High seroprevalence but short-lived immune response to SARS-CoV-2 infection in Paris.

Although the COVID-19 pandemic peaked in March/April 2020 in France, the prevalence of infection is barely known. Using high-throughput methods, we assessed herein the serological response against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of 1847 participants working in three sites of an institution in Paris conurbation. In May-July 2020, 11% (95% confidence interval [CI]: 9.7-12.6) of serums were positive for IgG against the SARS-CoV-2 N and S proteins, and 9.5% (95% CI: 8.2-11.0) were neutralizer in pseudo-typed virus assays. The prevalence of seroconversion was 11.6% (95% CI: 10.2-13.2) when considering positivity in at least one assay. In 5% of RT-qPCR positive individuals, no systemic IgGs were detected. Among immune individuals, 21% had been asymptomatic. Anosmia (loss of smell) and ageusia (loss of taste) occurred in 52% of the IgG-positive individuals and in 3% of the negative ones. In contrast, 30% of the anosmia-ageusia cases were seronegative, suggesting that the true prevalence of infection may have reached 16.6%. In sera obtained 4-8 weeks after the first sampling, anti-N and anti-S IgG titers and neutralization activity in pseudo-virus assay declined by 31%, 17%, and 53%, resulting thus in half-life of 35, 87, and 28 days, respectively. The population studied is representative of active workers in Paris. The short lifespan of the serological systemic responses suggests an underestimation of the true prevalence of infection.

Core-Modified Coelenterazine Luciferin Analogues: Synthesis and Chemiluminescence Properties.

In this work on the design and studies of luciferins related to the blue-hued coelenterazine, the synthesis of heterocyclic analogues susceptible to produce a photon, possibly at a different wavelength, is undertaken. Here, the synthesis of O-acetylated derivatives of imidazo[1,2-b]pyridazin-3(5 H)-one, imidazo[2,1-f][1,2,4]triazin-7(1 H)-one, imidazo[1,2-a]pyridin-3-ol, imidazo[1,2-a]quinoxalin-1(5 H)-one, benzo[f]imidazo[1,2-a]quinoxalin-3(11 H)-one, imidazo[1',2':1,6]pyrazino[2,3-c]quinolin-3(11 H)-one, and 5,11-dihydro-3 H-chromeno[4,3-e]imidazo[1,2-a]pyrazin-3-one is described thanks to extensive use of the Buchwald-Hartwig N-arylation reaction. The acidic hydrolysis of these derivatives then gave solutions of the corresponding luciferin analogues, which were studied. Not too unexpectedly, even if these were "dressed" with substituents found in actual substrates of the nanoKAZ/NanoLuc luciferase, no bioluminescence was observed with these compounds. However, in a phosphate buffer, all produced a light signal, by chemiluminescence, with extensive variations in their respective intensity and this could be increased by adding a quaternary ammonium salt in the buffer. This aspect was actually instrumental to determine the emission spectra of many of these luciferin analogues.

Bioluminescence Profiling of NanoKAZ/NanoLuc Luciferase Using a Chemical Library of Coelenterazine Analogues.

We describe here an extensive structure-bioluminescence relationship study of a chemical library of analogues of coelenterazine, using nanoKAZ/NanoLuc, a mutated luciferase originated from the catalytic subunit of the deep-sea shrimp Oplophorus gracilirostris. Out of the 135 O-acetylated precursors that were prepared by using our recently reported synthesis and following their hydrolysis to give solutions of the corresponding luciferins, notable bioluminescence improvements were achieved in comparison with furimazine, which is currently amongst the best substrates of nanoKAZ/NanoLuc. For instance, the rather more lipophilic analogue 8-(2,3-difluorobenzyl)-2-((5-methylfuran-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one provided a 1.5-fold improvement of the total light output over a 2 h period, a close to threefold increase of the initial signal intensity and a signal-to-background ratio five times greater than furimazine. The kinetic parameters for the enzymatic reaction were obtained for a selection of luciferin analogues and provided unexpected insights into the luciferase activity. Most prominently, along with a general substrate-dependent and irreversible inactivation of this enzyme, in the case of the optimized luciferin mentioned above, the consumption of 2664 molecules was found to be required for the detection of a single Relative Light Unit (RLU; a luminometer-dependent fraction of a photon).

