Interim estimates of 2013/14 influenza clinical severity and vaccine effectiveness in the prevention of laboratory-confirmed influenza-related hospitalisation, Canada, February 2014.

During the 2013/14 influenza season in Canada, 631 of 654 hospitalisations for laboratory-confirmed influenza enrolled in sentinel hospitals were due to Influenza A. Of the 375 with known subtype, influenza A(H1N1) accounted for 357. Interim unmatched vaccine effectiveness adjusted for age and presence of one or more medical comorbidities was determined by test-negative case-control design to be 58.5% (90% confidence interval (CI): 43.9-69.3%) overall and 57.9% (90% CI: 37.7-71.5) for confirmed influenza A(H1N1).

The persistence of seroprotective levels of antibodies after vaccination with PreHevbrio, a 3-antigen hepatitis B vaccine.

Prevention of hepatitis B virus (HBV) infection by vaccination can potentially eliminate HBV-related diseases. PreHevbrio™/PreHevbri® is a 3-antigen (S, preS1, preS2) HBV vaccine (3A-HBV) recently licensed for adults in the US, EU and Canada. This study evaluated antibody persistence in a subset of fully vaccinated and seroprotected (anti-HBs ≥ 10 mIU/mL) Finnish participants from the phase 3 trial (PROTECT) of 3A-HBV versus single-antigen HBV vaccine (1A-HBV). 465/528 eligible subjects were enrolled (3A-HBV: 244; 1A-HBV: 221). Baseline characteristics were balanced. After 2.5 years, more 3A-HBV subjects remained seroprotected (88.1 % [95 %CI: 84.1,92.2]) versus 1A-HBV (72.4 % [95 %CI: 66.6,78.3)], p < 0.0001) and had higher mean anti-HBs [1382.9 mIU/mL (95 %CI: 1013.8,1751.9) versus 252.6 mIU/mL (95 %CI: 127.5,377.6), p < 0.0001]. In multiple variable logistic regression analysis including age, vaccine, initial vaccine response, sex and BMI, only higher post dose 3 (Day 196) antibody titers significantly reduced the odds of losing seroprotection.

Safety and immunogenicity of an investigational quadrivalent meningococcal conjugate vaccine after one or two doses given to infants and toddlers.

With the emergence of multiple meningococcal serogroups in different geographic areas, broad vaccine protection from infancy is desirable. One hundred and seventy-five infants received either two doses of a meningococcal quadrivalent (A, C, W-135, Y) conjugate vaccine (MenACWY-CRM) at 6 and 12 months, one dose of MenACWY-CRM at 12 months, or MenC at 12 months and MenACWY-CRM at 18 months. Bactericidal antibody titers using human complement were measured before and 1 month after each dose. Injection-site reactions were reported by 22-45% of participants following MenACWY-CRM given at 6 or 12 months. Similar proportions of subjects had injection-site reactions following two doses of MenACWY-CRM (32-41%) or one dose of MenC (26-44%). The incidence of systemic adverse events was comparable between groups. After two doses of MenACWY-CRM, the percentages of participants reporting hSBA titers >or=8 were 100% for C, W-135, and Y, and 84% for A. Serogroup C titers were more than 10-fold higher after two doses of MenACWY-CRM than after one dose of MenC or MenACWY-CRM at 12 months. Serogroup C titers were comparable following a single dose of MenACWY-CRM or MenC at 12 months. MenACWY-CRM is well tolerated and immunogenic given at 12 months, or two doses at 6 and 12 months of age.

Expedient synthesis of functional single-component glycoliposomes using thiol-yne chemistry.

