New methods to unveil host-microbe interaction mechanisms along the microbiota-gut-brain-axis.

Links between the gut microbiota and human health have been supported throughout numerous studies, such as the development of neurological disease disorders. This link is referred to as the "microbiota-gut-brain axis" and is the focus of an emerging field of research. Microbial-derived metabolites and gut and neuro-immunological metabolites regulate this axis in health and many diseases. Indeed, assessing these signals, whether induced by microbial metabolites or neuro-immune mediators, could significantly increase our knowledge of the microbiota-gut-brain axis. However, this will require the development of appropriate techniques and potential models. Methods for studying the induced signals originating from the microbiota remain crucial in this field. This review discusses the methods and techniques available for studies of microbiota-gut-brain interactions. We highlight several much-debated elements of these methodologies, including the widely used in vivo and in vitro models, their implications, and perspectives in the field based on a systematic review of PubMed. Applications of various animal models (zebrafish, mouse, canine, rat, rabbit) to microbiota-gut-brain axis research with practical examples of in vitro methods and innovative approaches to studying gut-brain communications are highlighted. In particular, we extensively discuss the potential of "organ-on-a-chip" devices and their applications in this field. Overall, this review sheds light on the most widely used models and methods, guiding researchers in the rational choice of strategies for studies of microbiota-gut-brain interactions.

Serum and Salivary IgG and IgA Response After COVID-19 Messenger RNA Vaccination.

Importance: There is still considerable controversy in the literature regarding the capacity of intramuscular messenger RNA (mRNA) vaccination to induce a mucosal immune response. Objective: To compare serum and salivary IgG and IgA levels among mRNA-vaccinated individuals with or without previous SARS-CoV-2 infection. Design, Setting, and Participants: In this cohort study, SARS-CoV-2-naive participants and those with previous infection were consecutively included in the CoviCompare P and CoviCompare M mRNA vaccination trials and followed up to day 180 after vaccination with either the BNT162b2 (Pfizer-BioNTech) vaccine or the mRNA-1273 (Moderna) vaccine at the beginning of the COVID-19 vaccination campaign (from February 19 to June 8, 2021) in France. Data were analyzed from October 25, 2022, to July 13, 2023. Main Outcomes and Measures: An ultrasensitive digital enzyme-linked immunosorbent assay was used for the comparison of SARS-CoV-2 spike-specific serum and salivary IgG and IgA levels. Spike-specific secretory IgA level was also quantified at selected times. Results: A total of 427 individuals were included in 3 groups: participants with SARS-CoV-2 prior to vaccination who received 1 single dose of BNT162b2 (Pfizer-BioNTech) (n = 120) and SARS-CoV-2-naive individuals who received 2 doses of mRNA-1273 (Moderna) (n = 172) or 2 doses of BNT162b2 (Pfizer-BioNTech) (n = 135). The median age was 68 (IQR, 39-75) years, and 228 (53.4%) were men. SARS-CoV-2 spike-specific IgG saliva levels increased after 1 or 2 vaccine injections in individuals with previous infection and SARS-CoV-2-naive individuals. After vaccination, SARS-CoV-2-specific saliva IgA levels, normalized with respect to total IgA levels, were significantly higher in participants with previous infection, as compared with the most responsive mRNA-1273 (Moderna) recipients (median normalized levels, 155 × 10-5 vs 37 × 10-5 at day 29; 107 × 10-5 vs 54 × 10-5 at day 57; and 104 × 10-5 vs 70 × 10-5 at day 180 [P < .001]). In contrast, compared with day 1, spike-specific IgA levels in the BNT162b2-vaccinated SARS-CoV-2-naive group increased only at day 57 (36 × 10-5 vs 49 × 10-5 [P = .01]). Bona fide multimeric secretory IgA levels were significantly higher in individuals with previous infection compared with SARS-CoV-2-naive individuals after 2 antigenic stimulations (median optical density, 0.36 [IQR, 0.16-0.63] vs 0.16 [IQR, 0.10-0.22]; P < .001). Conclusions and Relevance: The findings of this cohort study suggest that mRNA vaccination was associated with mucosal immunity in individuals without prior SARS-CoV-2 infection, but at much lower levels than in previously infected individuals. Further studies are needed to determine the association between specific saliva IgA levels and prevention of infection or transmission.

