A Multidisciplinary Structural Approach to the Identification of the Haemophilus influenzae Type b Capsular Polysaccharide Protective Epitope.

Glycoconjugate vaccines so far licensed are generally composed of a native or size-reduced capsular polysaccharide conjugated to carrier proteins. Detailed information on the structural requirements necessary for CPS recognition is becoming the key to accelerating the development of next-generation improved glycoconjugate vaccines. Structural glycobiology studies using oligosaccharides (OS) complexed with functional monoclonal antibodies represent a powerful tool for gaining information on CPS immunological determinants at the atomic level. Herein, the minimal structural epitope of Haemophilus influenzae type b (Hib) CPS recognized by a functional human monoclonal antibody (hmAb) is reported. Short and well-defined Hib oligosaccharides originating from the depolymerization of the native CPS have been used to elucidate saccharide-mAb interactions by using a multidisciplinary approach combining surface plasmon resonance (SPR), saturation transfer difference-nanomagnetic resonance (STD-NMR), and X-ray crystallography. Our study demonstrates that the minimal structural epitope of Hib is comprised within two repeating units (RUs) where ribose and ribitol are directly engaged in the hmAb interaction, and the binding pocket fully accommodates two RUs without any additional involvement of a third one. Understanding saccharide antigen structural characteristics can provide the basis for the design of innovative glycoconjugate vaccines based on alternative technologies, such as synthetic or enzymatic approaches.

Impact of Protein Nanoparticle Shape on the Immunogenicity of Antimicrobial Glycoconjugate Vaccines.

Protein self-assembling nanoparticles (NPs) can be used as carriers for antigen delivery to increase vaccine immunogenicity. NPs mimic the majority of invading pathogens, inducing a robust adaptive immune response and long-lasting protective immunity. In this context, we investigated the potential of NPs of different sizes and shapes-ring-, rod-like, and spherical particles-as carriers for bacterial oligosaccharides by evaluating in murine models the role of these parameters on the immune response. Oligosaccharides from Neisseria meningitidis type W capsular polysaccharide were conjugated to ring-shape or nanotubes of engineered Pseudomonas aeruginosa Hemolysin-corregulated protein 1 (Hcp1cc) and to spherical Helicobacter pylori ferritin. Glycoconjugated NPs were characterized using advanced technologies such as High-Performance Liquid Chromatography (HPLC), Asymmetric Flow-Field Flow fractionation (AF4), and Transmission electron microscopy (TEM) to verify their correct assembly, dimensions, and glycosylation degrees. Our results showed that spherical ferritin was able to induce the highest immune response in mice against the saccharide antigen compared to the other glycoconjugate NPs, with increased bactericidal activity compared to benchmark MenW-CRM197. We conclude that shape is a key attribute over size to be considered for glycoconjugate vaccine development.

Proof of concept for a single-dose Group B Streptococcus vaccine based on capsular polysaccharide conjugated to Qß virus-like particles.

A maternal vaccine to protect neonates against Group B Streptococcus invasive infection is an unmet medical need. Such a vaccine should ideally be offered during the third trimester of pregnancy and induce strong immune responses after a single dose to maximize the time for placental transfer of protective antibodies. A key target antigen is the capsular polysaccharide, an anti-phagocytic virulence factor that elicits protective antibodies when conjugated to carrier proteins. The most prevalent polysaccharide serotypes conjugated to tetanus or diphtheria toxoids have been tested in humans as monovalent and multivalent formulations, showing excellent safety profiles and immunogenicity. However, responses were suboptimal in unprimed individuals after a single shot, the ideal schedule for vaccination during the third trimester of pregnancy. In the present study, we obtained and optimized self-assembling virus-like particles conjugated to Group B Streptococcus capsular polysaccharides. The resulting glyco-nanoparticles elicited strong immune responses in mice already after one immunization, providing pre-clinical proof of concept for a single-dose vaccine.

Neonatal invasive disease caused by Streptococcus agalactiae in Europe: the DEVANI multi-center study.