The preparation of a set of eight unprecedented amphiphilic neoglycolipids forming liposome nanoparticles is reported. The small library was readily obtained from various peracetylated propargyl glycopyranosides via efficient radical-initiated thiol-yne (TYC) coupling reactions using alkanethiols of different chain lengths. In addition, using sequential thiol-yne, both the nature and positioning of the lipophilic alkanethiols could be varied at will, thus providing unparalleled variability within the glycolipid structures. Two different classes of self-assemblies were prepared from the new neoglycolipids. First, liposomes of 150-300 nm were obtained by solvent injection of their ethanol or tetrahydrofuran (THF) solution in water. The resulting structures were analyzed by dynamic light scattering (DLS) and atomic force microscopy (AFM). The mannosylated lipid nanoparticle (compound 14) showed good stability in water. Alternatively, giant soft unilamellar vesicles were also obtained by film hydration and visualized by differential interference contrast microscopy (DIC). Incorporation of a hydrophobic dye to the solution prior to evaporation allowed visualization by confocal microscopy. Finally, the biological functions of the newly formed glycolipid vesicles were evaluated by multivalent carbohydrate-protein binding interactions using concanavalin A (ConA). Agglutination assays and the binding of glycolipid by dendritic cells (DCs) resulted in an increase in DCs immunostimulatory potential. Importantly, we did not see changes in cells viability at tested doses. This study provides a new, simple and highly efficient methodology to produce novel glyconanoparticle candidate as model in development of vaccine adjuvant and drug delivery system.

Comparison of antibody- and cell-mediated immune responses after intramuscular hepatitis C immunizations of BALB/c mice.

Current treatments for hepatitis C infection have limited efficacy, and there is no vaccine available. The goal of this study was to compare the immune response to several immunization combinations against hepatitis C virus (HCV). Six groups of mice were immunized at weeks 0, 4, and 8 with different combinations of a candidate HCV vaccine consisting of 100 microg recombinant HCV core/E1/E2 (rHCV) DNA plasmid and/or 25 microg rHCV polyprotein and 50 microL Montanide ISA- 51. Four weeks after the last injection, all groups of mice were sacrificed and blood samples and spleens were collected for measuring the levels of specific HCV antibodies (total IgG, IgG1, and IgG2a). Cell proliferation and intracellular interferon-gamma were also measured. Among the groups of immunized mice, only the mice immunized with rHCV DNA plasmid, rHCV polyprotein, and montanide (group D) and mice immunized with rHCV polyprotein and montanide (group F) demonstrated a significant increase in the total IgG titer after immunization. IgG1 was the predominant antibody detected in both groups D and F. No IgG2a was detected in any of the groups. Proliferation assays demonstrated that splenocytes from group D and group C (rHCV DNA primed/rHCV polyprotein boost) developed significant anti-HCV proliferative responses. The combination of an rHCV DNA plasmid, rHCV polyprotein, and montanide induced a high antibody titer with a predominance of IgG1 antibodies and recognized the major neutralization epitopes in HVR1. In contrast, group C did not show an increase in anti-HCV antibodies, but did show a proliferative response.

Safe use of the CXCR4 inhibitor ALX40-4C in humans.

ALX40-4C is a small peptide inhibitor of the chemokine receptor CXCR4 that can inhibit X4 strains of HIV-1. Prior to the discovery of chemokine receptors as the HIV coreceptors, ALX40-4C was used in phase I/II clinical trials to evaluate its therapeutic potential against HIV-1, making ALX40-4C the first anticoreceptor inhibitor to be tested in humans against HIV-1. Patients in the highest dose groups achieved ALX40-4C levels above the effective concentration of the drug for nearly the entire 1-month treatment period. ALX40-4C was well tolerated by 39 of 40 asymptomatic HIV-infected patients, despite the critical role of CXCR4 in normal development and hematopoiesis. No significant or consistent reductions in viral load were observed, but only 12 of the enrolled patients harbored virus types that used CXCR4. We also found that ALX40-4C interacts with the second extracellular loop of CXCR4 and inhibits infection exclusively by blocking direct virus-CXCR4 interactions.

Differential effect of heat shock on RNA metabolism in human Burkitt's lymphoma B-cell lines.