Reduction of product composition variability using pooled microbiome ecosystem therapy and consequence in two infectious murine models.

Growing evidence demonstrates the key role of the gut microbiota in human health and disease. The recent success of microbiotherapy products to treat recurrent Clostridioides difficile infection has shed light on its potential in conditions associated with gut dysbiosis, such as acute graft-versus-host disease, intestinal bowel diseases, neurodegenerative diseases, or even cancer. However, the difficulty in defining a "good" donor as well as the intrinsic variability of donor-derived products' taxonomic composition limits the translatability and reproducibility of these studies. Thus, the pooling of donors' feces has been proposed to homogenize product composition and achieve higher taxonomic richness and diversity. In this study, we compared the metagenomic profile of pooled products to corresponding single donor-derived products. We demonstrated that pooled products are more homogeneous, diverse, and enriched in beneficial bacteria known to produce anti-inflammatory short chain fatty acids compared to single donor-derived products. We then evaluated pooled products' efficacy compared to corresponding single donor-derived products in Salmonella and C. difficile infectious mouse models. We were able to demonstrate that pooled products decreased pathogenicity by inducing a structural change in the intestinal microbiota composition. Single donor-derived product efficacy was variable, with some products failing to control disease progression. We further performed in vitro growth inhibition assays of two extremely drug-resistant bacteria, Enterococcus faecium vanA and Klebsiella pneumoniae oxa48, supporting the use of pooled microbiotherapies. Altogether, these results demonstrate that the heterogeneity of donor-derived products is corrected by pooled fecal microbiotherapies in several infectious preclinical models.IMPORTANCEGrowing evidence demonstrates the key role of the gut microbiota in human health and disease. Recent Food and Drug Administration approval of fecal microbiotherapy products to treat recurrent Clostridioides difficile infection has shed light on their potential to treat pathological conditions associated with gut dysbiosis. In this study, we combined metagenomic analysis with in vitro and in vivo studies to compare the efficacy of pooled microbiotherapy products to corresponding single donor-derived products. We demonstrate that pooled products are more homogeneous, diverse, and enriched in beneficial bacteria compared to single donor-derived products. We further reveal that pooled products decreased Salmonella and Clostridioides difficile pathogenicity in mice, while single donor-derived product efficacy was variable, with some products failing to control disease progression. Altogether, these findings support the development of pooled microbiotherapies to overcome donor-dependent treatment efficacy.

Unmasking the potential of secretory IgA and its pivotal role in protection from respiratory viruses.

Mucosal immunity has regained its spotlight amidst the ongoing Coronavirus disease 19 (COVID-19) pandemic, with numerous studies highlighting the crucial role of mucosal secretory IgA (SIgA) in protection against Severe acute respiratory syndrome coronavirus-2 or SARS-CoV-2 infections. The observed limitations in the efficacy of currently authorized COVID-19 vaccines in inducing effective mucosal immune responses remind us of the limitations of systemic vaccination in promoting protective mucosal immunity. This resurgence of interest has motivated the development of vaccine platforms capable of enhancing mucosal responses, specifically the SIgA response, and the development of IgA-based therapeutics. Recognizing viral respiratory infections as a global threat, we would like to comprehensively review the existing knowledge on mucosal immunity, with a particular emphasis on SIgA, in the context of SARS-CoV-2, influenza, and Respiratory Syncytial Virus (RSV) infections. This review aims to describe the structural and functional specificities of SIgA, along with its nuanced role in combating influenza, RSV, and SARS-CoV-2 infections. Subsequent sections further elaborate promising vaccine strategies, including mucosal vaccines against Influenza, RSV, and SARS-CoV-2 respiratory viruses, currently undergoing preclinical and clinical development. Additionally, we address the challenges associated with mucosal vaccine development, concluding with a discussion on IgA-based therapeutics as a promising platform for the treatment of viral respiratory infections. This comprehensive review not only synthesizes current insights into mucosal immunity but also identifies critical knowledge gaps, strengthening the way for further advancements in our current understanding and approaches to combat respiratory viral threats.