PURPOSE: Group B streptococcus (GBS) remains a leading cause of invasive disease, mainly sepsis and meningitis, in infants < 3 months of age and of mortality among neonates. This study, a major component of the European DEVANI project (Design of a Vaccine Against Neonatal Infections) describes clinical and important microbiological characteristics of neonatal GBS diseases. It quantifies the rate of antenatal screening and intrapartum antibiotic prophylaxis among cases and identifies risk factors associated with an adverse outcome. METHODS: Clinical and microbiological data from 153 invasive neonatal cases (82 early-onset [EOD], 71 late-onset disease [LOD] cases) were collected in eight European countries from mid-2008 to end-2010. RESULTS: Respiratory distress was the most frequent clinical sign at onset of EOD, while meningitis is found in > 30% of LOD. The study revealed that 59% of mothers of EOD cases had not received antenatal screening, whilst GBS was detected in 48.5% of screened cases. Meningitis was associated with an adverse outcome in LOD cases, while prematurity and the presence of cardiocirculatory symptoms were associated with an adverse outcome in EOD cases. Capsular-polysaccharide type III was the most frequent in both EOD and LOD cases with regional differences in the clonal complex distribution. CONCLUSIONS: Standardizing recommendations related to neonatal GBS disease and increasing compliance might improve clinical care and the prevention of GBS EOD. But even full adherence to antenatal screening would miss a relevant number of EOD cases, thus, the most promising prophylactic approach against GBS EOD and LOD would be a vaccine for maternal immunization.

FHUSPA2/10 is a bactericidal monoclonal antibody targeting multiple repeated sequences of Moraxella catarrhalis UspA2.

Moraxella catarrhalis is an important and common respiratory pathogen that can cause Otitis Media, Community Acquired Pneumonia, and has been associated with an increased risk of exacerbations in chronic obstructive pulmonary disease in adults, leading to morbidity and mortality. Its ubiquitous surface protein A2 (UspA2) has been shown to interact with host structures and extracellular matrix proteins, suggesting a role at an early stage of infection and a contribution to bacterial serum resistance. The UspA proteins are homo-trimeric autotransporters that appear as a lollipop-shaped structure in electron micrographs. They are composed of an N-terminal head with adhesive properties, followed by a stalk, which ends by an amphipathic helix and a C-terminal membrane domain. The three family members UspA1, UspA2 and UspA2H, present different amino acid signatures both at the head and membrane-spanning regions. By combining electron microscopy, hydrogen deuterium exchange mass spectrometry and protein modeling, we identified a shared and repeated epitope recognized by FHUSPA2/10, a potent cross-bactericidal monoclonal antibody raised by UspA2 and deduced key amino acids involved in the binding. The finding strengthens the potential of UspA2 to be incorporated in a vaccine formulation against M. catarrhalis.

Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine candidates.

The introduction of glycoconjugate vaccines marks an important point in the fight against various infectious diseases. The covalent conjugation of relevant polysaccharide antigens to immunogenic carrier proteins enables the induction of a long-lasting and robust IgG antibody response, which is not observed for pure polysaccharide vaccines. Although there has been remarkable progress in the development of glycoconjugate vaccines, many crucial parameters remain poorly understood. In particular, the influence of the conjugation site and strategy on the immunogenic properties of the final glycoconjugate vaccine is the focus of intense research. Here, we present a comparison of two cysteine selective conjugation strategies, elucidating the impact of both modifications on the structural integrity of the carrier protein, as well as on the immunogenic properties of the resulting glycoconjugate vaccine candidates. Our work suggests that conjugation chemistries impairing structurally relevant elements of the protein carrier, such as disulfide bonds, can have a dramatic effect on protein immunogenicity.

Dissecting the Human Response to Staphylococcus aureus Systemic Infections.

Staphylococcus aureus is a common human commensal and the leading cause of diverse infections. To identify distinctive parameters associated with infection and colonization, we compared the immune and inflammatory responses of patients with a diagnosis of invasive S. aureus disease to healthy donors. We analyzed the inflammatory responses founding a pattern of distinctive cytokines significantly higher in the patients with invasive disease. The measure of antibody levels revealed a wide antibody responsiveness from all subjects to most of the antigens, with significantly higher response for some antigens in the invasive patients compared to control. Moreover, functional antibodies against toxins distinctively associated with the invasive disease. Finally, we examined the genomic variability of isolates, showing no major differences in genetic distribution compared to a panel of representative strains. Overall, our study shows specific signatures of cytokines and functional antibodies in patients with different primary invasive diseases caused by S. aureus. These data provide insight into human responses towards invasive staphylococcal infections and are important for guiding the identification of novel preventive and therapeutic interventions against S. aureus.

