Human cryptosporidiosis, caused primarily by Cryptosporidium hominis and a subset of Cryptosporidium parvum, is a major cause of moderate-to-severe diarrhea in children under 5 years of age in developing countries and can lead to nutritional stunting and death. Cryptosporidiosis is particularly severe and potentially lethal in immunocompromised hosts. Biological and technical challenges have impeded traditional vaccinology approaches to identify novel targets for the development of vaccines against C. hominis, the predominant species associated with human disease. We deemed that the existence of genomic resources for multiple species in the genus, including a much-improved genome assembly and annotation for C. hominis, makes a reverse vaccinology approach feasible. To this end, we sought to generate a searchable online resource, termed C. hominis gene catalog, which registers all C. hominis genes and their properties relevant for the identification and prioritization of candidate vaccine antigens, including physical attributes, properties related to antigenic potential and expression data. Using bioinformatic approaches, we identified ∼400 C. hominis genes containing properties typical of surface-exposed antigens, such as predicted glycosylphosphatidylinositol (GPI)-anchor motifs, multiple transmembrane motifs and/or signal peptides targeting the encoded protein to the secretory pathway. This set can be narrowed further, e.g. by focusing on potential GPI-anchored proteins lacking homologs in the human genome, but with homologs in the other Cryptosporidium species for which genomic data are available, and with low amino acid polymorphism. Additional selection criteria related to recombinant expression and purification include minimizing predicted post-translation modifications and potential disulfide bonds. Forty proteins satisfying these criteria were selected from 3745 proteins in the updated C. hominis annotation. The immunogenic potential of a few of these is currently being tested.Database URL: http://cryptogc.igs.umaryland.edu.

Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria's ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa) and might contribute (along with enterotoxins) to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them from the small to the large intestine where they invade colonocytes inducing a strong inflammatory response.

We performed serial Health Care Utilization and Attitudes Surveys (HUASs) among caretakers of children ages 0-59 months randomly selected from demographically defined populations participating in the Global Enteric Multicenter Study (GEMS), a case-control study of moderate-to-severe diarrhea (MSD) in seven developing countries. The surveys aimed to estimate the proportion of children with MSD who would present to sentinel health centers (SHCs) where GEMS case recruitment would occur and provide a basis for adjusting disease incidence rates to include cases not seen at the SHCs. The proportion of children at each site reported to have had an incident episode of MSD during the 7 days preceding the survey ranged from 0.7% to 4.4% for infants (0-11 months of age), from 0.4% to 4.7% for toddlers (12-23 months of age), and from 0.3% to 2.4% for preschoolers (24-59 months of age). The proportion of MSD episodes at each site taken to an SHC within 7 days of diarrhea onset was 15-56%, 17-64%, and 7-33% in the three age strata, respectively. High cost of care and insufficient knowledge about danger signs were associated with lack of any care-seeking outside the home. Most children were not offered recommended fluids and continuing feeds at home. We have shown the utility of serial HUASs as a tool for optimizing operational and methodological issues related to the performance of a large case-control study and deriving population-based incidence rates of MSD. Moreover, the surveys suggest key targets for educational interventions that might improve the outcome of diarrheal diseases in low-resource settings.

The 2014 west African Zaire Ebola virus epidemic prompted worldwide partners to accelerate clinical development of replication-defective chimpanzee adenovirus 3 vector vaccine expressing Zaire Ebola virus glycoprotein (ChAd3-EBO-Z). We aimed to investigate the safety, tolerability, and immunogenicity of ChAd3-EBO-Z in Malian and US adults, and assess the effect of boosting of Malians with modified vaccinia Ankara expressing Zaire Ebola virus glycoprotein and other filovirus antigens (MVA-BN-Filo).

Despite the heightened risk of serious influenza during infancy, vaccination is not recommended in infants younger than 6 months. We aimed to assess the safety, immunogenicity, and efficacy of maternal immunisation with trivalent inactivated influenza vaccine for protection of infants against a first episode of laboratory-confirmed influenza.

If individuals in a case/control study are subsequently observed as a cohort of cases and a cohort of controls, weighted regression analyses can be used to estimate the association between the exposures initially recorded and events occurring during the follow-up of the 2 cohorts. Such analyses can be conceptualized as being undertaken on a reconstructed source population from which cases and controls stem. To simulate this population, the cohort of cases is added to the cohort of controls expanded with the reciprocal of the case disease incidence odds (the sampling weight) to include all individuals in the source population who did not develop the case disease. We use a simulated dataset to illustrate how weighted generalized linear model regression can be used to estimate the association between an exposure captured during the case/control study component and an outcome that occurs during follow-up.