Thermal stress induces expression of a family of heat shock proteins which may regulate the synthesis of various cellular genes. We investigated the effect of heat shock on polyadenylation in Epstein-Barr Virus (EBV) negative and EBV transformed human Burkitt's lymphoma (BL) B-cell lines. Incubation of the BL B-cell line P3HR-1, carrying the defective EBV genome [EBV nuclear antigen-2 gene deletion] at 46 degrees C for 15 min increased nuclear poly(A) polymerase (PAP) activity. Thereafter, enzymatic activity declined and at 60 min it was reduced to about 50% of that observed in cells incubated at 37 degrees C. In contrast, no significant increase in PAP activity was observed at 15 min or thereafter in an EBV- BL cell line, ST-486, in response to elevated temperature. Furthermore, no heat shock mediated change in nuclear poly(A)-specific endonuclease activity was observed in either P3HR-1 or ST-486 cells suggesting a specific effect on PAP activity. However, thermal stress dependent increase in c-myc expression was detected only in P3HR-I cells. These results suggest an association between EBV transformation and enhanced expression of c-myc and PAP activity. To further determine the role of EBV, and EBV- BL cell line, BL-30, and BL-30 cells infected in vitro with a wild type strain of EBV, BL-30/B95-8, were investigated. BL-30/B-95-8, unlike the parental BL-30 cells, exhibited c-myc and PAP gene upregulation at 15 min but were downregulated at 60 min following exposure of cells to elevated temperatures. These results suggest that infection of human B-cells with EBV is associated with their ability to respond to thermal stress by increased PAP activity which may stabilize mRNA through enhanced polyadenylation.

Peripheral motor neuropathy associated with autonomic dysfunction in two sisters: new hereditary syndrome?

We describe two sisters with distal, slowly progressive muscular weakness and hypotrophy since childhood, autonomic dysfunction characterized by profuse sweating, distal cyanosis related to cold weather, orthostatic hypotension, and esophageal achalasia. Nerve conduction velocity of several motor nerves was slow, and although no sensory abnormalities were present, sural nerve biopsy revealed severe nonspecific demyelination. No similar patients could be found in the literature and we therefore suggest the possibility that these individuals have a newly recognized hereditary syndrome.

A combined vaccine against hepatitis A and B in children and adolescents.

BACKGROUND: Pediatric monocomponent formulations of vaccines against hepatitis A and B have been proved to be safe and immunogenic in children. OBJECTIVES: To investigate the safety and immunogenicity of a combined hepatitis A/B vaccine in children 1 to 15 years of age. METHODS: Three doses of a combined vaccine containing 360 enzyme-linked immunosorbent assay units of hepatitis A antigen and 10 microg of hepatitis B surface antigen were administered in a 0-, 1-, 6-month schedule to three groups of children: a group of 1- to 6-year-olds (n = 60); and two groups of 6- to 15-year-olds (both n = 60). RESULTS: Reactogenicity, assessed using diary cards, was not affected by the age of the subjects or the vaccine lot and was similar to that described with the monocomponent vaccines. Local and systemic reactions were mostly mild or moderate and resolved spontaneously. One month after the second dose 100 and 98.8% of all three groups had seroconverted against hepatitis A virus and hepatitis B virus, respectively. After the third dose all subjects were seropositive for both components, with geometric mean titers for anti-hepatitis A virus of 6518 to 8907 mIU/ml and for anti-hepatitis B surface antibody of 7255 to 11732 mIU/ml in the three groups. CONCLUSION: The combined pediatric hepatitis A/B vaccine formulation was well-tolerated and highly immunogenic in children 1 to 15 years old.

Transcriptional analysis of human peripheral blood mononuclear cells after influenza immunization.

Influenza A virus is a major cause of morbidity and mortality worldwide. There is a large knowledge base on the immune response to influenza. However, few studies have focused on global gene expression in immune cells after antigenic challenge. A better understanding of the host immune response is required for the development of more efficient means of prevention and treatment of influenza. In this study, global gene expression in peripheral blood mononuclear cells (PBMCs) after influenza immunization was analyzed. The differential gene expression in antigen-stimulated and non-stimulated PBMCs was determined by cDNA microarrays. To determine whether a specific gene profile was present during a proliferative memory cell response to influenza antigens, gene expression in response to PHA was compared with antigen-stimulated PBMCs. PHA induced the upregulation of 201 genes while influenza virus antigen upregulated more than triple that is 630 genes out of 1700 genes analyzed. Both influenza antigen and PHA commonly upregulated 138 genes. Interferon (IFN)-related genes were induced by influenza but not by PHA. The interferon-gamma induced protein precursor 10 (IP-10) was upregulated 27-fold while the interferon-induced 54 kDa protein exhibited a 13-fold increase. The following gene families were also selectively upregulated by influenza antigens: complement ligands and receptors, T cell activation genes, growth factors, genes related to antigen processing and inflammatory responses. With PHA, the genes TNF-R, CTSG, CD3 delta, C8B, CRF1 and CCR2 had higher expression compared with the viral antigen stimulation. Neutrophil defensins alpha-1 and two C-C chemokines, proteins MIP-1-beta and MIP-4, were among the genes upregulated by both PHA and influenza antigens. The results suggest that interferon-induced genes are one of the main transcriptional targets during the immune response to influenza virus.

