Neuroprotection by Preconditioning in Mice is Dependent on MyD88-Mediated CXCL10 Expression in Endothelial Cells.

The central nervous system (CNS) can be preconditioned to resist damage by peripheral pretreatment with low-dose gram-negative bacterial endotoxin lipopolysaccharide (LPS). Underlying mechanisms associated with transient protection of the cerebral cortex against traumatic brain injury include increased neuronal production of antiapoptotic and neurotrophic molecules, microglial-mediated displacement of inhibitory presynaptic terminals innervating the soma of cortical projection neurons, and synchronized firing of cortical projection neurons. However, the cell types and signaling responsible for these neuronal and microglial changes are unknown. A fundamental question is whether LPS penetrates the CNS or acts on the luminal surface of brain endothelial cells, thereby triggering an indirect parenchymal neuroprotective response. The present study shows that a low-dose intraperitoneal LPS treatment increases brain endothelial cell activation markers CD54, but does not open the blood-brain barrier or alter brain endothelial cell tight junctions as assessed by electron microscopy. NanoString nCounter transcript analyses of CD31-positive brain endothelial cells further revealed significant upregulation of Cxcl10, C3, Ccl2, Il1ß, Cxcl2, and Cxcl1, consistent with identification of myeloid differentiation primary response 88 (MyD88) as a regulator of these transcripts by pathway analysis. Conditional genetic endothelial cell gene ablation approaches demonstrated that both MyD88-dependent Toll-like receptor 4 (TLR4) signaling and Cxcl10 expression are essential for LPS-induced neuroprotection and microglial activation. These results suggest that C-X-C motif chemokine ligand 10 (CXCL10) production by endothelial cells in response to circulating TLR ligands may directly or indirectly signal to CXCR3 on neurons and/or microglia. Targeted activation of brain endothelial receptors may thus provide an attractive approach for inducing transient neuroprotection.

Viral delivery of a peptide-based immunomodulator enhances T cell priming during vaccination.

Modern, subunit-based vaccines have so far failed to induce significant T cell responses, contributing to ineffective vaccination against many pathogens. Importantly, while today's adjuvants are designed to trigger innate and non-specific immune responses, they fail to directly stimulate the adaptive immune compartment. Programmed cell death 1 (PD-1) partly regulates naïve-to-antigen-specific effector T cell transition and differentiation by suppressing the magnitude of activation. Indeed, we previously reported on a microbial-derived, peptide-based PD-1 checkpoint inhibitor, LD01, which showed potent T cell-stimulating activity when combined with a vaccine. Here we sought to improve the potency of LD01 by designing and testing new LD01 derivatives. Accordingly, we found that a modified version of an 18-amino acid metabolite of LD01, LD10da, improved T cell activation capability in a malaria vaccine model. Specifically, LD10da demonstrates improved antigen-specific CD8+ T cell expansion when combined prophylactically with an adenovirus-based malaria vaccine. A single dose of LD10da at the time of vaccination is sufficient to increase antigen-specific CD8+ T cell expansion in wild-type mice. Further, we show that LD10 can be encoded and delivered by a Modified Vaccinia Ankara viral vector and can enhance antigen-specific CD8+ T cell expansion comparable to that of synthetic peptide administration. Therefore, LD10da represents a promising biologic-based immunomodulator that can be genetically encoded and delivered, along with the antigen, by viral or other nucleic acid vectors to improve the efficacy and delivery of vaccines for ineradicable and emerging infectious diseases.

Identification and Immune Assessment of T Cell Epitopes in Five Plasmodium falciparum Blood Stage Antigens to Facilitate Vaccine Candidate Selection and Optimization.

