Primary progressive multiple sclerosis is a poorly understood disease entity with no specific prognostic biomarkers and scarce therapeutic options. We aimed to identify disease activity biomarkers in multiple sclerosis by performing an RNA sequencing approach in peripheral blood mononuclear cells from a discovery cohort of 44 untreated patients with multiple sclerosis belonging to different clinical forms and activity phases of the disease, and 12 healthy control subjects. A validation cohort of 58 patients with multiple sclerosis and 26 healthy control subjects was included in the study to replicate the RNA sequencing findings. The RNA sequencing revealed an interleukin 1 beta (IL1B) signature in patients with primary progressive multiple sclerosis. Subsequent immunophenotyping pointed to blood monocytes as responsible for the IL1B signature observed in this group of patients. Functional experiments at baseline measuring apoptosis-associated speck-like protein containing a CARD (ASC) speck formation showed that the NOD-leucine rich repeat and pyrin containing protein 3 (NLRP3) inflammasome was overactive in monocytes from patients with primary progressive multiple sclerosis, and canonical NLRP3 inflammasome activation with a combination of ATP plus lipopolysaccharide was associated with increased IL1B production in this group of patients. Primary progressive multiple sclerosis patients with high IL1B gene expression levels in peripheral blood mononuclear cells progressed significantly faster compared to patients with low IL1B levels based on the time to reach an EDSS of 6.0 and the Multiple Sclerosis Severity Score. In agreement with peripheral blood findings, both NLRP3 and IL1B expression in brain tissue from patients with primary progressive multiple sclerosis was mainly restricted to cells of myeloid lineage. Treatment of mice with a specific NLRP3 inflammasome inhibitor attenuated established experimental autoimmune encephalomyelitis disease severity and improved CNS histopathology. NLRP3 inflammasome-specific inhibition was also effective in reducing axonal damage in a model of lipopolysaccharide-neuroinflammation using organotypic cerebellar cultures. Altogether, these results point to a role of IL1B and the NLRP3 inflammasome as prognostic biomarker and potential therapeutic target, respectively, in patients with primary progressive multiple sclerosis.

Tandem mass spectrometry was used to identify naturally processed peptides bound to major histocompatibility complex (MHC) I and MHC II molecules in central nervous system (CNS) of eight patients with multiple sclerosis (MS). MHC molecules were purified from autopsy CNS material by immunoaffinity chromatography with monoclonal antibody directed against HLA-A, -B, -C, and -DR. Subsequently peptides were separated by reversedphase HPLC and analyzed by mass spectrometry. Database searches revealed 118 amino acid sequences from self-proteins eluted from MHC I molecules and 191 from MHC II molecules, corresponding to 174 identified source proteins. These sequences define previously known and potentially novel autoantigens in MS possibly involved in disease induction and antigen spreading. Taken together, we have initiated the characterization of the CNS-expressed MHC ligandome in CNS diseases and were able to demonstrate the presentation of naturally processed myelin basic protein peptides in the brain of MS patients.

Multiple sclerosis (MS) is a detrimental disease of the central nervous system (CNS) leading to long-term disability. In the course of animal models of multiple sclerosis (experimental autoimmune encephalomyelitis), we find enhanced activity of proteasome subunits b1i, b2, b2i and b5 in the CNS. We demonstrate that pharmacological inhibition of the proteasome by bortezomib ameliorates experimental autoimmune encephalomyelitis in mice and rats in prophylactic and therapeutic treatment with reduced numbers of T-cells secreting proinflammatory cytokines. The anti-inflammatory effect of proteasome inhibition was accompanied by reduced NF-jB activity in the CNS and lymphoid organs. The combined inhibition of proteasomes and lysosomal proteases involved in major histocompatibility complex II antigen presentation further improved therapeutic efficacy. We suggest proteasome inhibition alone or in combination with inhibition of lysosomal proteases as a novel therapeutic strategy against inflammation-induced neurodegeneration in the CNS. We demonstrate the impact of the proteasome and lysosomal proteases on development of autoimmunity.

Myxovirus A (MxA), a protein encoded by the MX1 gene with antiviral activity, has proven to be a sensitive measure of IFNβ bioactivity in multiple sclerosis (MS). However, the use of MxA as a biomarker of IFNβ bioactivity has been criticized for the lack of evidence of its role on disease pathogenesis and the clinical response to IFNβ. Here, we aimed to identify specific biomarkers of IFNβ bioactivity in order to compare their gene expression induction by type I IFNs with the MxA, and to investigate their potential role in MS pathogenesis. Gene expression microarrays were performed in PBMC from MS patients who developed neutralizing antibodies (NAB) to IFNβ at 12 and/or 24 months of treatment and patients who remained NAB negative. Nine genes followed patterns in gene expression over time similar to the MX1, which was considered the gold standard gene, and were selected for further experiments: IFI6, IFI27, IFI44L, IFIT1, HERC5, LY6E, RSAD2, SIGLEC1, and USP18. In vitro experiments in PBMC from healthy controls revealed specific induction of selected biomarkers by IFNβ but not IFNγ, and several markers, in particular USP18 and HERC5, were shown to be significantly induced at lower IFNβ concentrations and more selective than the MX1 as biomarkers of IFNβ bioactivity. In addition, USP18 expression was deficient in MS patients compared with healthy controls (p = 0.0004). We propose specific biomarkers that may be considered in addition to the MxA to evaluate IFNβ bioactivity, and to further explore their implication in MS pathogenesis.

