Jean-François Meritet

Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists.

A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means for correcting for serum matrix effects and allows residual drug levels or anti-drug neutralizing antibodies to be quantified even in serum samples with a relatively high degree of cytotoxicity.

Culture-proven herpetic keratitis after penetrating keratoplasty in patients with no previous history of herpes disease.

Objective To report three cases of herpetic infection in recipients of organ-cultured donor corneas among 586 consecutive corneal transplantation procedures. Methods Three patients with no history of symptomatic herpes infection underwent corneal transplantation for keratoconus (2 patients) and Fuchs dystrophy (1 patient). Two patients developed keratouveitis and primary graft failure. The third patient developed dendritic keratitis in the graft. Culture of corneal scrapings and the patients bandage contact lens were positive for herpes simplex virus type 1 (HSV-1). Donor and recipient sera were tested for HSV serology by EIA. Recipient corneal buttons were studied by means of transmission electron microscopy and immunohistochemistry. The three HSV-1 strains were genotyped by sequencing part of a variable antigenic domain of glycoprotein B (gB). Results None of the donor corneas showed endothelial cell necrosis after organ culture. All keratoplasties performed with the three mate donor corneas had an uncomplicated course. All three donor sera were positive for HSV. Preoperative recipient sera were positive for HSV. Analysis of the recipient corneal buttons showed no evidence of herpetic infection. Sequence analysis revealed three different gB genotypes. Conclusion Ascertaining that a postoperative herpetic infection in a corneal transplant originates from the donor tissue is still difficult. Although some features of the reported cases suggest donor-to-host transmission of herpes simplex virus, the recipients could have been the source of the virus.

Human bocavirus infection in hospitalized children during winter.

Human bocavirus (HBoV) has recently been described as a common agent of acute upper and lower respiratory tract infections in children. We screened by polymerase chain reaction for HBoV nucleic acid nasopharyngeal aspirates from hospitalized children with negative culture and immunofluorescence assay for respiratory syncytial virus, influenza viruses, adenovirus, and parainfluenza viruses. HBoV was detected in 32 children (5.5%) and was the second virus identified in nasopharyngeal aspirates after respiratory syncytial virus. Most of the children had severe disease.

One-step assay for quantification of neutralizing antibodies to biopharmaceuticals

Assessment of immunogenicity is an important part of biopharmaceutical drug safety evaluation and a prerequisite for the development of less immunogenic and safer biopharmaceuticals since anti-drug antibodies can impair the activity and compromise the safety of biopharmaceuticals. Although regulatory authorities recommend cell-based assays for detection of neutralizing antibodies (NAbs), such assays are difficult to standardize, and ill adapted to high-throughput analysis. These limitations have been overcome by the development of a unique one-step cell-based assay that allows both drug activity and drug NAbs to be quantified rapidly and with a high degree of precision simply be adding reporter cells to a sample. The reporter cells have been engineered to express firefly luciferase (FL) under the control of a drug-responsive promoter, and to express the drug of interest, the production of which is normalized relative to the expression of Renilla luciferase (RL) transcribed from a common doxycycline-inducible promoter. Residual drug levels present in a sample are first quantified by determination of FL expression, autocrine drug synthesis is then induced, and NAb activity is quantified from the difference in the ratio of FL/RL expression in the presence or absence of the sample. Since assay results are normalized relative to the expression of an internal standard, results are independent of cell number or differences in cell viability thus affording a high degree of assay precision and reducing serum matrix effects to a minimum. This unique assay platform is ideally suited for high-throughput analysis, is applicable to most biopharmaceuticals, and will facilitate standardization and comparison of immunogenicity data. The performance of the one-step assay is illustrated for interferon alpha2 (IFNalpha2) used widely to treated chronic hepatitis C (HCV) infection and neoplastic disease.