Gram-scale synthesis of luciferins derived from coelenterazine and original insights into their bioluminescence properties.

An original gram-scale synthesis of O-acetylated forms of coelenterazine, furimazine or hydroxy-bearing analogues of luciferins is described. The comparison over two hours of their bioluminescence, using the nanoKAZ/NanoLuc luciferase, provides remarkable insights useful for the selection of a substrate adapted for a given application.

Application of Machine Learning Prediction of Individual SARS-CoV-2 Vaccination and Infection Status to the French Serosurveillance Survey From March 2020 to 2022: Cross-Sectional Study.

BACKGROUND: The seroprevalence of SARS-CoV-2 infection in the French population was estimated with a representative, repeated cross-sectional survey based on residual sera from routine blood testing. These data contained no information on infection or vaccination status, thus limiting the ability to detail changes observed in the immunity level of the population over time. OBJECTIVE: Our aim is to predict the infected or vaccinated status of individuals in the French serosurveillance survey based only on the results of serological assays. Reference data on longitudinal serological profiles of seronegative, infected, and vaccinated individuals from another French cohort were used to build the predictive model. METHODS: A model of individual vaccination or infection status with respect to SARS-CoV-2 obtained from a machine learning procedure was proposed based on 3 complementary serological assays. This model was applied to the French nationwide serosurveillance survey from March 2020 to March 2022 to estimate the proportions of the population that were negative, infected, vaccinated, or infected and vaccinated. RESULTS: From February 2021 to March 2022, the estimated percentage of infected and unvaccinated individuals in France increased from 7.5% to 16.8%. During this period, the estimated percentage increased from 3.6% to 45.2% for vaccinated and uninfected individuals and from 2.1% to 29.1% for vaccinated and infected individuals. The decrease in the seronegative population can be largely attributed to vaccination. CONCLUSIONS: Combining results from the serosurveillance survey with more complete data from another longitudinal cohort completes the information retrieved from serosurveillance while keeping its protocol simple and easy to implement.

T cell migration and effector function differences in familial adenomatous polyposis patients with APC gene mutations.

Familial adenomatous polyposis (FAP) is an inherited disease characterized by the development of large number of colorectal adenomas with high risk of evolving into colorectal tumors. Mutations of the Adenomatous polyposis coli (APC) gene is often at the origin of this disease, as well as of a high percentage of spontaneous colorectal tumors. APC is therefore considered a tumor suppressor gene. While the role of APC in intestinal epithelium homeostasis is well characterized, its importance in immune responses remains ill defined. Our recent work indicates that the APC protein is involved in various phases of both CD4 and CD8 T cells responses. This prompted us to investigate an array of immune cell features in FAP subjects carrying APC mutations. A group of 12 FAP subjects and age and sex-matched healthy controls were studied. We characterized the immune cell repertoire in peripheral blood and the capacity of immune cells to respond ex vivo to different stimuli either in whole blood or in purified T cells. A variety of experimental approaches were used, including, pultiparamater flow cytometry, NanosString gene expression profiling, Multiplex and regular ELISA, confocal microscopy and computer-based image analyis methods. We found that the percentage of several T and natural killer (NK) cell populations, the expression of several genes induced upon innate or adaptive immune stimulation and the production of several cytokines and chemokines was different. Moreover, the capacity of T cells to migrate in response to chemokine was consistently altered. Finally, immunological synapses between FAP cytotoxic T cells and tumor target cells were more poorly structured. Our findings of this pilot study suggest that mild but multiple immune cell dysfunctions, together with intestinal epithelial dysplasia in FAP subjects, may facilitate the long-term polyposis and colorectal tumor development. Although at an initial discovery phase due to the limited sample size of this rare disease cohort, our findings open new perspectives to consider immune cell abnormalities into polyposis pathology.

Contribution of the glycolytic flux and hypoxia adaptation to efficient biofilm formation by Candida albicans.

