Early Induction of Cross-Reactive CD8+ T-Cell Responses in Tonsils After Live-Attenuated Influenza Vaccination in Children.

BACKGROUND: Live-attenuated influenza vaccine (LAIV) was licensed for prophylaxis of children 2-17 years old in Europe in 2012 and is administered as a nasal spray. Live-attenuated influenza vaccine induces both mucosal and systemic antibodies and systemic T-cell responses. Tonsils are the lymph nodes serving the upper respiratory tract, acting as both induction and effector site for mucosal immunity. METHODS: Here, we have studied the early tonsillar T-cell responses induced in children after LAIV. Thirty-nine children were immunized with trivalent LAIV (containing A/H1N1, A/H3N2, and B viruses) at days 3, 7, and 14 before tonsillectomy. Nonvaccinated controls were included for comparison. Tonsils and peripheral blood (pre- and postvaccination) were collected to study T-cell responses. RESULTS: Tonsillar and systemic T-cell responses differed between influenza strains, and both were found against H3N2 and B viruses, whereas only systemic responses were observed against A/H1N1. A significant increase in cross-reactive tonsillar CD8+ T cells recognizing conserved epitopes from a broad range of seasonal and pandemic viruses occurred at day 14. Tonsillar T cells showed significant cytokine responses (Th1, Th2, and granulocyte-macrophage colony-stimulating factor). CONCLUSIONS: Our findings support the use of LAIV in children to elicit broadly cross-reactive T cells, which are not induced by traditional inactivated influenza vaccines and may provide protection to novel virus strains.

Ductal epithelial expression of Ro52 correlates with inflammation in salivary glands of patients with primary Sjögren's syndrome.

Ro52 is an E3 ubiquitin ligase with a prominent regulatory role in inflammation. The protein is a common target of circulating autoantibodies in rheumatic autoimmune diseases, particularly Sjögren's syndrome (SS). In this study we aimed to investigate the expression of the SS target autoantigen Ro52 in salivary glands of patients with primary Sjögren's syndrome (pSS). Ro52 expression was assessed by immunohistochemical staining of paraffin-embedded and frozen salivary gland biopsies from 28 pSS patients and 19 non-pSS controls from Swedish and Norwegian registries, using anti-human Ro52 monoclonal antibodies. The degree and pattern of staining and inflammation was then evaluated. Furthermore, secreted Ro52 protein was measured in saliva and serum samples from the same individuals through a catch-enzyme-linked immunosorbent assay (ELISA). Ro52 was highly expressed in all the focal infiltrates in pSS patients. Interestingly, a significantly higher degree of Ro52 expression in ductal epithelium was observed in the patients compared to the non-pSS controls (P < 0·03). Moreover, the degree of ductal epithelial expression of Ro52 correlated with the level of inflammation (Spearman's r = 0·48, P < 0·0120). However, no secreted Ro52 protein could be detected in serum and saliva samples of these subjects. Ro52 expression in ductal epithelium coincides with degree of inflammation and is up-regulated in pSS patients. High expression of Ro52 might result in the breakage of tolerance and generation of Ro52 autoantibodies in genetically susceptible individuals. We conclude that the up-regulation of Ro52 in ductal epithelium might be a triggering factor for disease progression in SS.

Ro52- and Ro60-specific B cell pattern in the salivary glands of patients with primary Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is characterized by the presence of autoantibodies against the ribonucleoprotein (RNP) particles Ro/SSA and La/SSB, and mononuclear cell infiltration of exocrine tissues, especially salivary and lachrymal glands. Low numbers of autoantigen-specific memory B cells and elevated levels of plasma cells have been detected previously in the peripheral blood (PB) of pSS patients compared to controls. As both Ro52 and Ro60-specific cells have been detected in the salivary glands (SG) of pSS patients, we aimed to characterize the SSA-specific B cell pattern in SG biopsies. A series of double immunohistochemical stainings were performed on paraffin-embedded tissue from 10 well-characterized pSS patients for each Ro52 and Ro60 along with CD19, CD5, CD20 or CD27, respectively. Ro52 and Ro60-specific cells detected in SG tissue were found to be CD19(+) B cells located outside the CD19(+)/CD20(+) B cell zones (BCZ) and also interstitially. These SSA-specific cells were also quantified. No SSA-specific cells were CD5(+), indicating that they do not belong to the B-1 B cell subset. Furthermore, no SSA-specific cells were observed within the CD20(+) BCZ. Hence, no SSA-specific memory B cells were detected in these individuals. Contrary to this, SSA-specific cells were found to be CD19(+)/CD27(++), demonstrating that they are differentiating short or long-lived plasma cells. Taken together, our findings suggest that these lower levels of SSA-specific memory B cells in PB and absence of SSA-specific memory B cells in SG of pSS patients could result from activation of these cells into plasma cells at the site of inflammation.