The hurdles to effective blood stage malaria vaccine design include immune evasion tactics used by the parasite such as redundant invasion pathways and antigen variation among circulating parasite strains. While blood stage malaria vaccine development primarily focuses on eliciting optimal humoral responses capable of blocking erythrocyte invasion, clinically-tested Plasmodium falciparum (Pf) vaccines have not elicited sterile protection, in part due to the dramatically high levels of antibody needed. Recent development efforts with non-redundant, conserved blood stage antigens suggest both high antibody titer and rapid antibody binding kinetics are important efficacy factors. Based on the central role of helper CD4 T cells in development of strong, protective immune responses, we systematically analyzed the class II epitope content in five leading Pf blood stage antigens (RH5, CyRPA, RIPR, AMA1 and EBA175) using in silico, in vitro, and ex vivo methodologies. We employed in silico T cell epitope analysis to enable identification of 67 HLA-restricted class II epitope clusters predicted to bind a panel of nine HLA-DRB1 alleles. We assessed a subset of these for HLA-DRB1 allele binding in vitro, to verify the in silico predictions. All clusters assessed (40 clusters represented by 46 peptides) bound at least two HLA-DR alleles in vitro. The overall epitope prediction to in vitro HLA-DRB1 allele binding accuracy was 71%. Utilizing the set of RH5 class II epitope clusters (10 clusters represented by 12 peptides), we assessed stimulation of T cells collected from HLA-matched RH5 vaccinees using an IFN-γ T cell recall assay. All clusters demonstrated positive recall responses, with the highest responses - by percentage of responders and response magnitude - associated with clusters located in the N-terminal region of RH5. Finally, a statistically significant correlation between in silico epitope predictions and ex vivo IFN-γ recall response was found when accounting for HLA-DR matches between the epitope predictions and donor HLA phenotypes. This is the first comprehensive analysis of class II epitope content in RH5, CyRPA, RIPR, AMA1 and EBA175 accompanied by in vitro HLA binding validation for all five proteins and ex vivo T cell response confirmation for RH5.

Bridging Computational Vaccinology and Vaccine Development Through Systematic Identification, Characterization, and Downselection of Conserved and Variable Circumsporozoite Protein CD4 T Cell Epitopes From Diverse Plasmodium falciparum Strains.

An effective malaria vaccine must prevent disease in a range of populations living in regions with vastly different transmission rates and protect against genetically-diverse Plasmodium falciparum (Pf) strains. The protective efficacy afforded by the currently licensed malaria vaccine, Mosquirix™, promotes strong humoral responses to Pf circumsporozoite protein (CSP) 3D7 but protection is limited in duration and by strain variation. Helper CD4 T cells are central to development of protective immune responses, playing roles in B cell activation and maturation processes, cytokine production, and stimulation of effector T cells. Therefore, we took advantage of recent in silico modeling advances to predict and analyze human leukocyte antigen (HLA)-restricted class II epitopes from PfCSP - across the entire PfCSP 3D7 sequence as well as in 539 PfCSP sequence variants - with the goal of improving PfCSP-based malaria vaccines. Specifically, we developed a systematic workflow to identify peptide sequences capable of binding HLA-DR in a context relevant to achieving broad human population coverage utilizing cognate T cell help and with limited T regulatory cell activation triggers. Through this workflow, we identified seven predicted class II epitope clusters in the N- and C-terminal regions of PfCSP 3D7 and an additional eight clusters through comparative analysis of 539 PfCSP sequence variants. A subset of these predicted class II epitope clusters was synthesized as peptides and assessed for HLA-DR binding in vitro. Further, we characterized the functional capacity of these peptides to prime and activate human peripheral blood mononuclear cells (PBMCs), by monitoring cytokine response profiles using MIMIC® technology (Modular IMmune In vitro Construct). Utilizing this decision framework, we found sufficient differential cellular activation and cytokine profiles among HLA-DR-matched PBMC donors to downselect class II epitope clusters for inclusion in a vaccine targeting PfCSP. Importantly, the downselected clusters are not highly conserved across PfCSP variants but rather, they overlap a hypervariable region (TH2R) in the C-terminus of the protein. We recommend assessing these class II epitope clusters within the context of a PfCSP vaccine, employing a test system capable of measuring immunogenicity across a broad set of HLA-DR alleles.

