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We have studied the interference of soluble tumor necrosis factor-alpha receptor p55 (sTNF-R55) on the quantification of tumor necrosis factor-alpha (TNF-alpha) in three immunoassays: the commercially available enzyme amplified-sensitivity immunoassay (EASIA) of Medgenix and the ELISA of Boehringer Mannheim, and an assay based on electrochemiluminescence (ECL) technology. TNF-alpha measurements by EASIA and ELISA were not affected by the presence of sTNF-R55, but results by the ECL method were clearly influenced by the receptor. We then performed a second set of experiments with the ECL system to examine what factors might influence the importance of interference from sTNF-R55 and soluble TNF-alpha receptor p75 (sTNF-R75), which influences TNF-alpha measurements the same as sTNF-R55. We found that extending the incubation time and increasing the concentration of capture antibody decreased the interfering capacity of sTNF-R55 and sTNF-R75. A clear insight into these phenomena is of utmost importance for correct interpretation of cytokine quantification.

BACKGROUND: As only a few studies have examined the prevalence of various hepatitis C virus (HCV) subtypes in blood donors, information about the variability and route of infection in apparently healthy persons is limited. STUDY DESIGN AND METHODS: Blood donations collected at a large Parisian hospital (52,441) were investigated for antibodies to HCV. Serum samples were screened with an enzyme immunoassay. All HCV- positive donations were retested with a second enzyme immunoassay and confirmed by immunoblot. The HCV genotype was determined for all polymerase chain reaction-positive subjects. Untypable genotypes were sequenced in the NS5B region. RESULTS: In total, 83 (0.26%) blood donors were anti-HCV positive. Men (0.34%) were significantly more likely to be infected (p < 0.001) than women (0.19%). Prevalence rates in men between 20 and 39 years of age were higher than those in similar women (p = 0.01), but greater in women aged from 50 to 65 years (p = 0.05). Fifty-five sera were viremic, of which 49 could be genotyped by a line probe assay. One new HCV type 1 subtype and three new HCV type 2 subtypes were discovered. In total, 28, 10, 11, 5, and 1 serum samples were grouped into HCV types 1 through 5, respectively, involving a total of 13 subtypes. The mean age of HCV type 2-infected donors was 42 +/− 11 years, but that for type 3-infected subjects was only 30 +/− 4 years (p = 0.0048). Forty-nine subjects showed elevated alanine aminotransferase levels; 39 (80%) of these subjects were viremic (p < 0.05). CONCLUSION: Among the sampled population, an HCV prevalence rate of 0.26 percent was found, with the five most common European genotypes causing the infections. Four new subtypes were discovered. Correlation between genotype and risk factors was not apparent, but links with age, sex, and ethnic origin emerged.

A combination of hepatitis A and hepatitis B monovalent vaccines could offer advantages for disease control programs in terms of convenience, compliance and cost.Under randomized, double-blind conditions, 156 healthy young adults were divided into 3 groups to receive 1 of 3 lots of a combined hepatitis A/hepatitis B vaccine administered at months 0, 1, and 6. Safety and immunogenicity were assessed after each dose.Transient and predominantly mild reactions were reported by slightly more than half the vaccinees; no serious adverse effects were relate to vaccination. One month after dose 2, all subjects had converted to the hepatitis A component. Geometric mean titers (GMTs) of antibodies of hepatitis A virus varied from 4415 to 4882 mIU/ml in the three groups. For hepatitis B, most vaccinees (73%-92%) had protective levels of antibodies to hepatitis B virus (anti-HBs) after the second dose, and all were sero-protected after the booster. Anti-HBs GMTs at month 7 ranged from 1917 to 3298 mIU/ml.No statistically significant differences were observed between vaccine lots. The combined hepatitis A/hepatitis B vaccine was safe and clinically well tolerated and induced immune responses quantitatively similar to those obtained with the respective monovalent vaccines.

The quality of the hepatitis C virus (HCV)-specific T-cell response may greatly determine the course of an HCV infection. An adequate T-cell response may contribute to a successful clearance of the virus and a rapid recovery from the disease. An inadequate response may lead to viral persistence and may eventually contribute to the pathogenesis of hepatocellular damage in chronic disease. The effect of interferon alfa (IFN-alpha), presently the most popular therapeutic agent for chronic HCV infections, on HCV-specific T-cell responses is completely unknown. To demonstrate the presence of HCV-specific T lymphocytes during chronic HCV infections, to know their antigenic specificities, and to examine possible effects of IFN-alpha treatment on their presence and antigen recognition patterns, we have stimulated peripheral blood mononuclear cells (PBMC) from 35 chronic HCV patients with nine pools of synthetic peptides representing the HCV Core, E1, and E2 proteins as well as with a recombinant NS3 protein. The proliferative responses of PBMC from 16 healthy control subjects toward these antigens were measured for comparison. Lymphoproliferative responses of patients with chronic HCV infections were assayed either before (in 10 patients), during (in 13 patients), or after (in 21 patients) treatment with IFN-alpha. The analysis showed that PBMC from most HCV patients consistently recognized the COOH-terminal part of the core protein. E1, E2, and NS3 were recognized less frequently. This recognition pattern was not related to the therapy with IFN-alpha nor to the clinical response of the patient toward this therapy. The response to the Core protein could be fine-mapped to the COOH-terminal region encompassing amino acids (aa) 73 to 92, 121 to 140, 145 to 164, and 157 to 176. (Hepatology 1996 Jan;23(1):8-16)

Background Several pre-erythrocytic malaria vaccines based on the circumsporozoite protein (CSP) antigen of Plasmodium falciparum are in clinical development. Vaccine immunogenicity is commonly evaluated by the determination of anti-CSP antibody levels using IgG-based assays, but no standard assay is available to allow comparison of the different vaccines. Methods The validation of an anti-CSP repeat region enzyme-linked immunosorbent assay (ELISA) is described. This assay is based on the binding of serum antibodies to R32LR, a recombinant protein composed of the repeat region of P. falciparum CSP. In addition to the original recombinant R32LR, an easy to purify recombinant His-tagged R32LR protein has been constructed to be used as solid phase antigen in the assay. Also, hybridoma cell lines have been generated producing human anti-R32LR monoclonal antibodies to be used as a potential inexhaustible source of anti-CSP repeats standard, instead of a reference serum. Results The anti-CSP repeats ELISA was shown to be robust, specific and linear within the analytical range, and adequately fulfilled all validation criteria as defined in the ICH guidelines. Furthermore, the coefficient of variation for repeatability and intermediate precision did not exceed 23%. Non-interference was demonstrated for R32LR-binding sera, and the assay was shown to be stable over time. Conclusions This ELISA, specific for antibodies directed against the CSP repeat region, can be used as a standard assay for the determination of humoral immunogenicity in the development of any CSP-based P. falciparum malaria vaccine.

Background:Many cases of refractory hypertension cannot be attributed to specific identifiable factors. Haptoglobin polymorphism has been suggested as a candidate genetic marker in essential hypertension. The aim of this study was to investigate the distribution of haptoglobin types in patients with

Currently licensed influenza vaccines rely predominantly on the induction of strain-matched hemagglutination inhibition antibody responses. These vaccines have a proven record of safety and efficacy in preventing influenza-induced illness and complications. However, they do not confer protection to all vaccinated individuals, and the protection they afford is short-lived, particularly in older adults. Hemagglutination inhibition titers induced by these vaccines are considered correlates of protection, but recent data demonstrate that this is not always the case. It is clear that better insight is needed into the immune responses that correlate with protection against human influenza. Influenza vaccines that can induce cross-reactive cellular immune responses (CD4(+) and/or CD8(+) T-cell responses) might correct some of the shortcomings of currently used influenza vaccines. In the future, the use of infection-permissive and disease-modifying vaccines that allow for the induction of cross-reactive T-cell responses may become a valuable complement to the administration of trivalent inactivated influenza vaccines.

