[mRNA vaccination, a model of transition from basic biology to medicine]. / Vaccination ARN messager (ARNm), modèle de transition de la biologie fondamentale à la médecine.

Sixty years elapsed between the discovery of messenger RNA (mRNA) and the use of this molecule in an unprecedented global vaccination campaign that brought the Covid-19 pandemic under control. Sixty years of doubts for some and certainties for others about the possibility of using mRNA-an example of synthetic biology-in therapeutic medicine and vaccinology. Years of "translational" research and development have culminated in the success of anti-Covid-19 mRNA vaccines and the promise of more to come against emerging pathogens. A new paradigm in vaccinology, enabling pandemics to be tackled as they emerge. A lesson to be learned: medical progress is less a question of time than of the critical nature of the biological discovery that underpins it. Before leaving us, François Gros, who played a key role in the discovery of mRNA, was able to appreciate the relevance of this obvious fact.

Soixante ans ont séparé la découverte de l'ARN messager (ARNm) et l'utilisation de cette molécule dans une campagne planétaire inédite de vaccination ayant permis le contrôle de la pandémie de Covid-19. Soixante ans de doutes chez certains et de certitudes chez d'autres sur la possibilité d'utiliser l'ARNm ­ un exemple de biologie de synthèse ­ en médecine thérapeutique et en vaccinologie. Des années de recherche et de développement «  translationnels  ¼ pour aboutir au succès de vaccins à ARNm anti-Covid-19 et à la promesse d'autres à venir contre de nouveaux pathogènes émergents. Un nouveau paradigme de la vaccinologie permettant d'attaquer les pandémies dans le temps de leur émergence. Une leçon à tirer, le progrès médical est moins affaire de temps que de la nature décisive de la découverte biologique qui le sous-tend. François Gros, acteur de la découverte de l'ARNm a pu, avant de nous quitter, juger de la pertinence de cette évidence.

Parvimonas micra, an oral pathobiont associated with colorectal cancer, epigenetically reprograms human colonocytes.

Recently, an intestinal dysbiotic microbiota with enrichment in oral cavity bacteria has been described in colorectal cancer (CRC) patients. Here, we characterize and investigate one of these oral pathobionts, the Gram-positive anaerobic coccus Parvimonas micra. We identified two phylotypes (A and B) exhibiting different phenotypes and adhesion capabilities. We observed a strong association of phylotype A with CRC, with its higher abundance in feces and in tumoral tissue compared with the normal homologous colonic mucosa, which was associated with a distinct methylation status of patients. By developing an in vitro hypoxic co-culture system of human primary colonic cells with anaerobic bacteria, we show that P. micra phylotype A alters the DNA methylation profile promoters of key tumor-suppressor genes, oncogenes, and genes involved in epithelial-mesenchymal transition. In colonic mucosa of CRC patients carrying P. micra phylotype A, we found similar DNA methylation alterations, together with significant enrichment of differentially expressed genes in pathways involved in inflammation, cell adhesion, and regulation of actin cytoskeleton, providing evidence of P. micra's possible role in the carcinogenic process.

Ascorbate deficiency increases progression of shigellosis in guinea pigs and mice infection models.

Shigella spp. are the causative agents of bacterial dysentery and shigellosis, mainly in children living in developing countries. The study of Shigella entire life cycle in vivo and the evaluation of vaccine candidates' protective efficacy have been hampered by the lack of a suitable animal model of infection. None of the studies evaluated so far (rabbit, guinea pig, mouse) allowed the recapitulation of full shigellosis symptoms upon Shigella oral challenge. Historical reports have suggested that dysentery and scurvy are both metabolic diseases associated with ascorbate deficiency. Mammals, which are susceptible to Shigella infection (humans, non-human primates and guinea pigs) are among the few species unable to synthesize ascorbate. We optimized a low-ascorbate diet to induce moderate ascorbate deficiency, but not scurvy, in guinea pigs to investigate whether poor vitamin C status increases the progression of shigellosis. Moderate ascorbate deficiency increased shigellosis symptom severity during an extended period of time (up to 48 h) in all strains tested (Shigella sonnei, Shigella flexneri 5a, and 2a). At late time points, an important influx of neutrophils was observed both within the disrupted colonic mucosa and in the luminal compartment, although Shigella was able to disseminate deep into the organ to reach the sub-mucosal layer and the bloodstream. Moreover, we found that ascorbate deficiency also increased Shigella penetration into the colon epithelium layer in a Gulo-/- mouse infection model. The use of these new rodent models of shigellosis opens new doors for the study of both Shigella infection strategies and immune responses to Shigella infection.