Identification, Selection and Immune Assessment of Liver Stage CD8 T Cell Epitopes From Plasmodium falciparum.

Immunization with radiation-attenuated sporozoites (RAS) has been shown to protect against malaria infection, primarily through CD8 T cell responses, but protection is limited based on parasite strain. Therefore, while CD8 T cells are an ideal effector population target for liver stage malaria vaccine development strategies, such strategies must incorporate conserved epitopes that cover a large range of class I human leukocyte antigen (HLA) supertypes to elicit cross-strain immunity across the target population. This approach requires identifying and characterizing a wide range of CD8 T cell epitopes for incorporation into a vaccine such that coverage across a large range of class I HLA alleles is attained. Accordingly, we devised an experimental framework to identify CD8 T cell epitopes from novel and minimally characterized antigens found at the pre-erythrocytic stage of parasite development. Through in silico analysis we selected conserved P. falciparum proteins, using P. vivax orthologues to establish stringent conservation parameters, predicted to have a high number of T cell epitopes across a set of six class I HLA alleles representative of major supertypes. Using the decision framework, five proteins were selected based on the density and number of predicted epitopes. Selected epitopes were synthesized as peptides and evaluated for binding to the class I HLA alleles in vitro to verify in silico binding predictions, and subsequently for stimulation of human T cells using the Modular IMmune In-vitro Construct (MIMIC®) technology to verify immunogenicity. By combining the in silico tools with the ex vivo high throughput MIMIC platform, we identified 15 novel CD8 T cell epitopes capable of stimulating an immune response in alleles across the class I HLA panel. We recommend these epitopes should be evaluated in appropriate in vivo humanized immune system models to determine their protective efficacy for potential inclusion in future vaccines.

A Peptide-Based Checkpoint Immunomodulator Alleviates Immune Dysfunction in Murine Polymicrobial Sepsis.

ABSTRACT: Sepsis-induced immunosuppression involves both innate and adaptive immunity and is associated with the increased expression of checkpoint inhibitors, such as programmed cell-death protein 1 (PD-1). The expression of PD-1 is associated with poor outcomes in septic patients, and in models of sepsis, blocking PD-1 or its ligands with antibodies increased survival and alleviated immune suppression. While inhibitory antibodies are effective, they can lead to immune-related adverse events (irAEs), in part due to continual blockade of the PD-1 pathway, resulting in hyperactivation of the immune response. Peptide-based therapeutics are an alternative drug modality that provide a rapid pharmacokinetic profile, reducing the incidence of precipitating irAEs. We recently reported that the potent, peptide-based PD-1 checkpoint antagonist, LD01, improves T-cell responses. The goal of the current study was to determine whether LD01 treatment improved survival, bacterial clearance, and host immunity in the cecal-ligation and puncture (CLP)-induced murine polymicrobial sepsis model. LD01 treatment of CLP-induced sepsis significantly enhanced survival and decreased bacterial burden. Altered survival was associated with improved macrophage phagocytic activity and T-cell production of interferon-γ. Further, myeloperoxidase levels and esterase-positive cells were significantly reduced in LD01-treated mice. Taken together, these data establish that LD01 modulates host immunity and is a viable therapeutic candidate for alleviating immunosuppression that characterizes sepsis and other infectious diseases.

The Structure of the Cysteine-Rich Domain of Plasmodium falciparum P113 Identifies the Location of the RH5 Binding Site.