What Should We Know about Drug Levels and Therapeutic Drug Monitoring during Pregnancy and Breastfeeding in Inflammatory Bowel Disease under Biologic Therapy?

Data on the real long-term influences of in utero drug exposure in pregnant women on childhood development are scarce and remain not well determined and depend on the duration of in utero drug exposure and maternal drug levels. Therapeutic drug monitoring (TDM) during pregnancy may help limit fetal drug exposure while maintaining an effective dose for the treatment of the underlying inflammatory bowel disease (IBD) in women. Most antibody therapies used in patients with IBD are IgG molecules which are actively transported across the placenta, especially during the third trimester of the pregnancy. Here, we propose an up-to-date clinical review to summarize the available findings of serum drug levels in maternal blood during pregnancy, in the cord blood, infants at delivery and in breast milk of patients with IBD treated with biologics. Conversely, in comparison to adalimumab (ADA) levels, which are relatively stable during pregnancy, infliximab (IFX) drug clearance decreased significantly during the last two trimesters of the pregnancy, leading to increasing drug concentrations in the blood of the pregnant women. As most guidelines recommend using live vaccines in infants at the age of one or earlier in case of negative serum drug levels in newborns, statistical models could help clinicians in making a decision to adjust the last dose of the biologic during pregnancy and to determine the optimal date to vaccinate. Altogether, data from the literature offers strong reassurance in terms of safety for anti-TNFα therapies during pregnancy not only for IBD patients who intend to conceive, but also for pregnant women and for the physicians taking care of these patients. ADA and IFX levels in breast milk are detectable, but at very low levels, and therefore, it is recommended to pursue breast feeding under anti-TNFα therapy. Our knowledge on ustekinumab or vedolizumab levels in pregnant women remains unclear and scarce. These drugs are currently not recommended for patients with IBD in clinical practice. Therefore, TDM and proactive dose adjustment are not necessary during pregnancy since its impact on making a clinical decision have not yet been clearly demonstrated in routine practice. Overall, drug concentrations in the cord blood, an infant at birth and postpartum serum concentrations in infants, due to active placental drug transfer, may have a greater impact than the limited drug transfer in breast milk during lactation on the risk of infection and developmental outcomes. Ustekinumab and vedolizumab exposure during pregnancy and lactation are both considered low risk by the recent ECCO guidelines despite the limited data that are currently available.

Characterization of Canine Peyer's Patches by Multidimensional Analysis: Insights from Immunofluorescence, Flow Cytometry, and Single-Cell RNA Sequencing.

The oral route is effective and convenient for vaccine administration to stimulate a protective immune response. GALT plays a crucial role in mucosal immune responses, with Peyer's patches (PPs) serving as the primary site of induction. A comprehensive understanding of the structures and functions of these structures is crucial for enhancing vaccination strategies and comprehending disease mechanisms; nonetheless, our current knowledge of these structures in dogs remains incomplete. We performed immunofluorescence and flow cytometry studies on canine PPs to identify cell populations and structures. We also performed single-cell RNA sequencing (scRNA-seq) to investigate the immune cell subpopulations present in PPs at steady state in dogs. We generated and validated an Ab specifically targeting canine M cells, which will be a valuable tool for elucidating Ag trafficking into the GALT of dogs. Our findings will pave the way for future studies of canine mucosal immune responses to oral vaccination and enteropathies. Moreover, they add to the growing body of knowledge in canine immunology, further expanding our understanding of the complex immune system of dogs.

Higher serum infliximab concentrations following subcutaneous dosing are associated with deep remission in patients with inflammatory bowel disease.

