Vaccine development: obligate intracellular bacteria new tools, old pathogens: the current state of vaccines against obligate intracellular bacteria.

Obligate intracellular bacteria have remained those for which effective vaccines are unavailable, mostly because protection does not solely rely on an antibody response. Effective antibody-based vaccines, however, have been developed against extracellular bacteria pathogens or toxins. Additionally, obligate intracellular bacteria have evolved many mechanisms to subvert the immune response, making vaccine development complex. Much of what we know about protective immunity for these pathogens has been determined using infection-resolved cases and animal models that mimic disease. These studies have laid the groundwork for antigen discovery, which, combined with recent advances in vaccinology, should allow for the development of safe and efficacious vaccines. Successful vaccines against obligate intracellular bacteria should elicit potent T cell memory responses, in addition to humoral responses. Furthermore, they ought to be designed to specifically induce strong cytotoxic CD8+ T cell responses for protective immunity. This review will describe what we know about the potentially protective immune responses to this group of bacteria. Additionally, we will argue that the novel delivery platforms used during the Sars-CoV-2 pandemic should be excellent candidates to produce protective immunity once antigens are discovered. We will then look more specifically into the vaccine development for Rickettsiaceae, Coxiella burnetti, and Anaplasmataceae from infancy until today. We have not included Chlamydia trachomatis in this review because of the many vaccine related reviews that have been written in recent years.

Dendritic cell-mediated responses to secreted Cryptosporidium effectors promote parasite-specific CD8+ T cell responses.

Cryptosporidium causes debilitating diarrheal disease in patients with primary and acquired defects in T cell function. However, it has been a challenge to understand how this infection generates T cell responses and how they mediate parasite control. Here, Cryptosporidium was engineered to express a parasite effector protein (MEDLE-2) that contains the major histocompatibility complex-I restricted SIINFEKL epitope which is recognized by T cell receptor transgenic OT-I(OVA-TCR-I) clusters of differentiation (CD)8+ T cells. These modified parasites induced expansion of endogenous SIINFEKL-specific and OT-I CD8+ T cells that were a source of interferon-gamma (IFN-γ) that could restrict growth of Cryptosporidium. This T cell response was dependent on the translocation of the effector and similar results were observed with another secreted parasite effector (rhoptry protein 1). Although infection and these translocated effector proteins are restricted to intestinal epithelial cells, type 1 conventional dendritic cells were required to generate CD8+ T cell responses to these model antigens. These data sets highlight Cryptosporidium effectors as potential targets of the immune system and suggest that crosstalk between enterocytes and type 1 conventional dendritic cells is crucial for CD8+ T cell responses to Cryptosporidium.

Dendritic cell-mediated responses to secreted Cryptosporidium effectors are required for parasite-specific CD8 + T cell responses.

Cryptosporidium causes debilitating diarrheal disease in patients with primary and acquired defects in T cell function. However, it has been a challenge to understand how this infection generates T cell responses and how they mediate parasite control. Here, Cryptosporidium was engineered to express a parasite effector protein (MEDLE-2) that contains the MHC-I restricted SIINFEKL epitope which is recognized by TCR transgenic OT-I CD8 + T cells. These modified parasites induced expansion of endogenous SIINFEKL-specific and OT-I CD8 + T cells that were a source of IFN-γ that could restrict growth of Cryptosporidium . This T cell response was dependent on the translocation of the effector and similar results were observed with another secreted parasite effector (ROP1). Although infection and these translocated effector proteins are restricted to intestinal epithelial cells (IEC), type I dendritic cells (cDC1) were required to generate CD8 + T cell responses to these model antigens. These data sets highlight Cryptosporidium effectors as targets of the immune system and suggest that crosstalk between enterocytes and cDC1s is crucial for CD8 + T cell responses to Cryptosporidium .

A genetic screen identifies a protective type III interferon response to Cryptosporidium that requires TLR3 dependent recognition.

