SARS-CoV-2 remains a significant health threat due to its high infection and mutation rates. The emergence of new variants of concern poses challenges as they can lead to immune escape mutations, potentially reducing the efficacy of vaccines and antibody therapeutics. The receptor binding domain (RBD) of SARS-CoV-2 is particularly noteworthy as it is both the most rapidly evolving domain and the principal target of neutralizing antibodies. As an alternative to time-consuming and expensive neutralization assays, we have developed a bead-based multiplex surrogate virus neutralization test based on ACE2-RBD binding inhibition. We demonstrated how our high-throughput assay allows us to simultaneously assess anti-RBD neutralizing antibodies levels against multiple SARS-CoV-2 variants, providing data that is consistent with the gold-standard live virus neutralization assay. The utility of this assay was demonstrated by applying it to a large French population cohort to demonstrate that hybrid immunity (generated by a combination of vaccination and infection) is associated with protection against infection with the Omicron BA.1 and BA.2 lineages of SARS-COV-2.

Background The seasonal human coronaviruses (HCoV) NL63, 229E, OC43, and HKU1 are globally endemic, yet the majority of HCoV infections remain undiagnosed. Methods In a cross-sectional study, 2389 serum samples were collected from children and adults in France in 2020. In a longitudinal cohort study, 2520 samples were collected from 898 French individuals followed up between 2020 and 2021. Antibodies to HCoVs were measured using a bead-based multiplex assay. Results The rate of waning of anti-HCoV spike immunoglobulin G antibodies was estimated as 0.22-0.47 year(-1) for children, and 0.13-0.27 year(-1) for adults. Seroreversion was estimated as 0.31-1.37 year(-1) in children and 0.19-0.72 year(-1) in adults. The estimated seroconversion rate in children was consistent with 20%-39% of children being infected every year with each HCoV. Conclusions The high force of infection in children indicates that HCoVs may be responsible for a substantial proportion of fever episodes experienced by children.

BackgroundThe risk of SARS-CoV-2 (re-)infection remains present given waning of vaccine-induced and infection-acquired immunity, and ongoing circulation of new variants.AimTo develop a method that predicts virus neutralisation and disease protection based on variant-specific antibody measurements to SARS-CoV-2 antigens.MethodsTo correlate antibody and neutralisation titres, we collected 304 serum samples from individuals with either vaccine-induced or infection-acquired SARS-CoV-2 immunity. Using the association between antibody and neutralisation titres, we developed a prediction model for SARS-CoV-2-specific neutralisation titres. From predicted neutralising titres, we inferred protection estimates to symptomatic and severe COVID-19 using previously described relationships between neutralisation titres and protection estimates. We estimated population immunity in a French longitudinal cohort of 905 individuals followed from April 2020 to November 2021.ResultsWe demonstrated a strong correlation between anti-SARS-CoV-2 antibodies measured using a low cost high-throughput assay and antibody response capacity to neutralise live virus. Participants with a single vaccination or immunity caused by infection were especially vulnerable to symptomatic or severe COVID-19. While the median reduced risk of COVID-19 from Delta variant infection in participants with three vaccinations was 96% (IQR: 94-98), median reduced risk among participants with infection-acquired immunity was only 42% (IQR: 22-66).ConclusionOur results are consistent with data from vaccine effectiveness studies, indicating the robustness of our approach. Our multiplex serological assay can be readily adapted to study new variants and provides a framework for development of an assay that would include protection estimates.