BACKGROUND: The relationship between SC-IFX concentrations and favorable therapeutic outcomes in patients with Crohn's disease (CD) and ulcerative colitis (UC) remain elusive. PATIENTS AND METHODS: This cross-sectional trial study included consecutive IBD adult patients with IBD treated with SC-IFX at maintenance dose of 120mg/2 weeks. Investigated therapeutic outcomes included sustained clinical remission; composite clinical and biomarker remission [clinical remission and CRP < 5mg/L]; biochemical remission [FC < 250 µg/g]; and deep remission [clinical, biological and biochemical remission]. RESULTS: Of 91 patients identified, 71 patients qualified for inclusion in the study (70% with CD; 27% with concomitant immunomodulators). At the time of drug concentration measurement (median 13.5 months after switch), 55 (77%) patients had sustained clinical remission; n=44 (62%) composite clinical and biomarker remission; n=40 (56%) biochemical remission; and n=31 (43%) patients deep remission. The mean SC-IFX concentrations were significantly higher in patients with sustained clinical remission [p=0.014]; composite clinical and biomarker remission [p=0.003]; biochemical remission [p<0.001] and deep remission [p<0.001] compared to patients without having these outcomes. In multivariate analysis, SC-IFX concentration was the only factor independently associated with sustained clinical remission [odds ratio (OR): 4.7, 95% CI: 3.1-12.2, p=0.005)]; clinical and biomarker remission (OR: 9.21, 95%CI: 6.09-18.7, p=0.006); biochemical remission (OR: 37, 95%CI: 14-39.3), p<0.001); and deep remission (OR: 29, 95%CI:15.7-37.4, p<0.001). The optimal SC-IFX concentration cut-off associated with deep remission based on ROC analysis was 20µg/mL (sensitivity: 0.91, specificity: 0.80, accuracy: 0.85). Combination with an IMM failed to improve SC-IFX pharmacokinetics. CONCLUSION: Higher SC-IFX concentrations are associated with higher rates of favorable therapeutic outcomes in IBD patients. Serum SC-IFX concentrations higher than 20µg/mL were significantly associated with deep remission.

Proof-of-Concept Human Organ-on-Chip Study: First Step of Platform to Assess Neuro-Immunological Communication Involved in Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBD) are complex chronic inflammatory disorders of the gastrointestinal (GI) tract. Recent evidence suggests that the gut-brain axis may be pivotal in gastrointestinal and neurological diseases, especially IBD. Here, we present the first proof of concept for a microfluidic technology to model bilateral neuro-immunological communication. We designed a device composed of three compartments with an asymmetric channel that allows the isolation of soma and neurites thanks to microchannels and creates an in vitro synaptic compartment. Human-induced pluripotent stem cell-derived cortical glutamatergic neurons were maintained in soma compartments for up to 21 days. We performed a localized addition of dendritic cells (MoDCs) to either the soma or synaptic compartment. The microfluidic device was coupled with microelectrode arrays (MEAs) to assess the impact on the electrophysiological activity of neurons while adding dendritic cells. Our data highlight that an electrophysiologic signal is transmitted between two compartments of glutamatergic neurons linked by synapses in a bottom-up way when soma is exposed to primed dendritic cells. In conclusion, our study authenticates communication between dendritic cells and neurons in inflammatory conditions such as IBD. This platform opens the way to complexification with gut components to reach a device for pharmacological compound screening by blocking the gut-brain axis at a mucosal level and may help patients.

Bordetella bronchiseptica and Bordetella pertussis: Similarities and Differences in Infection, Immuno-Modulation, and Vaccine Considerations.

Bordetella pertussis and Bordetella bronchiseptica belong to the genus Bordetella, which comprises 14 other species. B. pertussis is responsible for whooping cough in humans, a severe infection in children and less severe or chronic in adults. These infections are restricted to humans and currently increasing worldwide. B. bronchiseptica is involved in diverse respiratory infections in a wide range of mammals. For instance, the canine infectious respiratory disease complex (CIRDC), characterized by a chronic cough in dogs. At the same time, it is increasingly implicated in human infections, while remaining an important pathogen in the veterinary field. Both Bordetella can evade and modulate host immune responses to support their persistence, although it is more pronounced in B. bronchiseptica infection. The protective immune responses elicited by both pathogens are comparable, while there are important characteristics in the mechanisms that differ. However, B. pertussis pathogenesis is more difficult to decipher in animal models than those of B. bronchiseptica because of its restriction to humans. Nevertheless, the licensed vaccines for each Bordetella are different in terms of formulation, route of administration and immune responses induced, with no known cross-reaction between them. Moreover, the target of the mucosal tissues and the induction of long-lasting cellular and humoral responses are required to control and eliminate Bordetella. In addition, the interaction between both veterinary and human fields are essential for the control of this genus, by preventing the infections in animals and the subsequent zoonotic transmission to humans.