Investigating a viral etiology for keratoconjunctivitis sicca among patients who are positive for human immunodeficiency virus.

PURPOSE: Herpesvirus infection of the lacrimal gland was investigated as an etiologic factor for keratoconjunctivitis sicca in patients who were positive for human immunodeficiency virus (HIV). METHODS: In this cross-sectional study, we recorded the Schirmer tests and tear break-up times (TBUTs) among 30 patients who were positive for HIV. Dry-eye state was defined as a Schirmer test of <10 mm of wetting at 5 min or a TBUT of <10 s. The polymerase chain reaction assay (PCR) for herpes family viruses [Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex virus (HSV), and varicella-zoster virus (VZV)] was performed on the conjunctival and tear specimens of the 30 HIV-positive patients by using virus-specific single primers. We compared the rates of virus DNA detection among dry-eye and non-dry-eye patients by calculating the odds ratio of detection for each virus. RESULTS: The odds ratio of viral DNA detection was adjusted for age, gender, race, CD4 count, and duration of HIV positivity. The adjusted odds ratios of EBV DNA detection among dry-eye to non-dry-eye patients were 1.30 (p = 0.79) and 0.97 (p = 0.98) by using Schirmer tests and TBUTs, respectively. For CMV, the adjusted odds ratios among dry-eye to non-dry-eye patients were 1.94 (p = 0.58) with Schirmer tests and 1.02 (p = 0.99) with TBUTs. HSV and VZV DNA were not detected in any samples. CONCLUSION: Our study does not support the role of herpesvirus infection of the lacrimal gland as a causative factor in the pathogenesis of dry eyes in patients positive for HIV.

Quantitation of HIV-1 RNA in Dried Plasma Spots (DPS) : A Field Approach to Therapeutic Monitoring.

The ability to measure accurately viral RNA in the plasma (1-3) and intracellular (4-7) compartments of HIV-1-infected persons has led to a dramatic improvement in our understanding of the natural history of HIV-1/AIDS. A number of recent studies have convincingly demonstrated that high levels of viral replication occur at all stages of disease (8-10), and that changes in viral RNA load are predictive of disease outcome (11,12) and response to therapy (13,14). These findings, combined with the introduction of potent new antivirals (15,16), have stimulated a growing interest in viral load monitoring, both as a function of disease status, and as a predictor of disease progression and therapeutic efficacy.

Quantification of HIV-1 RNA in Dried Plasma Spots (DPS) : A Field Approach to Therapeutic Monitoring.

The ability to accurately measure viral RNA in the plasma (1-3) and intracellular (4-7) compartments of HIV-1-infected persons has led to a dramatic improvement in the understanding of the natural history of HIV-1 and AIDS. A number of recent studies have convincingly demonstrated that high levels of viral replication occur at all stages of disease (8-10), and that changes in viral RNA load are predictive of disease outcome (11,12), and response to therapy (13,14). These findings, combined with the introduction of potent new antivirals (15,16), have stimulated a growing interest in viral load monitoring, both as a function of disease status, and as a predictor of disease progression and therapeutic efficacy.

Reactogenicity to a live attenuated varicella vaccine in Canadian children.