Group B Streptococcus chimeric capsular polysaccharides as novel multivalent vaccine candidates.

The capsular polysaccharide of the human pathogen Group B Streptococcus is a key virulence factor and vaccine candidate that induces protective antibodies when conjugated to carrier proteins. It consists of long polymeric chains of oligosaccharide repeating units, and each of the ten capsular serotypes described so far presents a unique chemical structure with distinct antigenic properties; therefore, broad protection against this pathogen could be achieved by a combination of ten glycoconjugates. Capsular polysaccharide biosynthesis and assembly follow a polymerase-dependent pathway that is widespread in encapsulated bacteria and is encoded by a polycistronic operon. Here we exploited the sequence similarity between the capsule operons of types V and IX to generate hybrid polysaccharides incorporating epitopes of both serotypes in a single molecule, by co-expressing their specific CpsM, O, I glycosyltransferases in a single isolate. Physicochemical and immunochemical methods confirmed that an engineered strain produced a high molecular weight chimeric polysaccharide, combining antigenic specificities of both type V and IX. By optimizing the copy number of key glycosyltransferase genes, we were able to modulate the ratio between type-specific epitopes. Finally, vaccination with chimeric glycoconjugates significantly decreased the incidence of disease in pups born from immunized mice challenged with either serotype. This study provides proof of concept for a new generation of glycoconjugate vaccines that combine the antigenic specificity of different polysaccharide variants in a single molecule, eliciting a protective immune response against multiple serotype variants.

Safety and immunogenicity of an investigational maternal trivalent group B streptococcus vaccine in pregnant women and their infants: Results from a randomized placebo-controlled phase II trial.

BACKGROUND: This study evaluated the safety and immunogenicity of an investigational trivalent group B streptococcus (GBS) vaccine in US pregnant women, transplacental serotype-specific antibody transfer and persistence in infants, and serotype-specific antibodies in breast milk. METHODS: This randomized, observer-blind, placebo-controlled trial administered one dose of trivalent GBS vaccine (n = 49) or placebo (n = 26) to healthy pregnant 18-40-year-old women at 240/7-346/7 weeks' gestation. Women were enrolled from March 2014 to August 2015. Safety follow-up continued through postpartum day 180. Primary immunogenicity objectives were to evaluate serotype Ia/Ib/III-specific immunoglobulin G (IgG) levels in sera from women on day 1 (pre-vaccination), day 31, delivery and postpartum days 42 and 90, and from infants at birth (cord blood), days 42 and 90. Antibody transfer ratios (cord blood/maternal sera at delivery) and serotype-specific secretory immunoglobulin A (sIgA) and IgG in breast milk after delivery and on postpartum days 42 and 90 were evaluated. The planned sample size was not based on statistical assumptions for this descriptive study. RESULTS: Baseline characteristics were similar between groups. Serious adverse events were reported for 16% of GBS-vaccinated women and 15% of their infants, and 15% of placebo recipients and 12% of their infants; none were fatal or deemed vaccine-related. Serotype-specific IgG geometric mean concentrations (GMCs) were 13-23-fold higher in vaccine vs placebo recipients on day 31 and persisted until postpartum day 90. Median antibody concentrations were substantially higher in women with detectable pre-vaccination antibody concentrations. Antibody transfer ratios in the vaccine group were 0.62-0.82. Infant IgG GMCs and breast milk sIgA GMCs were higher in the vaccine vs the placebo group at all timepoints. CONCLUSIONS: Maternal immunization with the trivalent GBS vaccine in US women had a favorable safety profile, elicited antibodies that were transplacentally transferred and persisted in infants for a minimum of 3 months. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov, NCT02046148.

Publisher Correction: Mice repeatedly exposed to Group-A ß-Haemolytic Streptococcus show perseverative behaviors, impaired sensorimotor gating, and immune activation in rostral diencephalon.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structure-Guided Design of a Group†B Streptococcus Type†III Synthetic Glycan-Conjugate Vaccine.

Invited for the cover of this issue is the group of Roberto Adamo at GlaxoSmithKline Research Center, Siena, and colleagues at The University of the Basque Country and Basque Research Technology Alliance. The image depicts a tactical plan with the different elements of the research as part of the team. Read the full text of the article at 10.1002/chem.202000284.