Mucosal antibodies harboring various antiviral activities may best protect mucosal surfaces against early HIV-1 entry at mucosal sites and they should be ideally induced by prophylactic HIV-1 vaccines for optimal prevention of sexually transmitted HIV-1. A phase I, double-blind, randomized, placebo-controlled trial was conducted in twenty-four healthy HIV-uninfected young women. The study objectives were to assess the safety, tolerability and immunogenicity of virosomes harboring surface HIV-1 gp41-derived P1 lipidated peptides (MYM-V101). Participants received placebo or MYM-V101 vaccine at 10 μg/dose or 50 μg/dose intramuscularly at week 0 and 8, and intranasally at week 16 and 24. MYM-V101 was safe and well-tolerated at both doses administered by the intramuscular and intranasal routes, with the majority of subjects remaining free of local and general symptoms. P1-specific serum IgGs and IgAs were induced in all high dose recipients after the first injection. After the last vaccination, vaginal and rectal P1-specific IgGs could be detected in all high dose recipients. Approximately 63% and 43% of the low and high dose recipients were respectively tested positive for vaginal P1-IgAs, while 29% of the subjects from the high dose group tested positive for rectal IgAs. Serum samples had total specific IgG and IgA antibody concentrations ≥0.4 μg/mL, while mucosal samples were usually below 0.01 μg/mL. Vaginal secretions from MYM-V101 vaccinated subjects were inhibiting HIV-1 transcytosis but had no detectable neutralizing activity. P1-specific Th1 responses could not be detected on PBMC. This study demonstrates the excellent safety and tolerability of MYM-V101, eliciting systemic and mucosal antibodies in the majority of subjects. Vaccine-induced mucosal anti-gp41 antibodies toward conserved gp41 motifs were harboring HIV-1 transcytosis inhibition activity and may contribute to reduce sexually-transmitted HIV-1. Trial Registration ClinicalTrials.gov NCT01084343

Background: A prophylactic human papillomavirus (HPV) vaccine targeting oncogenic HPV types in addition to HPV-16 and -18 may broaden protection against cervical cancer. Two Phase I/II, randomized, controlled studies were conducted to compare the immunogenicity and safety of investigational tetravalent HPV L1 virus-like particle (VLP) vaccines, containing VLPs from two additional oncogenic genotypes, with the licensed HPV-16/18 AS04-adjuvanted vaccine (control) in healthy 18-25 year-old women. Methods: In one trial (NCT00231413), subjects received control or one of 6 tetravalent HPV-16/18/31/45 AS04 vaccine formulations at months (M) 0,1,6. In a second trial (NCT00478621), subjects received control or one of 5 tetravalent HPV-16/18/33/58 vaccines formulated with different adjuvant systems (AS04, AS01 or AS02), administered on different schedules (M0,1,6 or M0,3 or M0,6). Results: One month after the third injection (Month 7), there was a consistent trend for lower anti-HPV-16 and -18 geometric mean antibody titers (GMTs) for tetravalent AS04-adjuvanted vaccines compared with control. GMTs were statistically significantly lower for an HPV-16/18/31/45 AS04 vaccine containing 20/20/10/10μg VLPs for both anti-HPV-16 and anti-HPV-18 antibodies, and for an HPV-16/18/33/58 AS04 vaccine containing 20/20/20/20μg VLPs for anti-HPV-16 antibodies. There was also a trend for lower HPV-16 and -18-specific memory B-cell responses for tetravalent AS04 vaccines versus control. No such trends were observed for CD4+ T-cell responses. Immune interference could not always be overcome by increasing the dose of HPV-16/18 L1 VLPs or by using a different adjuvant system. All formulations had acceptable reactogenicity and safety profiles. Reactogenicity in the 7-day post-vaccination period tended to increase with the introduction of additional VLPs, especially for formulations containing AS01. Conclusions: HPV-16 and -18 antibody responses were lower when additional HPV L1 VLPs were added to the HPV-16/18 AS04-adjuvanted vaccine. Immune interference is a complex phenomenon that cannot always be overcome by changing the antigen dose or adjuvant system. © 2014 Elsevier Ltd., SCOPUS: ar.j, info:eu-repo/semantics/published

Tyrosine phosphorylation is a hallmark for activation of STAT proteins, but their transcriptional activity also depends on other secondary modifications. Type I IFNs can activate both the ISGF3 (STAT1:STAT2:IRF9) complex and STAT3, but with cell-specific, selective triggering of only the ISGF3 transcriptional program. Following a genome-wide RNAi screen, we identified the SIN3 transcription regulator homolog A (Sin3a) as an important mediator of this STAT3-targeted transcriptional repression. Sin3a directly interacts with STAT3 and promotes its deacetylation. SIN3A silencing results in a prolonged nuclear retention of activated STAT3 and enhances its recruitment to the SOCS3 promoter, concomitant with histone hyperacetylation and enhanced STAT3-dependent transcription. Conversely, Sin3a is required for ISGF3-dependent gene transcription and for an efficient IFN-mediated antiviral protection against influenza A and hepatitis C viruses. The Sin3a complex therefore acts as a context-dependent ISGF3/STAT3 transcriptional switch.

MF59-adjuvanted influenza vaccines have superior immunogenicity in older adults compared with non-adjuvanted vaccines. We assessed whether changing formulation (i.e., increasing H3N2 antigen or decreasing the quantity of adjuvant) of the licensed, MF59-adjuvanted trivalent influenza subunit vaccine Fluad (®) (Novartis Vaccines and Diagnostics) improves the risk-benefit profile in vaccinees aged ≥ 65 years.A significant dose-response relationship was observed between antibody levels and MF59 dose; full dose formulations elicited the strongest immune responses, meeting immunogenicity licensure criteria by Day 8. Doubling H3N2 antigen content did not increase the response to this antigen. Increased frequency of circulating CD4+ T-cells specific for vaccine antigens were detected by Day 8; magnitude and functional profile of the CD4+ T-cell response was comparable across the different vaccination groups. Mild to moderate solicited local reactions were more common with vaccines formulated with higher doses of MF59 (®) , but there were no MF59- or antigen dose-related increase in the frequency of solicited systemic reactions or unsolicited adverse events and serious adverse events.We report on 357 subjects who received one of eight intramuscular vaccine formulations. Hemagglutination-inhibiting antibodies were assayed on Days 1, 8 and 22; magnitude and functional profile of CD4+ T-cell responses to vaccine antigens were assessed in subsets. Solicited adverse reactions were reported via diary cards for seven days after vaccination and spontaneous adverse events were monitored throughout the study.This study confirms that the current formulation is the optimal one for MF59-adjuvanted influenza vaccine for use in older adults.

Summary: Bone alkaline phosphatase is a marker of osteoblast activity. In order to study the posttranscriptional modification (glycosylation) of bone alkaline phosphatase in bone disease, we investigated the relationship between mass and catalytic activity of bone alkaline phosphatase in patients with osteoporosis and hyperthyroidism. Serum bone alkaline phosphatase aetivity was measured after lectin precipitation using the Iso-ALP test kit. Mass concentration of bone alkaline phosphatase was determined with an immunoradiome tric assay (Tandem-R Ostase). In general, serum bone alkaline phosphatase mass and activity concentration correlated well. The activity : mass ratio of bone alkaline phosphatase was low in hyperthyroidi sm. Activation energy of the reaction catalysed by bone alkaline phosphatase was high in osteoporosis and in hyperthyroidism. Experiments with neuraminidase digestion further demonstrated that the thermodynamic heterogeneity of bone alkaline phosphatase can be explained by a different glycosylation of the enzyme.

An in vitro model was developed that allowed the analysis of hepatitis B virus-specific cytotoxic T lymphocyte responses in patients suffering from acute and chronic hepatitis B virus infections. Since virus-specific cytotoxic T lymphocytes recognize endogenously synthesized and processed antigen only when it is presented in the context of autologous HLA class I molecules and since hepatitis B virus does not infect human cells in vitro , a panel of recombinant vaccinia viruses was constructed to induce the expression of hepatitis B virus envelope and nucleocapsid proteins in cultured primary cells or cell lines derived from the patients to be studied. In order for a cytotoxic T lymphocyte response to be detectable with the currently available techniques, a sufficient number of activated cytotoxic T lymphocytes is required. To meet this requirement, lymphocytes freshly isolated from venous blood were stimulated in vitro with recombinant vaccinia-infected and formaldehyde-fixed autologous T lymphoblasts. The presence of hepatitis B virus-specific cytotoxic T lymphocytes, amplified and activated during this induction culture, was demonstrated in a microcytotoxicity assay using 51 Cr-labeled, recombinant vaccinia-infected Epstein-Barr virus-immortalized, autologous B lymphocytes as target cells. Using this in vitro model, we were able to demonstrate the presence of hepatitis B virus envelope- and nucleocapsid-specific cytotoxic T lymphocytes in venous blood from one subject who had recently recovered from an acute hepatitis B virus infection and in three patients suffering from chronic hepatitis B virus infections. No hepatitis B virus-specific cytotoxic T lymphocytes activity was discernible in the venous blood from two vaccine recipients. Antibody-blocking experiments showed the HLA class I restricted nature of target cell recognition.

