Immunogenicity and protection against Mycobacterium avium with a heterologous RNA prime and protein boost vaccine regimen.

Prophylactic efficacy of two different delivery platforms for vaccination against Mycobacterium avium (M. avium) were tested in this study; a subunit and an RNA-based vaccine. The vaccine antigen, ID91, includes four mycobacterial antigens: Rv3619, Rv2389, Rv3478, and Rv1886. We have shown that ID91+GLA-SE is effective against a clinical NTM isolate, M. avium 2-151 smt. Here, we extend these results and show that a heterologous prime/boost strategy with a repRNA-ID91 (replicon RNA) followed by protein ID91+GLA-SE boost is superior to the subunit protein vaccine given as a homologous prime/boost regimen. The repRNA-ID91/ID91+GLA-SE heterologous regimen elicited a higher polyfunctional CD4+ TH1 immune response when compared to the homologous protein prime/boost regimen. More significantly, among all the vaccine regimens tested only repRNA-ID91/ID91+GLA-SE induced IFN-γ and TNF-secreting CD8+ T cells. Furthermore, the repRNA-ID91/ID91+GLA-SE vaccine strategy elicited high systemic proinflammatory cytokine responses and induced strong ID91 and an Ag85B-specific humoral antibody response a pre- and post-challenge with M. avium 2-151 smt. Finally, while all prophylactic prime/boost vaccine regimens elicited a degree of protection in beige mice, the heterologous repRNA-ID91/ID91+GLA-SE vaccine regimen provided greater pulmonary protection than the homologous protein prime/boost regimen. These data indicate that a prophylactic heterologous repRNA-ID91/ID91+GLA-SE vaccine regimen augments immunogenicity and confers protection against M. avium.

It Takes a Village: The Multifaceted Immune Response to Mycobacterium tuberculosis Infection and Vaccine-Induced Immunity.

Despite co-evolving with humans for centuries and being intensely studied for decades, the immune correlates of protection against Mycobacterium tuberculosis (Mtb) have yet to be fully defined. This lapse in understanding is a major lag in the pipeline for evaluating and advancing efficacious vaccine candidates. While CD4+ T helper 1 (TH1) pro-inflammatory responses have a significant role in controlling Mtb infection, the historically narrow focus on this cell population may have eclipsed the characterization of other requisite arms of the immune system. Over the last decade, the tuberculosis (TB) research community has intentionally and intensely increased the breadth of investigation of other immune players. Here, we review mechanistic preclinical studies as well as clinical anecdotes that suggest the degree to which different cell types, such as NK cells, CD8+ T cells, γ δ T cells, and B cells, influence infection or disease prevention. Additionally, we categorically outline the observed role each major cell type plays in vaccine-induced immunity, including Mycobacterium bovis bacillus Calmette-Guérin (BCG). Novel vaccine candidates advancing through either the preclinical or clinical pipeline leverage different platforms (e.g., protein + adjuvant, vector-based, nucleic acid-based) to purposefully elicit complex immune responses, and we review those design rationales and results to date. The better we as a community understand the essential composition, magnitude, timing, and trafficking of immune responses against Mtb, the closer we are to reducing the severe disease burden and toll on human health inflicted by TB globally.

Functional consequences of postnatal interventions in a mouse model of Fragile X syndrome.