Gut microbiota profile and the influence of nutritional status on bacterial distribution in diabetic and healthy Tunisian subjects.

Gut microbiota plays a key role in the regulation of metabolism and immunity. We investigated the profile of gut microbiota and the impact of dietary intake on gut bacterial distribution in diabetic and healthy Tunisian subjects, aiming to identify a dysbiotic condition, hence opening the way to restore eubiosis and facilitate return to health. In the present research, we enrolled 10 type 1 diabetic (T1D), 10 type 2 diabetic (T2D) patients and 13 healthy (H) subjects. Illumina Miseq technology was used to sequence V3-V4 hypervariable regions of bacterial 16SrRNA gene. Data were analyzed referring to QIIME 2 pipeline. RStudio software was used to explore the role of nutrition in gut bacterial distribution. At the phylum level, we identified an imbalanced gut microbiota composition in diabetic patients marked by a decrease in the proportion of Firmicutes and an increase in the abundance of Bacteroidetes compared with H subjects. We observed higher amounts of Fusobacteria and a decline in the levels of TM7 phyla in T1D patients compared with H subjects. However, we revealed a decrease in the proportions of Verrucomicrobia in T2D patients compared with H subjects. At the genus level, T2D subjects were more affected by gut microbiota alteration, showing a reduction in the relative abundance of Faecalibacterium, Akkermansia, Clostridium, Blautia and Oscillibacter, whereas T1D group shows a decrease in the proportion of Blautia. The gut bacteria distribution was mainly affected by fats and carbohydrates consumption. Gut microbiota composition was altered in Tunisian diabetic patients and affected by dietary habits.

The eukaryome of African children is influenced by geographic location, gut biogeography, and nutritional status.

Eukaryotes have historically been studied as parasites, but recent evidence suggests they may be indicators of a healthy gut ecosystem. Here, we describe the eukaryome along the gastrointestinal tract of children aged 2-5 years and test for associations with clinical factors such as anaemia, intestinal inflammation, chronic undernutrition, and age. Children were enrolled from December 2016 to May 2018 in Bangui, Central African Republic and Antananarivo, Madagascar. We analyzed a total of 1104 samples representing 212 gastric, 187 duodenal, and 705 fecal samples using a metabarcoding approach targeting the full ITS2 region for fungi, and the V4 hypervariable region of the 18S rRNA gene for the overall eukaryome. Roughly, half of all fecal samples showed microeukaryotic reads. We find high intersubject variability, only a handful of taxa that are likely residents of the gastrointestinal tract, and frequent co-occurrence of eukaryotes within an individual. We also find that the eukaryome differs between the stomach, duodenum, and feces and is strongly influenced by country of origin. Our data show trends towards higher levels of Fusarium equiseti, a mycotoxin producing fungus, and lower levels of the protist Blastocystis in stunted children compared to nonstunted controls. Overall, the eukaryome is poorly correlated with clinical variables. Our study is of one of the largest cohorts analyzing the human intestinal eukaryome to date and the first to compare the eukaryome across different compartments of the gastrointestinal tract. Our results highlight the importance of studying populations across the world to uncover common features of the eukaryome in health.

Global proteomic identifies multiple cancer-related signaling pathways altered by a gut pathobiont associated with colorectal cancer.

