Novel T cell interferon gamma release assay (IGRA) using spike recombinant protein for COVID19 vaccine response and Nucleocapsid for SARS-Cov2 response.

We explored the performance of a whole blood interferon gamma release assay (IGRA) based on the stimulation of SARS-Cov2-specific T cells by purified recombinant proteins. Twenty volunteers vaccinated with BNT162b2 were selected first for T cell response evaluation using an in-house IGRA, a commercial IGRA, and ELISpot showing a S2 > S1 poly-epitopic response. Next, 64 vaccinated and 103 non-vaccinated individuals were tested for humoral and T cell response (IGRA-Spike/-nucleocapsid recombinant proteins). Following the second vaccine injection, humoral (100%) and IGRA-Spike T cell (95.3%) responses took place irrespective of sex, age, and vaccine type. The humoral response declined first, followed by IGRA-Spike T cell response after the second vaccine injection. Altogether, this study confirms the utility of the IGRA-Spike/-nucleocapsid assay to complement serology in COVID19 vaccinated individuals and those who have recovered from SARS-Cov2.

Glucocorticoid use as a cause of non-cellular immune response to SARS-Cov2 Spike in patients with immune system diseases.

Disease modifying therapies compromise immune response to SARS-Cov2 or its vaccine in patients with immune system diseases (ISD). Therefore, analysis of the humoral and cellular responses against Spike is of utmost importance to manage ISD patients. A single-center retrospective study was conducted to evaluate the impact of COVID-19 immunization in 87 ISD patients and 81 healthy controls. We performed a whole blood interferon gamma release assay using SARS-Cov2 Spike and Nucleocapsid recombinant proteins in order to evaluate T-cell memory response, and an IgG anti-Spike ELISA to evaluate humoral response. Cellular (26.4%) and humoral (44.8%) responses were negative against Spike in ISD patients following COVID-19 immunization. In univariate analysis, an anti-Spike T cell defective response was associated with the use of glucocorticoids (Odds ratio [OR] = 10.0; p < 10-4), serum albumin level ≤40 g/L (OR = 18.9; p < 10-4), age over 55 years old (OR = 3.9, p = 0.009) and ≤2 vaccine injections (OR = 4.9; p = 0.001). The impact of glucocorticoids persisted after adjustment for age and number of vaccine injections (OR = 8.38, p < 0.001). In contrast, the humoral response was impacted by the use of anti-CD20 mAb (OR = 24.8, p < 10-4), and an extended time since immunization (≥75 days; OR = 4.3, p = 0.002). Double defective cellular/humoral responses (6.9%) were typically encountered in glucocorticoids and/or anti-CD20 mAb treated ISD with a serum albumin level ≤40 g/L (OR = 17.5; p = 0.002). Glucocorticoid usage, B cell depleting therapies, and a low serum albumin level were the main factors associated with a non-response to COVID-19 immunization in ISD patients. These results need further confirmation in larger studies.

Combining multi-antigenic immunodot with indirect immunofluorescence on HEp-2 cells improves the diagnosis of systemic sclerosis.

Systemic sclerosis (SSc) is associated, in nearly all patients, with autoantibodies (Ab). Accordingly, and in order to identify major (anti-CEN A/B and anti-Topo I) but also minor Abs, the usefulness of combining indirect immunofluorescence (IIF) on HEp-2 cells with an 11 multi-antigenic SSc immunodot was explored. 1689 samples tested at the request of clinicians, were evaluated retrospectively. The positivity rate was 28.8% and the diagnosis of SSc was supported for 232 samples. Two groups of Abs were considered: group 1, Abs (anti-CENP A/B, anti-Topo I) present at elevated levels in SSc patients; group 2, Abs for which the Ab specificity (odds ratio and/or positive predictive value) was improved by using IIF on HEp-2 cells (RNA-Polymerase III, fibrillarin, Th/T0, PM-Scl). Altogether, this study highlights the utility of combining IIF on HEp-2 cells with the SSc immunodot as the first line of an SSc Abs detection/SSc diagnostic strategy.