Plasmodium falciparum RH5 is a secreted parasite ligand that is essential for erythrocyte invasion through direct interaction with the host erythrocyte receptor basigin. RH5 forms a tripartite complex with two other secreted parasite proteins, CyRPA and RIPR, and is tethered to the surface of the parasite through membrane-anchored P113. Antibodies against RH5, CyRPA, and RIPR can inhibit parasite invasion, suggesting that vaccines containing these three components have the potential to prevent blood-stage malaria. To further explore the role of the P113-RH5 interaction, we selected monoclonal antibodies against P113 that were either inhibitory or noninhibitory for RH5 binding. Using a Fab fragment as a crystallization chaperone, we determined the crystal structure of the RH5 binding region of P113 and showed that it is composed of two domains with structural similarities to rhamnose-binding lectins. We identified the RH5 binding site on P113 by using a combination of hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis. We found that a monoclonal antibody to P113 that bound to this interface and inhibited the RH5-P113 interaction did not inhibit parasite blood-stage growth. These findings provide further structural information on the protein interactions of RH5 and will be helpful in guiding the development of blood-stage malaria vaccines that target RH5.IMPORTANCE Malaria is a deadly infectious disease primarily caused by the parasite Plasmodium falciparum It remains a major global health problem, and there is no highly effective vaccine. A parasite protein called RH5 is centrally involved in the invasion of host red blood cells, making it-and the other parasite proteins it interacts with-promising vaccine targets. We recently identified a protein called P113 that binds RH5, suggesting that it anchors RH5 to the parasite surface. In this paper, we use structural biology to locate and characterize the RH5 binding region on P113. These findings will be important to guide the development of new antimalarial vaccines to ultimately prevent this disease, which affects some of the poorest people on the planet.

A Peptide-Based PD1 Antagonist Enhances T-Cell Priming and Efficacy of a Prophylactic Malaria Vaccine and Promotes Survival in a Lethal Malaria Model.

The blockade of programmed cell death-1 (PD1) and its ligand PDL1 has been proven to be a successful immunotherapy against several cancers. Similar to cancer, PD1 contributes to the establishment of several chronic infectious diseases, including malaria. While monoclonal antibodies (mAbs) targeting checkpoint receptors are revolutionary in cancer treatment, the immune-related adverse events (irAEs) may prevent their utilization in prophylactic and therapeutic treatments of infectious diseases. The irAEs are, in part, due to the prolonged half-life of mAbs resulting in prolonged activation of the immune system. As an alternative modality to mAbs, peptides represent a viable option because they possess a shorter pharmacokinetic half-life and offer more formulation and delivery options. Here, we report on a 22-amino acid immunomodulatory peptide, LD01, derived from a Bacillus bacteria. When combined prophylactically with an adenovirus-based or irradiated sporozoite-based malaria vaccine, LD01 significantly enhanced antigen-specific CD8+ T cell expansion. Therapeutically, LD01 treatment of mice infected with a lethal malaria strain resulted in survival that was associated with lower numbers of FOXP3+Tbet+CD4+ regulatory T cells. Taken together, our results demonstrate that LD01 is a potent immunomodulator that acts upon the adaptive immune system to stimulate T cell responses both prophylactically and therapeutically.

Novel Peptide-Based PD1 Immunomodulators Demonstrate Efficacy in Infectious Disease Vaccines and Therapeutics.

Many pathogens use the same immune evasion mechanisms as cancer cells. Patients with chronic infections have elevated levels of checkpoint receptors (e.g., programed cell death 1, PD1) on T cells. Monoclonal antibody (mAb)-based inhibitors to checkpoint receptors have also been shown to enhance T-cell responses in models of chronic infection. Therefore, inhibitors have the potential to act as a vaccine "adjuvant" by facilitating the expansion of vaccine antigen-specific T-cell repertoires. Here, we report the discovery and characterization of a peptide-based class of PD1 checkpoint inhibitors, which have a potent adaptive immunity adjuvant capability for vaccines against infectious diseases. Briefly, after identifying peptides that bind to the recombinant human PD1, we screened for in vitro efficacy in reporter assays and human peripheral blood mononuclear cells (PBMC) readouts. We first found the baseline in vivo performance of the peptides in a standard mouse oncology model that demonstrated equivalent efficacy compared to mAbs against the PD1 checkpoint. Subsequently, two strategies were used to demonstrate the utility of our peptides in infectious disease indications: (1) as a therapeutic in a bacteria-induced lethal sepsis model in which our peptides were found to increase survival with enhanced bacterial clearance and increased macrophage function; and (2) as an adjuvant in combination with a prophylactic malaria vaccine in which our peptides increased T-cell immunogenicity and the protective efficacy of the vaccine. Therefore, our peptides are promising as both a therapeutic agent and a vaccine adjuvant for infectious disease with a potentially safer and more cost-effective target product profile compared to mAbs. These findings are essential for deploying a new immunomodulatory regimen in infectious disease primary and clinical care settings.