Structure, dynamics and immunogenicity of a catalytically inactive CXC chemokine-degrading protease SpyCEP from Streptococcus pyogenes.

Over 18 million disease cases and half a million deaths worldwide are estimated to be caused annually by Group A Streptococcus. A vaccine to prevent GAS disease is urgently needed. SpyCEP (Streptococcus pyogenes Cell-Envelope Proteinase) is a surface-exposed serine protease that inactivates chemokines, impairing neutrophil recruitment and bacterial clearance, and has shown promising immunogenicity in preclinical models. Although SpyCEP structure has been partially characterized, a more complete and higher resolution understanding of its antigenic features would be desirable prior to large scale manufacturing. To address these gaps and facilitate development of this globally important vaccine, we performed immunogenicity studies with a safety-engineered SpyCEP mutant, and comprehensively characterized its structure by combining X-ray crystallography, NMR spectroscopy and molecular dynamics simulations. We found that the catalytically-inactive SpyCEP antigen conferred protection similar to wild-type SpyCEP in a mouse infection model. Further, a new higher-resolution crystal structure of the inactive SpyCEP mutant provided new insights into this large chemokine protease comprising nine domains derived from two non-covalently linked fragments. NMR spectroscopy and molecular simulation analyses revealed conformational flexibility that is likely important for optimal substrate recognition and overall function. These combined immunogenicity and structural data demonstrate that the full-length SpyCEP inactive mutant is a strong candidate human vaccine antigen. These findings show how a multi-disciplinary study was used to overcome obstacles in the development of a GAS vaccine, an approach applicable to other future vaccine programs. Moreover, the information provided may also facilitate the structure-based discovery of small-molecule therapeutics targeting SpyCEP protease inhibition.

Structure-Guided Design of a Group†B Streptococcus Type†III Synthetic Glycan-Conjugate Vaccine.

Identification of glycan functional epitopes is of paramount importance for rational design of glycoconjugate vaccines. We recently mapped the structural epitope of the capsular polysaccharide from type III Group B Streptococcus (GBSIII), a major cause of invasive disease in newborns, by using a dimer fragment (composed of two pentasaccharide repeating units) obtained by depolymerization complexed with a protective mAb. Although reported data had suggested a highly complex epitope contained in a helical structure composed of more than four repeating units, we showed that such dimer conjugated to a carrier protein with a proper glycosylation degree elicited functional antibodies comparably to the full-length conjugated polysaccharide. Here, starting from the X-ray crystallographic structure of the polysaccharide fragment-mAb complex, we synthesized a hexasaccharide comprising exclusively the relevant positions involved in binding. Combining competitive surface plasmon resonance and saturation transfer difference NMR spectroscopy as well as in-silico modeling, we demonstrated that this synthetic glycan was recognized by the mAb similarly to the dimer. The hexasaccharide conjugated to CRM197 , a mutant of diphtheria toxin, elicited a robust functional immune response that was not inferior to the polysaccharide conjugate, indicating that it may suffice as a vaccine antigen. This is the first evidence of an X-ray crystallography-guided design of a synthetic carbohydrate-based conjugate vaccine.

Safety and Immunogenicity of a Second Dose of an Investigational Maternal Trivalent Group B Streptococcus Vaccine in Nonpregnant Women 4-6 Years After a First Dose: Results From a Phase 2 Trial.