Nomenclature report 2019: major histocompatibility complex genes and alleles of Great and Small Ape and Old and New World monkey species.

The major histocompatibility complex (MHC) is central to the innate and adaptive immune responses of jawed vertebrates. Characteristic of the MHC are high gene density, gene copy number variation, and allelic polymorphism. Because apes and monkeys are the closest living relatives of humans, the MHCs of these non-human primates (NHP) are studied in depth in the context of evolution, biomedicine, and conservation biology. The Immuno Polymorphism Database (IPD)-MHC NHP Database (IPD-MHC NHP), which curates MHC data of great and small apes, as well as Old and New World monkeys, has been upgraded. The curators of the database are responsible for providing official designations for newly discovered alleles. This nomenclature report updates the 2012 report, and summarizes important nomenclature issues and relevant novel features of the IPD-MHC NHP Database.

Correction to: Nomenclature report 2019: major histocompatibility complex genes and alleles of great and small ape and old and new world monkey species.

The original version of this article contained a spelling error in the Acknowledgments regarding the name of the funding organisation supporting GM and JAH. UKRI-BBSCR should have been UKRI-BBSRC, as is now indicated correctly below.

High rate of indeterminate results of the QuantiFERON-TB Gold in-tube test, third generation, in patients with systemic vasculitis.

OBJECTIVES: To describe the frequency of QuantiFERON-TB Gold in-tube test® (QFT-GIT) indeterminate results due to no response to phytohaemagglutinin A stimulation in the control tube in vasculitis patients prior to immunosuppressant therapy; and to compare it with other groups of patients. METHODS: This was a single-centre, retrospective study. Patients and controls were included between 1 January 2008 and 31 December 2015. We assessed the rate of indeterminate results of the QFT-GIT in 38 patients with systemic vasculitis prior to any corticosteroid or immunosuppressant therapy, compared with 40 non-vasculitis patients with biological inflammatory syndrome, and 310 non-immunosuppressed patients matched for gender and age. RESULTS: Indeterminate results due to no response to phytohaemagglutinin A were more frequent in vasculitis patients (21.1%) compared with non-vasculitis patients with biological inflammatory syndrome (7.5%) (Fisher's exact test: P = 0.11) and to anonymized controls (7%) (P = 0.009). Responses to phytohaemagglutinin A were significantly lower in vasculitis patients compared with other groups (Kruskal-Wallis test: P < 0.0001) and compared with non-vasculitis patients with biological inflammatory syndrome (P = 0.0015). The multivariable analysis identified as independent predictors of an indeterminate result of the QFT-GIT: the presence of systemic vasculitis (odds ratio 9.64 [1.14-81.3], P = 0.037) and a high neutrophil-to-lymphocyte ratio (odds ratio 1.70 [1.21-2.37], P = 0.002). One patient with an indeterminate result of QFT-GIT developed active tuberculosis after one year of corticosteroid therapy for giant cell arteritis. CONCLUSION: Our results question the reliability of QFT-GIT to rule out latent tuberculosis in vasculitis patients at diagnosis, prior to immunosuppressant therapy.

Gibbon genome and the fast karyotype evolution of small apes.

Gibbons are small arboreal apes that display an accelerated rate of evolutionary chromosomal rearrangement and occupy a key node in the primate phylogeny between Old World monkeys and great apes. Here we present the assembly and analysis of a northern white-cheeked gibbon (Nomascus leucogenys) genome. We describe the propensity for a gibbon-specific retrotransposon (LAVA) to insert into chromosome segregation genes and alter transcription by providing a premature termination site, suggesting a possible molecular mechanism for the genome plasticity of the gibbon lineage. We further show that the gibbon genera (Nomascus, Hylobates, Hoolock and Symphalangus) experienced a near-instantaneous radiation ∼5 million years ago, coincident with major geographical changes in southeast Asia that caused cycles of habitat compression and expansion. Finally, we identify signatures of positive selection in genes important for forelimb development (TBX5) and connective tissues (COL1A1) that may have been involved in the adaptation of gibbons to their arboreal habitat.