GAAP‐1: a transcriptional activator of p53 and IRF‐1 possesses pro‐apoptotic activity

The mechanisms that regulate the transcription of the tumour suppressor genes p53 and IRF‐1 are poorly understood. We have characterized a 68‐kDa transcription factor, GAAP‐1 (gatekeeper of apoptosis activating proteins), encoded by an alternative splice product of the PRDII‐BF1 gene, that recognizes a novel regulatory element within the p53 and IRF‐1 promoters. Transfection of U937 cells with GAAP‐1 activates p53 and IRF‐1 expression and leads to apoptosis, whereas over‐expression of GAAP‐1 in K562 cells that lack p53 and IRF‐1 induces cell differentiation. Alterations in the 6p24 locus containing the GAAP‐1 gene are frequent in acute myelogenous leukemia (AML), and AML‐derived cell lines display reduced GAAP‐1 mRNA levels. Together, these results suggest that GAAP‐1 acts as a gatekeeper at a critical point in the tumour suppressor gene pathway.

Oromucosal Interferon Therapy: Relationship Between Antiviral Activity and Viral Load

Intraperitoneal (i.p.) administration of 20,000 IU recombinant murine IFN-alpha (rMuIFN-alpha) was highly effective in protecting mice challenged i.p. with doses of encephalomyocarditis virus (EMCV) ranging from 44 to 440 LD(50) (p<0.001). Oromucosal (o.m.) IFN therapy was also found to be effective in protecting mice challenged with a lethal dose of EMCV. Thus, 40% of animals infected with 44 LD(50) of EMCV and treated o.m. with 20,000 IU rMuIFN-alpha survived infection with a mean survival time of 12.0 +/- 2.46 days relative to a mean of 6.11 +/- 0.38 days in the control group (p<0.05). Oromucosal IFN therapy was found to be ineffective, however, in animals infected with higher doses of EMCV (88-440 LD(50)), even though intraperitoneal administration of the same dose of rMuIFN-alpha resulted in the survival of 90%, 50%, and 60% of animals infected with 88, 220, and 440 LD(50) of EMCV, respectively. These results suggest that oromucosal IFN therapy is effective at relatively low viral load only and that the mechanism of action of oromucosal IFN therapy may be different from that of parenterally administered IFN. Our results suggest that oromucosal IFN therapy may be most effective in chronic viral infections as an alternative to parenterally administered IFN, which is clinically effective but poorly tolerated.

Diphtheria, tetanus, poliomyelitis, yellow fever and hepatitis B seroprevalence among HIV1-infected migrants. Results from the ANRS VIHVO vaccine sub-study.

BACKGROUND Few data are available on the seroprotection status of HIV1-infected patients with respect to vaccine-preventable diseases. OBJECTIVE To describe, in a population of HIV1-infected migrants on stable, effective ART therapy, the seroprevalence of diphtheria, poliomyelitis, tetanus, yellow fever antibodies and serostatus for hepatitis B, and to identify factors associated with seroprotection. Vaccine responses against diphtheria, tetanus, poliomyelitis and yellow fever were also studied. METHODS Sub-Saharan African patients participating in the ANRS-VIHVO cohort were enrolled prior to travel to their countries of origin. Serologic analyses were performed in a central laboratory before and after the trip. Univariate and multivariate logistic regression was used to identify factors associated with initial seroprotection. RESULTS 250 patients (99 men and 151 women) were included in the seroprevalence study. Median age was 45 years (IQR 39-52), median CD4 cell count was 440/μL (IQR 336-571), and 237 patients (95%) had undetectable HIV1 viral load. The initial seroprevalence rates were 69.0% (95%CI 63.2-74.7) for diphtheria, 70.7% (95%CI 65.0-76.3) for tetanus, and 85.9% (95%CI 81.6-90.2) for yellow fever. Only 64.4% (95%CI 58.5-70.3) of patients had protective antibody titers against all three poliomyelitis vaccine strains before travel. No serological markers of hepatitis B were found in 18.6% of patients (95%CI 13.7-23.3). Patient declaration of prior vaccination was the only factor consistently associated with initial seroprotection. CONCLUSIONS We found a low prevalence of seroprotection against diphtheria, poliomyelitis, tetanus and hepatitis B. HIV infected migrants living in France and traveling to their native countries need to have their vaccine schedule completed.