Background The Bacille Calmette–Guerin (BCG) tuberculosis (TB) vaccine provides incomplete protection, necessitating development of an effective vaccine against TB disease. The Mtb72F/AS02 candidate vaccine was previously shown to be clinically well tolerated and immunogenic in Purified Protein Derivative (PPD)-negative adults. To improve the stability of Mtb72F, a point mutation was introduced into a putative serine protease site to give the final M72 construct. AS01 is an Adjuvant System that can potentially improve both humoral and cellular immune responses compared to the AS02 Adjuvant System or unadjuvanted vaccine. This study evaluated the safety and immunogenicity in Mtb-naive adults of vaccines containing 40 μg of the M72 antigen with AS02 or AS01 and compared the results with Mtb72F/AS02 vaccine (40 μg dose), M72 in saline (40 μg dose) and AS01 alone. Methods In this Phase I/II observer-blind controlled trial, 110 participants were randomized (4:4:1:1:1) to receive M72/AS01, M72/AS02, Mtb72F/AS02, M72/saline or AS01, following a 0, 1-month schedule. Subjects receiving the adjuvanted M72 vaccines were followed up until 3 years post vaccination. Evaluation of the immune response and safety/reactogenicity was performed. Results For all vaccines, solicited adverse events (AEs) were predominantly mild to moderate and transient. No vaccine-related serious AEs occurred and no subject withdrew due to an AE. Immune responses induced by Mtb72F and M72 antigens combined with AS02 were similar. M72/AS01 and M72/AS02 induced robust polyfunctional M72-specific CD4 + T cell and antibody responses persisting at 3 years, with the highest CD4 + T cell responses found with M72/AS01. Conclusion This first clinical study with M72/AS01 and M72/AS02 showed that both vaccines were clinically well tolerated and induced high magnitude and persistent cell-mediated and humoral immune responses. The Mtb72F/AS02 and M72/AS02 vaccines were comparably immunogenic with significantly higher immune responses compared to the M72/saline control. Of the formulations tested, M72/AS01 demonstrated significantly higher vaccine specific Th1 CD4 + T cell responses supporting its further clinical evaluation.

BACKGROUND: Modern cell-culture production techniques and the use of adjuvants helps to ensure that the global demand for pandemic influenza vaccine can be met. This study aimed to assess the immunogenicty and safety profiles of various cell-culture-derived A/H1N1 pandemic vaccine formulations in healthy adult and elderly subjects. METHODS: Adult (18-60 years) subjects (n=544) received vaccine either containing 3.75mug of antigen with half the standard dose of MF59((R)) (Novartis Vaccines and Diagnostics) adjuvant, 7.5mug antigen with a full dose of MF59, or a non-adjuvanted vaccine containing 15mug of antigen. Elderly (

BACKGROUND & AIMS: Hepatitis C virus (HCV)-induced end-stage liver disease is currently the major indication for liver transplantation in the Western world. After transplantation, the donor liver almost inevitably becomes infected by the circulating virus and disease progression is accelerated in immune suppressed transplant patients. The current standard therapy, based on pegylated interferon and ribavirin, induces severe side effects and is often ineffective in this population. Therefore, new strategies to prevent graft re-infection are urgently needed. We have previously shown that monoclonal antibodies (mAbs) against the HCV co-receptor scavenger receptor class B type I (SR-BI/Cla1) inhibit infection by different HCV genotypes in cell culture. METHODS: Using phage display libraries, we have generated a large set of novel human mAbs against SR-BI and evaluated their effectiveness in preventing HCV infection and direct cell-to-cell spread in vitro and in vivo using uPA-SCID mice with a humanized liver. RESULTS: Eleven human monoclonal antibodies were generated that specifically recognize SR-BI. Two antibodies, mAb8 and mAb151, displayed the highest binding and inhibitory properties and also interfered with direct cell-to-cell spread in vitro. Studies in humanized mice showed that both antibodies were capable of preventing HCV infection and could block intrahepatic spread and virus amplification when administered 3 days after infection. Interestingly, anti-SR-BI therapy was effective against an HCV variant that escaped the control of the adaptive immune response in a liver transplant patient. CONCLUSIONS: The anti-SR-BI mAbs generated in this study may represent novel therapeutic tools to prevent HCV re-infection of liver allografts.

BACKGROUND: Vaccines against pandemic A/H1N1 influenza are required to protect the entire population. This dose range study aimed to identify priming antigen and adjuvant doses resulting in optimal levels of antibody-mediated protection after primary and one-year booster immunizations. METHODS: This randomised trial enrolled 410 healthy adult (18-60 years) and 251 healthy elderly (>60 years) participants. Subjects received vaccine containing either 3.75 μg or 7.5 μg antigen, adjuvanted with half the standard dose, or a standard dose of MF59(®) (Novartis Vaccines) adjuvant, respectively. An additional adult cohort received non-adjuvanted vaccine containing 15 μg antigen. Two doses of investigational vaccine were administered three weeks apart, followed by a single booster dose of adjuvanted seasonal influenza vaccine one year after priming. Immunogenicity was assessed by haemagglutination inhibition and microneutralization assays pre- and post-immunization, the safety profile of each vaccine was also evaluated. RESULTS: All of the vaccine formulations investigated were highly immunogenic and well tolerated in both adult and elderly subjects. The 7.5 μg formulation induced the highest antibody titres after primary and booster immunizations, and resulted in better long-term antibody persistence, in both age groups. Assessment according to European licensure criteria for influenza vaccines concluded that single adjuvanted priming doses containing 3.75 μg and 7.5 μg antigen were optimal for the adult and elderly populations, respectively. CONCLUSIONS: These data demonstrate that one priming dose of MF59-adjuvanted A/H1N1 vaccine provided healthy adult (3.75 μg or 7.5 μg formulations) and healthy elderly (7.5 μg formulation) individuals with adequate levels of seroprotection. Booster administration after two priming doses of either vaccine formulation resulted in the rapid development of seroprotective antibody titres.

Hepatitis C virus (HCV)-infected patients undergoing liver transplantation universally experience rapid reinfection of their new liver graft. Current treatment protocols do not prevent graft reinfection and, in addition, an accelerated disease progression is observed. In the present study, we have evaluated a novel strategy to prevent HCV infection using a lectin, griffithsin (GRFT) that specifically binds N-linked high-mannose oligosaccharides that are present on the viral envelope. The antiviral effect of GRFT was evaluated in vitro using the HCV pseudoparticle (HCVpp) and HCV cell culture (HCVcc) systems. We show here that preincubation of HCVpp and HCVcc with GRFT prevents infection of Huh-7 hepatoma cells. Furthermore, GRFT interferes with direct cell-to-cell transmission of HCV. GRFT acts at an early phase of the viral life cycle by interfering in a genotype-independent fashion with the interaction between the viral envelope proteins and the viral receptor CD81. The capacity of GRFT to prevent infection in vivo was evaluated using uPA +/+ -SCID mice (uPA stands for urokinase-type plasminogen activator) that harbor human primary hepatocytes in their liver (chimeric mice). In this proof-of-concept trial, we demonstrated that GRFT can mitigate HCV infection of chimeric mice. Treated animals that did become infected demonstrated a considerable delay in the kinetics of the viral infection. Our data demonstrate that GRFT can prevent HCV infection in vitro and mitigate HCV infection in vivo . GRFT treatment of chronically infected HCV patients undergoing liver transplantation may be a suitable strategy to prevent infection of the liver allograft.