Development of Antibodies to Ustekinumab Is Associated with Loss of Response in Patients with Inflammatory Bowel Disease.

Monitoring of anti-drug antibodies in patients on ustekinumab is not routinely recommended in patients with inflammatory bowel disease (IBD) due to low rates of immunogenicity. AIM OF STUDY: The purpose of this study was to investigate the relationship between anti-drug antibodies detected by a drug-tolerant assay and loss of response (LOR) to therapy in a cohort of patients with IBD being treated with ustekinumab. PATIENTS AND METHODS: This retrospective study consecutively enrolled all adult patients with moderate to severe active IBD who had at least 2 years of follow-up after ustekinumab was initiated. LOR was defined as CDAI > 220 or HBI > 4 for Crohn's disease (CD) and partial Mayo subscore > 3 for ulcerative colitis (UC) and with a modification in disease management. RESULTS: Ninety patients were included (78 CD and 12 UC; mean age 37 years). Median levels of anti-ustekinumab antibodies (ATU) were significantly higher in patients with LOR compared to those with ongoing clinical response (15.2 µg/mL-eq CI (7.9-21.5) and 4.7 µg/mL-eq CI (2.1-10.5), respectively; p = 0.04). The area under the ROC curve (AUROC) for ATU in predicting LOR was 0.76. The optimal cut-off point for identifying patients with LOR was 9.5 µg/mL-eq with a sensitivity of 80% and specificity of 85%. Uni- and multivariate analyses showed that serum ATU ≥ 9.5 µg/mL-eq (hazard ratio (HR) 2.54, 95%CI (1.80-5.93)), p = 0.022, prior vedolizumab (HR 2.78, 95%CI (1.09-3.34), p = 0.019) and prior azathioprine (HR 0.54, 95%CI (0.20-0.76), p = 0.014) exposures were the only factors independently associated with LOR to UST. CONCLUSION: In our real-life cohort, ATU was identified as an independent predictor of LOR to ustekinumab in patients with IBD.

Mucosal Vaccination with Live Attenuated Bordetella bronchiseptica Protects against Challenge in Wistar Rats.

Bordetella bronchiseptica (Bb) is a Gram-negative bacterium responsible for canine infectious respiratory disease complex (CIRDC). Several vaccines targeting this pathogen are currently licensed for use in dogs, but their mechanism of action and the correlates of protection are not fully understood. To investigate this, we used a rat model to examine the immune responses induced and the protection conferred by a canine mucosal vaccine after challenge. Wistar rats were vaccinated orally or intranasally on D0 and D21 with a live attenuated Bb vaccine strain. At D35, the rats of all groups were inoculated with 103 CFU of a pathogenic strain of B. bronchiseptica. Animals vaccinated via either the intranasal or the oral route had Bb-specific IgG and IgM in their serum and Bb-specific IgA in nasal lavages. Bacterial load in the trachea, lung, and nasal lavages was lower in vaccinated animals than in non-vaccinated control animals. Interestingly, coughing improved in the group vaccinated intranasally, but not in the orally vaccinated or control group. These results suggest that mucosal vaccination can induce mucosal immune responses and provide protection against a Bb challenge. This study also highlights the advantages of a rat model as a tool for studying candidate vaccines and routes of administration for dogs.

Anti-AGO1 Antibodies Identify a Subset of Autoimmune Sensory Neuronopathy.