In sub-Saharan Africa, invasive nontyphoidal Salmonella (iNTS) infections with serovars S. Enteritidis, S. Typhimurium and I 4,[5],12:i:- are widespread in children < 5 years old. Development of an efficacious vaccine would provide an important public health tool to prevent iNTS disease in this population. Glycoconjugates of S. Enteritidis core and O-polysaccharide (COPS) coupled to the homologous serovar phase 1 flagellin protein (FliC) were previously shown to be immunogenic and protected adult mice against death following challenge with a virulent Malian S. Enteritidis blood isolate. This study extends these observations to immunization of mice in early life and also assesses protection with partial and full regimens. Anti-COPS and anti-FliC serum IgG titers were assessed in infant and adult mice after immunization with 1, 2 or 3 doses of S. Enteritidis COPS:FliC alone or co-formulated with aluminum hydroxide or monophosphoryl lipid A (MPL) adjuvants. S. Enteritidis COPS:FliC was immunogenic in both age groups, although the immune responses were quantitatively lower in infants. Kinetics of antibody production were similar for the native and adjuvanted formulations after three doses; conjugates formulated with MPL elicited significantly increased anti-COPS IgG titers in adult but not infant mice. Nevertheless, robust protection against S. Enteritidis challenge was seen for all three formulations when three doses were given either during infancy or as adults. We further found that significant protection could be achieved with two COPS:FliC doses, despite elicitation of modest serum anti-COPS IgG antibody titers. These findings guide potential immunization strategies that may be translated to develop a human pediatric iNTS vaccine for sub-Saharan Africa.

Enteric fevers, caused by the Salmonella enterica serovars Typhi (ST), Paratyphi A (PA) and Paratyphi B (PB), are life-threatening illnesses exhibiting very similar clinical symptoms but with distinct epidemiologies, geographical distributions and susceptibilities to antimicrobial treatment. Nevertheless, the mechanisms by which the host recognizes pathogens with high levels of homology, such as these bacterial serovars, remain poorly understood. Using a three-dimensional organotypic model of the human intestinal mucosa and PA, PB, and ST, we observed significant differences in the secretion patterns of pro-inflammatory cytokines and chemokines elicited by these serovars. These cytokines/chemokines were likely to be co-regulated and influenced the function of epithelial cells, such as the production of IL-8. We also found differing levels of polymorphonuclear leukocyte (PMN) migration among various infection conditions that either included or excluded lymphocytes and macrophages (Mϕ), strongly suggesting feedback mechanisms among these cells. Blocking experiments showed that IL-1β, IL-6, IL-8, TNF-α and CCL3 cytokines were involved in the differential regulation of migration patterns. We conclude that the crosstalk among the lymphocytes, Mϕ, PMN and epithelial cells is cytokine/chemokine-dependent and bacterial-serotype specific, and plays a pivotal role in orchestrating the functional efficiency of the innate cells and migratory characteristics of the leukocytes.

The incidence of Salmonella enterica serovar Paratyphi A (PA) infection is on the rise and no licensed vaccines are available. We evaluated cell mediated immune (CMI) responses elicited in volunteers following immunization with a single dose (109 or 1010 cfu) of a novel attenuated live oral PA-vaccine strain (CVD 1902). Results showed increases in PA-lipopolysaccharide-specific IgG- and/or IgA B-memory cells and production of IFN-γ, TNF-α, IL-10, IL-23 and RANTES following stimulation with PA-antigens by peripheral blood mononuclear cells obtained 28 days post immunization. Flow cytometry assays revealed that vaccine elicited PA-specific CD8+ and/or CD4+ T effector/memory cells were predominantly multifunctional concomitantly expressing CD107a and/or producing IFN-γ, TNF-α and/or IL-2. Similar proportions of these MF cells expressed, or not, the gut homing marker integrin α4β7. The results suggest that immunization with CVD 1902 elicits CMI responses against PA supporting its further evaluation as a potential vaccine candidate against paratyphoid A fever.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhea worldwide. Among the 25 different ETEC adhesins, 22 are known as "colonization factors" (CFs), of which 17 are assembled by the chaperone-usher (CU) mechanism. Currently, there is no preventive therapy against ETEC, and CFs have been proposed as components for vaccine development. However, studies of diarrhea-causing ETEC strains worldwide indicate that between 15 and 50% of these are negative for known CFs, hindering the selection of the most widespread structures and suggesting that unknown adhesins remain to be identified. Here, we report the result of a comprehensive analysis of 35 draft genomes of ETEC strains which do not carry known adhesin genes; our goal was to find new CU pili loci. The phylogenetic profiles and serogroups of these strains were highly diverse, a majority of which produced only the heat-labile toxin. We identified 10 pili loci belonging to CU families β (1 locus), γ2 (7 loci), κ (1 locus), and π (1 locus), all of which contained the required number of open reading frames (ORFs) to encode functional structures. Three loci were variants of previously-known clusters, three had been only-partially described, and four are novel loci. Intra-loci genetic variability identified would allow the synthesis of up to 14 different structures. Clusters of putative γ2-CU pili were most common (23 strains), followed by putative β-CU pili (12 strains), which have not yet been fully characterized. Overall, our findings significantly increase the number of ETEC adhesion genes associated with human infections.