BACKGROUND: Maternal immunization against group B streptococcus (GBS) could protect infants from invasive GBS disease. Additional doses in subsequent pregnancies may be needed. We evaluated the safety and immunogenicity of a second dose of an investigational trivalent CRM197-glycoconjugate GBS vaccine (targeting serotypes Ia/Ib/III), administered to nonpregnant women 4-6 years postdose 1. METHODS: Healthy women either previously vaccinated with 1 dose of trivalent GBS vaccine 4-6 years before enrollment (n = 53) or never GBS vaccinated (n = 27) received a single trivalent GBS vaccine injection. Adverse events (AEs) were recorded. Serotype-specific (Ia/Ib/III) anti-GBS antibodies were measured by multiplex immunoassay prevaccination and 30/60 days postvaccination. RESULTS: AEs were reported with similar rates after a first or second dose; none were serious. Of previously GBS-vaccinated women, 92%-98% had anti-GBS concentrations that exceeded an arbitrary threshold (8 µg/mL) for each serotype 60 days postdose 2 vs 36%-56% postdose 1 in previously non-GBS-vaccinated women. Of previously GBS-vaccinated women with undetectable baseline (predose 1) anti-GBS levels, 90%-98% reached this threshold postdose 2. For each serotype, anti-GBS geometric mean concentrations (GMCs) 30/60 days postdose 2 in previously GBS-vaccinated women were ≥200-fold higher than baseline GMCs. Among women with undetectable baseline anti-GBS levels, postdose 2 GMCs in previously GBS-vaccinated women exceeded postdose 1 GMCs in previously non-GBS-vaccinated women (≥7-fold). CONCLUSIONS: A second trivalent GBS vaccine dose administered 4-6 years postdose 1 was immunogenic with a favorable safety profile. Women with undetectable preexisting anti-GBS concentrations may benefit from a sufficiently spaced second vaccine dose. CLINICAL TRIALS REGISTRATION: NCT02690181.

Evaluation of Immune Responses to Group B Streptococcus Type III Oligosaccharides Containing a Minimal Protective Epitope.

Recent structural studies demonstrated that the epitope recognized by a monoclonal antibody representative of the protective response against the type III group B Streptococcus polysaccharide was comprised within 2 of the repeating units that constitute the full-length native structure. In the current study, we took advantage of this discovery to design a novel vaccine based on multivalent presentation of the identified minimal epitope on a carrier protein. We show that highly glycosylated short oligosaccharide conjugates elicit functional immune responses comparable to those of the full-length native polysaccharide. The obtained results pave the way to the design of well-defined glycoconjugate vaccines based on short synthetic oligosaccharides.

Novel Multiplex Immunoassays for Quantification of IgG against Group B Streptococcus Capsular Polysaccharides in Human Sera.

Group B Streptococcus (GBS) infections constitute a major cause of invasive disease during the first three months of life and an unmet medical need that could be addressed by maternal vaccination. The GBS capsular polysaccharides (CPSs) have shown promise as vaccine targets in clinical studies. A highly specific serological assay to quantify maternal and neonatal anti-CPS antibody levels will be instrumental for GBS vaccine licensure. Here, we describe the development and comparison of two novel multiplex immunoassays (MIAs) based on the Luminex technology for the quantification of IgG antibodies recognizing the five most frequent GBS capsular variants (Ia, Ib, II, III, and V) out of the ten types identified. The first assay is based on the use of biotinylated CPSs coupled to streptavidin-derivatized magnetic microspheres (Biotin-CPS MIA), while the second is a sandwich assay with plain CPSs coupled to magnetic microspheres coated with polysaccharide-specific mouse monoclonal antibodies (Sandwich MIA). Both assays showed good specificity, linearity, and precision, although the Biotin-CPS MIA presented higher sensitivity and lower complexity than the Sandwich MIA. A panel of human sera representing a wide range of anti-CPS IgG concentrations was tested in parallel by the two assays, which resulted in comparable titers. Our data support the preservation of antigenic epitopes in the biotinylated polysaccharides and the suitability of the Biotin-CPS MIA for the precise determination of GBS anti-CPS IgG concentrations in human sera.IMPORTANCE Group B streptococcal infections can cause death in neonates up to 3 months of age. Intrapartum antibiotic prophylaxis in GBS-colonized mothers has limited early infections but has no impact after the first week of life. The development of a maternal vaccine to address this unmet medical need has been identified as a priority by the World Health Organization, and the GBS CPSs are considered the best antigen targets. However, to date there are no accepted standardized assays to measure immune responses to the investigational vaccines and for establishment of serocorrelates of protection. Here, we describe the performance of two microsphere-based pentaplex immunoassays for the determination of antibodies recognizing the five most frequent GBS serotypes. Our data confirm that an assay based on biotinylated polysaccharides coupled to streptavidin microspheres would be suitable for the intended purpose.

European Multicentre Tics in Children Studies (EMTICS): protocol for two cohort studies to assess risk factors for tic onset and exacerbation in children and adolescents.