The human papillomavirus type 16/18 (HPV-16/18) AS04-adjuvanted cervical cancer vaccine is licensed for females aged 10 years and above and is therefore likely to be coadministered with other licensed vaccines, such as hepatitis B. In this randomized, open-label study, we compared the immunogenicity of the hepatitis B vaccine administered alone (HepB group) or with the HPV-16/18 AS04-adjuvanted vaccine (HepB+HPV group) in healthy women aged 20 to 25 years (clinical trial NCT00637195). The hepatitis B vaccine was given at 0, 1, 2, and 12 months (an accelerated schedule which may be required by women at high risk), and the HPV-16/18 vaccine was given at 0, 1, and 6 months. One month after the third dose of hepatitis B vaccine, in the according-to-protocol cohort (n = 72 HepB+HPV; n = 76 HepB), hepatitis B seroprotection rates (titer of ≥10 mIU/ml) were 96.4% (95% confidence interval [CI], 87.5 to 99.6) and 96.9% (CI, 89.2 to 99.6) in the HepB+HPV and HepB groups, respectively, in women initially seronegative for anti-hepatitis B surface antigen (HBs) and anti-hepatitis B core antigen (HBc). Corresponding geometric mean titers of anti-HBs antibodies were 60.2 mIU/ml (CI, 40.0 to 90.5) and 71.3 mIU/ml (CI, 53.9 to 94.3). Anti-HBs antibody titers rose substantially after the fourth dose of hepatitis B vaccine. All women initially seronegative for anti-HPV-16 and anti-HPV-18 antibodies seroconverted after the second HPV-16/18 vaccine dose and remained seropositive up to 1 month after the third dose. Both vaccines were generally well tolerated, with no difference in reactogenicity between groups. In conclusion, coadministration of the HPV-16/18 AS04-adjuvanted vaccine did not affect the immunogenicity or safety of the hepatitis B vaccine administered in an accelerated schedule in young women. Copyright © 2011, American Society for Microbiology. All Rights Reserved., SCOPUS: ar.j, info:eu-repo/semantics/published

To facilitate genotype-specific high-throughput studies of hepatitis C virus (HCV), we have developed reporter viruses using JFH1-based recombinants expressing core-nonstructural protein 2 (NS2) of genotype 1 to 7 prototype isolates. We introduced enhanced green fluorescent protein (EGFP) into NS5A domain III of the genotype 2a virus J6/JFH1 [2a(J6)]. During Huh7.5 cell culture adaptation, 2a(J6)-EGFP acquired a 40-amino-acid (aa) (Δ40) or 25-aa (Δ25) deletion in NS5A domain II, rescuing the impairment of viral assembly caused by the EGFP insertion. Δ40 conferred efficient growth characteristics to 2a(J6) tagged with EGFP, DsRed-Express2, mCherry, or Renilla luciferase (RLuc), yielding peak supernatant infectivity titers of 4 to 5 log 10 focus-forming units (FFU)/ml. 2a(J6) with Δ40 or Δ25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFPΔ40 acquired various deletions in EGFP, while 2a(J6)Δ40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5AΔ40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those of the nontagged recombinants, with peak infectivity titers of 4 to 5 log 10 FFU/ml. RLuc recombinants showed slightly less efficient growth characteristics, with peak infectivity titers up to 10-fold lower. Overall, the EGFP and RLuc recombinants were genetically stable after one viral passage. The usefulness of these reporter viruses for high-throughput fluorescence- and luminescence-based studies of HCV-receptor interactions and serum-neutralizing antibodies was demonstrated. Finally, using RLuc viruses, we showed that the genotype-specific core-NS2 sequence did not influence the response to alfa-2b interferon (IFN-alfa-2b) and that genotype 1 to 7 viruses all responded to treatment with p7 ion channel inhibitors.

Endstage liver disease caused by chronic hepatitis C virus (HCV) infection is the leading indication for liver transplantation in the Western world. However, immediate reinfection of the grafted donor liver by circulating virus is inevitable and liver disease progresses much faster than the original disease. Standard antiviral therapy is not well tolerated and usually ineffective in liver transplant patients, whereas anti-HCV immunotherapy is hampered by the extreme genetic diversity of the virus and its ability to spread by way of cell-cell contacts. We generated a human monoclonal antibody against scavenger receptor class B type I (SR-BI), monoclonal antibody (mAb)16-71, which can efficiently prevent infection of Huh-7.5 hepatoma cells and primary hepatocytes by cell-culture-derived HCV (HCVcc). Using an Huh7.5 coculture system we demonstrated that mAb16-71 interferes with direct cell-to-cell transmission of HCV. Finally we evaluated the in vivo efficacy of mAb16-71 in “human liver urokinase-type plasminogen activator, severe combined immune deficiency (uPA-SCID) mice” (chimeric mice). A 2-week anti-SR-BI therapy that was initiated 1 day before viral inoculation completely protected all chimeric mice from infection with serum-derived HCV of different genotypes. Moreover, a 9-day postexposure therapy that was initiated 3 days after viral inoculation (when viremia was already observed in the animals) suppressed the rapid viral spread observed in untreated control animals. After cessation of anti-SR-BI-specific antibody therapy, a rise of the viral load was observed. Conclusion: Using in vitro cell culture and human liver-chimeric mouse models, we show that a human mAb targeting the HCV coreceptor SR-BI completely prevents infection and intrahepatic spread of multiple HCV genotypes. This strategy may be an efficacious way to prevent infection of allografts following liver transplantation in chronic HCV patients, and may even hold promise for the prevention of virus rebound during or following antiviral therapy. (HEPATOLOGY 2012)

Background Early detection of nonresponders to hepatitis C therapy limits unnecessary exposure to treatment and its side-effects. A recent algorithm combining baseline anti-NS4a antibodies and on-treatment quantitative PCR identified nonresponders to a combination of interferon and ribavirin after 1 week of treatment. Aim To validate a stopping rule based on baseline anti-NS4a antibody levels and early on-treatment virological response in treatment-naive genotype 1 chronic hepatitis C patients treated with the current standard pegylated interferon and ribavirin combination therapy. Methods Eighty-nine genotype 1 patients from the Dynamically Individualized Treatment of hepatitis C Infection and Correlates of Viral/Host dynamics Study treated for 48 weeks with standard 180 mu g pegylated interferon (PEG-IFN)-alpha-2a (weekly) and ribavirin 1000-1200mg (daily) were analysed. Baseline anti-NS4a antibody enzyme-linked immunosorbent assay (NS4a AA 1687-1718) was performed on pretreatment serum. Hepatitis C virus-RNA was assessed at days 0, 1, 4, 7, 8, 15, 22, 29, weeks 6, 7, 8, 10, 12 and 6 weekly thereafter until end of treatment. Multiple regression logistic analysis was performed. Results Overall 54 of 89 (61%) patients achieved sustained virological response. A baseline anti-NS4a antibody titre less than 1/1250 correlated with absence of favourable initial viral decline according to variable response types (P=0.015). The optimal algorithm was developed using the combination of the absence of anti-NS4a Ab (= 100.000 IU/ml at week 4. This algorithm has a specificity of 43% and negative predictive value of 100% to detect nonresponse to standard PEG-IFN-alpha-2a and ribavirin therapy at fourth week of therapy (intention-to-treat analysis). Conclusion The decision to stop the therapy in genotype 1 chronic hepatitis C patients treated with PEG-IFN-alpha-2a and ribavirin can be confidently made after 4 weeks of treatment based on the absence of baseline anti-NS4a Ab and a week-4 hepatitis C virus-RNA above 100.000 IU/ml. Eur J Gastroenterol Hepatol 22:1443-1448 (C) 2010 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral genome and has been a prime target for drug discovery efforts. Here, we report on the further development of tetracyclic indole inhibitors, binding to an allosteric site on the thumb domain. Structure-activity relationship (SAR) studies around an indolo-benzoxazocine scaffold led to the identification of compound 33 (MK-3281), an inhibitor with good potency in the HCV subgenomic replication assay and attractive molecular properties suitable for a clinical candidate. The compound caused a consistent decrease in viremia in vivo using the chimeric mouse model of HCV infection.

International audience; A distinctive feature of HCV is that its life cycle depends on lipoprotein metabolism. Viral morphogenesis and secretion follow the very low-density lipoprotein (VLDL) biogenesis pathway and, consequently, infectious HCV in the serum is associated with triglyceride-rich lipoproteins (TRL). Lipoprotein lipase (LPL) hydrolyzes TRL within chylomicrons and VLDL but, independently of its catalytic activity, it has a bridging activity, mediating the hepatic uptake of chylomicrons and VLDL remnants. We previously showed that exogenously added LPL increases HCV binding to hepatoma cells by acting as a bridge between virus-associated lipoproteins and cell surface heparan sulfate, while simultaneously decreasing infection levels. We show here that LPL efficiently inhibits cell infection with two HCV strains produced in hepatoma cells or in primary human hepatocytes transplanted into uPA-SCID mice with fully functional human ApoB-lipoprotein profiles. Viruses produced in vitro or in vivo were separated on iodixanol gradients into low and higher density populations, and the infection of Huh 7.5 cells by both virus populations was inhibited by LPL. The effect of LPL depended on its enzymatic activity. However, the lipase inhibitor tetrahydrolipstatin restored only a minor part of HCV infectivity, suggesting an important role of the LPL bridging function in the inhibition of infection. We followed HCV cell entry by immunoelectron microscopy with anti-envelope and anti-core antibodies. These analyses demonstrated the internalization of virus particles into hepatoma cells and their presence in intracellular vesicles and associated with lipid droplets. In the presence of LPL, HCV was retained at the cell surface. We conclude that LPL efficiently inhibits HCV infection by acting on TRL associated with HCV particles through mechanisms involving its lipolytic function, but mostly its bridging function. These mechanisms lead to immobilization of the virus at the cell surface. HCV-associated lipoproteins may therefore be a promising target for the development of new therapeutic approaches.