Background The protective immunity against omicron following a BNT162b2 Pfizer booster dose among elderly individuals (ie, those aged >65 years) is not well characterised. Methods In a community-based, prospective, longitudinal cohort study taking place in France in which 75 residents from three nursing homes were enrolled, we selected 38 residents who had received a two-dose regimen of mRNA vaccine and a booster dose of Pfizer BNT162b2 vaccine. We excluded individuals that did not receive three vaccine doses or did not have available sera samples. We measured anti-S IgG antibodies and neutralisation capacity in sera taken 56 (28-68) and 55 (48-64) days (median (range)) after the 2nd and 3rd vaccine doses, respectively. Antibodies targeting the SARS-CoV-2 Spike protein were measured with the S-Flow assay as binding antibody units per milliliter (BAU/mL). Neutralising activities in sera were measured as effective dilution 50% (ED50) with the S-Fuse assay using authentic isolates of delta and omicron BA.1. Findings Among the 38 elderly individuals recruited to the cohort study between November 23rd, 2020 and April 29th, 2021, with median age of 88 (range 72-101) years, 30 (78.95%) had been previously infected with SARS-CoV-2. After three vaccine doses, serum neutralising activity was lower against omicron BA.1 (median ED50 of 774.5, range 15.0-34660.0) than the delta variant (median ED50 of 4972.0, range 213.7-66340.0), and higher among previously infected (ie, convalescent; median ED50 against omicron: 1088.0, range 32.6-34660.0) compared with infection-naive residents (median ED50 against omicron: 188.4, range 15.0-8918.0). During the French omicron wave in December 2021-January 2022, 75% (6/8) of naive residents were infected, compared to 25% (7/30) of convalescent residents (P=0.0114). Anti-Spike antibody levels and neutralising activity against omicron BA.1 after a third BNT162b2 booster dose were lower in those with breakthrough BA.1 infection (n=13) compared with those without (n=25), with a median of 1429.9 (range 670.9-3818.3) BAU/mL vs 2528.3 (range 695.4-8832.0) BAU/mL (P=0.029) and a median ED50 of 281.1 (range 15.0-2136.0) vs 1376.0 (range 32.6-34660.0) (P=0.0013), respectively. Interpretation This study shows that elderly individuals who received three vaccine doses elicit neutralising antibodies against the omicron BA.1 variant of SARS-CoV-2. Elderly individuals who had also been previously infected showed higher neutralising activity compared with naive individuals. Yet, breakthrough infections with omicron occurred. Individuals with breakthrough infections had significantly lower neutralising titers compared to individuals without breakthrough infection. Thus, a fourth dose of vaccine may be useful in the elderly population to increase the level of neutralising antibodies and compensate for waning immunity. Copyright (C) 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Population-level immunity to SARS-CoV-2 is growing through vaccination as well as ongoing circulation. Given waning immunity and emergence of new variants, it is important to dynamically determine the risk of re-infection in the population. For estimating immune protection, neutralization titers are most informative, but these assays are difficult to conduct at a population level. Measurement of antibody levels can be implemented at high throughput, but has not been robustly validated as a correlate of protection. Here, we have developed a method that predicts neutralization and protection based on variant-specific antibody measurements to SARS-CoV-2 antigens. This approach allowed us to estimate population-immunity in a longitudinal cohort from France followed for up to 2 years. Participants with a single vaccination or immunity caused by infection only are especially vulnerable to COVID-19 or hospitalization due to SARS-CoV-2. While the median reduced risk to COVID-19 in participants with 3 vaccinations was 96%, the median reduced risk among participants with infection-acquired immunity only was 42%. The results presented here are consistent with data from vaccine-effectiveness studies indicating robustness of our approach. Our multiplex serological assay can be readily optimized and employed to study any new variant and provides a framework for development of an assay that would include protection estimates.