Autoantigen-specific memory B cells in primary Sjögren's syndrome.

Sjögren's syndrome (SS) is a systemic rheumatic autoimmune disease affecting the exocrine glandular function and is characterized by the presence of autoantibodies against the ribonucleoprotein particles, SS-A/Ro and SS-B/La, and mononuclear cell infiltration of exocrine tissues. Our aim is to characterize memory B cell pattern and function in relation to the progression of the disease, by analysing samples from a well-defined cohort of patients with primary SS. We have measured the number of Ro/La-specific plasma cells in peripheral blood mononuclear cells (PBMC) from 23 patients and 20 healthy controls by direct enzyme-linked immunospot (ELISPOT) assay. Furthermore, we quantified the Ro- and La-specific memory B cells in these individuals by a 6-day in vitro polyclonal stimulation of PBMC followed by an antigen-specific ELISPOT assay for the detection of memory B cells. In addition to this, ELISA profiling of autoantibodies was carried out using patients' plasma and supernatant, collected post-mitogen stimulation of PBMC. The average Ro60-, Ro52- and La48-specific plasma cells in PB was 9, 17 and 13 cells in 10(5) PBMC, respectively. After in vitro stimulation, these numbers increased to 43, 50 and 26 for Ro60, Ro52 and La48, correspondingly. However, the fraction of memory B cells activated into antibody-secreting cells was lower than the overall IgG B cell population. We conclude that these lower Ro/La-specific memory B cell levels may indicate that a greater portion of the Ro- and La-specific B cells are in an activated stage. This is in tune with previous reports.

Phenotypic diversity of peripheral blood plasma cells in primary Sjögren's syndrome.

Production of autoantibodies is one of the main features of primary Sjögren's syndrome (pSS). Long-lived plasma cells (PC) can produce autoantibodies for prolonged period of times without being affected by immunosuppressive therapies. As of today, little is known about the long-lived PC subset and their contribution to autoimmunity. We have characterized the phenotypic and migratory properties of peripheral blood PC isolated from pSS patients (grouped by focus score, FS) and compared them to PC from rheumatoid arthritis (RA) patients and normal non-autoimmune subjects. We observed two populations of PC in all study groups, CD19+ PC and CD19- PC. Interestingly, the CD19- PC subset was most prominent in autoimmune patients (pSS and RA) compared to normal controls. Further investigation of the PC phenotype revealed that a high percentage of both CD19+ and CD19- PC isolated from pSS and RA patients did not express the CD27 marker, which is normally highly expressed on all types of PC. Differences in the expression of markers such as IgM, IgG, CD95 and CXCR3 in the group with high FS compared to FS = 1, underscore the heterogeneity of pSS patient group and demonstrate that phenotypic pattern of circulating PC associates with the severity of inflammation in the salivary glands of these patients. Our migration experiments show that addition of CXCL12 to PC in vitro, do not alter the migration potential of PC in any group tested. However, we observed an overall higher spontaneous migration of PC from pSS compared to both RA and normal controls.

The human immunodeficiency virus type 1 Rev protein shuttles between the cytoplasm and nuclear compartments.

A retroviral regulatory protein, Rev (regulator of virion protein expression), is made in cells infected by human immunodeficiency virus (HIV). Rev is essential for the completion of the retroviral life cycle and interacts with the host cell at some posttranscriptional step in order to express the incompletely spliced HIV mRNAs from which HIV structural proteins are translated. Neither the host cell components nor the mechanisms responsible for this important regulation have been defined. We now report that Rev is a nucleocytoplasmic shuttle protein which is continuously transported between the cytoplasm, the nucleoli, and nucleoplasmic speckles enriched in RNA splicing and processing factors. The results show that Rev has the potential to interfere specifically with the splicing of the HIV pre-mRNA in the nucleoplasm and, next, guide such mRNAs to the cytoplasm for translation.