Objective: Salt sensitivity and the magnitude of systolic blood pressure have been linked to haptoglobin (Hp) polymorphism in normotensives. The aim of the present study was to investigate the indices of hypertension, the severity of complications and the occurrence of coronary and peripheral artery disease for the various haptoglobin phenotypes and their relation to the therapeutic needs (number and class of drugs) of established arterial hypertensives Design: Haptoglobin polymorphism was studied in 302 Caucasians with established essential arterial hypertension who had been treated for at least 1 year Methods: Haptoglobin polymorphism was studied using starch-gel electrophoresis of haemoglobin-supplemented serum Results: The relative allele frequencies of Hp 1 and Hp2 (0.360 and 0.640, respectively) in established hypertensives were comparable with those of the control population. Logistic regression analysis confirmed that Hp2-2 contributes to the therapeutic needs in hypertension. The most important factors determining therapeutic needs were coronary artery disease, Hp2-2 phenotype, body mass index (BMI) and left ventricular hypertrophy. Although no contributive effect of serum haptoglobin concentration could be derived from the logistic regression approach, analysis of serum haptoglobin concentration demonstrated a concentration-related effect on therapeutic needs for the Hp2-2 phenotype only Conclusions: The present study suggests that hypertensives with an Hp 2-2 phenotype need more complex combinations of antihypertensive drugs to reduce blood pressure to the same level. The hypertensive patient carrying Hp 2-2 is more likely to accumulate atherosclerotic lesions of the coronary or peripheral arteries, despite comparable lipid levels, smoking habits and BMI. Hp1-1 patients are characterized by a younger age at diagnosis and a lower complication rate. In view of the greater therapeutic needs and the higher complication rate, Hp2-2 hypertensives need more careful follow-up

Control of hepatitis C virus (HCV) infection remains a huge challenge of global medical importance. Using a variety of in vitro approaches, neutralizing antibodies (nAbs) have been identified in patients with acute and chronic hepatitis C. The exact role these nAbs play in the resolution of acute HCV infection still remains elusive. We have previously shown that purified polyclonal antibodies isolated from plasma obtained in 2003 from a chronic HCV patient (Patient H) can protect human liver chimeric mice from a subsequent challenge with the autologous HCV strain isolated from Patient H in 1977 (H77). In this study we investigated whether polyclonal antibodies isolated from Patient H in 2006 (H06), which display high cross-genotype neutralizing activity in both the HCV pseudoparticle (HCVpp) and HCV cell culture (HCVcc) systems, were also able to prevent HCV infection of different genotypes (gt) in vivo. Following passive immunization with H06-antibodies, chimeric mice were challenged with the consensus strains H77C (gt1a), ED43 (gt4a), or HK6a (gt6a). In accordance with previous results, H06-antibodies prevented infection of chimeric mice with the autologous virus. However, the outcome of a homologous challenge is highly influenced by the amount of challenge virus injected. Depending on the viral genotype used, H06-antibodies were able to protect up to 50% of chimeric mice from a heterologous challenge. Animals in which the antibody pretreatment failed displayed a clear delay in the kinetics of viral infection. Sequence analysis of the recovered viruses did not suggest antibody-induced viral escape. Conclusion: Polyclonal anti-HCV antibodies isolated from a chronic HCV patient can protect against an in vivo challenge with different HCV genotypes. However, the in vivo protective efficacy of cross-genotype neutralizing antibodies was less than predicted by cell culture experiments. (HEPATOLOGY 2011)

End-stage liver disease caused by chronic hepatitis C virus (HCV) infection is a leading cause for liver transplantation (LT). Due to viral evasion from host immune responses and the absence of preventive antiviral strategies, reinfection of the graft is universal. The mechanisms by which the virus evades host immunity to reinfect the liver graft are unknown. In a longitudinal analysis of six HCV-infected patients undergoing LT, we demonstrate that HCV variants reinfecting the liver graft were characterized by efficient entry and poor neutralization by antibodies present in pretransplant serum compared with variants not detected after transplantation. Monoclonal antibodies directed against HCV envelope glycoproteins or a cellular entry factor efficiently cross-neutralized infection of human hepatocytes by patient-derived viral isolates that were resistant to autologous host-neutralizing responses. These findings provide significant insights into the molecular mechanisms of viral evasion during HCV reinfection and suggest that viral entry is a viable target for prevention of HCV reinfection of the liver graft.

Combined HibMenCY and HibMenC conjugate vaccines may facilitate inclusion of vaccination against MenC and MenY into routine vaccination schedules, without additional injections. Immunogenicity and reactogenicity of vaccination with three different formulations of a novel HibMenCY-conjugate vaccine, or a HibMenC-conjugate vaccine was assessed. Infants were randomized to receive either Hib(2.5 µg)-MenC(5 µg)-MenY(5 µg)-TT, Hib(5 µg)-MenC(10 µg)-MenY(10 µg)-TT, Hib(5 µg)-MenC(5 µg)-MenY(5 µg)-TT or Hib(5 µg)-MenC(5 µg)-TT vaccines co-administered with DTPa-HBV-IPV at 2-3-4 months of age. Controls received licensed conjugate MenC-CRM197 vaccine co-administered with DTPa-HBV-IPV/Hib. A fourth dose was administered to a subset of children at age 12-18 months. Anti-PRP concentrations and meningococcal bactericidal (rSBA-MenC/Y) titres were measured prior to and one month post third and fourth vaccination dose. Solicited local, general symptoms and unsolicited adverse events were recorded for 7 and 30 days after each vaccination, respectively. Post dose 3, all subjects had anti-PRP antibody levels ≥ 0.15 µg/ml and rSBA-MenC ≥ 1:8. 97.0%-98.6% of HibMenCY recipients had rSBA-MenY ≥ 1:8. Pre-dose-4, 95.6%-100% of HibMenCY and HibMenC recipients had anti-PRP ≥ 0.15 µg/ml and 90.7%-97.6% recipients had rSBA-MenC titres ≥ 1:8. In HibMenCY groups, 78.6%-86.7% had persisting rSBA-MenY ≥ 1:8. The post-dose-4 response was robust after all vaccines with all subjects having anti-PRP ≥ 1 µg/ml and 92.3%-100% rSBA-MenC ≥ 1:128. All HibMenCY recipients had rSBA-MenY ≥ 1:128. Vaccination with the novel Hib-meningococcal vaccines had a safety profile similar to control. HibMenCY and HibMenC conjugate vaccine formulations given at 2-3-4 months of age with a fourth dose in the second year of life were immunogenic and had a comparable safety profile to licensed vaccines. (study 792014 and 100381;www.clinicaltrial.govID:NCT00129116)

Waning immunity to mumps after one or two doses of the measles, mumps and rubella (MMR) vaccine has been described. Using a human peripheral blood lymphocyte (PBL)–severe combined immunodeficiency (SCID) mouse model, MMR vaccine recipients with undetectable and high antibody titres against mumps were compared for the presence of circulating mumps-specific memory B cells. Peripheral blood mononuclear cells (PBMC) from six donors (three subjects with undetectable and three with high antibody titres against mumps) were injected into the spleens of non-obese diabetic (NOD)-SCID mice (three mice per subject). Mice were pretreated with TMβ1 and total body irradiation to improve engraftment. In vivo production of human antibodies against mumps was evaluated in mouse plasma on days 7, 10 and 13 with a commercial enzyme-linked immunosorbent assay (ELISA), functional reduction neutralization test. Three donors had mumps antibody titres below the detection limit (titre < 230) and three had high antibody titres (range 5700–7300). None of the mice injected with PBMC from subjects with undetectable antibody titres showed detectable human antibody titres, despite the presence of cell-mediated immunity in two of the three donors. Seven out of nine mice injected with PBMC from subjects with high antibody titres acquired detectable antibody titres for mumps in their plasma. PBMC from vaccinees without detectable serum antibodies against mumps virus were unable to induce secretion of anti-mumps antibodies in the blood of recipient mice, whereas PBMC from vaccinees with high antibody titres were able to do so. This observation suggests that the frequency of mumps-specific memory B cells is very low in vaccinees with undetectable antibody titres. These individuals may therefore be at risk of developing mumps disease upon encounter with wild-type virus.