Liposomes containing monophosphoryl lipid A and QS-21 serve as an effective adjuvant for soluble circumsporozoite protein malaria vaccine FMP013.

Malaria caused by Plasmodium falciparum continues to threaten millions of people living in the tropical parts of the world. A vaccine that confers sterile and life-long protection remains elusive despite more than 30years of effort and resources invested in solving this problem. Antibodies to a malaria vaccine candidate circumsporozoite protein (CSP) can block invasion and can protect humans against malaria. We have manufactured the Falciparum Malaria Protein-013 (FMP013) vaccine based on the nearly full-length P. falciparum CSP 3D7 strain sequence. We report here immunogenicity and challenge data on FMP013 antigen in C57BL/6 mice formulated with two novel adjuvants of the Army Liposome Formulation (ALF) series and a commercially available adjuvant Montanide ISA 720 (Montanide) as a control. ALF is a liposomal adjuvant containing a synthetic monophosphoryl lipid A (3D-PHAD®). In our study, FMP013 was adjuvanted with ALF alone, ALF containing aluminum hydroxide (ALFA) or ALF containing QS-21 (ALFQ). Adjuvants ALF and ALFA induced similar antibody titers and protection against transgenic parasite challenge that were comparable to Montanide. ALFQ was superior to the other three adjuvants as it induced higher antibody titers with improved boosting after the third immunization, higher serum IgG2c titers, and enhanced protection. FMP013+ALFQ also augmented the numbers of splenic germinal center-derived activated B-cells and antibody secreting cells compared to Montanide. Further, FMP013+ALFQ induced antigen-specific IFN-γ ELISPOT activity, CD4+ T-cells and a TH1-biased cytokine profile. These results demonstrate that soluble CSP can induce a potent and sterile protective immune response when formulated with the QS-21 containing adjuvant ALFQ. Comparative mouse immunogenicity data presented here were used as the progression criteria for an ongoing non-human primate study and a regulatory toxicology study in preparation for a controlled human malaria infection (CHMI) trial.

Rhesus macaque and mouse models for down-selecting circumsporozoite protein based malaria vaccines differ significantly in immunogenicity and functional outcomes.

BACKGROUND: Non-human primates, such as the rhesus macaques, are the preferred model for down-selecting human malaria vaccine formulations, but the rhesus model is expensive and does not allow for direct efficacy testing of human malaria vaccines. Transgenic rodent parasites expressing genes of human Plasmodium are now routinely used for efficacy studies of human malaria vaccines. Mice have however rarely predicted success in human malaria trials and there is scepticism whether mouse studies alone are sufficient to move a vaccine candidate into the clinic. METHODS: A comparison of immunogenicity, fine-specificity and functional activity of two Alum-adjuvanted Plasmodium falciparum circumsporozoite protein (CSP)-based vaccines was conducted in mouse and rhesus models. One vaccine was a soluble recombinant protein (CSP) and the other was the same CSP covalently conjugated to the Qß phage particle (Qß-CSP). RESULTS: Mice showed different kinetics of antibody responses and different sensitivity to the NANP-repeat and N-terminal epitopes as compared to rhesus. While mice failed to discern differences between the protective efficacy of CSP versus Qß-CSP vaccine following direct challenge with transgenic Plasmodium berghei parasites, rhesus serum from the Qß-CSP-vaccinated animals induced higher in vivo sporozoite neutralization activity. CONCLUSIONS: Despite some immunologic parallels between models, these data demonstrate that differences between the immune responses induced in the two models risk conflicting decisions regarding potential vaccine utility in humans. In combination with historical observations, the data presented here suggest that although murine models may be useful for some purposes, non-human primate models may be more likely to predict the human response to investigational vaccines.