Distinct effect of age, sex, and CMV seropositivity on dendritic cells and monocytes in human blood.

We analyzed the impact of age, sex, and CMV on blood monocyte and dendritic cell (DC) subpopulations in 256 healthy individuals aged from 19 to 96 years. Flow cytometry was performed on whole blood within the 4 h following blood drawing. Myeloid (mDC) and plasmacytoid DC (pDC), classical, intermediate, and nonclassical monocytes were enumerated by means of TruCount tubes (BD Biosciences). We provided reference values for mDC, pDC and the three monocyte subpopulations. The numbers of classical, intermediate, and nonclassical monocytes slightly increased with age while the numbers of mDC and pDC did not vary significantly. The level of expression of CD64 and CD163 on monocytes significantly increased with age while HLA-DR expression did not vary significantly. More precisely, CD163 expression level on intermediate monocyte slightly increased with age in women only (Spearman P = 0.019) while CD64 expression increased on monocytes in CMV-positive individuals only. We observed that sex had almost no impact on the numbers of monocytes and DC and on their expression level of CD64 and HLA-DR. We observed a significant decrease in the numbers of pDC with age in CMV-positive individuals, but not in CMV negative individuals. This suggests that the lifelong subclinical infection by CMV could influence the number of circulating DC of lymphoid origin. In contrast, CMV serostatus had no significant impact on absolute numbers of mDC and monocytes.

Comparative genomics of the human, macaque and mouse major histocompatibility complex.

The MHC is a highly polymorphic genomic region that encodes the transplantation and immune regulatory molecules. It receives special attention for genetic investigation because of its important role in the regulation of innate and adaptive immune responses and its strong association with numerous infectious and/or autoimmune diseases. The MHC locus was first discovered in the mouse and for the past 50 years it has been studied most intensively in both mice and humans. However, in recent years the macaque species have emerged as some of the more important and advanced experimental animal models for biomedical research into MHC with important human immunodeficiency virus/simian immunodeficiency virus and transplantation studies undertaken in association with precise MHC genotyping and haplotyping methods using Sanger sequencing and next-generation sequencing. Here, in this special issue on 'Macaque Immunology' we provide a short review of the genomic similarities and differences among the human, macaque and mouse MHC class I and class II regions, with an emphasis on the association of the macaque class I region with MHC polymorphism, haplotype structure and function.

Alternative complement pathway hemolytic assays reveal incomplete complement blockade in patients treated with eculizumab.

Eculizumab is a monoclonal anti-C5 antibody used in the treatment of atypical hemolytic uremic syndrome (aHUS). We monitored complement inhibition in 16 eculizumab-treated patients suffering from HUS or transplant rejection (not aHUS patients). Blood samples were obtained one to four weeks after the last eculizumab injection. We observed that eculizumab efficiently blocked the terminal pathway (TP) through classical pathway (CP) activation measured by kinetic hemolytic assay (HA) (<10%) but incompletely blocked the TP through alternative pathway (AP) activation measured by rabbit (APH50>23%) or chicken erythrocytes HA (AP100>15%). Conversely, functional ELISA revealed a complete blockade of TP through AP activation in all patients (<10%). C5a and sC5b9 levels were not correlated with residual APH50 or AP100. Similar results were obtained after in vitro addition of increasing amounts of eculizumab to a control serum (in vitro APH50>60% and AP100>20%). We also showed that ELISA was less sensitive than HA.

A microplate assay to measure classical and alternative complement activity.