BACKGROUND: Fragile X syndrome (FXS) is a leading genetic cause of autism and intellectual disability with cortical hyperexcitability and sensory hypersensitivity attributed to loss and hypofunction of inhibitory parvalbumin-expressing (PV) cells. Our studies provide novel insights into the role of excitatory neurons in abnormal development of PV cells during a postnatal period of inhibitory circuit refinement. METHODS: To achieve Fragile X mental retardation gene (Fmr1) deletion and re-expression in excitatory neurons during the postnatal day (P)14-P21 period, we generated CreCaMKIIa/Fmr1Flox/y (cOFF) and CreCaMKIIa/Fmr1FloxNeo/y (cON) mice, respectively. Cortical phenotypes were evaluated in adult mice using biochemical, cellular, clinically relevant electroencephalogram (EEG) and behavioral tests. RESULTS: We found that similar to global Fmr1 KO mice, the density of PV-expressing cells, their activation, and sound-evoked gamma synchronization were impaired in cOFF mice, but the phenotypes were improved in cON mice. cOFF mice also showed enhanced cortical gelatinase activity and baseline EEG gamma power, which were reduced in cON mice. In addition, TrkB phosphorylation and PV levels were lower in cOFF mice, which also showed increased locomotor activity and anxiety-like behaviors. Remarkably, when FMRP levels were restored in only excitatory neurons during the P14-P21 period, TrkB phosphorylation and mouse behaviors were also improved. CONCLUSIONS: These results indicate that postnatal deletion or re-expression of FMRP in excitatory neurons is sufficient to elicit or ameliorate structural and functional cortical deficits, and abnormal behaviors in mice, informing future studies about appropriate treatment windows and providing fundamental insights into the cellular mechanisms of cortical circuit dysfunction in FXS.

Acute pharmacological inhibition of matrix metalloproteinase-9 activity during development restores perineuronal net formation and normalizes auditory processing in Fmr1 KO mice.

Individuals with Fragile X Syndrome (FXS) and autism spectrum disorder (ASD) exhibit cognitive impairments, social deficits, increased anxiety, and sensory hyperexcitability. Previously, we showed that elevated levels of matrix metalloproteinase-9 (MMP-9) may contribute to abnormal development of parvalbumin (PV) interneurons and perineuronal nets (PNNs) in the developing auditory cortex (AC) of Fmr1 knock-out (KO) mice, which likely underlie auditory hypersensitivity. Thus, MMP-9 may serve as a potential target for treatment of auditory hypersensitivity in FXS. Here, we used the MMP-2/9 inhibitor, SB-3CT, to pharmacologically inhibit MMP-9 activity during a specific developmental period and to test whether inhibition of MMP-9 activity reverses neural oscillation deficits and behavioral impairments by enhancing PNN formation around PV cells in Fmr1 KO mice. Electroencephalography (EEG) was used to measure resting state and sound-evoked electrocortical activity in auditory and frontal cortices of postnatal day (P)22-23 male mice before and one-day after treatment with SB-3CT (25 mg/kg) or vehicle. At P27-28, animal behaviors were tested to measure the effects of the treatment on anxiety and hyperactivity. Results show that acute inhibition of MMP-9 activity improved evoked synchronization to auditory stimuli and ameliorated mouse behavioral deficits. MMP-9 inhibition enhanced PNN formation, increased PV levels and TrkB phosphorylation yet reduced Akt phosphorylation in the AC of Fmr1 KO mice. Our results show that MMP-9 inhibition during early postnatal development is beneficial in reducing some auditory processing deficits in the FXS mouse model and may serve as a candidate therapeutic for reversing sensory hypersensitivity in FXS and possibly other ASDs.

Deletion of Fmr1 from Forebrain Excitatory Neurons Triggers Abnormal Cellular, EEG, and Behavioral Phenotypes in the Auditory Cortex of a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is a leading genetic cause of autism with symptoms that include sensory processing deficits. In both humans with FXS and a mouse model [Fmr1 knockout (KO) mouse], electroencephalographic (EEG) recordings show enhanced resting state gamma power and reduced sound-evoked gamma synchrony. We previously showed that elevated levels of matrix metalloproteinase-9 (MMP-9) may contribute to these phenotypes by affecting perineuronal nets (PNNs) around parvalbumin (PV) interneurons in the auditory cortex of Fmr1 KO mice. However, how different cell types within local cortical circuits contribute to these deficits is not known. Here, we examined whether Fmr1 deletion in forebrain excitatory neurons affects neural oscillations, MMP-9 activity, and PV/PNN expression in the auditory cortex. We found that cortical MMP-9 gelatinase activity, mTOR/Akt phosphorylation, and resting EEG gamma power were enhanced in CreNex1/Fmr1Flox/y conditional KO (cKO) mice, whereas the density of PV/PNN cells was reduced. The CreNex1/Fmr1Flox/y cKO mice also show increased locomotor activity, but not the anxiety-like behaviors. These results indicate that fragile X mental retardation protein changes in excitatory neurons in the cortex are sufficient to elicit cellular, electrophysiological, and behavioral phenotypes in Fmr1 KO mice. More broadly, these results indicate that local cortical circuit abnormalities contribute to sensory processing deficits in autism spectrum disorders.