BackgroundDNA vaccines represent promising therapeutic strategies in autoimmune disorders such as multiple sclerosis (MS). However, the precise mechanisms by which DNA vaccines induce immune regulation remain largely unknown. Here, we aimed to expand previous knowledge existing on the mechanisms of action of DNA vaccines in the animal model of MS, experimental autoimmune encephalomyelitis (EAE), by treating EAE mice with a DNA vaccine encoding the myelin oligodendrocyte glycoprotein (MOG), and exploring the therapeutic effects on the disease-induced inflammatory and neurodegenerative changes.MethodsEAE was induced in C57BL6/J mice by immunization with MOG35-55 peptide. Mice were intramuscularly treated with a MOG-DNA vaccine or vehicle in prophylactic and therapeutic approaches. Histological studies were performed in central nervous system (CNS) tissue. Cytokine production and regulatory T cell (Treg) quantification were achieved by flow cytometry. Gene expression patterns were determined using microarrays, and the main findings were validated by real-time PCR.ResultsMOG-DNA treatment reduced the clinical and histopathological signs of EAE when administered in both prophylactic and therapeutic settings. Suppression of clinical EAE was associated with dampening of antigen (Ag)-specific proinflammatory Th1 and Th17 immune responses and, interestingly, expansion of Treg in the periphery and upregulation in the CNS of genes encoding neurotrophic factors and proteins involved in remyelination.ConclusionsThese results suggest for the first time that the beneficial effects of DNA vaccines in EAE are not limited to anti-inflammatory mechanisms, and DNA vaccines may also exert positive effects through hitherto unknown neuroprotective mechanisms.

The small hepatitis B surface antigen (HBsAg) of hepatitis B virus (HBV) has limited variability, but some serotypes and genotypes have been defined. Although no biological or pathogenetic differences could be traced to HBV serotypes, the clinical picture, response to treatment and long-term prognosis of HBV infection may vary with the HBV genotype, possibly due to differences in specific T cell recognition of HBV antigens from different genotypes. We analyzed murine CD8 + T cell responses to two K b -restricted HBsAg epitopes primed by four different HBsAg variants using protein-and DNA-based vaccination protocols. The K bbinding S 208-215 epitope 1 is processed from exogenous but not endogenous HBsAg. Variants of epitope 1 differing at two positions within the epitope (ILSPFLPL in ayw/adr versus IVSP-FIPL in adw2) efficiently primed cross-reactive CD8 + T cell responses. In contrast, the exchange of an N-terminal flanking residue (S to N) completely eliminated the immunogenicity of epitope 1. The K b -binding S 190-197 epitope 2 is processed from endogenous but not exogenous HBsAg. A single-residue exchange within the epitope (VWLSVIWM in ayw/adr versus VWLSAIWM in adw2) completely eliminated the immunogenicity of epitope 2. Single, conservative residue exchanges can thus give rise to diverging CD8 + T cell repertoires, suggesting an impressive complexity and flexibility of the CD8 + T cell repertoire to antigen variants from viruses with limited diversity.

The N-terminal domain of large tumor antigens (T-Ag) of polyomaviruses forms a DnaJ-like structure with a conserved J domain that associates with constitutively expressed stress protein heat shock protein (hsp)73. Mutant (but not wild-type) SV40 T-Ag show stable, ATP-dependent binding to the stress protein hsp73 when expressed in cells from different vertebrate tissues. Intracellular T/hsp73 complexes accumulate to high steady-state levels. From this observation, we designed a vector system that supports stable expression of a large variety of hsp73-capturing, chimeric antigens containing an N-terminal, T-Ag-derived domain, and different C-terminal antigenic domains from unrelated antigens. Most antigenic domains tested could be stably expressed only in eukaryotic cells as fusion protein/hsp73 complexes. The N-terminal 77 residues representing the J domain of T-Ag were required for stable hsp73 binding and efficient expression of chimeric antigens. Hsp73-bound chimeric antigens expressed by DNA vaccines showed strikingly enhanced immunogenicity evident in humoral (antibody) and cellular cytolytic T lymphocytes (CTL) responses. The described system supports efficient expression of chimeric, polyvalent antigens and their codelivery with hsp73 as a "natural adjuvant" for enhanced immunogenicity for T and B cells.

Little is known about the mechanisms leading to neurodegeneration in multiple sclerosis (MS) and the role of peripheral blood cells in this neurodegenerative component. We aimed to correlate brain radiological phenotypes defined by high and low neurodegeneration with gene expression profiling of peripheral blood mononuclear cells (PBMC) from MS patients. Magnetic resonance imaging (MRI) scans from 64 patients with relapsing-remitting MS (RRMS) were classified into radiological phenotypes characterized by low (N = 27) and high (N = 37) neurodegeneration according to the number of contrast-enhancing lesions, the relative volume of non-enhancing black holes on T1-weighted images, and the brain parenchymal fraction. Gene expression profiling was determined in PBMC using microarrays, and validation of selected genes was performed by polymerase chain reaction (PCR). B-cell immunophenotyping was conducted by flow cytometry. Microarray analysis revealed the B-cell specific genes FCRL1, FCRL2, FCRL5 (Fc receptor-like 1, 2 and 5 respectively), and CD22 as the top differentially expressed genes between patients with high and low neurodegeneration. Levels for these genes were significantly down-regulated in PBMC from patients with MRI phenotypes characterized by high neurodegeneration and microarray findings were validated by PCR. In patients with high neurodegeneration, immunophenotyping showed a significant increase in the expression of the B-cell activation markers CD80 in naïve B cells (CD45+/CD19+/CD27-/IgD+), unswitched memory B cells (CD45+/CD19+/CD27+/IgD+), and switched memory B cells (CD45+/CD19+/CD27+/IgD-), and CD86 in naïve and switched memory B cells. These results suggest that RRMS patients with radiological phenotypes showing high neurodegeneration have changes in B cells characterized by down-regulation of B-cell-specific genes and increased activation status.