BACKGROUND: We developed and validated a kinetic microplate hemolytic assay (HA) to quantify classical and alternative complement activity in a single dilution of human plasma or serum. METHODS: The assay is based on monitoring hemolysis of sensitized sheep (or uncoated rabbit) red blood cells by means of a 96-well microplate reader. The activity of the calibrator was evaluated by reference to 200 healthy adults. The conversion of 50% hemolysis time into a percentage of activity was obtained using a calibration curve plotted daily. RESULTS: The linearity of the assay as well as interference (by hemolysis, bilrubinemia and lipemia) was assessed for classical pathway (CP). The within-day and the between-day precision was satisfactory regarding the performance of commercially available liposome immunoassay (LIA) and ELISA. Patients with hereditary or acquired complement deficiencies were detected (activity was measured <30%). We also provided a reference range obtained from 200 blood donors. The agreement of CP evaluated on samples from 48 patients was 94% with LIA and 87.5% with ELISA. The sensitivity of our assay was better than that of LIA, and the cost was lower than either LIA or ELISA. In addition, this assay was less time consuming than previously reported HAs. CONCLUSIONS: This assay allows the simultaneous measurement of 36 samples in duplicate per run of a 96-well plate. The use of a daily calibration curve allows standardization of the method and leads to good reproducibility. The same technique was also adapted for the quantification of alternative pathway (AP) activity.

Discovery of novel MHC-class I alleles and haplotypes in Filipino cynomolgus macaques (Macaca fascicularis) by pyrosequencing and Sanger sequencing: Mafa-class I polymorphism.

Although the low polymorphism of the major histocompatibility complex (MHC) transplantation genes in the Filipino cynomolgus macaque (Macaca fascicularis) is expected to have important implications in the selection and breeding of animals for medical research, detailed polymorphism information is still lacking for many of the duplicated class I genes. To better elucidate the degree and types of MHC polymorphisms and haplotypes in the Filipino macaque population, we genotyped 127 unrelated animals by the Sanger sequencing method and high-resolution pyrosequencing and identified 112 different alleles, 28 at cynomolgus macaque MHC (Mafa)-A, 54 at Mafa-B, 12 at Mafa-I, 11 at Mafa-E, and seven at Mafa-F alleles, of which 56 were newly described. Of them, the newly discovered Mafa-A8*01:01 lineage allele had low nucleotide similarities (<86%) with primate MHC class I genes, and it was also conserved in the Vietnamese and Indonesian populations. In addition, haplotype estimations revealed 17 Mafa-A, 23 Mafa-B, and 12 Mafa-E haplotypes integrated with 84 Mafa-class I haplotypes and Mafa-F alleles. Of these, the two Mafa-class I haplotypes, F/A/E/B-Hp1 and F/A/E/B-Hp2, had the highest haplotype frequencies at 10.6 and 10.2%, respectively. This suggests that large scale genetic screening of the Filipino macaque population would identify these and other high-frequency Mafa-class I haplotypes that could be used as MHC control animals for the benefit of biomedical research.

Deleterious impact of feto-maternal MHC compatibility on the success of pregnancy in a macaque model.

The impact of feto-maternal histocompatibility on reproduction has inspired long-lasting debates. However, after the review of numerous articles, the impact of HLA allele sharing within couples on fecundity remains questionable. We decided to explore the impact of major histocompatibility complex (MHC) feto-maternal compatibility on reproduction in a cynomolgus macaque facility composed of animals of Mauritian descent. The Mauritian-derived macaque population presents a very restricted MHC polymorphism (only seven founding haplotypes) due to a strong founding bottleneck effect. The MHC polymorphism was investigated in 237 trios (male, female and offspring) using 17 microsatellite markers distributed across the MHC. Haplotypes were confirmed by segregation analysis. We evaluated the relative frequencies of MHC-compatible and MHC-semi-compatible offspring with the mothers. Among the 237 trios, we selected 42 trios for which the identity of the father is certain and for which the theoretical probabilities of fully compatible and semi-compatible offspring were equal. We found 11 offspring fully compatible and 31 offspring semi-compatible with their respective mother. The observed proportions were clearly outside the interval of confidence of 99 % and therefore most probably resulted from a selection of the semi-compatible offspring during pregnancy. We concluded that MHC fully compatible cynomolgus macaque offspring have a selective survival disadvantage in comparison with offspring inheriting a paternal MHC haplotype differing from maternal haplotypes.

