Modelling the association between neutralizing antibody levels and SARS-CoV-2 viral dynamics : implications to define correlates of protection against infection (preprint)

Background While anti-SARS-CoV-2 antibody kinetics have been well described in large populations of vaccinated individuals, we still poorly understand how they evolve during a natural infection and how this impacts viral clearance. Methods For that purpose, we analyzed the kinetics of both viral load and neutralizing antibody levels in a prospective cohort of individuals during acute infection by Alpha variant. Results Using a mathematical model, we show that the progressive increase in neutralizing antibodies leads to a shortening of the half-life of both infected cells and infectious viral particles. We estimated that the neutralizing activity reached 90% of its maximal level within 8 days after symptoms onset and could reduce the half-life of both infected cells and infectious virus by a 6-fold factor, thus playing a key role to achieve rapid viral clearance. Using this model, we conducted a simulation study to predict in a more general context the protection conferred by the existence of pre-existing neutralization, due to either vaccination or prior infection. We predicted that a neutralizing activity, as measured by ED50 >103, could reduce by 50% the risk of having viral load detectable by standard PCR assays and by 99% the risk of having viral load above the threshold of cultivable virus. Conclusions This threshold value for the neutralizing activity could be used to identify individuals with poor protection against disease acquisition.

Defective type I interferon immunity is associated with increasing COVID-19 severity (preprint)

Host immunity to infection with SARS-CoV-2 is highly variable, dictating diverse clinical outcomes ranging from asymptomatic to severe disease and death. We previously reported that reduced blood type I interferon (IFN-I) in severe COVID-19 patients preceded clinical worsening. These results were supported by studies which identified genetic mutations in loci of the TLR3- or TLR7-dependent IFN-I pathways, or autoantibodies neutralizing IFNα or IFNω, as major risk factors for development of severe and critical COVID-19 pneumonia. Here, we analyzed a range of IFN-I associated responses in patient cohorts with different severities of COVID-19, showing that baseline plasma IFNα measures differed significantly according to the immunoassay used, as well as timing of sampling, the IFNα subtype measured, and the presence of autoantibodies. We then compared immune responses induced by ex vivo stimulation between non-hospitalized moderate cases (n=27) and hospitalized (n=17) adult patients that required oxygen supplementation. This showed a consistently reduced induction of IFN-I proteins in hospitalized COVID-19 patients upon stimulation, that was not associated with detectable neutralizing autoantibodies against IFNα or IFNω. We confirmed the poor induction of IFN-I in an independent patient cohort (n=33), and showed it was more pronounced with severe disease. Intracellular proteomic analysis showed that while monocyte numbers were increased in hospitalized COVID-19 patients, they did not secrete IFN-I in response to stimulation. This was further confirmed by ex vivo whole blood stimulation with IFN-I which induced a transcriptomic response associated with inflammation in hospitalized COVID-19 patients, that was not seen in controls or non-hospitalized moderate cases. These results may explain the dichotomy of the poor clinical response to IFN-I based treatments in late stage COVID-19, despite the critical importance of IFN-I in early acute infection. An improved understanding of such variable responses to treatment may help to identify potential alternative therapeutic strategies.

The risk of COVID-19 death is much greater and age-dependent with type I IFN autoantibodies (preprint)