Stability and function of adult vasculature is sustained by Akt/Jagged1 signalling axis in endothelium.

The signalling pathways operational in quiescent, post-development vasculature remain enigmatic. Here we show that unlike neovascularization, endothelial Akt signalling in established vasculature is crucial not for endothelial cell (EC) survival, but for sustained interactions with pericytes and vascular smooth muscle cells (VSMCs) regulating vascular stability and function. Inducible endothelial-specific Akt1 deletion in adult global Akt2KO mice triggers progressive VSMC apoptosis. In hearts, this causes a loss of arteries and arterioles and, despite a high capillary density, diminished vascular patency and severe cardiac dysfunction. Similarly, endothelial Akt deletion induces retinal VSMC loss and basement membrane deterioration resulting in vascular regression and retinal atrophy. Mechanistically, the Akt/mTOR axis controls endothelial Jagged1 expression and, thereby, Notch signalling regulating VSMC maintenance. Jagged1 peptide treatment of Akt1ΔEC;Akt2KO mice and Jagged1 re-expression in Akt-deficient endothelium restores VSMC coverage. Thus, sustained endothelial Akt1/2 signalling is critical in maintaining vascular stability and homeostasis, thereby preserving tissue and organ function.

Sustained TNF production by central nervous system infiltrating macrophages promotes progressive autoimmune encephalomyelitis.

BACKGROUND: Tumor necrosis factor (TNF) has pleiotropic functions during both the demyelinating autoimmune disease multiple sclerosis (MS) and its murine model experimental autoimmune encephalomyelitis (EAE). How TNF regulates disability during progressive disease remains unresolved. Using a progressive EAE model characterized by sustained TNF and increasing morbidity, this study evaluates the role of unregulated TNF in exacerbating central nervous system (CNS) pathology and inflammation. METHODS: Progressive MS was mimicked by myelin oligodendrocyte glycoprotein (MOG) peptide immunization of mice expressing a dominant negative IFN-γ receptor alpha chain under the human glial fibrillary acidic protein promoter (GFAPγR1∆). Diseased GFAPγR1∆ mice were treated with anti-TNF or control monoclonal antibody during acute disease to monitor therapeutic effects on sustained disability, demyelination, CNS inflammation, and blood brain barrier (BBB) permeability. RESULTS: TNF was specifically sustained in infiltrating macrophages. Anti-TNF treatment decreased established clinical disability and mortality rate within 7 days. Control of disease progression was associated with a decline in myelin loss and leukocyte infiltration, as well as macrophage activation. In addition to mitigating CNS inflammation, TNF neutralization restored BBB integrity and enhanced CNS anti-inflammatory responses. CONCLUSIONS: Sustained TNF production by infiltrating macrophages associated with progressive EAE exacerbates disease severity by promoting inflammation and disruption of BBB integrity, thereby counteracting establishment of an anti-inflammatory environment required for disease remission.

CXCL13 promotes isotype-switched B cell accumulation to the central nervous system during viral encephalomyelitis.

Elevated CXCL13 within the central nervous system (CNS) correlates with humoral responses in several neuroinflammatory diseases, yet its role is controversial. During coronavirus encephalomyelitis CXCL13 deficiency impaired CNS accumulation of memory B cells and antibody-secreting cells (ASC) but not naïve/early-activated B cells. However, despite diminished germinal center B cells and follicular helper T cells in draining lymph nodes, ASC in bone marrow and antiviral serum antibody were intact in the absence of CXCL13. The data demonstrate that CXCL13 is not essential in mounting effective peripheral humoral responses, but specifically promotes CNS accumulation of differentiated B cells.

Distinct CD4 T-cell effects on primary versus recall CD8 T-cell responses during viral encephalomyelitis.