Beneficial effects of sound exposure on auditory cortex development in a mouse model of Fragile X Syndrome.

BACKGROUND: Fragile X syndrome (FXS) is the most common genetic cause of autism and intellectual disability. Fragile X mental retardation gene (Fmr1) knock-out (KO) mice display core deficits of FXS, including abnormally increased sound-evoked responses, and show a delayed development of parvalbumin (PV) cells. Here, we present the surprising result that sound exposure during early development reduces correlates of auditory hypersensitivity in Fmr1 KO mice. METHODS: Fmr1 KO and wild-type (WT) mice were raised in a sound-attenuated environment (AE) or sound-exposed (SE) to 14 kHz tones (5 Hz repetition rate) from P9 until P21. At P21-P23, event-related potentials (ERPs), dendritic spine density, PV expression and phosphorylation of tropomyosin receptor kinase B (TrkB) were analyzed in the auditory cortex of AE and SE mice. RESULTS: Enhanced N1 amplitude of ERPs, impaired PV cell development, and increased spine density in layers (L) 2/3 and L5/6 excitatory neurons were observed in AE Fmr1 KO compared to WT mice. In contrast, developmental sound exposure normalized ERP N1 amplitude, density of PV cells and dendritic spines in SE Fmr1 KO mice. Finally, TrkB phosphorylation was reduced in AE Fmr1 KO, but was enhanced in SE Fmr1 KO mice, suggesting that BDNF-TrkB signaling may be regulated by sound exposure to influence PV cell development. CONCLUSIONS: Our results demonstrate that sound exposure, but not attenuation, during early developmental window restores molecular, cellular and functional properties in the auditory cortex of Fmr1 KO mice, and suggest this approach as a potential treatment for sensory phenotypes in FXS.

Reduced perineuronal net expression in Fmr1 KO mice auditory cortex and amygdala is linked to impaired fear-associated memory.

Fragile X Syndrome (FXS) is a leading cause of heritable intellectual disability and autism. Humans with FXS show anxiety, sensory hypersensitivity and impaired learning. The mechanisms of learning impairments can be studied in the mouse model of FXS, the Fmr1 KO mouse, using tone-associated fear memory paradigms. Our previous study reported impaired development of parvalbumin (PV) positive interneurons and perineuronal nets (PNN) in the auditory cortex of Fmr1 KO mice. A recent study suggested PNN dynamics in the auditory cortex following tone-shock association is necessary for fear expression. Together these data suggest that abnormal PNN regulation may underlie tone-fear association learning deficits in Fmr1 KO mice. We tested this hypothesis by quantifying PV and PNN expression in the amygdala, hippocampus and auditory cortex of Fmr1 KO mice following fear conditioning. We found impaired tone-associated memory formation in Fmr1 KO mice. This was paralleled by impaired learning-associated regulation of PNNs in the superficial layers of auditory cortex in Fmr1 KO mice. PV cell density decreased in the auditory cortex in response to fear conditioning in both WT and Fmr1 KO mice. Learning-induced increase of PV expression in the CA3 hippocampus was only observed in WT mice. We also found reduced PNN density in the amygdala and auditory cortex of Fmr1 KO mice in all conditions, as well as reduced PNN intensity in CA2 hippocampus. There was a positive correlation between tone-associated memory and PNN density in the amygdala and auditory cortex, consistent with a tone-association deficit. Altogether our studies suggest a link between impaired PV and PNN regulation within specific regions of the fear conditioning circuit and impaired tone memory formation in Fmr1 KO mice.

Sensory Processing Phenotypes in Fragile X Syndrome.

