the energy sector are mostly responsible for the release of NOx and SOx, while the agricultural sector is mainly responsible for the emission of NH3 deriving from the barn, the manure storage, management and final field application. In this study, the emission of PM2.5, NOx and NH3 is analyzed in the main provinces of the Lombardy region in which livestock activities are carried out, comparing emissions of 2016-2019 and those of 2020 during the lockdown determined by the spread of Covid-19 disease. The aim is to understand if and how a change in air emissions can be identified. The results show that PM2.5 and NOx reduced, most of all in urban areas, whereas NH3 maintained the same trend of previous years. From the statistical analysis emerges also that NH3 has a different behavior respect to PM2.5 and NOx, these latter being much more correlated between each other than NH3. However, further studies should be carried out on a bigger spatial and temporal scale.