CD4 T-cell help is not a universal requirement for effective primary CD8 T cells but is essential to generate memory CD8 T cells capable of recall responses. This study examined how CD4 T cells affect primary and secondary anti-viral CD8 T-cell responses within the central nervous system (CNS) during encephalomyelitis induced by sublethal gliatropic coronavirus. CD4 T-cell depletion before infection did not impair peripheral expansion, interferon-γ production, CNS recruitment or initial CNS effector capacity of virus-specific CD8 T cells ex vivo. Nevertheless, impaired virus control in the absence of CD4 T cells was associated with gradually diminished CNS CD8 T-cell interferon-γ production. Furthermore, within the CD8 T-cell population short-lived effector cells were increased and memory precursor effector cells were significantly decreased, consistent with higher T-cell turnover. Transfer of memory CD8 T cells to reduce viral load in CD4-depleted mice reverted the recipient CNS CD8 T-cell phenotype to that in wild-type control mice. However, memory CD8 T cells primed without CD4 T cells and transferred into infected CD4-sufficient recipients expanded less efficiently and were not sustained in the CNS, contrasting with their helped counterparts. These data suggest that CD4 T cells are dispensable for initial expansion, CNS recruitment and differentiation of primary resident memory CD8 T cells as long as the duration of antigen exposure is limited. By contrast, CD4 T cells are essential to prolong primary CD8 T-cell function in the CNS and imprint memory CD8 T cells for recall responses.

IL-27 limits central nervous system viral clearance by promoting IL-10 and enhances demyelination.

IL-27 is a pleiotropic member of the IL-6 and IL-12 cytokine family composed of the IL-27p28 and the EBV-induced gene 3. IL-27 and its receptor mRNA are both upregulated in the CNS during acute encephalomyelitis induced by the JHM strain of mouse hepatitis virus (JHMV) and sustained during viral persistence. Contributions of IL-27 to viral pathogenesis were evaluated by infection of IL-27Rα-chain-deficient (IL-27Rα(-/-)) mice. The absence of IL-27 signaling accelerated virus control within the CNS associated with increased IFN-γ secreting virus-specific CD4+ and CD8+ T cells. Abrogation of IL-27 signaling did not affect virus-specific CD8+ T cell-mediated IL-10 production or cytolytic activity or Foxp3+ regulatory T cell populations. However, IL-10 production by virus-specific CD4+ T cells was reduced significantly. Despite increased T cell-mediated antiviral function in IL-27Rα(-/-) mice, the virus persisted in the CNS at similar levels as in wild-type mice. Nevertheless, IL-27Rα(-/-) mice exhibited decreased clinical disease during persistence, coincident with less severe demyelination, the hallmark tissue damage associated with JHMV infection. Overall, these data demonstrate that in contrast to viral infections at other sites, IL-27 does not play a proinflammatory role during JHMV-induced encephalomyelitis. Rather, it limits CNS inflammation and impairs control of CNS virus replication via induction of IL-10 in virus-specific CD4+ T cells. Furthermore, in contrast to its protective role in limiting CNS autoimmunity and preventing immunopathology, these data define a detrimental role of IL-27 in promoting demyelination by delaying viral control.

Progression from IgD+ IgM+ to isotype-switched B cells is site specific during coronavirus-induced encephalomyelitis.

