Low Prevalence of HLA-G Antibodies in Lung Transplant Patients Detected using MAIPA-Adapted Protocol.

Lung transplantation is often complicated by acute and/or chronic rejection leading to graft-function loss. In addition to the HLA donor-specific antibodies (HLA-DSA), a few autoantibodies are correlated with the occurrence of these complications. Recently, antibodies directed against non-classical HLA molecules, HLA-G, -E, and -F have been detected in autoimmune diseases, like systemic lupus erythematosus. Non-classical HLA molecules are crucial in the immunological acceptance of the lung graft, and some of their isoforms, like HLA-G*01:04 and -G*01:06, are associated with a negative clinical outcome. The aim of this study is to determine the frequency of detection of HLA-G antibodies in lung transplant recipients (LTRs) and their impact on the occurrence of clinical complications. After incubating the cell lines SPI-801, with and without three different HLA-G isoform expression, with sera from 90 healthy blood donors and 35 LTRs (before and after transplantation), HLA-G reactivity was revealed using reagents from commercial monoclonal antibody immobilization of platelet antigen assay (MAIPA ApDIA®). Only one serum from one blood donor had specific reactivity against the HLA-G transduced lines. Non-specific reactivity in many sera from LTRs was observed with transduced- and wild-type cell lines, which may suggest recognition of an autoantigen expressed by the SPI-801 cell line. In conclusion, this study allowed the development of a specific detection tool for non-denatured HLA-G antibodies. These antibodies seem uncommon, both in healthy subjects and in complicated LTRs. This study should be extended to patients suffering from autoimmune diseases as well as kidney and heart transplant recipients.

DO/ART4 gene sequencing in sub-Saharan cohorts and African migrants: useful data describing the diversity and spreading of rare variants.

BACKGROUND: Due to the unavailability of immunological reagents, the Dombrock blood group is insufficiently explored in African populations and can be a source of alloimmunization. A large study including pygmoid and nonpygmoid ethnic groups from East, Central, and West continental Africa, together with African migrants like Comorians, Afro-Caribbean from Martinique, and Maroons from French Guiana would be helpful to increase transfusion safety. STUDY DESIGN AND METHODS: Using genomic DNA extracted from blood samples collected from 336 nonpygmoid and 51 pygmoid Africans as well as 268 samples of African descent, DO coding regions were PCR-amplified and sequenced. RESULTS: DO*A and DO*B alleles were detected in almost all groups, with a clear predominance of DO*B in every cohort tested. DO*JO and DO*HY allele frequencies reached 10% or more in several ethnic groups. DO*B-SH-Gln149Lys, DO*B-Ile5Thr, and DO*DODE variants were identified both in African ethnic groups and outside Africa. Twelve novel variants were characterized on a DO*A or a DO*B background. Five of them were found in both African and migrant cohorts, the others were restricted to either within or outside Africa. No DO*DOYA, DO*DOLG, DO*DOLC, nor DO*DOMR variants were observed. A first phylogenetic tree was proposed including all variant alleles. CONCLUSION: This study across continental Africa and countries with African migrants provides a useful overview of Dombrock allele diversity and distribution. The identification of 12 new alleles underlines the importance of genotyping for Dombrock alleles, particularly to improve transfusion safety in countries hosting migrant populations of African descent.

Human pegivirus isolates characterized by deep sequencing from hepatitis C virus-RNA and human immunodeficiency virus-RNA-positive blood donations, France.

Human pegivirus (HPgV, formerly GBV-C) is a member of the genus Pegivirus, family Flaviviridae. Despite its identification more than 20 years ago, both natural history and distribution of this viral group in human hosts remain under exploration. Analysis of HPgV genomes characterized up to now points out the scarcity of French pegivirus sequences in databases. To bring new data regarding HPgV genomic diversity, we investigated 16 French isolates obtained from hepatitis C virus-RNA and human immunodeficiency virus-RNA-positive blood donations following deep sequencing and coupled molecular protocols. Initial phylogenetic analysis of 5'-untranslated region (5'-UTR)/E2 partial sequences permitted to assign HPgV isolates to genotypes 2 (n = 15) and 1 (n = 1), with up to 16% genetic diversity observed for both regions considered. Seven nearly full-length representative genomes were characterized subsequently, with complete polyprotein coding sequences exhibiting up to 13% genetic diversity; closest nucleotide (nt) divergence with available HPgV sequences was in the range 7% to 11%. A 36 nts deletion located on the NS4B coding region (N-terminal part, 12 amino acids) of the genotype 1 HPgV genome characterized was identified, along with single nucleotide deletions in two genotype 2, 5'-UTR sequences.