Background The protective immunity against Omicron following a BNT162b2 Pfizer booster dose among elderly is not well characterized. Methods Thirty-eight residents from three nursing homes were recruited for the study. Antibodies targeting the Spike protein of SARS-CoV-2 were measured with the S-Flow assay. Neutralizing activities in sera were measured as effective dilution 50% (ED50) with the S-Fuse assay using authentic isolates of Delta and Omicron. Results Among the 38 elderly included in the study, with median (inter-quartile range, IQR) age of 88 (81-92) years, 30 (78.9%) had been previously infected. The ED50 of neutralization were lower against Omicron than Delta, and higher among convalescent compared to naive residents. During an Omicron epidemic affecting two of the three nursing homes in December 2021-January 2022, 75% (6/8) of naive residents got infected, compared to 25% (6/24) of convalescents (P=0.03). Antibody levels to Spike and ED50 of neutralization against Omicron after the BNT162b2 booster dose were lower in those with breakthrough infection (n=12) compared to those without (n=20): median of 1256 vs 2523 BAU/mL (P=0.02) and median ED50 of 234 vs 1298 (P=0.0004), respectively. Conclusion This study confirmed the importance of receiving at least three antigenic exposures to the SARS-CoV-2 Spike protein for achieving satisfactory neutralizing antibody levels. In this population, protection against Omicron infection was increased in individuals who had been previously infected in addition to the three vaccine doses. Thus, a fourth antigenic exposure may be useful in the elderly population to prevent infection with Omicron, a variant known for its high escape immunity properties.

Background. 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. Methods. We developed a multiplex serological test for measuring antibodies to 5 SARS-CoV-2 antigens and the spike proteins of seasonal coronaviruses. We measured antibody responses in cohorts of hospitalized patients and healthcare workers followed for up to 11 months after symptoms. A mathematical model of antibody kinetics was used to quantify the duration of antibody responses. Antibody response data were used to train algorithms for estimating time since infection. Results. One year after symptoms, we estimate that 36% (95% range, 11%-94%) of anti-Spike immunoglobulin G (IgG) remains, 31% (95% range, 9%-89%) anti-RBD IgG remains, and 7% (1%-31%) of anti-nucleocapsid IgG remains. The multiplex assay classified previous infections into time intervals of 0-3 months, 3-6 months, and 6-12 months. This method was validated using data from a seroprevalence survey in France, demonstrating that historical SARS-CoV-2 transmission can be reconstructed using samples from a single survey. Conclusions. In addition to diagnosing previous SARS-CoV-2 infection, multiplex serological assays can estimate the time since infection, which can be used to reconstruct past epidemics.

ABSTRACT Direct interactions between bacterial and host glycans have been recently reported to be involved in the binding of pathogenic bacteria to host cells. In the case of Shigella, the Gram-negative enteroinvasive bacterium responsible for acute rectocolitis, such interactions contribute to bacterial adherence to epithelial cells. However, the role of glycans in the tropism of Shigella for immune cells whose glycosylation pattern varies depending on their activation state is unknown. We previously reported that Shigella targets activated, but not nonactivated, human CD4+ T lymphocytes. Here, we show that nonactivated CD4+ T lymphocytes can be turned into Shigella-targetable cells upon loading of their plasma membrane with sialylated glycosphingolipids (also termed gangliosides). The Shigella targeting profile of ganglioside-loaded nonactivated T cells is similar to that of activated T cells, with a predominance of injection of effectors from the type III secretion system (T3SS) not resulting in cell invasion. We demonstrate that gangliosides interact with the O-antigen polysaccharide moiety of lipopolysaccharide (LPS), the major bacterial surface antigen, thus promoting Shigella binding to CD4+ T cells. This binding step is critical for the subsequent injection of T3SS effectors, a step which we univocally demonstrate to be dependent on actin polymerization. Altogether, these findings highlight the critical role of glycan-glycan interactions in Shigella pathogenesis. IMPORTANCE Glycosylation of host cell surface varies with species and location in the body, thus contributing to species specificity and tropism of microorganisms. Cross talk by Shigella, the Gram-negative enteroinvasive bacterium responsible for bacillary dysentery, with its exclusively human host has been extensively studied. However, the molecular determinants of the step of binding to host cells are poorly defined. Taking advantage of the observation that human-activated CD4+ T lymphocytes, but not nonactivated cells, are targets of Shigella, we succeeded in rendering the refractory cells susceptible to targeting upon loading of their plasma membrane with sialylated glycosphingolipids (gangliosides) that are abundantly present on activated cells. We show that interactions between the sugar polar part of gangliosides and the polysaccharide moiety of Shigella lipopolysaccharide (LPS) promote bacterial binding, which results in the injection of effectors via the type III secretion system. Whereas LPS interaction with gangliosides was proposed long ago and recently extended to a large variety of glycans, our findings reveal that such glycan-glycan interactions are critical for Shigella pathogenesis by driving selective interactions with host cells, including immune cells.