Genetic predisposition, autoimmunity and environmental factors [e.g. pre- and perinatal difficulties, Group A Streptococcal (GAS) and other infections, stress-inducing events] might interact to create a neurobiological vulnerability to the development of tics and associated behaviours. However, the existing evidence for this relies primarily on small prospective or larger retrospective population-based studies, and is therefore still inconclusive. This article describes the design and methodology of the EMTICS study, a longitudinal observational European multicentre study involving 16 clinical centres, with the following objectives: (1) to investigate the association of environmental factors (GAS exposure and psychosocial stress, primarily) with the onset and course of tics and/or obsessive-compulsive symptoms through the prospective observation of at-risk individuals (ONSET cohort: 260 children aged 3-10 years who are tic-free at study entry and have a first-degree relative with a chronic tic disorder) and affected individuals (COURSE cohort: 715 youth aged 3-16 years with a tic disorder); (2) to characterise the immune response to microbial antigens and the host's immune response regulation in association with onset and exacerbations of tics; (3) to increase knowledge of the human gene pathways influencing the pathogenesis of tic disorders; and (4) to develop prediction models for the risk of onset and exacerbations of tic disorders. The EMTICS study is, to our knowledge, the largest prospective cohort assessment of the contribution of different genetic and environmental factors to the risk of developing tics in putatively predisposed individuals and to the risk of exacerbating tics in young individuals with chronic tic disorders.

The Streptococcus agalactiae complement interfering protein combines multiple complement-inhibitory mechanisms by interacting with both C4 and C3 ligands.

Group B Streptococcus (GBS) colonizes the human lower intestinal and genital tracts and constitutes a major threat to neonates from pregnant carrier mothers and to adults with underlying morbidity. The pathogen expresses cell-surface virulence factors that enable cell adhesion and penetration and that counteract innate and adaptive immune responses. Among these, the complement interfering protein (CIP) was recently described for its capacity to interact with the human C4b ligand and to interfere with the classical- and lectin-complement pathways. In the present study, we provide evidence that CIP can also interact with C3, C3b, and C3d. Immunoassay-based competition experiments showed that binding of CIP to C3d interferes with the interaction between C3d and the complement receptor 2/cluster of differentiation 21 (CR2/CD21) receptor on B cells. By B-cell intracellular signaling assays, CIP was confirmed to down-regulate CR2/CD21-dependent B-cell activation. The CIP domain involved in C3d binding was mapped via hydrogen deuterium exchange-mass spectrometry. The data obtained reveal a new role for this GBS polypeptide at the interface between the innate and adaptive immune responses, adding a new member to the growing list of virulence factors secreted by gram-positive pathogens that incorporate multiple immunomodulatory functions.-Giussani, S., Pietrocola, G., Donnarumma, D., Norais, N., Speziale, P., Fabbrini, M., Margarit, I. The Streptococcus agalactiae complement interfering protein combines multiple complement-inhibitory mechanisms by interacting with both C4 and C3 ligands.

Neonatal corticosterone mitigates autoimmune neuropsychiatric disorders associated with streptococcus in mice.

Increased glucocorticoid concentrations have been shown to favor resilience towards autoimmune phenomena. Here, we addressed whether experimentally induced elevations in circulating glucocorticoids mitigate the abnormalities exhibited by an experimental model of Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS). This is a pathogenic hypothesis linking repeated exposures to Group-A-beta-hemolytic streptococcus (GAS), autoantibodies targeting selected brain nuclei and neurobehavioral abnormalities. To persistently elevate glucocorticoid concentrations, we supplemented lactating SJL/J mice with corticosterone (CORT; 80 mg/L) in the drinking water. Starting in adolescence (postnatal day 28), developing offspring were exposed to four injections - at bi-weekly intervals - of a GAS homogenate and tested for behavioral, immunological, neurochemical and molecular alterations. GAS mice showed increased perseverative behavior, impaired sensorimotor gating, reduced reactivity to a serotonergic agonist and inflammatory infiltrates in the anterior diencephalon. Neonatal CORT persistently increased circulating glucocorticoids concentrations and counteracted these alterations. Additionally, neonatal CORT increased peripheral and CNS concentrations of the anti-inflammatory cytokine IL-9. Further, upstream regulator analysis of differentially expressed genes in the striatum showed that the regulatory effect of estradiol is inhibited in GAS-treated mice and activated in GAS-treated mice exposed to CORT. These data support the hypothesis that elevations in glucocorticoids may promote central immunomodulatory processes.