Cryptosporidium is a leading cause of severe diarrhea and diarrheal-related death in children worldwide. As an obligate intracellular parasite, Cryptosporidium relies on intestinal epithelial cells to provide a niche for its growth and survival, but little is known about the contributions that the infected cell makes to this relationship. Here we conducted a genome wide CRISPR/Cas9 knockout screen to discover host genes that influence Cryptosporidium parvum infection and/or host cell survival. Gene enrichment analysis indicated that the host interferon response, glycosaminoglycan (GAG) and glycosylphosphatidylinositol (GPI) anchor biosynthesis are important determinants of susceptibility to C. parvum infection and impact on the viability of host cells in the context of parasite infection. Several of these pathways are linked to parasite attachment and invasion and C-type lectins on the surface of the parasite. Evaluation of transcript and protein induction of innate interferons revealed a pronounced type III interferon response to Cryptosporidium in human cells as well as in mice. Treatment of mice with IFNλ reduced infection burden and protected immunocompromised mice from severe outcomes including death, with effects that required STAT1 signaling in the enterocyte. Initiation of this type III interferon response was dependent on sustained intracellular growth and mediated by the pattern recognition receptor TLR3. We conclude that host cell intrinsic recognition of Cryptosporidium results in IFNλ production critical to early protection against this infection.

Long-read assembly and comparative evidence-based reanalysis of Cryptosporidium genome sequences reveal expanded transporter repertoire and duplication of entire chromosome ends including subtelomeric regions.

Cryptosporidiosis is a leading cause of waterborne diarrheal disease globally and an important contributor to mortality in infants and the immunosuppressed. Despite its importance, the Cryptosporidium community has only had access to a good, but incomplete, Cryptosporidium parvum IOWA reference genome sequence. Incomplete reference sequences hamper annotation, experimental design, and interpretation. We have generated a new C. parvum IOWA genome assembly supported by Pacific Biosciences (PacBio) and Oxford Nanopore long-read technologies and a new comparative and consistent genome annotation for three closely related species: C. parvum, Cryptosporidium hominis, and Cryptosporidium tyzzeri We made 1926 C. parvum annotation updates based on experimental evidence. They include new transporters, ncRNAs, introns, and altered gene structures. The new assembly and annotation revealed a complete Dnmt2 methylase ortholog. Comparative annotation between C. parvum, C. hominis, and C. tyzzeri revealed that most "missing" orthologs are found, suggesting that the biological differences between the species must result from gene copy number variation, differences in gene regulation, and single-nucleotide variants (SNVs). Using the new assembly and annotation as reference, 190 genes are identified as evolving under positive selection, including many not detected previously. The new C. parvum IOWA reference genome assembly is larger, gap free, and lacks ambiguous bases. This chromosomal assembly recovers all 16 chromosome ends, 13 of which are contiguously assembled. The three remaining chromosome ends are provisionally placed. These ends represent duplication of entire chromosome ends including subtelomeric regions revealing a new level of genome plasticity that will both inform and impact future research.

The enteric pathogen Cryptosporidium parvum exports proteins into the cytosol of the infected host cell.

The parasite Cryptosporidium is responsible for diarrheal disease in young children causing death, malnutrition, and growth delay. Cryptosporidium invades enterocytes where it develops in a unique intracellular niche. Infected cells exhibit profound changes in morphology, physiology, and transcriptional activity. How the parasite effects these changes is poorly understood. We explored the localization of highly polymorphic proteins and found members of the Cryptosporidium parvum MEDLE protein family to be translocated into the cytosol of infected cells. All intracellular life stages engage in this export, which occurs after completion of invasion. Mutational studies defined an N-terminal host-targeting motif and demonstrated proteolytic processing at a specific leucine residue. Direct expression of MEDLE2 in mammalian cells triggered an ER stress response, which was also observed during infection. Taken together, our studies reveal the presence of a Cryptosporidium secretion system capable of delivering parasite proteins into the infected enterocyte.

Acute sleep fragmentation does not alter pro-inflammatory cytokine gene expression in brain or peripheral tissues of leptin-deficient mice.

