Biology subjects

Nastouli, E.

Publications and source records attributed to Nastouli, E..

9 recordsLinked to original sources

bioRxiv2021

Determinants of precocious B-cell aging in European adolescences living with perinatally acquired HIV-1 after over 10 years of suppressive therapy

HIV infection results in a state of chronic immune activation leading to premature immune aging, B-cells dysfunction, that persists despite prolonged virological suppression. In this scenario, adolescence living with perinatally acquired HIV (PHIV), deserve a peculiar attention since potentially exposed for their entire life to chronic immune activation. Here we identified determinants of precocious aging B cells in 40 PHIV undergoing suppressive antiretroviral therapy (ART) for median 13.5 years. All individuals started ART by 2nd year of life and achieved virus suppression within the 1st year of ART, with majority of patient maintaining suppression until analysis and 5/40 experiencing viral Spike (transient elevation of HIV-1 VL, 50-999 copies/ml). We employed a multiomics approach including deep immunological B and T cell phenotype in PBMC, with aging B cells defined by the expression of T-bet and CD11c; plasma proteomics analysis by mass spectrometry and serum level of anti-measles antibodies as correlates of humoral response. We found that individuals with expansion of aging B cell, defined by the expression of T-bet+CD11c+, were those starting treatment later, presenting detectable levels of cell-associated HIV-1 RNA, history of Spikes, and a higher frequency of exhausted T-cells, including those expressing PD-1, LAG3, TIGIT. Accordingly, the proteomic analysis revealed that subjects with expansion of aging B cells and exhausted T cells had enrichment of proteins involved in immune inflammation and complement activation pathways, such as CLU and APCS which are also involved in tumor progression. Signs of precocious aging were associated with a reduced capacity to maintain virological memory against measles vaccination. To our knowledge, this is the first study focusing on precocious B-cell aging and dysfunctionality in PHIV with long-term virological suppression. Our experimental strategy enabled identification of clinical, viral, cellular and plasma soluble markers associated with B-cells aging. Our results pave the way to further define risk of disease progression or lymphoproliferative disorders in PHIV. Author summaryDespite a successful antiretroviral therapy (ART), adolescence living with perinatally acquired HIV (PHIV) experience B-cells dysfunction, including loss of vaccine-induced immunological memory and higher risk of developing B-cells associated tumors. It is thus paramount to define novel and precise correlates of precious aging B cell for the definition of novel therapeutic strategies. Here, we studied 40 PHIV who started treatment by 2nd year of life and maintain virological suppression for 13.5 years, with 5/40 patients experiencing transient elevation of the HIV-1 load in the plasma (Spike). We applied a multi-omics approach including immunological B and T cell phenotype, plasma proteomics analysis and serum level of anti-measles antibodies as functional correlates of vaccine-induced immunity. We found that levels of aging B cells were positively associated with age at ART start, cell associated HIV-1 RNA (caHIV-1 RNA) and the presence of Spikes. Individuals with increased proportions of aging B cells had concomitant expansion of exhausted T cells and were unable to maintain vaccine-induced immunity over time. B-cell aging, and T-cell exhaustion were also associated with proteins involved in immune inflammation. The factors found here to be associated with aging B-cell could inform further therapeutic studies.

immunology↗

bioRxiv2021

Reduced antibody cross-reactivity following infection with B.1.1.7 than with parental SARS-CoV-2 strains

We examined the immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant B.1.1.7 that arose in the United Kingdom and spread globally. Antibodies elicited by B.1.1.7 infection exhibited significantly reduced recognition and neutralisation of parental strains or of the South Africa B.1.351 variant, than of the infecting variant. The drop in cross-reactivity was more pronounced following B.1.1.7 than parental strain infection, indicating asymmetric heterotypic immunity induced by SARS-CoV-2 variants.

immunology↗

bioRxiv2021

Favourable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases

Differences in humoral immunity to coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), between children and adults remain unexplained and the impact of underlying immune dysfunction or suppression unknown. Here, we examined the antibody immune competence of children and adolescents with prevalent inflammatory rheumatic diseases, juvenile idiopathic arthritis (JIA), juvenile dermatomyositis (JDM) and juvenile systemic lupus erythematosus (JSLE), against the seasonal human coronavirus (HCoV)-OC43 that frequently infects this age group. Despite immune dysfunction and immunosuppressive treatment, JIA, JDM and JSLE patients mounted comparable or stronger responses than healthier peers, dominated by IgG antibodies to HCoV-OC43 spike, and harboured IgG antibodies that cross-reacted with SARS-CoV-2 spike. In contrast, responses to HCoV-OC43 and SARS-CoV-2 nucleoproteins exhibited delayed age-dependent class-switching and were not elevated in JIA, JDM and JSLE patients, arguing against increased exposure. Consequently, autoimmune rheumatic diseases and their treatment were associated with a favourable ratio of spike to nucleoprotein antibodies.