In this work, we investigated the oncogenic role of Streptococcus gallolyticus subsp. gallolyticus (SGG), a gut bacterium associated with colorectal cancer (CRC). We showed that SGG UCN34 accelerates colon tumor development in a chemically induced CRC murine model. Full proteome and phosphoproteome analysis of murine colons chronically colonized by SGG UCN34 revealed that 164 proteins and 725 phosphorylation sites were differentially regulated. Ingenuity Pathway Analysis (IPA) indicates a pro-tumoral shift specifically induced by SGG UCN34, as ~ 90% of proteins and phosphoproteins identified were associated with digestive cancer. Comprehensive analysis of the altered phosphoproteins using ROMA software revealed up-regulation of several cancer hallmark pathways such as MAPK, mTOR and integrin/ILK/actin, affecting epithelial and stromal colonic cells. Importantly, an independent analysis of protein arrays of human colon tumors colonized with SGG showed up-regulation of PI3K/Akt/mTOR and MAPK pathways, providing clinical relevance to our findings. To test SGG's capacity to induce pre-cancerous transformation of the murine colonic epithelium, we grew ex vivo organoids which revealed unusual structures with compact morphology. Taken together, our results demonstrate the oncogenic role of SGG UCN34 in a murine model of CRC associated with activation of multiple cancer-related signaling pathways.

Factors Associated with Carriage of Enteropathogenic and Non-Enteropathogenic Viruses: A Reanalysis of Matched Case-Control Data from the AFRIBIOTA Site in Antananarivo, Madagascar.

Environmental Enteric Dysfunction (EED) is an associate driver of stunting in poor settings, and intestinal infections indirectly contribute to the pathophysiology underlying EED. Our work aimed at assessing whether enteric viral carriage is determinant to stunting. A total of 464 healthy and asymptomatic children, aged 2 to 5 years, were recruited, and classified as non-stunted, moderately stunted, or severely stunted. Among the recruited children, 329 stool samples were obtained and screened for enteric and non-enteric viruses by real-time polymerase chain reaction. We statistically tested for the associations between enteric viral and potential risk factors. Approximately 51.7% of the stool samples were positive for at least one virus and 40.7% were positive for non-enteric adenoviruses. No statistical difference was observed between virus prevalence and the growth status of the children. We did not find any statistically significant association between viral infection and most of the socio-demographic risk factors studied, except for having an inadequate food quality score or an over-nourished mother. In addition, being positive for Ascaris lumbricoides was identified as a protective factor against viral infection. In conclusion, we did not find evidence of a direct link between stunting and enteropathogenic viral carriage in our population.

Metagenomics revealed a correlation of gut phageome with autism spectrum disorder.

The human gut bacteriome is believed to have pivotal influences on human health and disease while the particular roles associated with the gut phageome have not been fully characterized yet with few exceptions. It is argued that gut microbiota can have a potential role in autism spectrum disorders (ASD). The public microbiota database of ASD and typically developing (TD) Chinese individuals were analyzed for phage protein-coding units (pPCU) to find any link between the phageome and ASD. The gut phageome of ASD individuals showed a wider diversity and higher abundance compared to TD individuals. The ASD phageome was associated with a significant expansion of Caudoviricetes bacteriophages. Phages infecting Bacteroidaceae and prophages encoded within Faecalibacterium were more frequent in ASD than in TD individuals. The expansion and diversification of ASD phageome can influence the bacterial homeostasis by imposing pressure on the bacterial communities. In conclusion, the differences of phages community in in ASD and TD can be used as potential diagnosis biomarkers of ASD. Further investigations are needed to verify the role of gut phage communities in the pathogenesis of ASD.

Salmonella effector SopB reorganizes cytoskeletal vimentin to maintain replication vacuoles for efficient infection.

A variety of intracellular bacteria modulate the host cytoskeleton to establish subcellular niches for replication. However, the role of intermediate filaments, which are crucial for mechanical strength and resilience of the cell, and in bacterial vacuole preservation remains unclear. Here, we show that Salmonella effector SopB reorganizes the vimentin network to form cage-like structures that surround Salmonella-containing vacuoles (SCVs). Genetic removal of vimentin markedly disrupts SCV organization, significantly reduces bacterial replication and cell death. Mechanistically, SopB uses its N-terminal Cdc42-binding domain to interact with and activate Cdc42 GTPase, which in turn recruits vimentin around SCVs. A high-content imaging-based screening identified that MEK1/2 inhibition led to vimentin dispersion. Our work therefore elucidates the signaling axis SopB-Cdc42-MEK1/2 as mobilizing host vimentin to maintain concrete SCVs and identifies a mechanism contributing to Salmonella replication. Importantly, Trametinib, a clinically-approved MEK1/2 inhibitor identified in the screen, displayed significant anti-infection efficacy against Salmonella both in vitro and in vivo, and may provide a therapeutic option for treating drug-tolerant salmonellosis.