HLA-H: Transcriptional Activity and HLA-E Mobilization.

Little attention is paid to pseudogenes from the highly polymorphic HLA genetic region. The pseudogene HLA-H is defined as a non-functional gene because it is deleted at different frequencies in humans and because it encodes a potentially non-functional truncated protein. However, different studies have shown HLA-H transcriptional activity. We formerly identified 13 novel HLA-H alleles, including the H*02:07 allele, which reaches 19.6% in East Asian populations and encodes a full-length HLA protein. The aims of this study were to explore the expression and possible function of the HLA-H molecule. HLA-H may act as a transmembrane molecule and/or indirectly via its signal peptide by mobilizing HLA-E to the cell surface. We analyzed HLA-H RNA expression in Peripheral Blood Mononuclear Cells (PBMC), Human Bronchial Epithelial Cells (HBEC), and available RNA sequencing data from lymphoblastoid cell lines, and we looked to see whether HLA-E was mobilized at the cell surface by the HLA-H signal peptide. Our data confirmed that HLA-H is transcribed at similar levels to HLA-G. We characterized a hemizygous effect in HLA-H expression, and expression differed according to HLA-H alleles; most interestingly, the HLA-H*02:07 allele had the highest level of mRNA expression. We showed that HLA-H signal peptide incubation mobilized HLA-E molecules at the cell surface of T-Lymphocytes, monocytes, B-Lymphocytes, and primary epithelial cells. Our results suggest that HLA-H may be functional but raises many biological issues that need to be addressed.

HLA-G Haplotypes Are Differentially Associated with Asthmatic Features.

Human leukocyte antigen (HLA)-G, a HLA class Ib molecule, interacts with receptors on lymphocytes such as T cells, B cells, and natural killer cells to influence immune responses. Unlike classical HLA molecules, HLA-G expression is not found on all somatic cells, but restricted to tissue sites, including human bronchial epithelium cells (HBEC). Individual variation in HLA-G expression is linked to its genetic polymorphism and has been associated with many pathological situations such as asthma, which is characterized by epithelium abnormalities and inflammatory cell activation. Studies reported both higher and equivalent soluble HLA-G (sHLA-G) expression in different cohorts of asthmatic patients. In particular, we recently described impaired local expression of HLA-G and abnormal profiles for alternatively spliced isoforms in HBEC from asthmatic patients. sHLA-G dosage is challenging because of its many levels of polymorphism (dimerization, association with ß2-microglobulin, and alternative splicing), thus many clinical studies focused on HLA-G single-nucleotide polymorphisms as predictive biomarkers, but few analyzed HLA-G haplotypes. Here, we aimed to characterize HLA-G haplotypes and describe their association with asthmatic clinical features and sHLA-G peripheral expression and to describe variations in transcription factor (TF) binding sites and alternative splicing sites. HLA-G haplotypes were differentially distributed in 330 healthy and 580 asthmatic individuals. Furthermore, HLA-G haplotypes were associated with asthmatic clinical features showed. However, we did not confirm an association between sHLA-G and genetic, biological, or clinical parameters. HLA-G haplotypes were phylogenetically split into distinct groups, with each group displaying particular variations in TF binding or RNA splicing sites that could reflect differential HLA-G qualitative or quantitative expression, with tissue-dependent specificities. Our results, based on a multicenter cohort, thus support the pertinence of HLA-G haplotypes as predictive genetic markers for asthma.

Evidence for two phylogenetic clusters within hepatitis C virus (HCV) genotype 2 inferred from analysis of complete coding sequences of 15 HCV strains.

The aim of this study was to gain further insight into the evolution and classification of hepatitis C virus (HCV) by assessing the subtype distribution of 273 genotype 2 strains isolated from French blood donors from 1990 to 2010 and by determining complete coding sequences in subtype 2 strains. These classified into 7 of the established subtypes and into 15 additional lineages not yet assigned to a known subtype. Phylogenetic tree construction showed two well-supported clusters. Cluster 1 included most subtype 2 strains while cluster 2 included subtype 2l and one unassigned subtype 2. Full genome sequencing was performed on 15 genotype 2 strains belonging to both clusters, that is, one subtype 2b, two subtype 2c, three subtype 2i, two subtype 2j, one subtype 2k, two subtype 2l, and four unassigned strains. Genomes included a 9042- to 9108-nucleic acid open reading frame coding for a polyprotein comprising 3014-3036 amino acids. Mean nucleotide distances between subtypes belonging to the first cluster was 20.2 ± 1.4% while the mean distance between the two clusters was 25.9 ± 0.3%. Analysis indicated that the bifurcation between subtype 2l and other subtype 2 strains occurred early in the evolutionary process. Subtype 2l retained a genomic feature characteristic of non-genotype 2, that is, absence of the 60-nucleotide insertion in the NS5A region. This finding suggests that appearance and fixation of this insertion occurred late in the evolutionary history of HCV type 2 and that its absence is an ancestral feature of HCV.