Microbial pathogens possess a diversity of weapons that disrupt host homeostasis and immune defenses, thus resulting in the establishment of infection. The best-characterized system mediating bacterial protein delivery into target eukaryotic cells is the type III secretion system (T3SS) expressed by Gram-negative bacteria, including the human enteric pathogens Shigella, Salmonella, Yersinia, and enteropathogenic/enterohemorragic Escherichia coli (EPEC/EHEC). The emerging global view is that these T3SS-bearing pathogens share similarities in their ability to target key cellular pathways such as the cell cytoskeleton, trafficking, cell death/survival, and the NF-κB and MAPK signaling pathways. In particular, multiple host proteins are targeted in a given pathway, and different T3SS effectors from various pathogens share functional similarities.

Microbial pathogens possess a diversity of weapons that disrupt host homeostasis and immune defenses, thus resulting in the establishment of infection. The best-characterized system mediating bacterial protein delivery into target eukaryotic cells is the type III secretion system (T3SS) expressed by Gram-negative bacteria, including the human enteric pathogens Shigella, Salmonella, Yersinia, and enteropathogenic/enterohemorragic Escherichia coli (EPEC/EHEC). The emerging global view is that these T3SS-bearing pathogens share similarities in their ability to target key cellular pathways such as the cell cytoskeleton, trafficking, cell death/survival, and the NF-κB and MAPK signaling pathways. In particular, multiple host proteins are targeted in a given pathway, and different T3SS effectors from various pathogens share functional similarities.

Many human Gram-negative bacterial pathogens express a Type Three Secretion Apparatus (T3SA), including among the most notorious Shigella spp., Salmonella enterica, Yersinia enterocolitica and enteropathogenic Escherichia coli (EPEC). These bacteria express on their surface multiple copies of the T3SA that mediate the delivery into host cells of specific protein substrates critical to pathogenesis. Shigella spp. are Gram-negative bacterial pathogens responsible for human bacillary dysentery. The effector function of several Shigella T3SA substrates has largely been studied but their potential cellular targets are far from having been comprehensively delineated. In addition, it is likely that some T3SA substrates have escaped scrutiny as yet. Indeed, sequencing of the virulence plasmid of Shigella flexneri has revealed numerous open reading frames with unknown functions that could encode additional T3SA substrates. Taking advantage of label-free mass spectrometry detection of proteins secreted by a constitutively secreting strain of S. flexneri, we identified five novel substrates of the T3SA. We further confirmed their secretion through the T3SA and translocation into host cells using β-lactamase assays. The coding sequences of two of these novel T3SA substrates (Orf13 and Orf131a) have a guanine-cytosine content comparable to those of T3SA components and effectors. The three other T3SA substrates identified (Orf48, Orf86 and Orf176) have significant homology with antitoxin moieties of type II Toxin-Antitoxin systems usually implicated in the maintenance of low copy plasmids. While Orf13 and Orf131a might constitute new virulence effectors contributing to S. flexneri pathogenicity, potential roles for the translocation into host cells of antitoxins or antitoxin-like proteins during Shigella infection are discussed.