Human milk nutritional composition across lactational stages in Central Africa.

The African region encompasses the highest undernutrition burden with the highest neonatal and infant mortality rates globally. Under these circumstances, breastfeeding is one of the most effective ways to ensure child health and development. However, evidence on human milk (HM) composition from African women is scarce. This is of special concern, as we have no reference data from HM composition in the context of food insecurity in Africa. Furthermore, data on the evolution of HM across lactational stages in this setting lack as well. In the MITICA study, we conducted a cohort study among 48 Central-African women and their 50 infants to analyze the emergence of gut dysbiosis in infants and describe the mother-infant transmission of microbiota between birth and 6 months of age. In this context, we assessed nutritional components in HM of 48 lactating women in Central Africa through five sampling times from week 1 after birth until week 25. Unexpectedly, HM-type III (Secretor + and Lewis genes -) was predominant in HM from Central African women, and some nutrients differed significantly among HM-types. While lactose concentration increased across lactation periods, fatty acid concentration did not vary significantly. The overall median level of 16 detected individual human milk oligosaccharides (HMOs; core structures as well as fucosylated and sialylated ones) decreased from 7.3 g/l at week 1 to 3.5 g/l at week 25. The median levels of total amino acids in HM dropped from 12.8 mg/ml at week 1 to 7.4 mg/ml at week 25. In contrast, specific free amino acids increased between months 1 and 3 of lactation, e.g., free glutamic acid, glutamine, aspartic acid, and serine. In conclusion, HM-type distribution and certain nutrients differed from Western mother HM.

Food Insecurity and Maternal Diet Influence Human Milk Composition between the Infant's Birth and 6 Months after Birth in Central-Africa.

Although the World Health Organization (WHO) and UNICEF recommend that infants should be exclusively breastfed for the first 6 months of life, evidence is scarce on how the mother's undernourishment status at delivery and maternal dietary factors influence human milk (HM) composition during the first 6 months of life in regions with high food insecurity. The maternal undernourishment status at delivery, maternal diet, and HM nutrients were assessed among 46 women and their 48 vaginally born infants in Bangui at 1, 4, 11, 18, and 25 weeks after birth through 24-h recalls and food consumption questionnaires from December 2017 to June 2019 in the context of the "Mother-to-Infant TransmIssion of microbiota in Central-Africa" (MITICA) study. High food insecurity indexes during the follow-up were significantly associated with them having lower levels of many of the human milk oligosaccharides (HMOs) that were measured and with lower levels of retinol (aß-coef = −0.2, p value = 0.04), fatty acids (aß-coef = −7.2, p value = 0.03), and amino acids (aß-coef = −2121.0, p value < 0.001). On the contrary, women from food-insecure households displayed significantly higher levels of lactose in their HM (aß-coef = 3.3, p value = 0.02). In parallel, the consumption of meat, poultry, and fish was associated with higher HM levels of many of the HMOs that were measured, total amino acids (aß-coef = 5484.4, p value < 0.001), and with lower HM levels of lactose (aß-coef = −15.6, p value = 0.01). Food insecurity and maternal diet had a meaningful effect on HM composition with a possible impact being an infant undernourishment risk. Our results plead for consistent actions on food security as an effective manner to influence the nutritional content of HM and thereby, potentially improve infant survival and healthy growth.

Stunted children display ectopic small intestinal colonization by oral bacteria, which cause lipid malabsorption in experimental models.