The fungal pathogen Candida albicans forms therapeutically challenging biofilms on biomedical implants. Using a transcript profiling approach genes whose expression is favoured upon biofilm growth compared with planktonic growth have been previously identified. Knock-out mutants for 38 of these genes were constructed, six of which showed a specific defect in biofilm formation. Among these genes, TYE7 that encodes a transcriptional activator of glycolytic genes in planktonic and biofilm growth conditions was identified as being required for the cohesiveness of biofilms. Biofilms formed by the tye7Δ knock-out mutant showed a hyperfilamentous morphology, and growth of this mutant on solid medium under hypoxia was also associated with the production of hyphae. Similar to TYE7 inactivation, inhibition of glycolysis or ATP synthesis using oxalate or an uncoupler, respectively, triggered morphogenesis when a wild-type strain was grown under hypoxia. These treatments also induced the formation of weakly cohesive, hyper-filamentous biofilms by a wild-type strain. Our data indicate that a hypoxic environment is generated within C. albicans biofilms and that continued biofilm development requires a Tye7p-dependent upregulation of glycolytic genes necessary to adapt to hypoxia and prevent uncontrolled hyphal formation. Thus, adaptation to hypoxia is an integral component of biofilm formation in C. albicans.

The tumor suppressor adenomatous polyposis coli regulates T lymphocyte migration.

Adenomatous polyposis coli (APC) is a tumor suppressor whose mutations underlie familial adenomatous polyposis (FAP) and colorectal cancer. Although its role in intestinal epithelial cells is well characterized, APC importance in T cell biology is ill defined. APC regulates cytoskeleton organization, cell polarity, and migration in various cell types. Here, we address whether APC plays a role in T lymphocyte migration. Using a series of cell biology tools, we unveiled that T cells from FAP patients carrying APC mutations display impaired adhesion and motility in constrained environments. We further dissected the cellular mechanisms underpinning these defects in APC-depleted CEM T cell line that recapitulate the phenotype observed in FAP T cells. We found that APC affects T cell motility by modulating integrin-dependent adhesion and cytoskeleton reorganization. Hence, APC mutations in FAP patients not only drive intestinal neoplasms but also impair T cell migration, potentially contributing to inefficient antitumor immunity.

A lentiviral vector expressing a dendritic cell-targeting multimer induces mucosal anti-mycobacterial CD4+ T-cell immunity.

Most viral vectors, including the potently immunogenic lentiviral vectors (LVs), only poorly direct antigens to the MHC-II endosomal pathway and elicit CD4+ T cells. We developed a new generation of LVs encoding antigen-bearing monomers of collectins substituted at their C-terminal domain with the CD40 ligand ectodomain to target and activate antigen-presenting cells. Host cells transduced with such optimized LVs secreted soluble collectin-antigen polymers with the potential to be endocytosed in vivo and reach the MHC-II pathway. In the murine tuberculosis model, such LVs induced efficient MHC-II antigenic presentation and triggered both CD8+ and CD4+ T cells at the systemic and mucosal levels. They also conferred a significant booster effect, consistent with the importance of CD4+ T cells for protection against Mycobacterium tuberculosis. Given the pivotal role of CD4+ T cells in orchestrating innate and adaptive immunity, this strategy could have a broad range of applications in the vaccinology field.

Seroprevalence and risk factors of exposure to COVID-19 in homeless people in Paris, France: a cross-sectional study.

BACKGROUND: During the COVID-19 lockdown period from March 17 to May 11, 2020, French authorities in Paris and its suburbs relocated people experiencing recurrent homelessness to emergency shelters, hotels, and large venues. A serological survey was done at some of these locations to assess the COVID-19 exposure prevalence in this group. METHODS: We did a cross-sectional seroprevalence study at food distribution sites, emergency shelters, and workers' residences that were provided medical services by Médecins Sans Frontières in Paris and Seine-Saint-Denis in the Ile-de-France region. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody seropositivity was detected by Luciferase-Linked Immunosorbent Assay and Pseudo Neutralization Test. Sociodemographic and exposure related information was collected via a verbal questionnaire to analyse risk factors and associations with various COVID-19 symptoms. FINDINGS: Between June 23 and July 2, 2020, 426 (52%) of 818 individuals recruited tested positive in 14 sites. Seroprevalence varied significantly by type of recruitment site (χ2 p<0·0001), being highest among those living in workers' residences (88·7%, 95% CI 81·8-93·2), followed by emergency shelters (50·5%, 46·3-54·7), and food distribution sites (27·8%, 20·8-35·7). More than two thirds of COVID-19 seropositive individuals (68%, 95% CI 64·2-72·2; 291 of 426) did not report any symptoms during the recall period. COVID-19 seropositivity was strongly associated with overcrowding (medium density: adjusted odds ratio [aOR] 2·7, 95% CI 1·5-5·1, p=0·0020; high density: aOR 3·4, 1·7-6·9, p<0·0001). INTERPRETATION: These results show high exposure to SARS-CoV-2 with important variations between those at different study sites. Living in crowded conditions was the strongest factor associated with exposure level. This study underscores the importance of providing safe, uncrowded accommodation, alongside adequate testing and public health information. FUNDING: Médecins Sans Frontières, Epicentre, Institut Pasteur's URGENCE nouveau coronavirus fund, Total Foundation.