The enteroinvasive bacterium Shigella is a facultative intracellular bacterium known, in vitro, to invade a large diversity of cells through the delivery of virulence effectors into the cell cytoplasm via a type III secretion system (T3SS). Here, we provide evidence that the injection of T3SS effectors does not necessarily result in cell invasion. Indeed, we demonstrate through optimization of a T3SS injection reporter that effector injection without subsequent cell invasion, termed the injection-only mechanism, is the main strategy used by Shigella to target human immune cells. We show that in vitro-activated human peripheral blood B, CD4(+) T, and CD8(+) T lymphocytes as well as switched memory B cells are mostly targeted by the injection-only mechanism. B and T lymphocytes residing in the human colonic lamina propria, encountered by Shigella upon its crossing of the mucosal barrier, are also mainly targeted by injection-only. These findings reveal that cells refractory to invasion can still be injected, thus extending the panel of host cells manipulated to the benefit of the pathogen. Future analysis of the functional consequences of the injection-only mechanism toward immune cells will contribute to the understanding of the priming of adaptive immunity, which is known to be altered during the course of natural Shigella infection.

Antibody-mediated immunity to Shigella, the causative agent of bacillary dysentery, requires several episodes of infection to get primed and is short-lasting, suggesting that the B cell response is functionally impaired. We show that upon ex vivo infection of human colonic tissue, invasive S. flexneri interacts with and occasionally invades B lymphocytes. The induction of a type three secretion apparatus (T3SA)-dependent B cell death is observed in the human CL-01 B cell line in vitro, as well as in mouse B lymphocytes in vivo. In addition to cell death occurring in Shigella-invaded CL-01 B lymphocytes, we provide evidence that the T3SA needle tip protein IpaD can induce cell death in noninvaded cells. IpaD binds to and induces B cell apoptosis via TLR2, a signaling receptor thus far considered to result in activation of B lymphocytes. The presence of bacterial co-signals is required to sensitize B cells to apoptosis and to up-regulate tlr2, thus enhancing IpaD binding. Apoptotic B lymphocytes in contact with Shigella-IpaD are detected in rectal biopsies of infected individuals. This study therefore adds direct B lymphocyte targeting to the diversity of mechanisms used by Shigella to dampen the host immune response.