Environmental enteric dysfunction (EED) is an inflammatory syndrome postulated to contribute to stunted child growth and to be associated with intestinal dysbiosis and nutrient malabsorption. However, the small intestinal contributions to EED remain poorly understood. This study aimed to assess changes in the proximal and distal intestinal microbiota in the context of stunting and EED and to test for a causal role of these bacterial isolates in the underlying pathophysiology. We performed a cross-sectional study in two African countries recruiting roughly 1,000 children aged 2 to 5 years and assessed the microbiota in the stomach, duodenum, and feces. Upper gastrointestinal samples were obtained from stunted children and stratified according to stunting severity. Fecal samples were collected. We then investigated the role of clinical isolates in EED pathophysiology using tissue culture and animal models. We find that small intestinal bacterial overgrowth (SIBO) is extremely common (>80%) in stunted children. SIBO is frequently characterized by an overgrowth of oral bacteria, leading to increased permeability and inflammation and to replacement of classical small intestinal strains. These duodenal bacterial isolates decrease lipid absorption in both cultured enterocytes and mice, providing a mechanism by which they may exacerbate EED and stunting. Further, we find a specific fecal signature associated with the EED markers fecal calprotectin and alpha-antitrypsin. Our study shows a causal implication of ectopic colonization of oral bacterial isolated from the small intestine in nutrient malabsorption and gut leakiness in vitro. These findings have important therapeutic implications for modulating the microbiota through microbiota-targeted interventions.

Putative Biomarkers of Environmental Enteric Disease Fail to Correlate in a Cross-Sectional Study in Two Study Sites in Sub-Saharan Africa.

Environmental enteric dysfunction (EED) is an elusive, inflammatory syndrome of the small intestine thought to be associated with enterocyte loss and gut leakiness and lead to stunted child growth. To date, the gold standard for diagnosis is small intestine biopsy followed by histology. Several putative biomarkers for EED have been proposed and are widely used in the field. Here, we assessed in a cross-sectional study of children aged 2-5 years for a large set of biomarkers including markers of protein exudation (duodenal and fecal alpha-1-antitrypsin (AAT)), inflammation (duodenal and fecal calprotectin, duodenal, fecal and blood immunoglobulins, blood cytokines, C-reactive protein (CRP)), gut permeability (endocab, lactulose-mannitol ratio), enterocyte mass (citrulline) and general nutritional status (branched-chain amino acids (BCAA), insulin-like growth factor) in a group of 804 children in two Sub-Saharan countries. We correlated these markers with each other and with anemia in stunted and non-stunted children. AAT and calprotectin, CRP and citrulline and citrulline and BCAA correlated with each other. Furthermore, BCAA, citrulline, ferritin, fecal calprotectin and CRP levels were correlated with hemoglobin levels. Our results show that while several of the biomarkers are associated with anemia, there is little correlation between the different biomarkers. Better biomarkers and a better definition of EED are thus urgently needed.

Factors associated with anaemia among preschool- age children in underprivileged neighbourhoods in Antananarivo, Madagascar.

BACKGROUND: Anaemia occurs in children when the haemoglobin level in the blood is less than the normal (11 g/dL), the consequence is the decrease of oxygen quantity in the tissues. It is a prevalent public health problem in many low-income countries, including Madagascar, and data on risk factors are lacking. We used existing data collected within the pathophysiology of environmental enteric dysfunction (EED) in Madagascar and the Central African Republic project (AFRIBIOTA project) conducted in underprivileged neighbourhoods of Antananarivo to investigate the factors associated with anaemia in children 24 to 59 months of age. METHODS: Children included in the AFRIBIOTA project in Antananarivo for whom data on haemoglobin and ferritin concentrations were available were included in the study. Logistic regression modelling was performed to identify factors associated with anaemia. RESULTS: Of the 414 children included in this data analysis, 24.4% were found to suffer from anaemia. We found that older children (adjusted OR: 0.95; 95% CI: 0.93-0.98) were less likely to have anaemia. Those with iron deficiency (adjusted OR: 6.1; 95% CI: 3.4-11.1) and those with a high level of faecal calprotectin (adjusted OR: 2.5; 95% CI: 1.4-4.4) were more likely to have anaemia than controls. CONCLUSIONS: To reduce anaemia in the children in this underprivileged area, more emphasis should be given to national strategies that improve children's dietary quality and micronutrient intake. Furthermore, existing measures should be broadened to include measures to reduce infectious disease burden.

Changes in Systemic Regulatory T Cells, Effector T Cells, and Monocyte Populations Associated With Early-Life Stunting.