Labeling of RNA transcripts of eukaryotic cells in culture with BrUTP using a liposome transfection reagent (DOTAP).

Bromouridine-triphosphate (BrUTP), when introduced into eukaryotic cells in culture, substitutes for UTP during transcription, thereby labeling pre-mRNA for detection by immunochemical methods. In earlier studies, BrUTP was internalized by means of microinjection or by exposing isolated nuclei or permeable cells to BrUTP. We describe here a simple method for the extensive uptake of BrUTP into monolayers of growing cells using a cationic liposome transfectant (DOTAP). Within minutes, DOTAP mediates uptake of BrUTP both at 37 degrees and 4 degrees C. This is followed by incorporation into RNA in the nucleus upon further incubation under culture conditions. In this way, large numbers of actively growing cells may be subjected to biochemical experiments.

The postvaccination antibody response to influenza virus proteins.

The radio-immunoblot (RIB) assay was used to examine the antibody response to proteins of the vaccine strains induced after influenza vaccination. Vaccination stimulated an antibody response to the surface glycoproteins (HA and NA) and to the internal antigens (NP and M) of the three vaccine strains. Antibodies were detected to both the monomeric form of the haemagglutinin (HA) and its two subunits HA1 and HA2. In addition, antibody to the monomeric form of NA was detected. A wide range of response patterns was observed to the viral proteins. All three major antibody classes (IgG, IgA and IgM) were induced after vaccination and in the majority of volunteers the antibody reactivity increased one week after vaccination. IgM antibodies had a wider reactivity pattern, recognising proteins and subunits which were not fully processed or slightly degraded. The varied antibody response induced after influenza vaccination reflects the differing infection histories of the volunteers with influenza. We show some of the practical limitations of studying the antibody response to influenza vaccination.

Serum antibodies from MS patients do not recognize HTLV-I, HIV-1, HIV-2 and SIV.

A retroviral aetiology has been proposed for multiple sclerosis (MS). Although there is as yet no definitive evidence of viral involvement, there have been preliminary reports of antiretroviral antibody detection in sera from MS patients. Such sera have, for example, been found to react with HTLV-I. We here describe investigations involving various immunological techniques which attempt to confirm the virus-specific nature of these antibodies against a range of human and macaque retroviruses. Sera from 25 MS patients, 25 patients with non-associated neurological diseases and 16 patients with non-neurological conditions were tested by immunoblotting methods using lysates of HIV-1-, HIV-2-, HTLV-I- and SIV-infected cells as antigens. None of the sera reacted against any of these retroviral antigens but each serum demonstrated a distinctive and reproducible reaction pattern against cellular components of the cells in which the viruses were propagated. Further examination of the sera was carried out by ELISA using synthetic oligopeptides covering the HIV-1 Gag p24 protein as antigens. None of the sera reacted with the peptides. Our results suggest that in some MS patients the repeated seropositivity to HTLV-I may be due to the reaction with host cell proteins.

Diisopropylfluorophosphate-binding proteins in the outer membrane of Fusobacterium nucleatum: strain variations.

Six strains of Fusobacterium nucleatum were tested for their ability to react with [3H]diisopropylfluorophosphate (DFP), a serine protease inhibitor. Several cytoplasmic proteins were labelled but the strongest labelling was regularly observed in a few outer membrane proteins. The number and the molecular mass of the proteins detected varied according to the strain tested. A 61 kDa protein was labelled in all strains tested, including the two type strains ATCC 10953 and ATCC 25586. A 65 kDa protein was particularly strongly labelled in strains Fev1 and F6.

A comparison of the humoral and cellular immune responses at different immunological sites after split influenza virus vaccination of mice.