SummaryWaning immunity to mumps after one or two doses of the measles,mumps and rubella (MMR) vaccine has been described. Using a humanperipheral blood lymphocyte (PBL)–severe combined immunodeficiency(SCID) mouse model, MMR vaccine recipients with undetectable and highantibody titres against mumps were compared for the presence of circu-lating mumps-specific memory B cells. Peripheral blood mononuclear cells(PBMC) from six donors (three subjects with undetectable and three withhigh antibody titres against mumps) were injected into the spleens ofnon-obese diabetic (NOD)-SCID mice (three mice per subject). Mice werepretreated with TMb1 and total body irradiation to improve engraftment.In vivo production of human antibodies against mumps was evaluated inmouse plasma on days 7, 10 and 13 with a commercial enzyme-linkedimmunosorbent assay (ELISA), functional reduction neutralization test.Three donors had mumps antibody titres below the detection limit (titre< 230) and three had high antibody titres (range 5700–7300). None of themice injected with PBMC from subjects with undetectable antibody titresshowed detectable human antibody titres, despite the presence of cell-mediated immunity in two of the three donors. Seven out of nine miceinjected with PBMC from subjects with high antibody titres acquireddetectable antibody titres for mumps in their plasma. PBMC from vacci-nees without detectable serum antibodies against mumps virus wereunable to induce secretion of anti-mumps antibodies in the blood of reci-pient mice, whereas PBMC from vaccinees with high antibody titres wereable to do so. This observation suggests that the frequency of mumps-spe-cific memory B cells is very low in vaccinees with undetectable antibodytitres. These individuals may therefore be at risk of developing mumpsdisease upon encounter with wild-type virus.Keywords: B cell; memory; mumps; vaccination; viral

Anabolic steroids are among the most frequently detected compounds in doping analysis. They are extensively metabolized and therefore an in-depth knowledge about steroid metabolism is needed. In this study, a liquid chromatography tandem mass spectometry (LC-MS/MS) method based on a precursor ion scan with a uPA-SCID mouse with humanized liver (a chimeric mouse) was explored for the detection of steroid metabolism. Methandienone was used as a model compound. The application of the precursor ion scan method in positive human samples and chimeric mice samples after methandienone administration allowed the detection of most steroid metabolites without any structural restriction. Three hitherto unreported metabolites were found using this approach. These metabolites were characterized using LC-MS/MS and feasible structures were proposed. The structure of one of them, 6-ene-epimethandienone, was confirmed by the synthesis of the reference compound. A selected reaction monitoring (SRM) method for the specific detection of all these metabolites has been developed. The application of this method to several human and chimeric mouse samples confirmed that more than 80% of the steroid metabolites were found in both samples. Only metabolites that are poorly detectable by LC-MS/MS were not detected in some urine samples. The metabolic nature of the unreported metabolites was also confirmed. A global strategy for the detection of steroid metabolites combining both human and chimeric mouse urine is proposed.

Tuberculosis (TB) remains a major cause of illness and death worldwide, making a new TB vaccine an urgent public health priority. Purified protein derivative (PPD)-negative adults ( n = 50) were equally randomized to receive 3 doses at 1-month intervals (at 0, 1, and 2 months) of one of the following vaccines: Mtb72F/AS02 A (10 or 40 μg antigen), Mtb72F/saline (10 or 40 μg antigen), or AS02 A . Mtb72F/AS02 A recipients received an additional dose 1 year after the first dose to evaluate if the elicited immune response could be boosted. Mtb72F/AS02 A vaccines were locally reactogenic but clinically well tolerated, with transient adverse events (usually lasting between 1 and 4 days) that resolved without sequelae being observed. No vaccine-related serious adverse events were reported. Vaccination with Mtb72F/AS02 A induced a strong Mtb72F-specific humoral response and a robust Mtb72F-specific CD4 + T-cell response, both of which persisted at 9 months after primary immunization and for 1 year after the booster immunization. There was no significant difference between the magnitude of the CD4 + T-cell response induced by the 10-μg and 40-μg Mtb72F/AS02 A vaccines. The Mtb72F-specific CD4 + T cells predominantly expressed CD40L; CD40L and interleukin-2 (IL-2); CD40L and tumor necrosis factor alpha (TNF-α); CD40L, IL-2, and TNF-α; and CD40L, IL-2, TNF-α, and gamma interferon (IFN-γ). Serum IFN-γ, but not TNF-α, was detected 1 day after doses 2 and 3 for the Mtb72F/AS02 A vaccine but did not persist. Vaccine-induced CD8 + T-cell responses were not detected, and no immune responses were elicited with AS02 A alone. In conclusion, Mtb72F/AS02 A is clinically well tolerated and is highly immunogenic in TB-naïve adults. The 10- and 40-μg Mtb72F/AS02 A vaccines show comparable safety and immunogenicity profiles.

Human creatine kinase (CK) was demonstrated to be partly present as a glycated molecule. Sialic acid, galactose and sulfate were also found to be present on the molecule. The glycated forms were characterized by higher activation energies and were thermally unstable. Skeletal muscle CK showed lower relative binding towards the lectin concanavalin A (Con A) in comparison with the heart tissue forms. After skeletal muscle trauma, CK in serum was found to be less glycated than in tissue. After acute myocardial infarction (AMI), no glycated CK could be detected in serum. Following injury, it appears that the transfer from tissue to plasma is accompanied by a loss of glycated isoforms. High-voltage electrophoresis showed no differences in the distribution of CK isoforms between the glycated and non-glycated forms.

SummaryWe have examined the prognostic value of the levels in the blood of granulocyte elastase-α1-proteinase inhibitor (E-α1-PI) complex, tumor necrosis factor-α (TNF-α) and urokinase-type plasminogen activator (u-PA) in 35 patients with severe infection upon admission to an Intensive Care Unit. Fourteen patients died.No differences for E-α1-PI complex were found between survivors and nonsurvivors, but in all patients the levels on admission were eight-fold higher than the reference value.TNF-α levels, measured by immunoassay, on admission were four times higher in the nonsurvivors than in the survivors (p = 0.0003) and correlated with the severity of the disease (APACHE II score, r = 0.43, p Total u-PA antigen (u-PA Ag), plasmin-activatable single-chain u-PA (scu-PA) and inactive, nonactivatable u-PA (u-PA#) were on admission all two-fold higher in the nonsurvivors (p = 0.0006, 0.003 and 0.0003, respectively), while normal in the survivors. In both, survivors and nonsurvivors, the ratio between scu-PA and u-PA Ag was significantly decreased (p In a stepwise logistic regression analysis, documentation of infection and plasma levels of u-PA Ag contributed most significantly to prediction of patient outcome. Serum levels of TNF-α did not. These results suggest that, in addition to a number of other clinical and laboratory parameters, u-PA Ag can be used as a prognostic marker in patients with severe infection admitted to an Intensive Care Unit.

Background & Aims: The human liver-uPA(+/+)-SCID mouse is currently the best small animal model available for viral hepatitis infection studies. Methods: We identify critical factors affecting animal survival, engraftment efficacy, kinetics of liver repopulation and virological outcome by analysing the data from 400 human hepatocyte transplantations and 115 subsequent HBV and/or HCV inoculations in this mouse model. Results: Almost one third of animals succumbed during the first week after hepatocyte transplantation. Only during this critical period, liver necrosis due to embolization of donor cells in the portal vein was observed. This may have caused a fatal acute liver failure that complicated the pre-existing chronic liver disease. From the second week onwards, confluent hepatocyte clusters repopulated the liver and restored its synthetic functions as evidenced by increasing human albumin levels in plasma. Xenogenic repopulation by human cells proceeded approximately 4-times slower compared to allogenic mouse hepatocytes. All HBV inoculations were successful, even in animals with low graft take. HCV infection rate varied substantially, although every donor cell type yielded infectable animals. A reproducible 100% HCV infectivity was reached with high quality inocula in animals with human albumin plasma levels >1 mg/ml. Superior animal survival, adequate liver engraftment and a high viral infection rate were observed after transplanting cryopreserved commercial human hepatocytes. Conclusions: Our findings favour the use of commercially available, cryopreserved human hepatocytes for the humanization of the uPA(+/+)-SCID liver. While HBV infectivity criteria are less stringent, human albumin plasma levels exceeding 1 mg/ml are required for a consistent HCV infection in chimeric mice. (C) 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

We evaluated the humoral and cellular immunogenicity of adjuvanted and non-adjuvanted H5N1 influenza vaccine in two groups of 300 adults: aged 18-60 and >60 years in a randomized, open-label, uncontrolled phase 2 trial. Participants received two injections (D0, D21) of 7.5 microg hemagglutinin without adjuvant or 30 microg with aluminum hydroxide adjuvant. Antibody responses and cytokine secretion were assessed before and after vaccination. Excluding the 6/300 non-elderly and 47/300 elderly participants with pre-existing antibodies, geometric mean titers (dil(-1)) on D42 were higher with 30 microg+Ad and were comparable between age groups. Participants with pre-existing antibodies responded strongly to the first vaccination (GMTs in the range 147-228 on D21). Vaccination increased both Th1 and Th2 T-cell responses. The predominantly Th1 profile observed before vaccination was unaffected by vaccination. H5N1 influenza vaccine is no less immunogenic in elderly adults than in younger adults and, due to a higher proportion non-naïve elderly, immunogenicity was higher in this latter group.