SARS-CoV-2 infection fatality rate (IFR) doubles with every five years of age from childhood onward. Circulating autoantibodies neutralizing IFN-α, IFN-ω, and/or IFN-β are found in ~20% of deceased patients across age groups. In the general population, they are found in ~1% of individuals aged 20-70 years and in >4% of those >70 years old. With a sample of 1,261 deceased patients and 34,159 uninfected individuals, we estimated both IFR and relative risk of death (RRD) across age groups for individuals carrying autoantibodies neutralizing type I IFNs, relative to non-carriers. For autoantibodies neutralizing IFN-α2 or IFN-ω, the RRD was 17.0[95% CI:11.7-24.7] for individuals under 70 years old and 5.8[4.5-7.4] for individuals aged 70 and over, whereas, for autoantibodies neutralizing both molecules, the RRD was 188.3[44.8-774.4] and 7.2[5.0-10.3], respectively. IFRs increased with age, from 0.17%[0.12-0.31] for individuals <40 years old to 26.7%[20.3-35.2] for those ≥80 years old for autoantibodies neutralizing IFN-α2 or IFN-ω, and from 0.84%[0.31-8.28] to 40.5%[27.82-61.20] for the same two age groups, for autoantibodies neutralizing both molecules. Autoantibodies against type I IFNs increase IFRs, and are associated with high RRDs, particularly those neutralizing both IFN-α2 and -ω. Remarkably, IFR increases with age, whereas RRD decreases with age. Autoimmunity to type I IFNs appears to be second only to age among common predictors of COVID-19 death.

Targeting the chemokine receptor CXCR4 with histamine analogue to reduce inflammation in juvenile arthritis: a proof of concept for COVID-19 therapeutic approach (preprint)

Among immune cells, activated monocytes play a detrimental role in chronic and viral-induced inflammatory pathologies. The uncontrolled activation of monocytes and the subsequent excessive production of inflammatory factors damage bone-cartilage joints in Juvenile Idiopathic Arthritis (JIA), a childhood rheumatoid arthritis (RA) disease. Inflammatory monocytes also exert a critical role in the cytokine storm induced by SARS-CoV2 infection in severe COVID-19 patients. The moderate beneficial effect of current therapies and clinical trials highlights the need of alternative strategies targeting monocytes to treat RA and COVID-19 pathologies. Here, we show that targeting CXCR4 with small amino compound such as the histamine analogue clobenpropit (CB) inhibits spontaneous and induced-production of a set of key inflammatory cytokines by monocytes isolated from blood and synovial fluids of JIA patients. Moreover, daily intraperitoneal CB treatment of arthritic mice results in significant decrease in circulating inflammatory cytokine levels, immune cell infiltrates, joints erosion, and bone resorption leading to reduction of disease progression. Finally, we provide the prime evidence that the exposure of whole blood from hospitalized COVID-19 patients to CB significantly reduces levels of key cytokine-storm-associated factors including TNF-, IL-6 and IL-1{beta}. These overall data show that targeting CXCR4 with CB-like molecules may represent a promising therapeutic option for chronic and viral-induced inflammatory diseases.

Constitutive IFNα protein production in bats (preprint)

Bats are the only mammals with self-powered flight and account for 20% of all extant mammalian diversity. In addition, they harbor many emerging and reemerging viruses, including multiple coronaviruses, several of which are highly pathogenic in other mammals, but cause no disease in bats. How this relationship between bats and viruses exists is not yet fully understood. Existing evidence supports a specific role for the innate immune system, in particular type I interferon (IFN) responses, a major component of antiviral immunity. Previous studies in bats have shown that components of the IFN pathway are constitutively activated at the transcriptional level. In this study, we tested the hypothesis that the type I IFN response in bats is also constitutively activated at the protein level. For this we utilized highly sensitive Single Molecule (Simoa) digital ELISA assays, previously developed for humans that we adapted to bat samples. We prospectively sampled four non-native chiroptera species from French zoos. We identified a constitutive expression of IFNα protein in the circulation of healthy bats, and concentrations that are physiologically active in humans. Expression levels differed according to the species examined, but was not associated with age, sex, or health status suggesting constitutive IFNα protein expression independent of disease. These results confirm a unique IFN response in bat species that may explain their ability to coexist with multiple viruses in the absence of pathology. These results may help to manage potential zoonotic viral reservoirs and potentially identify new anti-viral strategies.

