Hélène Jeulin

Rapid generation of full clinical-grade human antiadenovirus cytotoxic T cells for adoptive immunotherapy.

Adenovirus (ADV) infections are one of the major causes of morbidity and mortality after hematopoietic stem cell transplantation, despite new antiviral treatment strategies. We describe here a complete clinical-grade generation of human anti-ADV cytotoxic T cells to propose an adoptive immunotherapy. Peripheral blood mononuclear cells (PBMC) from 7 healthy donors, known for their good cellular immunity against ADV, were stimulated for 6 hours with a synthetic peptide pool covering the ADV5 Hexon protein interferon-γ (IFN-γ) secreting cells were isolated on a clinical device. After immunoselection, a mean number of 1.01±0.84×106 total nucleated cells was obtained. The isolated ADV-specific T cells were mainly CD4+ (mean=56%±20.8%, yield=51%±32.4%) but also CD8+ (mean=42%±27%, yield=56%±39.3%). Isolated T lymphocytes (CTL) were expanded to carry out functional tests. Ability of the expanded CTL to secrete IFN-γ and to proliferate after restimulation with the ADV peptide pool was confirmed. A high cytotoxicity against autologous target cells loaded with ADV antigens was observed but not against nonloaded target cells. We observed a decrease of 1.27 log of the allogeneic reaction against non HLA identical healthy donor PBMC with CTL compared with the PBMC before selection. Clinical-grade generation of ADV-specific T cells was achieved with a synthetic antigen. This technology has the advantage of being fast, and is sufficiently reactive to be proposed for immunotherapy if antiviral treatment fails.

Comparison of In-House Real-Time Quantitative PCR to the Adenovirus R-Gene Kit for Determination of Adenovirus Load in Clinical Samples

ABSTRACT In the context of hematopoietic stem cell transplantation, adenovirus infections are associated with relevant mortality and morbidity. Detection of adenovirus DNA by quantitative PCR is the “gold standard” for these patients. A total of 150 samples, namely, 78 whole-blood, 22 cerebrospinal fluid, 24 digestive biopsy, and 26 stool samples, from 29 patients, including 24 hematopoietic stem cell transplant recipients, were tested for the detection of adenovirus using an in-house real-time quantitative PCR assay (A. Heim, C. Ebnet, G. Harste, and P. Pring-Akerblom, J. Med. Virol. 70:228-239, 2003) and the commercially available Adenovirus R-Gene kit. Adenovirus DNA was automatically isolated from whole-blood samples (Magna Pure LC system; Roche) or was manually extracted from other specimens (QIAamp; Qiagen) using the appropriate kit. The intra- and interassay reproducibilities and sensitivities were evaluated with cell culture supernatant dilutions. Of the 150 samples tested, 86 were found to be positive and 55 were found to be negative using both techniques. Nine (6%) discordant results were obtained. In most cases, discrepant results concerned samples with low viral loads. Quantitative results for all concordant positive samples were analyzed using the Spearman correlation test. A good correlation between the results of the in-house assay and those of the kit assay was obtained (r = 0.95; P < 0.001). Regarding the threshold cycle value for internal control spiked samples, none of the 150 samples tested contained a PCR inhibitor. In conclusion, a relevant correlation of results between the in-house assay and the kit assay, as well as the high-quality reproducibility and sensitivity of the kit assay, warranted its use for follow-up of hematopoietic stem cell transplantation recipients.

Contribution of human herpesvirus 6 (HHV-6) viral load in whole blood and serum to investigate integrated HHV-6 transmission after haematopoietic stem cell transplantation.

BACKGROUND Human herpesvirus 6 (HHV-6) is susceptible to latency and recurrence. A less-frequent form of HHV-6 persistence is the integration of viral DNA into host chromosomes. OBJECTIVES To investigate HHV-6 viral load after haematopoietic stem cell transplantation (HSCT) in whole blood (WB) and serum with regard to integrated HHV-6 transmission diagnosis. STUDY DESIGN HHV-6 DNA quantitation in serum and WB was performed using quantitative polymerase chain reaction for the follow-up of a 16-year-old girl after HSCT. In whole blood, results were expressed as HHV-6 genomic equivalent copies (gec) per milliliter of WB or per million cells. RESULTS HHV-6 viral load (undetectable before HSCT) increased up to 3.05 x 10(7)gec/10(6)cells. HHV-6 viral load in the donor sample (3.44 x 10(6)gec/10(6)cells) was in favor of viral transmission through HSCT. The correlation between viral load in WB and serum was significant (p=0.0005). Viral load results expressed as gec/10(6)cells in WB was more reliable than results expressed as gec/ml of whole blood. CONCLUSION These findings indicate that HHV-6 may be transmitted during HSCT as integrated virus contained in the graft. This reiterates that in the setting of HSCT, HHV-6 viral load must be correctly interpreted. Using HHV-6 viral load expressed as gec/10(6) cells may be more suitable for the follow-up of patients with integrated HHV-6.