BACKGROUND AND OBJECTIVES: Autoantibodies (Abs) improve diagnosis and treatment decisions of idiopathic neurologic disorders. Recently, we identified Abs against Argonaute (AGO) proteins as potential autoimmunity biomarkers in neurologic disorders. In this study, we aim to reveal (1) the frequency of AGO1 Abs in sensory neuronopathy (SNN), (2) titers and IgG subclasses, and (3) their clinical pattern including response to treatment. METHODS: This retrospective multicentric case/control study screened 132 patients with SNN, 301 with non-SNN neuropathies, 274 with autoimmune diseases (AIDs), and 116 healthy controls (HCs) for AGO1 Abs through ELISA. Seropositive cases were also tested for IgG subclasses, titers, and conformation specificity. RESULTS: AGO1 Abs occurred in 44 patients, comprising significantly more of those with SNN (17/132 [12.9%]) than those with non-SNN neuropathies (11/301 [3.7%]; p = 0.001), those with AIDs (16/274 [5.8%]; p = 0.02), or HCs (0/116; p < 0.0001). Ab titers ranged from 1:100 to 1:100,000. IgG subclass was mainly IgG1, and 11/17 AGO1 Ab-positive SNN (65%) had a conformational epitope. AGO1 Ab-positive SNN was more severe than AGO1 Ab-negative SNN (e.g., SNN score: 12.2 vs 11.0, p = 0.004), and they more frequently and more efficiently responded to immunomodulatory treatments than AGO1 Ab-negative SNN (7/13 [54%] vs 6/37 [16%], p = 0.02). Regarding the type of treatments more precisely, this significant difference was confirmed for the use of IV immunoglobulins (IVIg) but not for steroids or second-line treatments. Multivariate logistic regression adjusted for potential confounders showed that AGO1 Ab positivity was the only predictor of response to treatment (OR 4.93, 1.10-22.24 95% CI, p = 0.03). DISCUSSION: Although AGO Abs are not specific for SNN, based on our retrospective data, they may identify a subset of cases with SNN with more severe features and a possibly better response to IVIg. The significance of AGO1 Abs in clinical practice needs to be explored on a larger series.

Prior COVID-19 Immunization Does Not Cause IgA- or IgG-Dependent Enhancement of SARS-CoV-2 Infection.

Antibody-dependent enhancement (ADE) can increase the rates and severity of infection with various viruses, including coronaviruses, such as MERS. Some in vitro studies on COVID-19 have suggested that prior immunization enhances SARS-CoV-2 infection, but preclinical and clinical studies have demonstrated the contrary. We studied a cohort of COVID-19 patients and a cohort of vaccinated individuals with a heterologous (Moderna/Pfizer) or homologous (Pfizer/Pfizer) vaccination scheme. The dependence on IgG or IgA of ADE of infection was evaluated on the serum samples from these subjects (twenty-six vaccinated individuals and twenty-one PCR-positive SARS-CoV-2-infected patients) using an in vitro model with CD16- or CD89-expressing cells and the Delta (B.1.617.2 lineage) and Omicron (B.1.1.529 lineage) variants of SARS-CoV-2. Sera from COVID-19 patients did not show ADE of infection with any of the tested viral variants. Some serum samples from vaccinated individuals displayed a mild IgA-ADE effect with Omicron after the second dose of the vaccine, but this effect was abolished after the completion of the full vaccination scheme. In this study, FcγRIIIa- and FcαRI-dependent ADE of SARS-CoV-2 infection after prior immunization, which might increase the risk of severe disease in a second natural infection, was not observed.

Long-term humoral response following Delta and Omicron BA.1 co-infection.

This study reports the 6-month humoral immune response in vaccinated patients concomitantly infected with Delta and Omicron BA.1 variants of SARS-CoV-2. Interestingly, the simultaneous exposure to the Delta and BA.1 S proteins does not confer an additional immune advantage compared to exposure to the BA.1 S protein alone.

Prior SARS-CoV-2 infection enhances and reshapes spike protein-specific memory induced by vaccination.

The diversity of vaccination modalities and infection history are both variables that have an impact on the immune memory of individuals vaccinated against SARS-CoV-2. To gain more accurate knowledge of how these parameters imprint on immune memory, we conducted a long-term follow-up of SARS-CoV-2 spike protein-specific immune memory in unvaccinated and vaccinated COVID-19 convalescent individuals as well as in infection-naïve vaccinated individuals. Here, we report that individuals from the convalescent vaccinated (hybrid immunity) group have the highest concentrations of spike protein-specific antibodies at 6 months after vaccination. As compared with infection-naïve vaccinated individuals, they also display increased frequencies of an atypical mucosa-targeted memory B cell subset. These individuals also exhibited enhanced TH1 polarization of their SARS-CoV-2 spike protein-specific follicular T helper cell pool. Together, our data suggest that prior SARS-CoV-2 infection increases the titers of SARS-CoV-2 spike protein-specific antibody responses elicited by subsequent vaccination and induces modifications in the composition of the spike protein-specific memory B cell pool that are compatible with enhanced functional protection at mucosal sites.