Fragile X syndrome (FXS) is a neurodevelopmental disorder that causes intellectual disability. It is a leading known genetic cause of autism. In addition to cognitive, social, and communication deficits, humans with FXS demonstrate abnormal sensory processing including sensory hypersensitivity. Sensory hypersensitivity commonly manifests as auditory, tactile, or visual defensiveness or avoidance. Clinical, behavioral, and electrophysiological studies consistently show auditory hypersensitivity, impaired habituation to repeated sounds, and reduced auditory attention in humans with FXS. Children with FXS also exhibit significant visuospatial impairments. Studies in infants and toddlers with FXS have documented impairments in processing texture-defined motion stimuli, temporal flicker, perceiving ordinal numerical sequence, and the ability to maintain the identity of dynamic object information during occlusion. Consistent with the observations in humans with FXS, fragile X mental retardation 1 ( Fmr1) gene knockout (KO) rodent models of FXS also show seizures, abnormal visual-evoked responses, auditory hypersensitivity, and abnormal processing at multiple levels of the auditory system, including altered acoustic startle responses. Among other sensory symptoms, individuals with FXS exhibit tactile defensiveness. Fmr1 KO mice also show impaired encoding of tactile stimulation frequency and larger size of receptive fields in the somatosensory cortex. Since sensory deficits are relatively more tractable from circuit mechanisms and developmental perspectives than more complex social behaviors, the focus of this review is on clinical, functional, and structural studies that outline the auditory, visual, and somatosensory processing deficits in FXS. The similarities in sensory phenotypes between humans with FXS and animal models suggest a likely conservation of basic sensory processing circuits across species and may provide a translational platform to not just develop biomarkers but also to understand underlying mechanisms. We argue that preclinical studies in animal models of FXS can facilitate the ongoing search for new therapeutic approaches in FXS by understanding mechanisms of basic sensory processing circuits and behaviors that are conserved across species.

Inflammatory Determinants of Pregravid Obesity in Placenta and Peripheral Blood.

Pre-pregnancy (pregravid) obesity has been linked to several adverse health outcomes for both mother and offspring. Complications during pregnancy include increased risk for gestational diabetes, hypertension, preeclampsia, placental abruption, and difficulties during delivery. Several studies suggest that these negative outcomes are mediated by heightened systemic inflammation as well as changes in placental development and function. However, the molecular mechanisms by which pregravid obesity affects these processes are poorly understood. In this study, we aimed to address this question by carrying out a comprehensive analysis of the systemic maternal immune system coupled with placental gene expression and microbial profiling at term delivery (11 lean and 14 obese). Specifically, we examined the impact of pregravid obesity on circulating cytokines, chemokine, adipokines, and growth factors using multiplex Luminex assay. Innate and adaptive immune cell frequencies and their cytokine production in response to stimuli were measured using flow cytometry. Finally, changes in placental transcriptome and microbiome were profiled using RNA- and 16S-sequencing, respectively. Pregravid obesity is characterized by insulin and leptin resistance, high levels of circulating inflammatory markers IL-6 and CRP, in addition to chemokine IL-8 (p < 0.01). Moreover, pregravid obesity was associated with lower frequency of naïve CD4+ T-cells (p < 0.05), increased frequency of memory CD4+ T-cells (p < 0.01), and a shift towards Th2 cytokine production (p = 0.05). Myeloid cells from the obese cohort produced higher levels of pro-inflammatory cytokines but lower levels of chemokines following TLR stimulation (p < 0.05). Lastly, pregravid obesity is associated with increased abundance of Bacteroides and changes in the expression of genes important for nutrient transport and immunity (FDR < 0.05). Collectively, these data indicate that pregravid obesity is associated with heightened systemic inflammation and of dysregulated nutrient transport in the placenta and provide insight into the basis of fetal reprogramming.

Maternal Pregravid Obesity Remodels the DNA Methylation Landscape of Cord Blood Monocytes Disrupting Their Inflammatory Program.