Chromosomally integrated HHV-6: slow decrease of HHV-6 viral load after hematopoietic stem-cell transplantation.

Human herpesvirus (HHV)-6 belongs to the Betaherpesvirinae subfamily. Similar to other herpesviruses, HHV-6 has a latent stage (1) with problematic reactivation in immunocompromised subjects, leading to fatal infections including meningoencephalitis (2, 3). A less-frequent HHV-6 persistence form consists in integration in host chromosomes (4). The prevalence of HHV-6 integration, characterized by high viral load in healthy individuals, has been estimated at approximately 0.2% to 2% in the whole population (5–7) and can thus concern up to 2% of hemotopoieticstem-cell transplantation (HSCT) donors or recipients (8). Some previous publications have described cases of integrated HHV-6 transmission through HSCT (9 –11). In those cases, the recipient has undetectable HHV-6 viral load before the transplantation and becomes positive to high values ( 10 genomic equivalent copies [gec]/10cells) for HHV-6 DNA early after transplantation ( 10 days) (9, 12). The presence of high HHV-6 viral load in an HSCT recipient could also be misinterpreted as HHV-6 reactivation and lead to unnecessary treatment with antiviral compounds, such as ganciclovir, which have drawbacks (13). Here, we describe another type of case in which high HHV-6 viral load is also caused by integrated HHV-6 and could be misinterpreted as an active infection leading to unnecessary treatment. The patient is a 25-year-old woman diagnosed with Hodgkin’s lymphoma having experienced failure after a prior autologous transplant. The disease before transplantation was chemosensitive and in good partial remission. She was given an allogeneic HSCT mobilized from peripheral blood on July 11, 2008, from a 38-year-old unrelated matched (10 of 10 loci) female donor. The reduced-intensity conditioning regimen included Fludarabine (25 mg/m/ day) on days 7 to 3, Melphalan (140 mg/m) on day 2, and ATG Fresenius (13 mg/kg/day) on days 5, 3, and 1. The prophylaxis of graft-versushost disease included cyclosporin and mycophenolate mofetil. The recipient’s whole blood (WB) samples were tested weekly for adenovirus (14), Epstein-Barr virus, cytomegalovirus (CMV), and HHV-6 (EBV R-geneTM and CMV HHV6,7,8 R-geneTM; Argène, Varilhes, France) by quantitative polymerase chain reaction (qPCR) after DNA isolation (Magna Pure LC system®, Roche, Meylan, France); the results were expressed as genomic equivalent copies per milliliter of WB. Moreover, albumin gene DNA was retrospectively amplified according to Laurendeau et al. (15) to quantify HHV-6 gec/10 cells, considering that each cell contains two copies of albumin genome (16). Retrospectively, HHV-6 DNA was tested in the donor’s peripheral blood mononuclear cells. Before the graft, a high HHV-6 viral load (1.30 10 gec/10 cells) led to treatment with intravenous foscarnet (180 mg/kg/day) from day 7 pretransplant to day 14 posttransplant, without decrease in the viral load. Other infectious events included fever of undocumented origin on day 1 and CMV infection on day 45, successfully treated with valganciclovir. The high HHV-6 viral load before transplantation was in favor of integrated HHV-6 and should have prevented patient treatment. Retrospectively, HHV-6 integration in the recipient was confirmed by HHV-6 DNA isolation in hair follicles (17) and mouth swab specimens. The recipient’s parents could not be tested for integrated HHV-6 (6). Besides, HHV-6 DNA was undetectable in donor peripheral blood mononuclear cells. Unexpectedly, 37 days after the transplantation, the HHV-6 viral load in WB remained up to 8.45 10 gec/10 cells. Quantitative analysis of hematopoietic chimerism was performed by real-time qPCR using TaqMan technology

Characterization of hepatitis B virus surface antigen variability and impact on HBs antigen clearance under nucleos(t)ide analogue therapy