UNLABELLED: Various infections in the central nervous system (CNS) trigger B cell accumulation; however, the relative dynamics between viral replication and alterations in distinct B cell subsets are largely unknown. Using a glia-tropic coronavirus infection, which is initiated in the brain but rapidly spreads to and predominantly persists in the spinal cord, this study characterizes longitudinal changes in B cell subsets at both infected anatomical sites. The phase of T cell-dependent, antibody-independent control of infectious virus was associated with a similar recruitment of naive/early-activated IgD(+) IgM(+) B cells into both the brain and spinal cord. This population was progressively replaced by CD138(-) IgD(-) IgM(+) B cells, isotype-switched CD138(-) IgD(-) IgM(-) memory B cells (B(mem)), and CD138(+) antibody-secreting cells (ASC). A more rapid transition to B(mem) and ASC in spinal cord than in brain was associated with higher levels of persisting viral RNA and transcripts encoding factors promoting B cell migration, differentiation, and survival. The results demonstrate that naive/early-activated B cells are recruited early during coronavirus CNS infection but are subsequently replaced by more differentiated B cells. Furthermore, viral persistence, even at low levels, is a driving force for accumulation of isotype-switched B(mem) and ASC. IMPORTANCE: Acute and chronic human CNS infections are associated with an accumulation of heterogeneous B cell subsets; however, their influence on viral load and disease is unclear. Using a glia-tropic coronavirus model, we demonstrate that the accumulation of B cells ranging from early-activated to isotype-switched differentiation stages is both temporally and spatially orchestrated. Acutely infected brains and spinal cords indiscriminately recruit a homogeneous population of early-activated B cells, which is progressively replaced by diverse, more differentiated subsets. The latter process is accelerated by elevated proinflammatory responses associated with viral persistence. The results imply that early-recruited B cells do not have antiviral function but may contribute to the inflammatory environment or act as antigen-presenting cells. Moreover, CNS viral persistence is a driving force promoting differentiated B cells with protective potential.

Role of CD25(+) CD4(+) T cells in acute and persistent coronavirus infection of the central nervous system.

The influence of CD25(+)CD4(+) regulatory T cells (Treg) on acute and chronic viral infection of the central nervous system (CNS) was examined using a glial tropic murine coronavirus. Treg in the CNS were highest during initial T cell mediated virus control, decreased and then remained relatively stable during persistence. Anti-CD25 treatment did not affect CNS recruitment of inflammatory cells. Viral control was initially delayed; however, neither the kinetics of viral control nor viral persistence were affected. By contrast, the absence of Treg during the acute phase resulted in increased demyelination during viral persistence. These data suggest that CNS inflammation, progression of viral control and viral persistence are relatively independent of CD25(+)CD4(+) Treg. However, their absence during acute infection alters the ability of the host to limit tissue damage.

IL-21 optimizes T cell and humoral responses in the central nervous system during viral encephalitis.

Acute coronavirus encephalomyelitis is controlled by T cells while humoral responses suppress virus persistence. This study defines the contribution of interleukin (IL)-21, a regulator of T and B cell function, to central nervous system (CNS) immunity. IL-21 receptor deficiency did not affect peripheral T cell activation or trafficking, but dampened granzyme B, gamma interferon and IL-10 expression by CNS T cells and reduced serum and intrathecal humoral responses. Viral control was already lost prior to humoral CNS responses, but demyelination remained comparable. These data demonstrate a critical role of IL-21 in regulating CNS immunity, sustaining viral persistence and preventing mortality.

Intrathecal humoral immunity to encephalitic RNA viruses.

The nervous system is the target for acute encephalitic viral infections, as well as a reservoir for persisting viruses. Intrathecal antibody (Ab) synthesis is well documented in humans afflicted by infections associated with neurological complications, as well as the demyelinating disease, multiple sclerosis. This review focuses on the origin, recruitment, maintenance, and biological relevance of Ab-secreting cells (ASC) found in the central nervous system (CNS) following experimental neurotropic RNA virus infections. We will summarize evidence for a highly dynamic, evolving humoral response characterized by temporal alterations in B cell subsets, proliferation, and differentiation. Overall local Ab plays a beneficial role via complement-independent control of virus replication, although cross or self-reactive Ab to CNS antigens may contribute to immune-mediated pathogenesis during some infections. Importantly, protective Ab exert anti-viral activity not only by direct neutralization, but also by binding to cell surface-expressed viral glycoproteins. Ab engagement of viral glycoproteins blocks budding and mediates intracellular signaling leading to restored homeostatic and innate functions. The sustained Ab production by local ASC, as well as chemokines and cytokines associated with ASC recruitment and retention, are highlighted as critical components of immune control.