Release of infectious virus and cytokines in nasopharyngeal swabs from individuals infected with non-B.1.1.7 or B.1.1.7 SARS-CoV-2 variants. (preprint)

The mechanisms that allowed for the SARS-CoV-2 B.1.1.7 variant to rapidly outcompete pre-existing variants in many countries remain poorly characterized. Here, we analyzed viral release, anti-SARS-CoV-2 antibodies and cytokine production in a retrospective series of 427 RTqPCR+ nasopharyngeal swabs collected in COVID-19 patients harbouring either non-B.1.1.7 or B.1.17 variants. We utilized a novel rapid assay, based on S-Fuse-T reporter cells, to quantify infectious SARS-CoV-2. With both non-B.1.1.7 and B.1.1.7 variants, viral titers were highly variable, ranging from 0 to >106 infectious units, and correlated with viral RNA levels. Lateral flow antigenic rapid diagnostic tests (RDTs) were positive in 96% of the samples harbouring infectious virus. About 67 % of individuals carried detectable infectious virus within the first two days after onset of symptoms. This proportion decreased overtime, and viable virus was detected up to 14 days. Samples containing anti-SARS-CoV-2 IgG or IgA did not generally harbour infectious virus. The proportion of individuals displaying viable virus or being RDT-positive was not higher with B.1.1.7 than with non- B.1.1.7 variants. Ct values were slightly but not significantly lower with B.1.1.7. The variant was characterized by a fast decrease of infectivity overtime and a marked release of 17 cytokines (including IFN-b, IP-10, IL-10 and TRAIL). Our results highlight differences between non-B.1.1.7 and B.1.1.7 variants. B.1.1.7 is associated with modified viral decays and cytokine profiles at the nasopharyngeal mucosae during symptomatic infection.

A Monocyte/Dendritic Cell Molecular Signature of SARS-CoV-2 Related Multisystem Inflammatory Syndrome in Children (MIS-C) with Severe Myocarditis (preprint)

SARS-CoV-2 infection in children is generally milder than in adults, yet a proportion of cases result in hyperinflammatory conditions often including myocarditis. To better understand these cases, we applied a multi-parametric approach to the study of blood cells of 56 children hospitalized with suspicion of SARS-CoV-2 infection. The most severe forms of MIS-C (multisystem inflammatory syndrome in children related to SARS-CoV-2), that resulted in myocarditis, were characterized by elevated levels of pro-angiogenesis cytokines and several chemokines. Single-cell transcriptomic analyses identified a unique monocyte/dendritic cell gene signature that correlated with the occurrence of severe myocarditis, characterized by sustained NF-κ B activity, TNF-α signaling, associated with decreased gene expression of NF-κ B inhibitors. We also found a weak response to type-I and type-II interferons, hyperinflammation and response to oxidative stress related to increased HIF-1α and VEGF signaling. These results provide potential for a better understanding of disease pathophysiology.Funding: The study was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), by the “URGENCE COVID-19” fundraising campaign of Institut Pasteur, by the Atip-Avenir, Emergence ville de Paris program and fond de dotation Janssen Horizon and by government grants managed by the Agence National de la Recherche as part of the “Investment for the Future” program (Institut Hospitalo-Universitaire Imagine, grant ANR-10-IAHU-01, Recherche Hospitalo-Universitaire, grant ANR-18-RHUS-0010, Laboratoire d’Excellence ‘‘Milieu Intérieur”, grant ANR-10-LABX-69-01), the Centre de Référence Déficits Immunitaires Héréditaires (CEREDIH), the Agence National de la Recherche (ANR-flash Covid19 “AIROCovid” to FRL and “CoVarImm” to DD and JDS), and by the FASTFoundation (French Friends of Sheba Tel Hashomer Hospital). The LabTech Single-Cell@Imagine is supported by the Paris Region and the “Investissements d’avenir” program through the 2019 ATF funding – Sésame Filières PIA (Grant N°3877871).CdC is the recipient of a CIFRE-PhD (Sanofi). L.B. was a recipient of an Imagine institute PhD international program supported by the Fondation Bettencourt Schueller. L.B. was also supported by the EUR G.E.N.E. (reference #ANR-17-EURE-0013) and is part of the Université de Paris IdEx #ANR-18-IDEX-0001 funded by the French Government through its“Investments for the Future” program. S.M. was a recipient of an INSERM and Institut Imagine post-doctorat program supported by the Fondation pour la Recherche Médicale (FRMN°SPF20170938825). NS was a recipient of the Pasteur-Roux-Cantarini Fellowship. VGP obtained an Imagine international PhD fellowship program supported by the Fondation Bettencourt Schueller. BPP is the recipient of an ANRS post-doctoral fellowship.Conflict of Interest: DD, FRL, JT and MMM are listed as inventors on a patent application related to this technology (European Patent Application no. EP21305197, entitled “Methods of predicting multisystem inflammatory syndrome (MIS-C) with severe myocarditis in subjects suffering from a SARS-CoV-2 infection”).Ethical Approval: The study was approved by the Ethics Committee (Comité de Protection des Personnes Ouest IV, n° DC-2017-2987).