For hepatitis B virus (HBV)‐related chronic infection under treatment by nucleos(t)ide analogues (NUCs), HBsAg clearance is the ultimate therapeutic goal but very infrequent. We investigated how HBV envelope protein variability could lead to differential HBsAg clearance on NUCs. For 12 HBV genotype D patients receiving NUCs, six resolvers (HBsAg clearance) were compared to six matched nonresolvers (HBsAg persistence). PreS/S amino acid (aa) sequences were analysed with bioinformatics to predict HBV envelope antigenicity and aa covariance. To enrich our analyses on very rare resolvers, these were compared with other HBV genotype D strains in three characterized clinical cohorts including common chronically infected patients. The sT125M+sP127T combination was observed in four nonresolvers of six, corroborated by aa covariance analysis, associated with a lower predicted antigenicity than sT125T+sP127P. Concordant features within this HBV key functional domain, at positions 125 and 127, were reported from two of the three comparative cohorts. In our hands, a lower ELISA reactivity of HBV‐vaccinated mice sera was observed against the sT125M mutant. In the S gene, 56 aa changes in minor variants were detected in non‐resolvers, mainly in the major hydrophilic region, vs 28 aa changes in resolvers. Molecular features in patients showing HBsAg persistence on NUCs argue in favour of a different aa pattern in the HBV S gene compared to those showing HBsAg clearance. In nonresolvers, a decrease in HBs ‘a’ determinant antigenicity and more frequent mutations in the S gene suggest a role for the HBV envelope characteristics in HBsAg persistence.

BRIP1 coding variants are associated with a high risk of hepatocellular carcinoma occurrence in patients with HCV- or HBV-related liver disease

The molecular mechanisms of hepatocellular carcinoma (HCC) carcinogenesis are still not fully understood. DNA repair defects may influence HCC risk. The aim of the study was to look for potential genetic variants of DNA repair genes associated with HCC risk among patients with alcohol- or viral-induced liver disease. We performed four case-control studies on 2,006 European- (Derivation#1 and #2 studies) and African-ancestry (Validation#1 and #2 studies) patients originating from several cohorts in order to assess the association between genetic variants on DNA repair genes and HCC risk using a custom array encompassing 94 genes. In the Derivation#1 study, the BRIP1 locus reached array-wide significance (Chi-squared SV-Perm, P=5.00×10−4) among the 253 haplotype blocks tested for their association with HCC risk, in patients with viral cirrhosis but not among those with alcoholic cirrhosis. The BRIP1 haplotype block included three exonic variants (rs4986763, rs4986764, rs4986765). The BRIP1 ‘AAA’ haplotype was significantly associated with an increased HCC risk [odds ratio (OR), 2.01 (1.19–3.39); false discovery rate (FDR)-P=1.31×10−2]. In the Derivation#2 study, results were confirmed for the BRIP1 ‘GGG’ haplotype [OR, 0.53 (0.36–0.79); FDR-P=3.90×10−3]. In both Validation#1 and #2 studies, BRIP1 ‘AAA’ haplotype was significantly associated with an increased risk of HCC [OR, 1.71 (1.09–2.68); FDR-P=7.30×10−2; and OR, 6.45 (4.17–9.99); FDR-P=2.33×10−19, respectively]. Association between the BRIP1 locus and HCC risk suggests that impaired DNA mismatch repair might play a role in liver carcinogenesis, among patients with HCV- or HBV-related liver disease.

Impact of deletions and mutations in Hepatitis B virus envelope proteins on serological profile and clinical evolution

The Hepatitis B virus (HBV) envelope glycoproteins are essential for viral entry into the hepatocyte and are also targets for host immune response. The study of these proteins could allow us to highlight molecular hot points influencing HBV fitness, which would subsequently modify the clinical evolution of the disease, both under anti-viral therapy or without treatment. The present short communication underlines the importance of the high variability in HBV envelope proteins, in regard with the literature and in our hands, for HBV-infected patients either on anti-HBV treatment or not. We report mutations in antigenic areas of S protein, i.e. CD8+/CD4+ T-cell epitopes and B-cell epitopes in the major hydrophilic region (MHR), such as sI126N and sG145R possibly involved in the rare coexisting Hepatitis B surface Antigen (HBsAg)/anti-HBs serological pattern. We mostly report serial mutations in preS region including preS1 deletion (aa 1-6, 31-71, 38-73, 72-104) and preS2 deletion (aa132-141) in patients with various clinical evolutions. Some of these viral envelope mutations, due to immune selection pressure, may result in a worsening of the hepatic disease.