Prevalence of SARS-CoV-2 antibodies in France: results from nationwide serological surveillance.

Assessment of the cumulative incidence of SARS-CoV-2 infections is critical for monitoring the course and extent of the COVID-19 epidemic. Here, we report estimated seroprevalence in the French population and the proportion of infected individuals who developed neutralising antibodies at three points throughout the first epidemic wave. Testing 11,000 residual specimens for anti-SARS-CoV-2 IgG and neutralising antibodies, we find nationwide seroprevalence of 0.41% (95% CI: 0.05-0.88) mid-March, 4.14% (95% CI: 3.31-4.99) mid-April and 4.93% (95% CI: 4.02-5.89) mid-May 2020. Approximately 70% of seropositive individuals have detectable neutralising antibodies. Infection fatality rate is 0.84% (95% CI: 0.70-1.03) and increases exponentially with age. These results confirm that the nationwide lockdown substantially curbed transmission and that the vast majority of the French population remained susceptible to SARS-CoV-2 in May 2020. Our study shows the progression of the first epidemic wave and provides a framework to inform the ongoing public health response as viral transmission continues globally.

Single-Cell Acoustic Force Spectroscopy: Resolving Kinetics and Strength of T Cell Adhesion to Fibronectin.

Assessing the strength and kinetics of molecular interactions of cells with the extracellular matrix is fundamental to understand cell adhesion processes. Given the relevance of these processes, there is a strong need for physical methods to quantitatively assess the mechanism of cell adhesion at the single-cell level, allowing discrimination of cells with different behaviors. Here we introduce single-cell acoustic force spectroscopy (scAFS), an approach that makes use of acoustic waves to exert controlled forces, up to 1 nN, to hundreds of individual cells in parallel. We demonstrate the potential of scAFS by measuring adhesion forces and kinetics of CD4+ T lymphocytes (CD4) to fibronectin. We determined that CD4 adhesion is accelerated by interleukin-7, their main regulatory cytokine, whereas CD4 binding strength remains the same. Activation of these cells likely increases their chance to bind to the vessel wall in the blood flow to infiltrate inflamed tissues and locally coordinate the immune response.

Designed mono- and di-covalent inhibitors trap modeled functional motions for Trypanosoma cruzi proline racemase in crystallography.

Chagas disease, caused by Trypanosoma cruzi, affects millions of people in South America and no satisfactory therapy exists, especially for its life threatening chronic phase. We targeted the Proline Racemase of T. cruzi, which is present in all stages of the parasite life cycle, to discover new inhibitors against this disease. The first published crystal structures of the enzyme revealed that the catalytic site is too small to allow any relevant drug design. In previous work, to break through the chemical space afforded to virtual screening and drug design, we generated intermediate models between the open (ligand free) and closed (ligand bound) forms of the enzyme. In the present work, we co-crystallized the enzyme with the selected inhibitors and found that they were covalently bound to the catalytic cysteine residues in the active site, thus explaining why these compounds act as irreversible inhibitors. These results led us to the design of a novel, more potent specific inhibitor, NG-P27. Co-crystallization of this new inhibitor with the enzyme allowed us to confirm the predicted protein functional motions and further characterize the chemical mechanism. Hence, the catalytic Cys300 sulfur atom of the enzyme attacks the C2 carbon of the inhibitor in a coupled, regiospecific-stereospecific Michael reaction with trans-addition of a proton on the C3 carbon. Strikingly, the six different conformations of the catalytic site in the crystal structures reported in this work had key similarities to our intermediate models previously generated by inference of the protein functional motions. These crystal structures span a conformational interval covering roughly the first quarter of the opening mechanism, demonstrating the relevance of modeling approaches to break through chemical space in drug design.