The spleen, bone marrow and lymph nodes are all known to be important organs for the initiation and maintenance of an immune response after vaccination. To investigate the differences and similarities in the humoral and cellular immune responses between these tissues, we vaccinated mice once or twice with the conventional human dose (15 microg HA) of influenza A (H3N2) split virus vaccine and analysed the sera and lymphocytes collected from the different sites. We found that the response of antibody secreting cells (ASC) in the lymph nodes appeared to be more transient than in the spleen, possibly because the influenza-specific IgM ASC in particular might have migrated from the lymph nodes immediately after activation. The serum antibody response was found to initially correspond with the ASC response elicited in the spleen and the lymph nodes, whereas the later serum IgG reflected the ASC response in the bone marrow. Proliferation of influenza-specific CD4(+) and CD8(+) cells was predominantly observed in the spleen and was associated with higher concentrations of cytokines than in the lymph nodes. The finding of influenza-specific CD8(+) cell proliferation in the spleen indicates that a split influenza virus vaccine may stimulate a cytotoxic T-cell response. Our results also showed that the primary response elicited a mixed Th1/Th2 profile, whereas the secondary response was skewed towards a Th2 type. Each of the three tissues had a different immunological pattern, suggesting that in preclinical vaccine studies, there is a case for investigating a range of immunological sites.

Human cell lines used in a micro neutralization test for measuring influenza-neutralizing antibodies.

An in situ neutralization test (NT) including ELISA for the measurement of influenza antigen was developed and evaluated. Two human cell lines, fibroblasts (HS27) cells and salivary gland epithelial duct (HSG) cells, were compared with Madin-Darby Canine Kidney (MDCK) cells. The viral production in the human cell lines was lower than that for MDCK cells, which influenced the results of the assay in the HSG and HS27 cells. However, when lowering the infectious dose, the NT using HS27 cells gave a sensitive and stable assay with low background in the ELISA. The NT titres were very low when using HSG cells compared to MDCK cells. The HS27 NT was used to analyze the humoral response after an influenza A infection in patients from a placebo-controlled zanamivir study. We found no differences in NT titres between patients treated with zanamivir or placebo. The MDCK and HS27 NT gave higher titres and more pronounced titre differences than the gold standard haemagglutinin inhibition (HAI) assay. Compared to the HAI assay, the sensitive NT using HS27 cells also revealed heterologous NT-titre rises after influenza infection in the patients.

The serological pattern of autoantibodies to the Ro52, Ro60, and La48 autoantigens in primary Sjögren's syndrome patients and healthy controls.

OBJECTIVE: Sjögren's syndrome (SS) is characterized by exocrine secretion dysfunction. Hallmarks of the chronic autoimmune disease are cellular infiltration of the exocrine glands and the presence of serum autoantibodies against Ro and La. The purpose of this study was to perform a detailed characterisation of the serological pattern against the Ro and La autoantigens in terms of antigen specificity and antibody isotype. METHODS: Serum samples from 100 patients with primary SS and 100 matched healthy controls were tested by enzyme-linked immunosorbent assay (ELISA) with recombinant human Ro and La proteins as antigens. RESULTS: There were higher frequencies of Ro and La positive serum in the SS patients than in the control sera, and the titres were higher in the positive sera from SS patients than the controls. The SS patients often had antibodies against two or three of the antigens tested, while the positive control sera often reacted against only one of the autoantigens. The SS patients had a broader immunoglobulin isotype repertoire in their autoantibodies while the controls when positive usually had one antigen specific isotype. CONCLUSION: We found a distinct and significant difference in the serum antibody specificity and immunoglobulin isotype pattern between SS patients and matched controls. This variance may point to different mechanisms by which these autoantibodies are generated.

Purification and characterization of a 65-kilodalton diisopropylfluorophosphate-binding protein in the outer membrane of Fusobacterium nucleatum Fev1.

A 65-kilodalton protein was identified in the outer membrane of Fusobacterium nucleatum Fev1 by SDS-PAGE. The relative amount of the protein varied during growth, being greatest in stationary phase. The protein was radio-labeled by [125I]-lactoperoxidase treatment of live cells and was only partially extractable with 2% sodium dodecylsulfate (SDS) at room temperature, and therefore assumed to be both exposed to the cell surface and peptidoglycan associated. In intact cells the protein bound diisopropylfluorophosphate (DFP), indicating that it may be a serine protease. DFP-binding activity depended apparently on proper localization of the proteins in the membrane. Several methods were tried in attempts to purify the 65-kilodalton protein; the method that gave best results was preparative SDS-polyacrylamide gel electrophoresis. The isoelectric point was determined to about pH 5. Amino acid composition analysis showed that the 65-kilodalton protein possesses an excess of hydrophilic over hydrophobic residues, polarity index 52%. The N-terminal end of the protein was apparently blocked.

Subcellular redistribution and surface exposure of the Ro52, Ro60 and La48 autoantigens during apoptosis in human ductal epithelial cells: a possible mechanism in the pathogenesis of Sjögren's syndrome.