With the advent of subgenomic hepatitis C virus (HCV) replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs), previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A), but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I) as well as NS5A (S2204R), whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes., Author Summary The hepatitis C virus (HCV) is a major cause of acute and chronic liver disease. Unusual for a positive strand RNA virus, HCV has the high propensity to establish persistent infection, which increases the risk for liver cirrhosis and hepatocellular carcinoma. No selective therapy is available thus far and its development has been hampered by the lack of adequate cell culture systems. With the advent of subgenomic replicons, i.e. RNAs containing only the viral replicase genes and that self-amplify in the human liver cell line Huh-7, this hurdle has been overcome to some extent. However, save for a single genotype 2a isolate, efficient replication of all HCV isolates described thus far requires replication enhancing mutations (REMs), but genomes with REMs do not support production of infectious virus particles. In this study we show that except for one mutation in non-structural protein 4B, REMs interfere with the assembly of infectious virus particles, whereas an unaltered HCV genome supports production of cell culture–derived virus that is infectious in vitro and in vivo. Our observations provide an explanation for the attenuation of cell culture adapted HCV genomes and open new perspectives for the development of culture systems for difficult to treat HCV genotypes.

In 2006 an outbreak of mumps occurred in the US and amazingly the majority of cases were individuals that had received two vaccine doses. This unexpected outbreak may be due to the low immunogenicity of the mumps vaccine, insufficient vaccination coverage or a combination of both. Both humoral and cellular immune responses induced by the mumps vaccine are considered to contribute to the vaccine-induced protection. The magnitude and persistence of these responses determine the overall efficacy of the vaccine and ultimately the degree of vaccination coverage that needs to be reached to eliminate the mumps virus. Although Finland and the US have used the same mumps vaccine for decades, a mumps outbreak has been reported in the US while in Finland this virus appears to be eliminated. In this commentary we speculate on the factors that may be responsible for this striking difference.

1University of Medicine “Gr T Popa” Iasi and Hospital “C. I. Parhon” Iasi, 2Hopital Erasme, Departement Medico-Chirurgical de Nephrologie, Dialyse et Transplantation, 808 Route de Lennik Brussels, Belgium, 3Huddinge University Hospital, Department of Clinical Science, Karolinska Institutet, Division of Renal Medicine, Huddinge/Stockholm, Sweden, 4University Hospital Ghent, Internal Medicine, De Pintelaan 185, 9000 Ghent, Belgium, 5Centre Hepato-Biliaire, Assistance Publique-Hopitaux de Paris Hopital Paul Brousse and Institut National de la Sante et la Recherche Medicale 785, Villejuif, France, 6University Hospital Ghent, Nephrology Department, De Pintelaan 185, 9000 Ghent, Belgium, 7Ospedali Riuniti, CNR IBIM Epidemiologia Clinica delle Malattie Renali e dellIpertensione Arteriosa e Unita Operativa, 89124 Reggio Cal, Italy and 8INSERM Unit 271, Internal Medicine, Lyon, France

The study evaluated and compared the prevalence of anti-HBs and exposure to hepatitis B virus (HBV) in vaccinated South African babies aged between 5 and 24 months from the Expanded Programme on Immunisation clinic [EPI group] and paediatric outpatient clinic [OPD group], and results were stratified by HIV status. A total of 303 (243 EPI group and 60 OPD group) babies were studied. All sera were tested for anti-HBs, HBsAg and anti-HBc, while IgM anti-HBc and HBV DNA were only tested in samples positive for HBsAg and/or anti-HBc. Overall, there was a gross difference in the prevalence of anti-HBs marker between the EPI and OPD groups. The EPI group demonstrated higher levels of seroconversion (89.3% vs. 81.7%; p = 0.105) and seroprotection rates (86.0% vs. 75.0%; p = 0.038), compared to the OPD babies. When the overall results were stratified by HIV status, seroprotection was 85.7% for the HIV-negatives and 78.1% for the HIV-positives, although this was not statistically significant (p = 0.125). The seroprotection rates were almost comparable between the HIV-positives (84.3%; n = 51) and the HIV-negatives (86.5%; n = 192) (p = 0.695) in the EPI group. In contrast, reduced seroprotection rates were observed between the HIV-positives (63.6%; n = 22) and HIV-negatives (81.6%; n = 38) in the OPD group, although this was not statistically significant (p = 0.123). Interestingly, no HBsAg or anti-HBc marker was detected in the OPD group, compared to total exposure rate of 4.9% (HBsAg carriage was 1.2%) in the EPI group.

The viral life cycle of the hepatitis C virus (HCV) has been studied mainly using different in vitro cell culture models. Studies using pseudoviral particles (HCVpp) and more recently cell culture–derived virus (HCVcc) suggest that at least three host cell molecules are important for HCV entry in vitro: the tetraspanin CD81, the scavenger receptor class B member I, and the tight junction protein Claudin-1. Whether these receptors are equally important for an in vivo infection remains to be demonstrated. We show that CD81 is indispensable for an authentic in vivo HCV infection. Prophylactic treatment with anti-CD81 antibodies completely protected human liver-uPA-SCID mice from a subsequent challenge with HCV consensus strains of different genotypes. Administration of anti-CD81 antibodies after viral challenge had no effect. Conclusion: Our experiments provide evidence for the critical role of CD81 in a genuine HCV infection in vivo and open new perspectives for the prevention of allograft reinfection after orthotopic liver transplantation in chronically infected HCV patients. (HEPATOLOGY 2008;48:1761–1768.)

Four conserved RNA stem-loop structures designated SL47, SL87, SL248, and SL443 have been predicted in the hepatitis C virus (HCV) core encoding region. Moreover, alternative translation products have been detected from a reading frame overlapping the core gene (core+1/ARFP/F). To study the importance of the core+1 frame and core-RNA structures for HCV replication in cell culture and in vivo, a panel of core gene silent mutations predicted to abolish core+1 translation and affecting core-RNA stem-loops were introduced into infectious-HCV genomes of the isolate JFH1. A mutation disrupting translation of all known forms of core+1 and affecting SL248 did not alter virus production in Huh7 cells and in mice xenografted with human liver tissue. However, a combination of mutations affecting core+1 at multiple codons and at the same time, SL47, SL87, and SL248, delayed RNA replication kinetics and substantially reduced virus titers. The in vivo infectivity of this mutant was impaired, and in virus genomes recovered from inoculated mice, SL87 was restored by reversion and pseudoreversion. Mutations disrupting the integrity of this stem-loop, as well as that of SL47, were detrimental for virus viability, whereas mutations disrupting SL248 and SL443 had no effect. This phenotype was not due to impaired RNA stability but to reduced RNA translation. Thus, SL47 and SL87 are important RNA elements contributing to HCV genome translation and robust replication in cell culture and in vivo.

Influenza vaccines remain largely underused. A promising alternative to Current intramuscular vaccines is a trivalent inactivated influenza vaccine (TIV) delivered using a microinjection system to offer a less invasive and possibly more acceptable vaccination. A phase 11, multicentre, randomised open-label study in 978 healthy adults (18-57 years) evaluated the immunogenicity and safety of intradermal TIV. Subjects received a 0.1 ml injection of intradermal TIV, containing 9 mu g of haemagglutinin (HA) per strain (it = 588) or a conventional 0.5 ml intramuscular vaccine (15 mu g of HA/strain: n = 390). Intradermal,11 TIV induced non-inferior humoral immune responses against all three strains and superior responses against both A strains (H1N1, H3N2) compared with the control. Both vaccines were well tolerated. (C) 2008 Elsevier Ltd. All rights reserved.