The World Health Organization recently prequalified a typhoid conjugate vaccine (TCV), recommending its use in persons ≥6 months to 45 years residing in typhoid fever (TF)-endemic areas. We now need to consider how TCVs can have the greatest impact in the most vulnerable populations.

Cryptosporidium is a leading cause of childhood diarrhoea and associated physical and cognitive impairment in low-resource settings. Cryptosporidium-positive faecal samples (n = 190) from children aged ≤ 5 years enrolled in the Global Enteric Multicenter Study (GEMS) in Mozambique detected by ELISA (11.5%, 430/3754) were successfully PCR-amplified and sequenced at the gp60 or ssu rRNA loci for species determination and genotyping. Three Cryptosporidium species including C. hominis (72.6%, 138/190), C. parvum (22.6%, 43/190), and C. meleagridis (4.2%, 8/190) were detected. Children ≤ 23 months were more exposed to Cryptosporidium spp. infections than older children. Both C. hominis and C. parvum were more prevalent among children with diarrhoeal disease compared to those children without it (47.6% vs. 33.3%, p = 0.007 and 23.7% vs. 11.8%, p = 0.014, respectively). A high intra-species genetic variability was observed within C. hominis (subtype families Ia, Ib, Id, Ie, and If) and C. parvum (subtype families IIb, IIc, IIe, and IIi) but not within C. meleagridis (subtype family IIIb). No association between Cryptosporidium species/genotypes and child's age was demonstrated. The predominance of C. hominis and C. parvum IIc suggests that most of the Cryptosporidium infections were anthroponotically transmitted, although zoonotic transmission events also occurred at an unknown rate. The role of livestock, poultry, and other domestic animal species as sources of environmental contamination and human cryptosporidiosis should be investigated in further molecular epidemiological studies in Mozambique.

The association between childhood diarrheal disease and linear growth faltering in developing countries is well described. However, the impact attributed to specific pathogens has not been elucidated, nor has the impact of recommended antibiotic treatment.

Vibriocidal antibodies are currently the best characterised correlate of protection against cholera and are used to gauge immunogenicity in vaccine trials. Although other circulating antibody responses have been associated with a decreased risk of infection, the correlates of protection against cholera have not been comprehensively compared. We aimed to analyse antibody-mediated correlates of protection from both V cholerae infection and cholera-related diarrhoea.

Approximately 90% of chronic typhoid carriers with persistent Salmonella enterica serovar Typhi (S. Typhi) gallbladder infection have gallstones. In Samoa, where typhoid fever has been endemic for many decades, risk factors predisposing to the development of gallstones are increasing among adults. The Samoa Typhoid Fever Control Program dispatches a "Typhoid Epidemiologic SWAT Team" to perform a household investigation of every blood culture-confirmed case of acute typhoid fever. Investigations include screening household contacts to detect chronic carriers. Following limited training, two nonexpert ultrasound operators performed point-of-care ultrasound (POCUS) on 120 Samoan adults from August to September 2019 to explore the feasibility of POCUS to detect individuals with gallstones during household investigations and community screenings. POCUS scans from 120 Samoan adults in three cohorts (28 food handlers, two typhoid cases and their 18 household contacts, and 72 attendees at an ambulatory clinic) were reviewed by a board-certified radiologist who deemed 96/120 scans (80%) to be interpretable. Compared with the radiologist (gold standard), the nonexpert operators successfully detected 6/7 Samoans with gallstones (85.7% sensitivity) and correctly identified 85/89 without gallstones (95.5% specificity). The proportion (24/120) of uninterpretable scans from this pilot that used minimally trained clinicians (who are neither radiologists nor ultrasound technicians) indicates the need for additional training of POCUS operators. Nevertheless, this pilot feasibility study engenders optimism that in the Samoan setting nonexperts can be trained to use POCUS to diagnose cholelithiasis, thereby helping (along with stool cultures and Vi serology) to identify possible chronic S. Typhi carriers.