High diversity of MIC genes in non-human primates.

The human MHC class I (MHC-I) chain-related genes A and B (MICA and MICB) encode stress-induced glycoproteins, ligands for the activating receptor NKG2D. They display an unusually high degree of polymorphism, next only to that of classical MHC-I. The functional relevance and selective pressure behind this peculiar polymorphism, which is quite distinct from that of classical MHC-I, remain largely unknown. This study increases the repertoire of allelic sequences determined for the MIC genes of non-human primates. Sequencing (mainly exons 2, 3, 4, 5) MIC genes of 72 Macaca fascicularis (Mafa), 63 Pan troglodytes (Patr), and 18 Gorilla gorilla (Gogo) individuals led to the identification of 35, 14, and 3 new alleles, respectively. Additionally, we confirm the existence of three independent MIC genes in M. fascicularis, i.e., Mafa-MICA, Mafa-MICB, and Mafa-MICB/A, the latter being a hybrid of Mafa-MICB and Mafa-MICA. By multiple sequence alignment and phylogenetic analysis, we further demonstrate that the present day MIC genes most likely derive from a single human MICB-like ancestral gene.

Copy number variation analysis in the great apes reveals species-specific patterns of structural variation.

Copy number variants (CNVs) are increasingly acknowledged as an important source of evolutionary novelties in the human lineage. However, our understanding of their significance is still hindered by the lack of primate CNV data. We performed intraspecific comparative genomic hybridizations to identify loci harboring copy number variants in each of the four great apes: bonobos, chimpanzees, gorillas, and orangutans. For the first time, we could analyze differences in CNV location and frequency in these four species, and compare them with human CNVs and primate segmental duplication (SD) maps. In addition, for bonobo and gorilla, patterns of CNV and nucleotide diversity were studied in the same individuals. We show that CNVs have been subject to different selective pressures in different lineages. Evidence for purifying selection is stronger in gorilla CNVs overlapping genes, while positive selection appears to have driven the fixation of structural variants in the orangutan lineage. In contrast, chimpanzees and bonobos present high levels of common structural polymorphism, which is indicative of relaxed purifying selection together with the higher mutation rates induced by the known burst of segmental duplication in the ancestor of the African apes. Indeed, the impact of the duplication burst is noticeable by the fact that bonobo and chimpanzee share more CNVs with gorilla than expected. Finally, we identified a number of interesting genomic regions that present high-frequency CNVs in all great apes, while containing only very rare or even pathogenic structural variants in humans.

NOVEOS and ImmunoCAP Have Similar Performances for Diagnosing Food Allergies.

BACKGROUND: The clinical significance of newly available platforms for specific IgE measurement must be evaluated. However, data are lacking for NOVEOS (Hycor), especially for food allergens. OBJECTIVE: We compared the technical and clinical performance of two platforms (ImmunoCAP and NOVEOS) to measure specific IgE to 10 food allergens. METHODS: Sera from 289 clinically characterized patients were tested for IgE specific for six food allergen extracts (egg white, cow's milk, peanut, hazelnut, fish, and shrimp) and four molecular allergens (Gal d 1, Bos d 8, Ara h 2, and Cor a 14). Specific IgE measurements were carried out using ImmunoCAP and NOVEOS methods. Food allergy diagnoses were established according to international guidelines. RESULTS: A strong correlation (ρ > 0.9) was present between the two platforms whereas specific IgE concentrations measured with NOVEOS were consistently lower (mean, -15%) than with ImmunoCAP. NOVEOS and ImmunoCAP provided similar overall odds ratios and relative risks for food allergy diagnosis with both allergen extracts and molecular allergens. When all 10 allergens were considered, NOVEOS provided better receiver operating characteristic curves (P = .04). Finally, we found that the most discordant results were observed with hazelnut and peanut extracts and were related to cross-reactive carbohydrate determinants for these two with ImmunoCAP. CONCLUSIONS: Specific IgE determination by either ImmunoCAP or NOVEOS (odds ratios of allergy, 25.1 or 33.0, respectively) is highly informative regarding the risk of allergy in the selected population. The NOVEOS platform presents the advantage of being less affected by unwanted reactivity owing to carbohydrate determinant-specific IgE while requiring a 10-fold lower test sample volume.