Analysis of hepatitis C virus strains circulating in Republic of the Congo.

The aim of this study was to assess the seroprevalence, viremia, genotype distribution, and demographic history of hepatitis C virus (HCV) in the Republic of the Congo. Testing was carried out on sera samples collected in 2005 from 807 Bantus belonging to the Kongo, Teke, and Ngala subgroups and 80 Pygmies. Positive HCV serology was found in 50 (5.6%) individuals including 31 (60%) who were viremic. Seroprevalence increased with age with a cutoff at 50 years: 2.8% <50 versus 12% >50. Twenty-one strains belonged to four described subtypes, that is, 4c in eight cases, 4h in two, 4k in three, and 4r in eight. Ten strains could not be assigned to any known subtype and may represent six new variants, that is, subtype 4 in five cases and subtype 2 in one. Evolutionary analysis of subtype 4c and 4r sequences indicated a period of enhanced transmission in the mid-twentieth century probably due to iatrogenic causes. This study underlines the high genetic diversity of strains in the Republic of the Congo with nine subtypes 4 and one subtype 2.

Molecular characterization of genotype 2 and 4 hepatitis C virus isolates in French blood donors.

The subtype distribution of 142 genotype 2 and 97 genotype 4 hepatitis C virus (HCV) isolates from the sera of 1,319 volunteer blood donors in France was determined by gene sequencing and by phylogenetic analysis of the NS5B region and E1 envelope. Findings underlined a wide range of subtypes in both genotypes, that is, 20 in HCV-2 and 11 in HCV-4. Eighteen of these 31 subtypes had not been defined previously. Some subtypes, that is, 2a, 2b, 2c, 2i, 2k, 4a, and 4d, showed numerous strains while subtypes in donors from West Africa or Central Africa showed an endemic profile with only a few strains. A Bayesian coalescence approach was used to estimate the demographic history of each HCV subtype. The estimated mean dates of the most recent common ancestors (MRCA) were 1,889 (confidence interval (CI), 1,842-1,930) for HCV-2a, 1,886 (CI, 1,843-1,921) for HCV-2b, 1,791 (CI, 1,699-1,848) for HCV-2c, 1,846 (CI, 1,803-1,878) for HCV-2i, 1,911 (CI, 1,879-1,937) for HCV-4a, and 1,957 (CI, 1,943-1,967) for HCV-4d. The period of spread for subtype 2b, 2c, and 2i was between 1900 and 1960 whereas rapid exponential spread for subtype 2a, 4a, and 4d occurred in the 1960s. The inferred histories of population growth indicated that transmission rates differed according to HCV subtype. These results may help to predict the future burden of HCV in France.

Lentiviral-mediated gene delivery in human monocyte-derived dendritic cells: optimized design and procedures for highly efficient transduction compatible with clinical constraints.

Gene delivery to dendritic cells (DCs) could represent a powerful method of inducing potent, long-lasting immunity. Although recent studies underline the intense interest in lentiviral vector-mediated monocyte-derived DC transduction, efficient gene transfer methods currently require high multiplicities of infection and are not compatible with clinical constraints. We have designed a strategy to optimize the efficiency and clinical relevance of this approach. Initially, using a third generation lentiviral vector expressing green fluorescent protein, we found that modifying the vector design, the DC precursor cell type, and the DC differentiation stage for transduction results in sustained transgene expression in 75-85% of immature DCs (transduction at a multiplicity of infection of 8). This high efficiency was reproducible among different donors irrespective of whether DCs were expanded from fresh or cryopreserved CD14(+) precursors. We then developed procedures that bypass the need for highly concentrated lentiviral preparations and the addition of polybrene to achieve efficient transduction. DCs transduced under these conditions retain their immature phenotype and immunostimulatory potential in both autologous and allogeneic settings. Furthermore, genetically modified DCs maintain their ability to respond to maturation signals and secrete bioactive IL-12, indicating that they are fully functional. Finally, the level of transgene expression is preserved in the therapeutically relevant mature DCs, demonstrating that there is neither promoter-silencing nor loss of transduced cells during maturation. The novel approach described should advance lentiviral-mediated monocyte-derived DC transduction towards a clinical reality.