Serological reconstruction of COVID-19 epidemics through analysis of antibody kinetics to SARS-CoV-2 proteins (preprint)

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces a complex antibody response that varies by orders of magnitude between individuals and over time. Waning antibody levels lead to reduced sensitivity of serological diagnostic tests over time. This undermines the utility of serological surveillance as the SARS-CoV-2 pandemic progresses into its second year. Here we develop a multiplex serological test for measuring antibodies of three isotypes (IgG, IgM, IgA) to five SARS-CoV-2 antigens (Spike (S), receptor binding domain (RBD), Nucleocapsid (N), Spike subunit 2, Membrane-Envelope fusion) and the Spike proteins of four seasonal coronaviruses. We measure antibody responses in several cohorts of French and Irish hospitalized patients and healthcare workers followed for up to eleven months after symptom onset. The data are analysed with a mathematical model of antibody kinetics to quantify the duration of antibody responses accounting for inter-individual variation. One year after symptoms, we estimate that 36% (95% range: 11%, 94%) of anti-S IgG remains, 31% (9%, 89%) anti-RBD IgG remains, and 7% (1%, 31%) anti-N IgG remains. Antibodies of the IgM isotype waned more rapidly, with 9% (2%, 32%) anti-RBD IgM remaining after one year. Antibodies of the IgA isotype also waned rapidly, with 10% (3%, 38%) anti-RBD IgA remaining after one year. Quantitative measurements of antibody responses were used to train machine learning algorithms for classification of previous infection and estimation of time since infection. The resulting diagnostic test classified previous infections with 99% specificity and 98% (95% confidence interval: 94%, 99%) sensitivity, with no evidence for declining sensitivity over the time scale considered. The diagnostic test also provided accurate classification of time since infection into intervals of 0 - 3 months, 3 - 6 months, and 6 - 12 months. Finally, we present a computational method for serological reconstruction of past SARS-CoV-2 transmission using the data from this test when applied to samples from a single cross-sectional sero-prevalence survey.

Distinct systemic and mucosal immune responses to SARS-CoV-2 (preprint)

Coordinated local mucosal and systemic immune responses following SARS-CoV-2 infection protect against COVID-19 pathologies or fail leading to severe clinical outcomes. To understand this process, we performed an integrated analysis of SARS-CoV-2 spike-specific antibodies, cytokines, viral load and 16S bacterial communities in paired nasopharyngeal swabs and plasma samples from a cohort of clinically distinct COVID-19 patients during acute infection. Plasma viral load was associated with systemic inflammatory cytokines that were elevated in severe COVID-19, and also with spike-specific neutralizing antibodies. In contrast, nasopharyngeal viral load correlated with SARS-CoV-2 humoral responses but inversely with interferon responses, the latter associating with protective microbial communities. Potential pathogenic microrganisms, often implicated in secondary respiratory infections, were associated with mucosal inflammation and elevated in severe COVID-19. Our results demonstrate distinct tissue compartmentalization of SARS-CoV-2 immune responses and highlight a role for the nasopharyngeal microbiome in regulating local and systemic immunity that determines COVID-19 clinical outcomes.