The Ro52, Ro60 and La48 autoantigens are associated with Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). The mechanisms behind tolerance breakdown of these self-peptides remain unclear; however, apoptosis has been proposed to cause their presentation to the immune system. We have examined the localization of transiently expressed enhanced green fluorescent protein (EGFP)-tagged Ro52, Ro60 and La48 autoantigens in a human salivary gland (HSG) cell line by laser confocal microscopy under normal growth conditions and during apoptosis. Surface exposure of Ro52, Ro60 and La48 was demonstrated on nonfixed apoptotic cells with monoclonal antibodies (MoAbs) or with primary SS patient antisera. Laser scanning cytometry determined the apoptotic frequency. EGFP alone was studied as control. We found that Ro52 mainly is cytoplasmic, Ro60 both nuclear and cytoplasmic, while La48 only resides in the nucleus under normal conditions. During early apoptosis, La48 is dramatically redistributed to the cytoplasm, while the localization of Ro52 and Ro60 is maintained. All three autoantigens filled apoptotic blebs and covered TUNEL (terminal-deoxynucleotidyl-transferase-mediated dUTP-digoxigenin nick end labelling)-positive apoptotic bodies. Identical results were obtained in COS-7 cells. We have developed a transfection system to study the intracellular localization of the three autoantigens Ro52, Ro60 and La48, without antibody detection. During apoptosis, there is an intracellular redistribution of endogenous and EGFP-tagged Ro52, Ro60 and La48, leading to surface exposure. These findings may indicate a role for apoptosis in the induction and facilitation of humoral responses to Ro52, Ro60 and La48 in the autoimmune exocrinopathy of SS.

Influenza virus: immunity and vaccination strategies. Comparison of the immune response to inactivated and live, attenuated influenza vaccines.

Influenza virus is a globally important respiratory pathogen which causes a high degree of morbidity and mortality annually. The virus is continuously undergoing antigenic change and thus bypasses the host's acquired immunity to influenza. Despite the improvement in antiviral therapy during the last decade, vaccination is still the most effective method of prophylaxis. Vaccination induces a good degree of protection (60-90% efficacy) and is well tolerated by the recipient. For those at risk of complications from influenza, annual vaccination is recommended due to the antigenic changes in circulating strains. However, there is still room for improvement in vaccine efficacy, long-lasting effect, ease of administration and compliance rates. The mucosal tissues of the respiratory tract are the main portal entry of influenza, and the mucosal immune system provides the first line of defence against infection. Secretory immunoglobulin A (SIgA) and IgM are the major neutralizing antibodies directed against mucosal pathogens. These antibodies work to prevent pathogen entry and can function intracellularly to inhibit replication of virus. This review describes influenza virus infection, epidemiology, clinical presentation and immune system response, particularly as it pertains to mucosal immunity and vaccine use. Specifically, this review provides an update of the current status on influenza vaccination and concentrates on the two main types of influenza vaccines currently in use, namely the cold-adapted vaccine (CAV) given intranasally/orally, and the inactivated vaccine (IV) delivered subcutanously or intramuscularly. The commercially available trivalent IV (TIV) elicits good serum antibody responses but induces poorly mucosal IgA antibody and cell-mediated immunity. In contrast, the CAV may elicit a long-lasting, broader immune (humoral and cellular) response, which more closely resembles natural immunity. The immune response induced by these two vaccines will be compared in this review.

Parenteral influenza vaccination induces a rapid systemic and local immune response.

The kinetics of the local immune response in the upper respiratory tract to parenterally administered inactivated split trivalent influenza vaccine were examined in 19 healthy subjects. Influenza virus-specific antibody-secreting cells (ASC) could be detected as early as 2 days after vaccination in peripheral blood and tonsils, with a peak at approximately 1 week after vaccination and a decline to insignificant levels after 6 weeks. Circulating ASC produced IgG, IgA, and IgM, whereas ASC in tonsils produced mainly IgA and IgM. Influenza virus-specific antibodies were predominantly IgG and IgM in serum and IgA in oral fluid; they rose after 1 week and were elevated at 6 weeks. This may indicate a secretory involvement of the anti-influenza virus response in the upper respiratory tract. Parenteral influenza vaccination induced an immediate and significant immune response in both the upper respiratory tract and peripheral blood.