The functional A allele was resurrected via recombination in the human ABO blood group gene.

Functional A and B alleles are distinguished at two critical sites in exon 7 of the human ABO blood group gene. The most frequent nonfunctional O alleles have one-base deletion in exon 6 producing a frameshift, and it has the A type signature in two critical sites in exon 7. Previous studies indicated that B and O alleles were derived from A allele in human lineage. In this study, we conducted a phylogenetic network analysis using six representative haplotypes: A101, A201, B101, O01, O02, and O09. The result indicated that the A allele, possibly once extinct in the human lineage a long time ago, was resurrected by a recombination between B and O alleles less than 300,000 years ago.

Glucocorticoids selectively affect the memory T cell response to SARS-Cov2 spike in vaccinated and post-infected patients with systemic lupus erythematosus.

Immune response to vaccines and pathogens remains unclear in patients with systemic lupus erythematosus (SLE). To investigate this, a single-center retrospective study was conducted with 47 SLE patients vaccinated against COVID-19, including 13 who subsequently developed an asymptomatic/mild disease. As compared to controls, post-vaccine response against Spike was reduced in SLE patients when considering both memory T-cells in a whole blood interferon gamma release assay (IGRA-S) and IgG anti-Spike antibody (Ab) responses. The SLE-associated defective IGRA-S response was associated with a serum albumin level below 40 g/L and with the use of glucocorticoids, while a defective IgG anti-Spike Ab response was associated with lower levels of anti-dsDNA and anti-SSA/Ro 52 kDa Abs. IGRA-S and IgG anti-Spike responses were independent from SLE activity and clinical phenotype, low complement, hypergammaglobulinemia, and lymphopenia. As compared to controls, SLE patients showed a rapid decay of anti-Spike T-cell memory and stable IgG anti-Spike Ab responses. In conclusion, both T cell and humoral anti-Spike responses were independently affected in our SLE patients cohort, which supports the exploration of both responses in the follow-up of SLE patients and especially in those receiving glucocorticoids.

Expression sequence tag library derived from peripheral blood mononuclear cells of the chlorocebus sabaeus.

BACKGROUND: African Green Monkeys (AGM) are amongst the most frequently used nonhuman primate models in clinical and biomedical research, nevertheless only few genomic resources exist for this species. Such information would be essential for the development of dedicated new generation technologies in fundamental and pre-clinical research using this model, and would deliver new insights into primate evolution. RESULTS: We have exhaustively sequenced an Expression Sequence Tag (EST) library made from a pool of Peripheral Blood Mononuclear Cells from sixteen Chlorocebus sabaeus monkeys. Twelve of them were infected with the Simian Immunodeficiency Virus. The mononuclear cells were or not stimulated in vitro with Concanavalin A, with lipopolysacharrides, or through mixed lymphocyte reaction in order to generate a representative and broad library of expressed sequences in immune cells. We report here 37,787 sequences, which were assembled into 14,410 contigs representing an estimated 12% of the C. sabaeus transcriptome. Using data from primate genome databases, 9,029 assembled sequences from C. sabaeus could be annotated. Sequences have been systematically aligned with ten cDNA references of primate species including Homo sapiens, Pan troglodytes, and Macaca mulatta to identify ortholog transcripts. For 506 transcripts, sequences were quasi-complete. In addition, 6,576 transcript fragments are potentially specific to the C. sabaeus or corresponding to not yet described primate genes. CONCLUSIONS: The EST library we provide here will prove useful in gene annotation efforts for future sequencing of the African Green Monkey genomes. Furthermore, this library, which particularly well represents immunological and hematological gene expression, will be an important resource for the comparative analysis of gene expression in clinically relevant nonhuman primate and human research.