Control of humoral immunity and auto-immunity by the CXCR4/CXCL12 axis in lupus patients following influenza vaccine

BACKGROUND CXCR4 is a chemokine receptor with multiple effects on the immune system, upregulated in patients with SLE, and correlated with disease severity. OBJECTIVE This study has investigated whether the levels of CXCR4 expressed on leucocyte subsets in lupus patients are correlated with the efficacy and the safety of the influenza vaccine. METHODS Twenty-seven patients were vaccinated and vaccine immunogenicity and tolerance were evaluated. CXCR4 was assayed on leucocyte subsets and correlated with clinical and immunological signs of diseases activity. RESULTS A significant increase in the titres of antibodies to the three viral strains was observed along with trends towards an increased vaccine efficacy in patients with quiescent disease vs patients with active disease. Recent flu vaccine history and, to a lesser extent, immunosuppressive treatment may influence vaccine immunogenicity. Influenza immunization was not associated with clinical side-effects or clinical lupus flare but with an increase in rheumatoid factor levels. Our study also confirms the correlation of CXCR4 expression with biological autoimmunity as shown by the correlation between the percentage of CXCR4-positive T cells and the ANA titres at D0, and the reverse correlation between CXCR4 expression and vaccine immunogenicity as demonstrated by the higher percentage of CXCR4-positive T cells at D0 and D30 in non-responders vs responders. CONCLUSION Altogether, our study confirms the efficacy and the safety of flu vaccine in SLE patients, highlights the role of CXCR4 as a surrogate marker for autoimmunity in lupus and shows that CXCR4 expression on T cells is predictive of vaccine efficacy in SLE patients.

Recognition of core-derived epitopes from a novel HBV-targeted immunotherapeutic by T-cells from patients infected by different viral genotypes.

Hepatitis B virus (HBV) infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular carcinoma. Current therapies based on nucleos(t)ide analogs or pegylated-interferon-α lead to control of viral replication in most patients but rarely achieve cure. A potential strategy to control chronic hepatitis B is to restore or induce functional anti-HBV T-cell immune responses using HBV-specific immunotherapeutics. However, viral diversity is a challenge to the development of this class of products as HBV genotypes display a sequence diversity of up to 8%. We have developed a novel HBV-targeted immunotherapeutic, TG1050, based on a non-replicative Adenovirus vector encoding a unique and large fusion protein composed of multiple antigenic regions derived from a HBV genotype D sequence. Using peripheral blood mononuclear cells from 23 patients chronically infected by five distinct genotypes (gt A, B, C, D and E) and various sets of peptides encompassing conserved versus divergent regions of HBV core we have measured ability of TG1050 genotype D core-derived peptides to be recognized by T-cells from patients infected by various genotypes. Overall, PBMCs from 78% of genotype B or C- and 100% genotype A or E-infected patients lead to detection of HBV core-specific T-cells recognizing genotype D antigenic domains located both in conserved and variable regions. This proof-of-concept study supports the clinical development of TG1050 in large patient populations independently of infecting genotypes.

Production of TNF-alpha ex vivo is predictive of an immune response to flu vaccination in a frail elderly population.

ObjectiveTo investigate the relationship between the response to influenza vaccination and the ability to produce proinflamatory cytokines in elderly subjects.MethodsWhole blood samples from 25 elderly subjects collected before influenza vaccination were stimulated with the influenza vaccine in order to evaluate the secretion of five specific cytokines: TNFα, IFNα, IFNγ, IL2 and IL10. The results were correlated with the increased HAI antibody titres two weeks after vaccination.ResultsOnly 30% of elderly individuals seroconverted after vaccination. Although 50 to 70% of the cohort did not produce TNFα, IFNα, IFNγ, IL2 or IL10, all of the individuals who seroconverted were able to produceTNFα. Furthermore production of IFNγ, with or without production of IFNα/β, was not associated with a better response to the vaccine.ConclusionProduction of TNFα appears to be primordial for an efficient vaccine response, and may provide a predictive marker for the humoral response to vaccination. It may also provide the basis for evaluating agents designed to rescue TNFα-producing cells. This study emphasises a need to rescue TNF-producing cell function.