Unveiling Cerebral Leishmaniasis: parasites and brain inflammation in Leishmania donovani infected mice.

Visceral leishmaniasis (VL) is a systemic disease with multifaceted clinical manifestations, including neurological signs, however, the involvement of the nervous system during VL is underestimated. Accordingly, we investigated both brain infection and inflammation in a mouse model of VL. Using bioluminescent Leishmania donovani and real-time 2D-3D imaging tools, we strikingly detected live parasites in the brain, where we observed a compartmentalized dual-phased inflammation pattern: an early phase during the first two weeks post-infection, with the prompt arrival of neutrophils and Ly6Chigh macrophages in an environment presenting a variety of pro-inflammatory mediators (IFN-γ, IL-1ß, CXCL-10/CXCR-3, CCL-7/CCR-2), but with an intense anti-inflammatory response, led by IL-10; and a re-inflammation phase three months later, extremely pro-inflammatory, with novel upregulation of mediators, including IL-1ß, TNF-α and MMP-9. These new data give support and corroborate previous studies connecting human and canine VL with neuroinflammation and blood-brain barrier disruption, and conclusively place the brain among the organs affected by this parasite. Altogether, our results provide convincing evidences that Leishmania donovani indeed infects and inflames the brain.

Imaging visceral leishmaniasis in real time with golden hamster model: Monitoring the parasite burden and hamster transcripts to further characterize the immunological responses of the host.

Characterizing the clinical, immunological and parasitological features associated with visceral leishmaniasis is complex. It involves recording in real time and integrating quantitative multi-parametric data sets from parasite infected host tissues. Although several models have been used, hamsters are considered the bona fide experimental model for Leishmania donovani studies. To study visceral leishmaniasis in hamsters we generated virulent transgenic L. donovani that stably express a reporter luciferase protein. Two complementary methodologies were combined to follow the infectious process: in vivo imaging using luciferase-expressing Leishmania and real time RT-PCR to quantify both Leishmania and host transcripts. This approach allows us: i) to assess the clinical outcome of visceral leishmaniasis by individual monitoring of hamster weight, ii) to follow the parasite load in several organs by real time analysis of the bioluminescence in vivo and through real time quantitative PCR analysis of amastigote parasite transcript abundance ex vivo, iii) to evaluate the immunological responses triggered by the infection by quantifying hamster transcripts on the same samples and iv) to limit the number of hamsters selected for further analysis. The overall data highlight a correlation between the transcriptional cytokine signatures of hamster affected tissues and the amastigote burden fluctuations, thus providing new insights into the immunopathological process driven by L. donovani in the tissues of mammalian hosts. Finally, they suggest organ-specific immune responses.

New insights into experimental visceral leishmaniasis: Real-time in vivo imaging of Leishmania donovani virulence.

Visceral leishmaniasis is an insidious neglected disease with worldwide distribution. It is caused by parasites from the Leishmania donovani complex, which are able to be transmitted by different species of phlebotomine sand flies and to infect numerous mammal hosts. Despite the high number of people infected or at risk, and the remarkable quantity of studies focusing on this disease, a proper experimental model to efficiently decipher the infectious process of visceral leishmaniasis taking into account the nuances of parasite's virulence and the duration of the infection is still lacking. Therefore, using golden Syrian hamsters and BALB/c mice, state-of-the-art genetic manipulation applied on a fully virulent L. donovani strain and in vivo imaging approaches, we describe herein three benefits for experimental visceral leishmaniasis: (i) the development of a double transfected bioluminescent (firefly luciferase) and fluorescent (E2-crimson) virulent strain of L. donovani (Ld1S_luci_E2-crimson), favoring a wide range of both in vivo and in vitro investigations, (ii) the establishment of a non-invasive mouse model to evaluate the infectious process during visceral leishmaniasis and the parasite's virulence in real time, allowing longitudinal studies with the same animals, and (iii) the elaboration of a suitable method to reinstate (and verify anew) the virulence in a population of attenuated parasites, by recovering persistent parasites from chronic infected mice. Consequently, these results open up new perspectives on the study of visceral leishmaniasis, especially in the fields of therapeutics and vaccinology, since the model described herein renders now possible long-lasting follow up studies, with easy and accurate day-by-day verifications of the infection status along with a reduced number of laboratory animals. TRIAL REGISTRATION: ClinicalTrials.gov 2013-0047.