Stunting and environmental enteric dysfunction (EED) may be responsible for altered gut and systemic immune responses. However, their impact on circulating immune cell populations remains poorly characterized during early life. A detailed flow cytometry analysis of major systemic immune cell populations in 53 stunted and 52 non-stunted (2 to 5 years old) children living in Antananarivo (Madagascar) was performed. Compared to age-matched non-stunted controls, stunted children aged 2-3 years old had a significantly lower relative proportion of classical monocytes. No significant associations were found between stunting and the percentages of effector T helper cell populations (Th1, Th2, Th17, Th1Th17, and cTfh). However, we found that HLA-DR expression (MFI) on all memory CD4+ or CD8+ T cell subsets was significantly lower in stunted children compared to non-stunted controls. Interestingly, in stunted children compared to the same age-matched non-stunted controls, we observed statistically significant age-specific differences in regulatory T cells (Treg) subsets. Indeed, in 2- to 3-year-old stunted children, a significantly higher percentage of memory Treg, whilst a significantly lower percentage of naive Treg, was found. Our results revealed that both innate and adaptive systemic cell percentages, as well as activation status, were impacted in an age-related manner during stunting. Our study provides valuable insights into the understanding of systemic immune system changes in stunted children.

High prevalence of small intestine bacteria overgrowth and asymptomatic carriage of enteric pathogens in stunted children in Antananarivo, Madagascar.

Environmental Enteric Dysfunction (EED) refers to an incompletely defined syndrome of inflammation, reduced absorptive capacity, and reduced barrier function in the small intestine. It is widespread among children and adults in low- and middle-income countries and is also associated with poor sanitation and certain gut infections possibly resulting in an abnormal gut microbiota, small intestinal bacterial overgrowth (SIBO) and stunting. We investigated bacterial pathogen exposure in stunted and non-stunted children in Antananarivo, Madagascar by collecting fecal samples from 464 children (96 severely stunted, 104 moderately stunted and 264 non-stunted) and the prevalence of SIBO in 109 duodenal aspirates from stunted children (61 from severely stunted and 48 from moderately stunted children). SIBO assessed by both aerobic and anaerobic plating techniques was very high: 85.3% when selecting a threshold of ≥105 CFU/ml of bacteria in the upper intestinal aspirates. Moreover, 58.7% of the children showed more than 106 bacteria/ml in these aspirates. The most prevalent cultivated genera recovered were Streptococcus, Neisseria, Staphylococcus, Rothia, Haemophilus, Pantoea and Branhamella. Feces screening by qPCR showed a high prevalence of bacterial enteropathogens, especially those categorized as being enteroinvasive or causing mucosal disruption, such as Shigella spp., enterotoxigenic Escherichia coli, enteropathogenic E. coli and enteroaggregative E. coli. These pathogens were detected at a similar rate in stunted children and controls, all showing no sign of severe diarrhea the day of inclusion but both living in a highly contaminated environment (slum-dwelling). Interestingly Shigella spp. was the most prevalent enteropathogen found in this study (83.3%) without overrepresentation in stunted children.

The First-in-Human Synthetic Glycan-Based Conjugate Vaccine Candidate against Shigella.

Shigella, the causative agent of shigellosis, is among the main causes of diarrheal diseases with still a high morbidity in low-income countries. Relying on chemical synthesis, we implemented a multidisciplinary strategy to design SF2a-TT15, an original glycoconjugate vaccine candidate targeting Shigella flexneri 2a (SF2a). Whereas the SF2a O-antigen features nonstoichiometric O-acetylation, SF2a-TT15 is made of a synthetic 15mer oligosaccharide, corresponding to three non-O-acetylated repeats, linked at its reducing end to tetanus toxoid by means of a thiol-maleimide spacer. We report on the scale-up feasibility under GMP conditions of a high yielding bioconjugation process established to ensure a reproducible and controllable glycan/protein ratio. Preclinical and clinical batches complying with specifications from ICH guidelines, WHO recommendations for polysaccharide conjugate vaccines, and (non)compendial tests were produced. The obtained SF2a-TT15 vaccine candidate passed all toxicity-related criteria, was immunogenic in rabbits, and elicited bactericidal antibodies in mice. Remarkably, the induced IgG antibodies recognized a large panel of SF2a circulating strains. These preclinical data have paved the way forward to the first-in-human study for SF2a-TT15, demonstrating safety and immunogenicity. This contribution discloses the yet unreported feasibility of the GMP synthesis of conjugate vaccines featuring a unique homogeneous synthetic glycan hapten fine-tuned to protect against an infectious disease.