OBJECTIVE: To assess the reactogenicity and safety of a thermostable, high titre, varicella vaccine in healthy infants and children. DESIGN: Open study of 505 children monitored for 42 days after vaccination. SETTING: Three urban Canadian centres (Halifax, Ottawa and Vancouver). PARTICIPANTS: 505 healthy children one to 12 years of age were enrolled and 504 completed the study. All were susceptible to varicella by history. INTERVENTIONS: All participants received one dose of live attenuated varicella vaccine (1x10(4.5) plaque forming units/dose) subcutaneously. MAIN OUTCOME MEASURES: The children were monitored from the day of vaccine administration (day 0) until day 42. All local and general symptoms and signs were recorded on diary cards by the patients' parents, who were encouraged to fill in the cards on days 2 to 3 and 18 to 24 via telephone calls from study personnel. RESULTS: Most of the symptoms noted after vaccine administration were mild and transient, and all resolved within the respective follow-up periods. Injection site symptoms included pain (17.5%, 13.9% and 30.4% in centres 1, 2 and 3 respectively), redness (21.1%, 32.1% and 48.8%) and swelling (7%, 10.3% and 29.2%). The general symptoms reported were fever 37.5 degrees C or higher (3.5%, 4.8% and 3.0%) and varicella-like rashes (6.4%, 2.4% and 0%). Two subjects had severe symptoms (one with cervical lymphadenopathy, and one with a fever higher than 39 degrees C) probably related to vaccine administration. No serious adverse events were reported during the entire study. CONCLUSION: The vaccine was well tolerated.

Interferon-gamma inhibits CD44-hyaluronan interactions in normal human B lymphocytes.

The interaction of CD44 with its ligand hyaluronan (HA) plays a vital role in lymphopoiesis, lymphocyte homing, T cell activation, and metastasis. This study addresses the effect of cytokines involved in B cell growth on CD44-HA interactions in normal human B cells. Activation of B lymphocytes with LPS, pokeweed mitogen, or anti-IgM antibodies with or without IL-2 or IL-4 failed to induce HA adhesion. Stimulation of B cells with the phorbol ester PMA, however, induced strong HA recognition, which was inhibited by IFN-gamma and to some extent by IL-4. Investigation of the potential molecular mechanism involved revealed that PMA-induced HA adhesion correlated with enhanced expression of CD44-H- and V6-containing isoforms, as determined by flow cytometry, and the differential induction of V4- and V5-containing isoforms, as determined by reverse transcriptase-based polymerase chain reaction analysis. The inhibition of PMA-induced adhesion by IFN-gamma and IL-4 correlated with the downregulation of CD44 H expression and altered usage of exons V4 and V5. However, changes in the electrophoretic mobility of CD44 proteins, as a measure of posttranslational modifications, were not detected in response to PMA and IFN-gamma or PMA and IL-4. These results suggest that the inhibition of PMA-induced HA adhesion by IFN-gamma and IL-4 may influence B cell migration through their ability to downregulate CD44-HA interactions.

Regulation of CD44-hyaluronan interactions in Burkitt's lymphoma and Epstein-Barr virus-transformed lymphoblastoid B cells by PMA and interleukin-4.

In this study, we investigated the regulation of CD44-hyaluronan (HA) interactions in a panel of EBV+ Burkitt's lymphoma (BL) and lymphoblastoid B cell lines (B-LCL) generated by in vitro EBV transformation of normal human B cells. The results show that among B cell mitogens, phorbol 12-myristate 13-acetate (PMA) alone induced strong HA recognition in EBV+ BL-30/B95-8 cells. Among the cytokines that affect B cell growth and differentiation, IL-4 alone induced HA recognition in BL-30/B95-8 cells. Attempts to delineate the molecular mechanism for this increased HA adhesion in BL-30/B95-8 cells revealed an enhanced expression of CD44 H, isoforms containing V3, V6, and V9 exons, alterations in the splicing pattern of the V4 exon, and the increased electrophoretic mobility of the CD44 H protein. In contrast, the ability to recognize HA was not observed in B-LCL cells stimulated with either PMA or IL-4, even though these cells respond to IL-4, as observed by upregulation of CD23 expression. The molecular pathways that regulate CD44 expression and CD44-mediated HA binding may be selectively inactivated in B-LCL cells. These results may have implications with respect to the generation and spread of B cell tumors.

Increased frequency of Epstein-Barr virus excretion in patients with new daily persistent headaches.

