Immunoprofiling Identifies Functional B and T Cell Subsets Induced by an Attenuated Whole Parasite Malaria Vaccine as Correlates of Sterile Immunity

Immune correlates of protection remain elusive for most vaccines. An identified immune correlate would accelerate the down-selection of vaccine formulations by reducing the need for human pathogen challenge studies that are currently required to determine vaccine efficacy. Immunization via mosquito-delivered, radiation-attenuated P. falciparum sporozoites (IMRAS) is a well-established model for efficacious malaria vaccines, inducing greater than 90% sterile immunity. The current immunoprofiling study utilized samples from a clinical trial in which vaccine dosing was adjusted to achieve only 50% protection, thus enabling a comparison between protective and non-protective immune signatures. In-depth immunoprofiling was conducted by assessing a wide range of antigen-specific serological and cellular parameters and applying our newly developed computational tools, including machine learning. The computational component of the study pinpointed previously un-identified cellular T cell subsets (namely, TNFα-secreting CD8+CXCR3−CCR6− T cells, IFNγ-secreting CD8+CCR6+ T cells and TNFα/FNγ-secreting CD4+CXCR3−CCR6− T cells) and B cell subsets (i.e., CD19+CD24hiCD38hiCD69+ transitional B cells) as important factors predictive of protection (92% accuracy). Our study emphasizes the need for in-depth immunoprofiling and subsequent data integration with computational tools to identify immune correlates of protection. The described process of computational data analysis is applicable to other disease and vaccine models.

Genome-Wide Identification of the Thaumatin-like Protein Family Genes in Gossypium barbadense and Analysis of Their Responses to Verticillium dahliae Infection

(1) Background: Plants respond to pathogen challenge by activating a defense system involving pathogenesis-related (PR) proteins. The PR-5 family includes thaumatin, thaumatin-like proteins (TLPs), and other related proteins. TLPs play an important role in response to biotic and abiotic stresses. Many TLP-encoding genes have been identified and functionally characterized in the model plant species. (2) Results: We identified a total of 90 TLP genes in the G. barbadense genome. They were phylogenetically classified into 10 subfamilies and distributed across 19 chromosomes and nine scaffolds. The genes were characterized by examining their exon–intron structures, promoter cis-elements, conserved domains, synteny and collinearity, gene family evolution, and gene duplications. Several TLP genes were predicted to be targets of miRNAs. Investigation of expression changes of 21 GbTLPs in a G. barbadense cultivar (Hai7124) resistance to Verticillium dahliae revealed 13 GbTLPs being upregulated in response to V. dahliae infection, suggesting a potential role of these GbTLP genes in disease response. (3) Conclusions: The results of this study allow insight into the GbTLP gene family, identify GbTLP genes responsive to V. dahliae infection, and provide candidate genes for future studies of their roles in disease resistance.

Colonization and local host response following intramammary Staphylococcus chromogenes challenge in dry cows

Although extensive research has been performed on bovine non-aureus staphylococci (NAS), several aspects such as bacteria-host interaction remain largely unstudied. Moreover, only a few mastitis pathogen challenge studies in cows have been conducted in the dry period, an important period that allows intramammary infection (IMI) to cure and new IMI to occur. We challenged 16 quarters of 4 Holstein Friesian cows at dry off with 100; 100 000 or 10 000 000 CFU of the udder-adapted S. chromogenes IM strain. Four quarters from one cow served as negative controls. Internally sealed quarters remained untouched, whereas non-sealed quarters were sampled 3 times during the dry period. After parturition, colostrum and daily milk samples were taken during the first week of lactation of all quarters. In total, 8 quarters appeared to be colonized, since S. chromogenes IM was recovered at least once during the experiment, as substantiated using Multilocus Sequence Typing. S. chromogenes IM shedding was highest in dry quarters inoculated with 10 000 000 CFU. Colonized quarters had the highest quarter somatic cell count (qSCC) in early lactation. Inoculated quarters (both colonized and non-colonized) had lower IL-6 and IL-10 concentrations in the dry period, whilst IFN-γ levels tended to be higher in colonized quarters compared to non-inoculated quarters. Also, IgG2 levels were higher in inoculated compared to non-inoculated quarters and the IgG2/IgG1 ratio was on average above 1. To conclude, we showed that dry quarters can be colonized with S. chromogenes IM, resulting in a shift towards a Th1 response in late gestation and early lactation characterised by an increased IgG2 concentration. However, further research is needed to confirm our findings.

α-galactosylceramide-reactive NKT cells increase IgG1 class switch against a C. difficile polysaccharide antigen and enhance immunity against a live pathogen challenge

All clinical Clostridioides difficile strains identified to date express a surface capsule-like polysaccharide structure known as polysaccharide II (PSII). The PSII antigen is immunogenic and when conjugated to a protein carrier induces a protective antibody response in animal models. Given that CD1d-restricted Natural Killer T (NKT) cells promote antibody responses, including those against carbohydrates, we tested the hypothesis that immunization with PSII and a CD1d-binding glycolipid adjuvant could lead to enhanced protection against a live C. difficile challenge. We purified PSII from a clinical isolate of C. difficile and immunized B6 mice with PSII alone or PSII plus the CD1d-binding glycolipid α-galactosylceramide (α-GC). PSII-specific IgM and IgG titers were evident in sera from immunized mice. The inclusion of α-GC had a modest influence on isotype switch but increasing the ratio of IgG1/IgG2c. Enhanced protection against C. difficile disease was achieved by inclusion of the α-GC ligand and was associated with reduced bacterial numbers in fecal pellets. In contrast, NKT-deficient Traj18 -/- mice were not protected by the PSII/α-GC immunization modality. Absence of NKT cells similarly had a modest effect on isotype switch but ratios of IgG1/IgG2c decreased. These results indicate that α-GC-driven NKT cells move the humoral immune response against C. difficile PSII antigen towards Th2-driven IgG1 and may contribute to augmented protection. This study suggests that NKT activation represents a pathway for additional B cell help that could be used to supplement existing efforts to develop vaccines against polysaccharides derived from C. difficile and other pathogens.