Event Abstract Back to Event B lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection mediated by the virulence factor IpaD Katharina Nothelfer1*, Ellen T. Arena1, Laurie Pinaud1, Michel Neunlist2, Ilia Belotserkovsky1, Claude Parsot1, Rubhana Raqib3, Brian Mozeleski1, Philippe J. Sansonetti1 and Armelle Phalipon1* 1 Institut Pasteur, France 2 Centre Hospitalier Universitaire (CHU) Hôtel Dieu, France 3 International Centre for Diarrhoeal Diseases Research, Bangladesh (ICDDR,B), Bangladesh Shigella is a gram-negative enteroinvasive bacterium and the causative agent of bacillary dysentery, an acute recto-colitis. Antibody-mediated natural immunity to Shigella requires several episodes of infection to get primed and is short-lasting, suggesting that the B cell response is functionally impaired. Here we show that upon ex vivo infection of human colonic tissue, invasive S. flexneri interacts with and invades B lymphocytes. We observe the induction of a type three secretion apparatus (T3SA)-dependent B cell death in vitro, both in lamina propria B lymphocytes and the human CL-01 B cell line. This cell death and the parallel reduction of the B cell pool can also be observed in an in vivo mouse infection model. Intriguingly, Shigella-induced B cell death does not require bacterial invasion or injection of virulence effectors via the T3SA in vitro. Instead, the virulence factor IpaD triggers mitochondrial B cell apoptosis in the presence of bacterial co-signals that render B lymphocytes prone to die. We provide evidence that IpaD binds to and induces apoptosis via TLR2, a signaling pathway that has thus far only been considered as a mitogenic stimulus for B lymphocytes. Apoptotic B lymphocytes in close contact with Shigella displaying IpaD are also detected in isolated lymphoid follicles of rectal biopsies of naturally-infected individuals. These findings reveal a novel mechanism of T3SA action to induce B cell death by the binding of a virulence factor and reveal an efficient strategy by which entero-invasive pathogens could impair the priming of a protective immune response. Keywords: Shigella flexneri, B lymphocytes, Apoptosis, TLR2, virulence factor Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Nothelfer K, Arena ET, Pinaud L, Neunlist M, Belotserkovsky I, Parsot C, Raqib R, Mozeleski B, Sansonetti PJ and Phalipon A (2013). B lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection mediated by the virulence factor IpaD. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01169 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Katharina Nothelfer, Institut Pasteur, Paris, France, katharina.nothelfer@pasteur.fr Dr. Armelle Phalipon, Institut Pasteur, Paris, France, armelle.phalipon@pasteur.fr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Katharina Nothelfer Ellen T Arena Laurie Pinaud Michel Neunlist Ilia Belotserkovsky Claude Parsot Rubhana Raqib Brian Mozeleski Philippe J Sansonetti Armelle Phalipon Google Katharina Nothelfer Ellen T Arena Laurie Pinaud Michel Neunlist Ilia Belotserkovsky Claude Parsot Rubhana Raqib Brian Mozeleski Philippe J Sansonetti Armelle Phalipon Google Scholar Katharina Nothelfer Ellen T Arena Laurie Pinaud Michel Neunlist Ilia Belotserkovsky Claude Parsot Rubhana Raqib Brian Mozeleski Philippe J Sansonetti Armelle Phalipon PubMed Katharina Nothelfer Ellen T Arena Laurie Pinaud Michel Neunlist Ilia Belotserkovsky Claude Parsot Rubhana Raqib Brian Mozeleski Philippe J Sansonetti Armelle Phalipon Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Au-delà de l’invasion cellulaire, la stratégie du touché-coulé : un nouveau paradigme pour la pathogénicité de Shigella L’invasion de la muqueuse du côlon humain par les entérobactéries à Gram négatif du genre Shigella aboutit à une rectocolite aigue appelée dysenterie bacillaire qui reste un problème de santé publique majeur. Shigella possède un Système de Sécrétion de Type Trois (SST3) codé par un plasmide de virulence qui permet la translocation d’effecteurs bactériens dans le cytoplasme des cellules eucaryotes pour manipuler leurs fonctions. Ces effecteurs détournent les cellules épithéliales pour constituer une niche de réplication intracellulaire et interagissent avec les cellules immunes pour affecter l’initiation de la réponse immune adaptative. En conséquence, plusieurs épisodes infectieux sont nécessaires afin d’établir une protection immunitaire humorale qui est toutefois de courte durée. Ces travaux de thèse avaient pour but (i) d’approfondir les connaissances sur l’interaction de Shigella avec l’hôte en se concentrant sur les mécanismes dépendant du SST3 interférant avec les lymphocytes et (ii) de déterminer si des effecteurs du SST3 non encore identifiés sont codés par le plasmide de virulence. Nos résultats démontrent que la translocation d’effecteur du SST3 peut être découplée de l’invasion cellulaire, conduisant à des cellules « injectées-seulement ». Nous rapportons que Shigella induit l’apoptose des lymphocytes B par un mécanisme dépendant de la protéine située à l’extrémité du SST3 mais indépendant de la translocation d’effecteurs. Ces résultats établissent un nouveau paradigme concernant la pathogénicité de Shigella au-delà de l’invasion cellulaire, basé sur des mécanismes de type « touché-coulé » qui sont au cœur des interactions entre ce pathogène et les cellules immunes. Par ailleurs, nous décrivons la capacité de Shigella jusque-là inconnue d’interférer avec la sécrétion d’anticorps par les lymphocytes B, ce qui pourrait contribuer à moduler la réponse spécifique humorale de l’hôte. Enfin, nous avons identifié cinq nouveaux effecteurs potentiels de Shigella codés par le plasmide de virulence et injectés par le SST3 dans les cellules eucaryotes. Ces travaux de thèse apportent donc de nouveaux éléments concernant la pathogénicité de Shigella par la découverte de nouveaux mécanismes ciblant les cellules immunes et l’identification de nouvelles protéines bactériennes injectées dans le cytoplasme des cellules de l’hôte.