A monocyte/dendritic cell molecular signature of SARS-CoV2-related multisystem inflammatory syndrome in children (MIS-C) with severe myocarditis (preprint)

SARS-CoV-2 infection in children is generally milder than in adults, yet a proportion of cases result in hyperinflammatory conditions often including myocarditis. To better understand these cases, we applied a multi-parametric approach to the study of blood cells of 56 children hospitalized with suspicion of SARS-CoV-2 infection. The most severe forms of MIS-C (multisystem inflammatory syndrome in children related to SARS-CoV-2), that resulted in myocarditis, were characterized by elevated levels of pro-angiogenesis cytokines and several chemokines. Single-cell transcriptomic analyses identified a unique monocyte/dendritic cell gene signature that correlated with the occurrence of severe myocarditis, characterized by sustained NF-kB activity, TNF-a signaling, associated with decreased gene expression of NF-kB inhibitors. We also found a weak response to type-I and type-II interferons, hyperinflammation and response to oxidative stress related to increased HIF-1a and VEGF signaling. These results provide potential for a better understanding of disease pathophysiology.

Dysregulated primary hemostasis in critically ill COVID-19 patients (preprint)

Background: Microvascular thrombosis, as well as arterial and venous thrombotic events, have been largely described during severe Coronavirus disease 19 (COVID-19). Therapeutic anticoagulation has been proposed in critical patients, however mechanisms underlying hemostasis dysregulation remain unclear. Methods: We explored two independent cross-sectional cohorts to identify soluble markers and gene-expression signatures that discriminated COVID-19 severity and outcomes. Results: We found that elevated soluble (s) P-selectin at admission was associated with disease severity. Elevated sP-selectin was predictive of intubation and death (ROC AUC = 0.67, p = 0.028 and AUC = 0.74, p = 0.0047, respectively). An optimal cutoff value of 150 NC (normalized concentration) was predictive of intubation with 66% negative predictive value (NPV) and 61% positive predictive value (PPV), and of death with 90% NPV and 55% PPV. Next, an unbiased gene set enrichment analysis revealed that critically ill patients had increased expression of genes related to primary hemostasis. Hierarchical clustering identified ITG2AB, GP1BB, PPBP and SELPLG to be upregulated in a grade-dependent manner. ROC curve analysis for the prediction of mechanical ventilation was significant for SELPLG and PPBP (AUC = 0.8, p = 0.046 for both markers). An optimal cutoff value for PBPP was predictive of mechanical ventilation with 100% NPV and 45% PPV, and for SELPLG was predictive of mechanical ventilation with 100% NPV and 50% PPV.Conclusion: We provide evidence that platelets contribute to disease severity with the identification of sP-selectin as a biomarker for poor outcome. Transcriptional analysis identified PPBP and SELPLG RNA count as biomarkers for mechanical ventilation. These findings provide rationale for novel therapeutic approaches with antiplatelet agents.

SARS-CoV-2 infection damages airway motile cilia and impairs mucociliary clearance (preprint)

Understanding how SARS-CoV-2 spreads within the respiratory tract is important to define the parameters controlling the severity of COVID-19. We examined the functional and structural consequences of SARS-CoV-2 infection in a reconstituted human bronchial epithelium model. SARS-CoV-2 replication caused a transient decrease in epithelial barrier function and disruption of tight junctions, though viral particle crossing remained limited. Rather, SARS-CoV-2 replication led to a rapid loss of the ciliary layer, characterized at the ultrastructural level by axoneme loss and misorientation of remaining basal bodies. The motile cilia function was compromised, as measured in a mucociliary clearance assay. Epithelial defense mechanisms, including basal cell mobilization and interferon-lambda induction, ramped up only after the initiation of cilia damage. Analysis of SARS-CoV-2 infection in Syrian hamsters further demonstrated the loss of motile cilia in vivo. This study identifies cilia damage as a pathogenic mechanism that could facilitate SARS-CoV-2 spread to the deeper lung parenchyma.