The role of the humoral immune response in the natural course of hepatitis C virus (HCV) infection is widely debated. Most chronically infected patients have immunoglobulin G (IgG) antibodies capable of neutralizing HCV pseudoparticles (HCVpp) in vitro. It is, however, not clear whether these IgG can prevent a de novo HCV infection in vivo and contribute to the control of viremia in infected individuals. We addressed this question with homologous in vivo protection studies in human liver–urokinase-type plasminogen activator (uPA)+/+ severe combined immune deficient (SCID) mice. Chimeric mice were loaded with chronic phase polyclonal IgG and challenged 3 days later with a 100% infectious dose of the acute phase H77C virus, both originating from patient H. Passive immunization induced sterilizing immunity in five of eight challenged animals. In the three nonprotected animals, the HCV infection was attenuated, as evidenced by altered viral kinetics in comparison with five control IgG-treated animals. Plasma samples obtained from the mice at viral challenge neutralized H77C-HCVpp at dilutions as high as 1/400. Infection was completely prevented when, before administration to naive chimeric mice, the inoculum was pre-incubated in vitro at an IgG concentration normally observed in humans. Conclusion: Polyclonal IgG from a patient with a long-standing HCV infection not only displays neutralizing activity in vitro using the HCVpp system, but also conveys sterilizing immunity toward the ancestral HCV strain in vivo, using the human liver–chimeric mouse model. Both experimental systems will be useful tools to identify neutralizing antibodies for future clinical use. (HEPATOLOGY 2008.)

The hepatitis B virus core (HBc) virus-like particle (VLP) is known as one of the most immunogenic antigens and carrier vehicles in different immunization strategies. Recent findings are suggesting the potential of the HBc VLPs as an oral immunogen. Here, we focus on the induction of serum humoral responses by oral administration of HBc VLPs in preparations substantially free of lipopolysaccharide and immunomodulating encapsidated RNA. The full-length HBc antigen was used, because the C-terminal arginine-rich tail may contribute to the immunogenicity of the antigen as the region is involved in cell surface heparan sulfate binding and internalization of the protein. Serum antibody levels and isotypes were determined following oral administration of the HBc VLPs with the perspective of using the HBc VLP as an immunostimulatory and carrier molecule for epitopes of blood-borne diseases in oral immunization vaccination strategies. Following oral administration of the HBc VLP preparations to mice, a strong serum humoral response was induced with mainly immunoglobulin G2a (IgG2a) antibodies, pointing toward a Th1 response which is essential in the control of intracellular pathogens. Intraperitoneal immunization with the HBc VLP induced a stronger, mixed Th1/Th2 response. Finally, a comparison was made with the induced serum humoral response following oral administration of the recombinant cholera toxin B pentamer, a commonly used oral immunogen. These immunizations, in contrast, induced predominantly antibodies of the IgG1 isotype, indicative of a Th2 response. These data suggest that the HBc VLP can be an interesting carrier molecule in oral vaccine development.

BackgroundThe availability of H5N1 vaccines that can elicit a broad cross-protective immunity against different currently circulating clade 2 H5N1 viruses is a pre-requisite for the development of a successful pre-pandemic vaccination strategy. In this regard, it has recently been shown that adjuvantation of a recombinant clade 1 H5N1 inactivated split-virion vaccine with an oil-in-water emulsion-based adjuvant system also promoted cross-immunity against a recent clade 2 H5N1 isolate (A/Indonesia/5/2005, subclade 2.1). Here we further analyse the cross-protective potential of the vaccine against two other recent clade 2 isolates (A/turkey/Turkey/1/2005 and A/Anhui/1/2005 which are, as defined by WHO, representatives of subclades 2.2 and 2.3 respectively).Methods and findingsTwo doses of the recombinant A/Vietnam/1194/2004 (H5N1, clade 1) vaccine were administered 21 days apart to volunteers aged 18-60 years. We studied the cross-clade immunogenicity of the lowest antigen dose (3.8 microg haemagglutinin) given with (N = 20) or without adjuvant (N = 20). Immune responses were assessed at 21 days following the first and second vaccine doses and at 6 months following first vaccination. Vaccination with two doses of 3.8 microg of the adjuvanted vaccine induced four-fold neutralising seroconversion rates in 85% of subjects against A/turkey/Turkey/1/2005 (subclade 2.2) and 75% of subjects against A/Anhui/1/2005 (subclade 2.3) recombinant strains. There was no response induced against these strains in the non-adjuvanted group. At 6 months following vaccination, 70% and 60% of subjects retained neutralising antibodies against the recombinant subclade 2.2 and 2.3 strains, respectively and 40% of subjects retained antibodies against the recombinant subclade 2.1 A/Indonesia/5/2005 strain.ConclusionsIn addition to antigen dose-sparing, adjuvantation of inactivated split H5N1 vaccine promotes broad and persistent cross-clade immunity which is a pre-requisite for a pre-pandemic vaccine.Trial registrationClinicalTrials.gov NCT00309634.

Production of infectious hepatitis C virus in cell culture has become possible because of the unique properties of the JFH1 isolate. However, virus titers are rather low, limiting the utility of this system. Here we describe the generation of cell culture-adapted JFH1 variants yielding higher titers of infectious particles and enhanced spread of infection in cultured cells. Sequence analysis of adapted genomes revealed a complex pattern of mutations that differed in two independent experiments. Adaptive mutations were observed both in the structural and in the nonstructural regions, with the latter having the highest impact on enhancement of virus titers. The major adaptive mutation was identified in NS5A, and it enhanced titers of three intergenotypic chimeras consisting of the structural region of a genotype 1a, 1b, or 3a isolate and the remainder of the JFH1 isolate. The mutation resides at the P3 position of the NS5A-B cleavage site and slows down processing, implying that subtle differences in replication complex formation appear to determine the efficiency of virus formation. Highly adapted JFH1 viruses carrying six mutations established a robust infection in uPA-transgenic SCID mice xenografted with human hepatocytes. However, the mutation in NS5A which enhanced virus titers in cell culture the most had reverted to wild type in nearly half of the viral genomes isolated from these animals at 15 weeks postinoculation. These results argue for some level of impaired fitness of this mutant in vivo.

Hepatitis C virus (HCV) enters cells via a pH- and clathrin-dependent endocytic pathway. Scavenger receptor BI (SR-BI) and CD81 are important entry factors for HCV internalization into target cells. The SR-BI gene gives rise to at least two mRNA splice variants, SR-BI and SR-BII, which differ in their C termini. SR-BI internalization remains poorly understood, but SR-BII is reported to endocytose via a clathrin-dependent pathway, making it an attractive target for HCV internalization. We demonstrate that HCV soluble E2 can interact with human SR-BI and SR-BII. Increased expression of SR-BI and SR-BII in the Huh-7.5 hepatoma cell line enhanced HCV strain J6/JFH and JFH infectivity, suggesting that endogenous levels of these receptors limit infection. Elevated expression of SR-BI, but not SR-BII, increased the rate of J6/JFH infection, which may reflect altered intracellular trafficking of the splice variants. In human plasma, HCV particles have been reported to be complexed with lipoproteins, suggesting an indirect interaction of the virus with SR-BI and other lipoprotein receptors. Plasma from J6/JFH-infected uPA-SCID mice transplanted with human hepatocytes demonstrates an increased infectivity for SR-BI/II-overexpressing Huh-7.5 cells. Plasma-derived J6/JFH infectivity was inhibited by an anti-E2 monoclonal antibody, suggesting that plasma virus interaction with SR-BI was glycoprotein dependent. Finally, anti-SR-BI antibodies inhibited the infectivity of cell culture- and plasma-derived J6/JFH, suggesting a critical role for SR-BI/II in HCV infection.

Although a limited number of HIV-infected patients have broadly neutralizing antibodies, it has not been examined whether these antibodies can protect against infection with primary virus in vivo. Here we screened the plasma of 23 HIV-1-infected patients for broadly neutralizing antibodies. Purified antibodies from subjects with broad and more narrow responses were administered to huPBL-NOD/Scid mice that were subsequently challenged with primary viruses of clade A, B and CRF01_AE. Although we observed a lack of correlation between the data from the in vitro neutralization assay and the results from the passive immunization experiments, we report for the first time that antibodies from HIV-infected persons can inhibit replication of primary virus isolates in an animal model.