The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000).

Entamoeba histolytica, Giardia, and Cryptosporidium are common intestinal protozoan parasites that contribute to a high burden of childhood morbidity and mortality. Our study quantified the association between intestinal protozoan parasites and child anthropometric outcomes among children under-5.

Although there are many overlapping features, pediatric diarrheal diseases can vary in severity, duration, clinical manifestations, and sequelae according to the causal pathogen, which in turn can impact the economic burden on patients and their families. We aimed to evaluate the household costs of diarrheal disease by pathogen in 7 countries.

Although immunity induced by typhoid fever is moderated and short-lived, typhoid vaccination with the attenuated Ty21a oral vaccine generates long-lasting protection rates reaching up to 92%. Thus, there are important differences on how wild-type Salmonella and typhoid vaccine strains stimulate host immunity. We hypothesize that vaccine strains with different mutations might affect gut inflammation and intestinal permeability by different mechanisms. To test this hypothesis, we used an in vitro organotypic model of the human intestinal mucosa composed of human intestinal epithelial cells, lymphocytes/monocytes, endothelial cells, and fibroblasts. We also used six Salmonella enterica serovar Typhi (S. Typhi) strains: the licensed Ty21a oral vaccine, four typhoid vaccine candidates (i.e., CVD 908, CVD 909, CVD 910, and CVD 915) and the wild-type Ty2 strain. We found that genetically engineered S. Typhi vaccine strains elicit differential host changes not only in the intestinal permeability and secretion of inflammatory cytokines, but also in the phenotype and activation pathways of innate cells. These changes were distinct from those elicited by the parent wild-type S. Typhi and depended on the genetic manipulation. In sum, these results emphasize the importance of carefully selecting specific manipulations of the Salmonella genome in the development of typhoid vaccines.

Enteric fever is caused by three Salmonella enterica serovars: Typhi, Paratyphi A, and Paratyphi B sensu stricto Although vaccines against two of these serovars are licensed (Typhi) or in clinical development (Paratyphi A), as yet there are no candidates for S. Paratyphi B. To gain genomic insight into these serovars, we sequenced 38 enteric fever-associated strains from Chile and compared these with reference genomes. Each of the serovars was separated genomically based on the core genome. Genomic comparisons identified loci that were aberrant between serovars Paratyphi B sensu stricto and Paratyphi B Java, which is typically associated with gastroenteritis; however, the majority of these were annotated as hypothetical or phage related and thus were not ideal vaccine candidates. With the genomic information in hand, we engineered a live attenuated S. Paratyphi B sensu stricto vaccine strain, CVD 2005, which was capable of protecting mice from both homologous challenge and heterologous challenge with S. Paratyphi B Java. These findings extend our understanding of S. Paratyphi B and provide a viable vaccine option for inclusion in a trivalent live attenuated enteric fever vaccine formulation.IMPORTANCE We developed a live attenuated Salmonella enterica serovar Paratyphi B vaccine that conferred protection in mice against challenge with S Paratyphi B sensu stricto and S Paratyphi B Java, which are the causes of enteric fever and gastroenteritis, respectively. Currently, the incidence of invasive S. Paratyphi B sensu stricto infections is low; however, the development of new conjugate vaccines against other enteric fever serovars could lead to the emergence of S. Paratyphi B to fill the niche left by these other pathogens. As such, an effective S. Paratyphi B vaccine would be a useful tool in the armamentarium against Salmonella infections. Comparative genomics confirmed the serovar-specific groupings of these isolates and revealed that there are a limited number of genetic differences between the sensu stricto and Java strains, which are mostly hypothetical and phage-encoded proteins. The observed level of genomic similarity likely explains why we observe some cross-protection.