Herpes simplex virus type 1 hepatitis due to primary infection in a pancreas-kidney transplant recipient.

Herpes simplex Virus (HSV) hepatitis is a rare complication of HSV-1 primary infection, with a delayed diagnosis, affecting mainly immunocompromised patients. We describe a case of HSV-1 hepatitis after primary infection occurring in the postoperative days after a pancreas-kidney transplantation. The patient presented with an unusual evolution of a persistent severe hepatitis associated with a persistent viremia (Quantitative Polymerase Chain Reaction) despite an adequate intravenous (iv) antiviral treatment. Abdominal computed tomography scan showed a miliary hepatitis. The diagnosis of HSV-1 hepatitis was confirmed by immuno-chemistry on liver biopsy. The donor was negative for anti-HSV antibodies, excluding contamination by the graft. This case report emphasizes a rather seldom risk of care-associated viral infections, predominantly in immunocompromised patients.

Molecular features of Hepatitis E Virus circulation in environmental and human samples

BACKGROUND AND OBJECTIVES Hepatitis E virus (HEV) is emerging but its circulation between humans and the environment remains misunderstood. HEV ORF2 gene encodes the capsid playing a key role in viral interactions with surfaces, ORF3 products are involved in the viral cycle. Our aim was to study the molecular characteristics of ORF2 and ORF3 which could favor HEV fitness in patients and the environment. STUDY DESIGN Samples from 69 patients with hepatitis (blood/stools), 20 urban wastewaters, 20 effluents of a pig slaughterhouse, 22 farm pigs (stools), 20 wild boars (liver/stools) were collected in North-Eastern France. HEV strains were analyzed by direct sequencing within the ORF2 M region, of ORF2/ORF3, for phylogeny and physicochemical prediction and for ORF2 by ultra-deep sequencing. RESULTS The results showed frequent HEV-positive samples: 9.1% of the patient bloods, 23.1% of their stools; 25.0% of wastewaters, 75.0% for the slaughterhouse, 10.0% of the boar livers, 5.3% of their stools. The strains were classified as HEV genotype 3. In ORF2, HEV highlighted one homogeneous major viral variant within quasispecies and a decrease in predicted antigenicity for two minor mutations (D442G, V402A). A cysteine signature at position 81 in ORF3 was observed in the boars. CONCLUSIONS HEV RNA genotype 3 was detected in patients and in animals, in a slaughterhouse effluent and in wastewater. Moreover, the low variability of amino acids in the ORF2 M region and molecular features in ORF2 and ORF3 suggested that HEV strains could be advantageous for key properties.

Emerging resistance mutations in PI-naive patients failing an atazanavir-based regimen (ANRS multicentre observational study)

Background Atazanavir is a PI widely used as a third agent in combination ART. We aimed to determine the prevalence and the patterns of resistance in PI-naive patients failing on an atazanavir-based regimen. Methods We analysed patients failing on an atazanavir-containing regimen used as a first line of PI therapy. We compared the sequences of reverse transcriptase and protease before the introduction of atazanavir and at failure [two consecutive viral loads (VLs) >50 copies/mL]. Resistance was defined according to the 2014 Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) algorithm. Results Among the 113 patients, atazanavir was used in the first regimen in 71 (62.8%) patients and in the first line of a PI-based regimen in 42 (37.2%). Atazanavir was boosted with ritonavir in 95 (84.1%) patients and combined with tenofovir/emtricitabine or lamivudine (n = 81) and abacavir/lamivudine or emtricitabine (n = 22). At failure, median VL was 3.05 log10 copies/mL and the median CD4+ T cell count was 436 cells/mm3. The median time on atazanavir was 21.2 months. At failure, viruses were considered resistant to atazanavir in four patients (3.5%) with the selection of the following major atazanavir-associated mutations: I50L (n = 1), I84V (n = 2) and N88S (n = 1). Other emergent PI mutations were L10V, G16E, K20I/R, L33F, M36I/L, M46I/L, G48V, F53L, I54L, D60E, I62V, A71T/V, V82I/T, L90M and I93L/M. Emergent NRTI substitutions were detected in 21 patients: M41L (n = 2), D67N (n = 3), K70R (n = 1), L74I/V (n = 3), M184V/I (n = 16), L210W (n = 1), T215Y/F (n = 3) and K219Q/E (n = 2). Conclusions Resistance to atazanavir is rare in patients failing the first line of an atazanavir-based regimen according to the ANRS. Emergent NRTI resistance-associated mutations were reported in 18% of patients.