Pathogen Challenge and Dietary Shift Alter Microbiota Composition and Activity in a Mucin-Associated in vitro Model of the Piglet Colon (MPigut-IVM) Simulating Weaning Transition

Enterotoxigenic Escherichia coli (ETEC) is the principal pathogen responsible for post-weaning diarrhea in newly weaned piglets. Expansion of ETEC at weaning is thought to be the consequence of various stress factors such as transient anorexia, dietary change or increase in intestinal inflammation and permeability, but the exact mechanisms remain to be elucidated. As the use of animal experiments raise more and more ethical concerns, we used a recently developed in vitro model of piglet colonic microbiome and mucobiome, the MPigut-IVM, to evaluate the effects of a simulated weaning transition and pathogen challenge at weaning. Our data suggested that the tested factors impacted the composition and functionality of the MPigut-IVM microbiota. The simulation of weaning transition led to an increase in relative abundance of the Prevotellaceae family which was further promoted by the presence of the ETEC strain. In contrast, several beneficial families such as Bacteroidiaceae or Ruminococcaceae and gut health related short chain fatty acids like butyrate or acetate were reduced upon simulated weaning. Moreover, the incubation of MPigut-IVM filtrated effluents with porcine intestinal cell cultures showed that ETEC challenge in the in vitro model led to an increased expression of pro-inflammatory genes by the porcine cells. This study provides insights about the etiology of a dysbiotic microbiota in post-weaning piglets.

Honeybees, Bumblebees and Leaf-cutter Bees Vary in the Effect of a Simulated Pathogen Challenge on Individual Thermoregulation

Bees regulate their individual body temperatures by non-flight thermogenesis (NFT). The effects of a pathogen challenge on thermoregulation in bees generally is unknown, although honeybees have displayed opposing responses between two studies. To establish whether bees in general experience disruption of thermoregulation under pathogen challenge, we investigated a representative species of each of three major bee social backgrounds (honeybees, Apis mellifera; bumblebees, Bombus impatiens; and solitary bees, Megachile rotundata) and measured the body surface temperatures of individual bees as they recovered from cold torpor by NFT after injection with lipopolysaccharide (LPS) solution, which simulated a pathogen challenge. We found that LPS injection affected rewarming in the annually eusocial B. impatiens, but not A. mellifera or the solitary M. rotundata. Specifically, the pathogen challenge increased post-recovery body temperatures by 2 oC in B. impatiens individuals. Our findings indicate that immune responses by individual bees can interfere with thermoregulation, but this effect is not consistent among major bee species.

Fatty Acid Desaturases: Uncovering Their Involvement in Grapevine Defence against Downy Mildew

Grapevine downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most severe and devastating diseases in viticulture. Unravelling the grapevine defence mechanisms is crucial to develop sustainable disease control measures. Here we provide new insights concerning fatty acid’s (FA) desaturation, a fundamental process in lipid remodelling and signalling. Previously, we have provided evidence that lipid signalling is essential in the establishment of the incompatible interaction between grapevine and Plasmopara viticola. In the first hours after pathogen challenge, jasmonic acid (JA) accumulation, activation of its biosynthetic pathway and an accumulation of its precursor, the polyunsaturated α-linolenic acid (C18:3), were observed in the leaves of the tolerant genotype, Regent. This work was aimed at a better comprehension of the desaturation processes occurring after inoculation. We characterised, for the first time in Vitis vinifera, the gene family of the FA desaturases and evaluated their involvement in Regent response to Plasmopara viticola. Upon pathogen challenge, an up-regulation of the expression of plastidial FA desaturases genes was observed, resulting in a higher content of polyunsaturated fatty acids (PUFAs) of chloroplast lipids. This study highlights FA desaturases as key players in membrane remodelling and signalling in grapevine defence towards biotrophic pathogens.

Probiotic Bacteria and Their Cell Walls Induce Th1-Type Immunity Against Salmonella Typhimurium Challenge

Probiotics have been associated with a variety of health benefits. They can act as adjuvant to enhance specific immune response. Bacterial cell wall (CW) molecules are key structures that interact with host receptors promoting probiotic effects. The adjuvant capacity underlying this sub-cellular fraction purified from Lactobacillus casei CRL431 and L. paracasei CNCMI-1518 remains to be characterized. We interrogated the molecular and cellular events after oral feeding with probiotic-derived CW in addition to heat-inactivated Salmonella Typhimurium and their subsequent protective capacity against S. Typhimurium challenge. Intact probiotic bacteria were orally administered for comparison. We find that previous oral feeding with probiotics or their sub-cellular fraction reduce bacterial burden in spleen and liver after Salmonella challenge. Antibody responses after pathogen challenge were negligible, characterized by not major changes in the antibody-mediated phagocytic activity, and in the levels of total and Salmonella-specific intestinal sIgA and serum IgG, respectively. Conversely, the beneficial effect of probiotic-derived CW after S. Typhimurium challenge were ascribed to a Th1-type cell-mediated immunity which was characterized by augmentation of the delayed-type hypersensitivity response. The cell-mediated immunity associated with the oral feeding with probiotic-derived CW was accompanied with a Th1-cell polarizing cytokines, distinguished by increase IFN-γ/IL-4 ratio. Similar results were observed with the intact probiotics. Our study identified molecular events associated with the oral administration of sub-cellular structures derived from probiotics and their adjuvant capacity to exert immune modulatory function.