Prepregnancy maternal obesity is associated with adverse outcomes for the offspring, including increased incidence of neonatal bacterial sepsis and necrotizing enterocolitis. We recently reported that umbilical cord blood (UCB) monocytes from babies born to obese mothers generate a reduced IL-6/TNF-α response to TLR 1/2 and 4 ligands compared to those collected from lean mothers. These observations suggest altered development of the offspring's immune system, which in turn results in dysregulated function. We therefore investigated transcriptional and epigenetic differences within UCB monocytes stratified by prepregnancy maternal body mass index. We show that UCB monocytes from babies born to obese mothers generate a dampened response to LPS stimulation compared with those born to lean mothers, at the level of secreted immune mediators and transcription. Because gene expression profiles of resting UCB monocytes from both groups were comparable, we next investigated the role of epigenetic differences. Indeed, we detected stark differences in methylation levels within promoters and regulatory regions of genes involved in TLR signaling in resting UCB monocytes. Interestingly, the DNA methylation status of resting cells was highly predictive of transcriptional changes post-LPS stimulation, suggesting that cytosine methylation is one of the dominant mechanisms driving functional inadequacy in UCB monocytes obtained from babies born to obese mothers. These data highlight a potentially critical role of maternal pregravid obesity-associated epigenetic changes in influencing the function of an offspring's monocytes at birth. These findings further our understanding of mechanisms that explain the increased risk of infection in neonates born to mothers with high prepregnancy body mass index.

Androgen supplementation improves some but not all aspects of immune senescence in aged male macaques.

Aging leads to a progressive decline in immune function commonly referred to as immune senescence, which results in increased incidence and severity of infection. In addition, older males experience a significant disruption in their levels of circulating androgens, notably testosterone and dehydroepiandrosterone (DHEA), which has been linked to sarcopenia, osteoporosis, cardiovascular disease, and diabetes. Since sex steroid levels modulate immune function, it is possible that the age-related decline in androgen levels can also affect immune senescence. Therefore, in this study, we evaluated the pleiotropic effects of physiological androgen supplementation in aged male rhesus macaques (n = 7/group) on immune cell subset frequency and response to vaccination. As expected, frequency of naïve CD4 and CD8 T cells declined in aged non-treated macaques, while that of memory T cells increased. In contrast, frequency of naïve and memory T cells remained stable in androgen-supplemented males. In addition, levels of inflammatory cytokines increased less steeply in supplemented aged males compared to the aged controls. Despite these changes, androgen-supplemented animals only showed modest improvement in antibody responses following vaccination compared to age non-treated controls. These data indicate that short-term physiological androgen supplementation can improve some but not all aspects of immune senescence.

Long-lasting effect of obesity on skeletal muscle transcriptome.

BACKGROUND: Reduced physical activity and increased intake of calorically-dense diets are the main risk factors for obesity, glucose intolerance, and type 2 diabetes. Chronic overnutrition and hyperglycemia can alter gene expression, contributing to long-term obesity complications. While caloric restriction can reduce obesity and glucose intolerance, it is currently unknown whether it can effectively reprogram transcriptome to a pre-obesity level. The present study addressed this question by the preliminary examination of the transcriptional dynamics in skeletal muscle after exposure to overnutrition and following caloric restriction. RESULTS: Six male rhesus macaques of 12-13 years of age consumed a high-fat western-style diet for 6 months and then were calorically restricted for 4 months without exercise. Skeletal muscle biopsies were subjected to longitudinal gene expression analysis using next-generation whole-genome RNA sequencing. In spite of significant weight loss and normalized insulin sensitivity, the majority of WSD-induced (n = 457) and WSD-suppressed (n = 47) genes remained significantly dysregulated after caloric restriction (FDR ≤0.05). The MetacoreTM pathway analysis reveals that western-style diet induced the sustained activation of the transforming growth factor-ß gene network, associated with extracellular matrix remodeling, and the downregulation of genes involved in muscle structure development and nutritional processes. CONCLUSIONS: Western-style diet, in the absence of exercise, induced skeletal muscle transcriptional programing, which persisted even after insulin resistance and glucose intolerance were completely reversed with caloric restriction.

CD103+ CD8 T Cells in the Toxoplasma-Infected Brain Exhibit a Tissue-Resident Memory Transcriptional Profile.