Sub-second heat inactivation of coronavirus (preprint)

Heat treatment denatures viral proteins that comprise the virion, making virus incapable of infecting a host. Coronavirus (CoV) virions contain single-stranded RNA genomes with a lipid envelope and 4 proteins, 3 of which are associated with the lipid envelope and thus are thought to be easily denatured by heat or surfactant-type chemicals. Prior studies have shown that a temperature of as low as 75 oC and treatment duration of 15 min can effectively inactivate CoV. The applicability of a CoV heat inactivation method greatly depends on the length of time of a heat treatment and the temperature needed to inactivate the virus. With the goal of finding conditions where sub-second heat exposure of CoV can sufficiently inactivate CoV, we designed and developed a simple system that can measure sub-second heat inactivation of CoV. The system is composed of capillary stainless-steel tubing immersed in a temperature-controlled oil bath followed by an ice bath, through which virus solution can be flowed at various speeds. Flowing virus solution at different speeds, along with a real-time temperature monitoring system, allows the virus to be accurately exposed to a desired temperature for various durations of time. Using mouse hepatitis virus (MHV), a beta-coronavirus, as a model system, we identified that 85.2 oC for 0.48 s exposure is sufficient to obtain > 5 Log10 reduction in viral titer (starting titer: 5 x 107 PFU/mL), and that when exposed to 83.4 oC for 0.95 s, the virus was completely inactivated (zero titer, > 6 Log10 reduction).

A genetic variant protective for COVID-19 is inherited from Neanderthals (preprint)

It was recently shown that the major genetic risk factor associated with becoming severely ill with COVID-19 when infected by SARS-CoV-2 is inherited from Neandertals. Thanks to new genetic association studies additional risk factors are now being discovered. Using data from a recent genome-wide associations from the Genetics of Mortality in Critical Care (GenOMICC) consortium, we show that a haplotype at a region associated with requiring intensive care is inherited from Neandertals. It encodes proteins that activate enzymes that are important during infections with RNA viruses. As compared to the previously described Neandertal risk haplotype, this Neandertal haplotype is protective against severe COVID-19, is of more moderate effect, and is found at substantial frequencies in all regions of the world outside Africa.

Impaired type I interferon activity and exacerbated inflammatory responses in severe Covid-19 patients (preprint)

Background: Coronavirus disease 2019 (Covid-19) is a major global threat that has already caused more than 100,000 deaths worldwide. It is characterized by distinct patterns of disease progression implying a diverse host immune response. However, the immunological features and molecular mechanisms involved in Covid-19 severity remain so far poorly known. Methods: We performed an integrated immune analysis that included in-depth phenotypical profiling of immune cells, whole-blood transcriptomic and cytokine quantification on a cohort of fifty Covid19 patients with a spectrum of disease severity. All patient were tested 8 to 12 days following first symptoms and in absence of anti-inflammatory therapy. Results: A unique phenotype in severe and critically ill patients was identified. It consists in a profoundly impaired interferon (IFN) type I response characterized by a low interferon production and activity, with consequent downregulation of interferon-stimulated genes. This was associated with a persistent blood virus load and an exacerbated inflammatory response that was partially driven by the transcriptional factor NF{kappa}B. It was also characterized by increased tumor necrosis factor (TNF)- and interleukin (IL)-6 production and signaling as well as increased innate immune chemokines. Conclusion: We propose that type-I IFN deficiency in the blood is a hallmark of severe Covid-19 and could identify and define a high-risk population. Our study provides a rationale for testing IFN administration combined with adapted anti-inflammatory therapy targeting IL-6 or TNF- in most severe patients. These data also raise concern for utilization of drugs that interfere with the IFN pathway.