Lipid Droplet Formation, Their Localization and Dynamics during Leishmania major Macrophage Infection.

Leishmania, the causative agent of vector-borne diseases, known as leishmaniases, is an obligate intracellular parasite within mammalian hosts. The outcome of infection depends largely on the activation status of macrophages, the first line of mammalian defense and the major target cells for parasite replication. Understanding the strategies developed by the parasite to circumvent macrophage defense mechanisms and to survive within those cells help defining novel therapeutic approaches for leishmaniasis. We previously showed the formation of lipid droplets (LDs) in L. major infected macrophages. Here, we provide novel insights on the origin of the formed LDs by determining their cellular distribution and to what extent these high-energy sources are directed to the proximity of Leishmania parasites. We show that the ability of L. major to trigger macrophage LD accumulation is independent of parasite viability and uptake and can also be observed in non-infected cells through paracrine stimuli suggesting that LD formation is from cellular origin. The accumulation of LDs is demonstrated using confocal microscopy and live-cell imagin in parasite-free cytoplasmic region of the host cell, but also promptly recruited to the proximity of Leishmania parasites. Indeed LDs are observed inside parasitophorous vacuole and in parasite cytoplasm suggesting that Leishmania parasites besides producing their own LDs, may take advantage of these high energy sources. Otherwise, these LDs may help cells defending against parasitic infection. These metabolic changes, rising as common features during the last years, occur in host cells infected by a large number of pathogens and seem to play an important role in pathogenesis. Understanding how Leishmania parasites and different pathogens exploit this LD accumulation will help us define the common mechanism used by these different pathogens to manipulate and/or take advantage of this high-energy source.

The Cyclical Development of Trypanosoma vivax in the Tsetse Fly Involves an Asymmetric Division.

Trypanosoma vivax is the most prevalent trypanosome species in African cattle. It is thought to be transmitted by tsetse flies after cyclical development restricted to the vector mouthparts. Here, we investigated the kinetics of T. vivax development in Glossina morsitans morsitans by serial dissections over 1 week to reveal differentiation and proliferation stages. After 3 days, stable numbers of attached epimastigotes were seen proliferating by symmetric division in the cibarium and proboscis, consistent with colonization and maintenance of a parasite population for the remaining lifespan of the tsetse fly. Strikingly, some asymmetrically dividing cells were also observed in proportions compatible with a continuous production of pre- metacyclic trypomastigotes. The involvement of this asymmetric division in T. vivax metacyclogenesis is discussed and compared to other trypanosomatids.

Differentiation of Leishmania donovani in host-free system: analysis of signal perception and response.

Leishmania donovani are the causative agents of kala-azar in humans. They undergo a developmental program following changes in the environment, resulting in the reversible transformation between the extracellular promastigote form in the sand fly vector and the obligatory intracellular amastigote form in phagolysosomes of macrophages. A host-free differentiation system for L. donovani was used to investigate the initial process of promastigote to amastigote differentiation. Within an hour after exposing promastigotes to differentiation signal (concomitant exposure to 37 degrees C and pH 5.5), they expressed the amastigote-specific A2 protein family. At 5 h they started to transform to amastigote-shaped cells, a process that was completed 7 h later. This morphological transformation occurred synchronously, while cells arrested at G1. By sequential exposure to elevated temperature (for 24 h) and then acidic pH, we found that heat was responsible for the growth arrest and acidic pH to its release and subsequent route to differentiation into amastigotes. Lastly, ethanol and Azetidine 2 carboxylic acid (a synthetic proline analog) that induced heat shock response in promastigotes were capable of replacing heat in the differentiation signal.