In a case-control study 27 (84%) of 32 patients with new daily persistent headaches (NDPH) and 8 (25%) of 32 controls had evidence of Epstein-Barr virus (EBV) "active" infection, as demonstrated by EBV excretion and/or early antigen titre above 1:32. 20 (62%) patients and 4 (12%) controls were excreting EBV in the oropharynx, as determined by a dot hybridisation assay. The mean titre of IgG antibodies to early antigen was significantly higher in patients than controls. EBV reactivation may be important in the pathogenesis of NDPH. Alternatively, patients with NDPH may be unusually prone to EBV reactivation.

Comparison of the VIDAS RSV assay and the Abbott Testpack RSV with direct immunofluorescence for detection of respiratory syncytial virus in nasopharyngeal aspirates.

The sensitivity and accuracy of the VIDAS RSV assay in testing fresh specimens were 82.7 and 87.1%, respectively, whereas specimens previously frozen at -70 degrees C gave a sensitivity of 96.2% and an accuracy of 95.4%. The sensitivity and accuracy of Abbott Testpack RSV were 92.6 and 91.3% for fresh specimens and 86.8 and 88.1% for frozen specimens. The advantages and drawbacks of the two assays are discussed.

CD44 isoforms containing exons V6 and V7 are differentially expressed on mitogenically stimulated normal and Epstein-Barr virus-transformed human B cells.

CD44, a cell adhesion molecule, exists in multiple isoforms that are generated by RNA alternative splicing. CD44 isoforms containing exon V6 (CD44 V6) have been associated with tumorigenesis and metastasis. We investigated the association between human B-cell activation and CD44 V6 isoform expression by analysing its expression in resting and mitogenically stimulated B cells. Results showed that resting B cells expressed the CD44 H (no variable exon) isoform alone. Activation of B cells [phorbol myristate acetate (PMA), surface immunoglobulin cross-linking alone or in the presence of interleukin-2 (IL-2)] induced CD44E (variable exon V8-10), R2 (VIO) and CD44 isoforms containing exons V6 and/or V7 (CD44 V6/V7). Epstein-Barr virus (EBV) infection of B cells, an alternative method of B-cell activation, induced the expression of CD44 E and R2 but not CD44 V6/V7. These results indicate that CD44 V6/V7 expression depends on the mode of activation. CD44 isoform expression was also investigated in a panel of EBV-negative and EBV-positive Burkitt's lymphoma (BL) B-cell lines. EBV-negative BL cells did not express CD44. In contrast, EBV-positive BL cells expressed CD44 H, R2 and E but not CD44 V6/V7 isoforms, suggesting an association between EBV infection and CD44 isoform induction. To determine directly the role of EBV in CD44 isoform induction, an EBV-negative BL cell line, BL30 (negative for all isoforms of CD44), BL30 infected in vitro with the EBNA-2-defective P3HR1 (BL30/P3HR1), and the wild-type B95-8 strain of EBV (BL30/B95-8) were examined. The parental BL30 cells infected with the wild-type EBV strain, but not with the P3HR-1 strain, expressed CD44 H, R2 and E isoforms, as seen in EBV-immortalized B cells. These studies suggest that (1) alternative splicing of CD44 isoforms is differentially regulated depending on the mode and state of cell activation, and that (2) the CD44 V6/V7 isoforms may represent B-cell activation antigens that are induced by mitogenic stimulation but not following EBV infection.

Role of XIAP protein, a human member of the inhibitor of apoptosis (IAP) protein family, in phytohemagglutinin-induced apoptosis of human T cell lines.

A new family of human genes xiap, hiap-1 and hiap-2, which are homologous to the baculovirus iap (inhibitor of apoptosis) genes cp-iap and op-iap, has been recently cloned and shown to suppress apoptosis after serum withdrawal or exposure to a free radical inducer. In order to examine the role of one of these human genes, namely xiap, in lymphoid cells, we studied XIAP expression, after PHA stimulation in three different human T cell lines. We report here that stimulation with PHA resulted in the human T cell lines undergoing apoptosis, as assessed by DNA fragmentation and by propidium iodide (PI) staining and flow cytometry. When XIAP protein expression was evaluated by Western blot, we observed that the induction of apoptosis by PHA was associated with a parallel decrease of XIAP expression. We also provide evidence that stably transfected Jurkat cells containing the xiap open reading frame became resistant to PHA-induced apoptosis. These data suggest a role for XIAP protein in the regulation of apoptosis in lymphoid cells.