Obesity and sleep fragmentation (SF) are often co-occurring pro-inflammatory conditions in patients with obstructive sleep apnea. Leptin is a peptide hormone produced by adipocytes that has anorexigenic effects upon appetite while regulating immunity. The role of leptin in mediating inflammatory responses to SF is incompletely understood. Male C57BL/6j (lean) and ob/ob mice (leptin-deficient mice exhibiting obese phenotype) were subjected to SF or control conditions for 24 h using an automated SF chamber. Trunk blood and tissue samples from the periphery (liver, spleen, fat, and heart) and brain (hypothalamus, prefrontal cortex, and hippocampus) were collected. Quantitative PCR was used to determine relative cytokine gene expression of pro-inflammatory (IL-1ß, TNF-α) and anti-inflammatory (TGF-ß1) cytokines. Enzyme-linked immunosorbent assay (ELISA) was used to determine serum corticosterone concentration. Ob/ob mice exhibited elevated cytokine gene expression in liver (TNF-α, TGF-ß1), heart (TGF-ß1), fat (TNF-α), and brain (hippocampus, hypothalamus, prefrontal cortex: IL-1ß, TNF-α) compared with wild-type mice. Conversely, leptin deficiency decreased pro-inflammatory cytokine gene expression in heart (IL-1ß, TNF-α). SF significantly increased IL-1ß and TNF-α gene expression in fat and TGF-ß1 expression in spleen relative to controls, but only in wild-type mice. SF increased basal serum corticosterone regardless of genotype. Taken together, these findings suggest that leptin deficiency affects cytokine gene expression differently in the brain compared to peripheral tissues with minimal interaction from acute SF.

Novel environment influences the effect of paradoxical sleep deprivation upon brain and peripheral cytokine gene expression.

Sleep loss increases inflammatory mediators in brain and peripheral tissues, but the mechanisms underlying this association are not fully understood. Male C57BL/6j mice were exposed to paradoxical sleep deprivation (PSD) for 24h using the modified multiple platform (MMP) technique (platforms over water) or two different controls: home cage or a dry platform cage, which constituted a novel environment. PSD mice exhibited increased IL-1ß and TNF-α pro-inflammatory gene expression in brain (hypothalamus, hippocampus, pre-frontal cortex), as well as in peripheral tissues (liver, spleen), when compared with home-cage controls. In addition, among PSD mice, TGFß1, an anti-inflammatory cytokine, was increased in pre-frontal cortex, liver, and spleen in conjunction with elevated serum corticosterone concentration relative to home-cage controls. However, these differences were nearly abolished when PSD mice were compared with control mice subjected to a dry MMP cage, suggesting that simply exposing mice to a novel environment can induce an acute inflammatory response.

Acute sleep fragmentation induces tissue-specific changes in cytokine gene expression and increases serum corticosterone concentration.

Sleep deprivation induces acute inflammation and increased glucocorticosteroids in vertebrates, but effects from fragmented, or intermittent, sleep are poorly understood. Considering the latter is more representative of sleep apnea in humans, we investigated changes in proinflammatory (IL-1ß, TNF-α) and anti-inflammatory (TGF-ß1) cytokine gene expression in the periphery (liver, spleen, fat, and heart) and brain (hypothalamus, prefrontal cortex, and hippocampus) of a murine model exposed to varying intensities of sleep fragmentation (SF). Additionally, serum corticosterone was assessed. Sleep was disrupted in male C57BL/6J mice using an automated sleep fragmentation chamber that moves a sweeping bar at specified intervals (Lafayette Industries). Mice were exposed to bar sweeps every 20 s (high sleep fragmentation, HSF), 120 s (low sleep fragmentation, LSF), or the bar remained stationary (control). Trunk blood and tissue samples were collected after 24 h of SF. We predicted that HSF mice would exhibit increased proinflammatory expression, decreased anti-inflammatory expression, and elevated stress hormones in relation to LSF and controls. SF significantly elevated IL-1ß gene expression in adipose tissue, heart (HSF only), and hypothalamus (LSF only) relative to controls. SF did not increase TNF-α expression in any of the tissues measured. HSF increased TGF-ß1 expression in the hypothalamus and hippocampus relative to other groups. Serum corticosterone concentration was significantly different among groups, with HSF mice exhibiting the highest, LSF intermediate, and controls with the lowest concentration. This indicates that 24 h of SF is a potent inducer of inflammation and stress hormones in the periphery, but leads to upregulation of anti-inflammatory cytokines in the brain.