During chronic infection, memory T cells acquire a unique phenotype and become dependent on different survival signals than those needed for memory T cells generated during an acute infection. The distinction between the role of effector and memory T cells in an environment of persistent antigen remains unclear. Here, in the context of chronic Toxoplasma gondii infection, we demonstrate that a population of CD8 T cells exhibiting a tissue-resident memory (TRM) phenotype accumulates within the brain. We show that this population is distributed throughout the brain in both parenchymal and extraparenchymal spaces. Furthermore, this population is transcriptionally distinct and exhibits a transcriptional signature consistent with the TRM observed in acute viral infections. Finally, we establish that the CD103+ TRM population has an intrinsic capacity to produce both IFN-γ and TNF-α, cytokines critical for parasite control within the central nervous system (CNS). The contribution of this population to pro-inflammatory cytokine production suggests an important role for TRM in protective and ongoing immune responses in the infected CNS. Accession number: GSE95105.

Genomic and functional analysis of the host response to acute simian varicella infection in the lung.

Varicella Zoster Virus (VZV) is the causative agent of varicella and herpes zoster. Although it is well established that VZV is transmitted via the respiratory route, the host-pathogen interactions during acute VZV infection in the lungs remain poorly understood due to limited access to clinical samples. To address these gaps in our knowledge, we leveraged a nonhuman primate model of VZV infection where rhesus macaques are intrabronchially challenged with the closely related Simian Varicella Virus (SVV). Acute infection is characterized by immune infiltration of the lung airways, a significant up-regulation of genes involved in antiviral-immunity, and a down-regulation of genes involved in lung development. This is followed by a decrease in viral loads and increased expression of genes associated with cell cycle and tissue repair. These data provide the first characterization of the host response required to control varicella virus replication in the lung and provide insight into mechanisms by which VZV infection can cause lung injury in an immune competent host.

Transcriptome Profiling Reveals Disruption of Innate Immunity in Chronic Heavy Ethanol Consuming Female Rhesus Macaques.

It is well established that heavy ethanol consumption interferes with the immune system and inflammatory processes, resulting in increased risk for infectious and chronic diseases. However, these processes have yet to be systematically studied in a dose and sex-dependent manner. In this study, we investigated the impact of chronic heavy ethanol consumption on gene expression using RNA-seq in peripheral blood mononuclear cells isolated from female rhesus macaques with daily consumption of 4% ethanol available 22hr/day for 12 months resulting in average ethanol consumption of 4.3 g/kg/day (considered heavy drinking). Differential gene expression analysis was performed using edgeR and gene enrichment analysis using MetaCore™. We identified 1106 differentially expressed genes, meeting the criterion of ≥ two-fold change and p-value ≤ 0.05 in expression (445 up- and 661 down-regulated). Pathway analysis of the 879 genes with characterized identifiers showed that the most enriched gene ontology processes were "response to wounding", "blood coagulation", "immune system process", and "regulation of signaling". Changes in gene expression were seen despite the lack of differences in the frequency of any major immune cell subtype between ethanol and controls, suggesting that heavy ethanol consumption modulates gene expression at the cellular level rather than altering the distribution of peripheral blood mononuclear cells. Collectively, these observations provide mechanisms to explain the higher incidence of infection, delay in wound healing, and increase in cardiovascular disease seen in subjects with Alcohol use disorder.

microRNAs Regulate Host Immune Response and Pathogenesis During Influenza Infection in Rhesus Macaques.

microRNAs (miRNAs) are small noncoding RNAs that are key regulators of biological processes, including the immune response to viral infections. Differential expression levels of cellular miRNAs and their predicted targets have been described in the lungs of H1N1-infected BALB/c mice, the lungs of H5N1 influenza-infected cynomolgus macaques, and in peripheral blood mononuclear cells (PBMCs) of critically ill patients infected with 2009 pandemic H1N1. However, a longitudinal analysis of changes in the expression of miRNAs and their targets during influenza infection and how they relate to viral replication and host response has yet to be carried out. In the present study, we conducted a comprehensive analysis of innate and adaptive immune responses as well as the expression of several miRNAs and their validated targets in both peripheral blood and bronchoalveolar lavage (BAL) collected from rhesus macaques over the course of infection with the 2009 H1N1 virus A/Mexico/4108/2009 (MEX4108). We describe a distinct set of differentially expressed miRNAs in BAL and PBMCs, which regulate the expression of genes involved in inflammation, immune response, and regulation of cell cycle and apoptosis.