immunology↗

bioRxiv2021

The impact of Spike mutations on SARS-CoV-2 neutralization

Multiple SARS-CoV-2 vaccines have shown protective efficacy, which is most likely mediated by neutralizing antibodies recognizing the viral entry protein, Spike. Antibodies from SARS-CoV-2 infection neutralize the virus by focused targeting of Spike and there is limited serum cross-neutralization of the closely-related SARS-CoV. As new SARS-CoV-2 variants are rapidly emerging, exemplified by the B.1.1.7, 501Y.V2 and P.1 lineages, it is critical to understand if antibody responses induced by infection with the original SARS-CoV-2 virus or the current vaccines will remain effective against virus variants. In this study we evaluate neutralization of a series of mutated Spike pseudotypes including a B.1.1.7 Spike pseudotype. The analyses of a panel of Spike-specific monoclonal antibodies revealed that the neutralizing activity of some antibodies was dramatically reduced by Spike mutations. In contrast, polyclonal antibodies in the serum of patients infected in early 2020 remained active against most mutated Spike pseudotypes. The majority of serum samples were equally able to neutralize the B.1.1.7 Spike pseudotype, however potency was reduced in a small number of samples (3 of 36) by 5-10-fold. This work highlights that changes in the SARS-CoV-2 Spike can alter neutralization sensitivity and underlines the need for effective real-time monitoring of emerging mutations and their impact on vaccine efficacy.

immunology↗

bioRxiv2020

Real-time colorimetric LAMP methodology for quantitative nucleic acids detection at the point-of-care

Loop-mediated isothermal amplification is known for its high sensitivity, specificity and tolerance to inhibiting-substances. We developed a device for performing real-time colorimetric LAMP combining the accuracy of lab-based quantitative molecular diagnosis with the simplicity of point-of-care testing. This handheld device employs a single reaction-pot for amplification and a mini-camera for detection. Competitive features are the rapid analysis (<30min), quantification over 9 log-units, crude sample-compatibility (saliva, tissue, swabs), low detection limit (<5copies/reaction), smartphone-operation and fast prototyping (3D-printing). The devices clinical utility is demonstrated in cancer-mutations and COVID-19 testing. Excellent performance includes: detection of 0.01% of BRAF-V600E-to-wild-type molecules; 97% sensitivity to SARS-CoV-2 RNA detection (89 samples); 83% (Ct<34), 98% (Ct<30) and 100% (Ct<25) to 163 nasopharyngeal-swabs; 100% specificity in all cases. The device high technology-readiness-level makes it a suitable platform for performing any colorimetric LAMP assay; moreover, its simple and inexpensive fabrication holds promise for fast deployment and application in global diagnostics.

bioengineering↗

bioRxiv2020

Pre-existing and de novo humoral immunity to SARS-CoV-2 in humans

Several related human coronaviruses (HCoVs) are endemic in the human population, causing mild respiratory infections1. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiologic agent of Coronavirus disease 2019 (COVID-19), is a recent zoonotic infection that has quickly reached pandemic proportions2,3. Zoonotic introduction of novel coronaviruses is thought to occur in the absence of pre-existing immunity in the target human population. Using diverse assays for detection of antibodies reactive with the SARS-CoV-2 spike (S) glycoprotein, we demonstrate the presence of pre-existing humoral immunity in uninfected and unexposed humans to the new coronavirus. SARS-CoV-2 S-reactive antibodies were readily detectable by a sensitive flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents. These were predominantly of the IgG class and targeted the S2 subunit. In contrast, SARS-CoV-2 infection induced higher titres of SARS-CoV-2 S-reactive IgG antibodies, targeting both the S1 and S2 subunits, as well as concomitant IgM and IgA antibodies, lasting throughout the observation period of 6 weeks since symptoms onset. SARS-CoV-2-uninfected donor sera also variably reacted with SARS-CoV-2 S and nucleoprotein (N), but not with the S1 subunit or the receptor binding domain (RBD) of S on standard enzyme immunoassays. Notably, SARS-CoV-2-uninfected donor sera exhibited specific neutralising activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes, according to levels of SARS-CoV-2 S-binding IgG and with efficiencies comparable to those of COVID-19 patient sera. Distinguishing pre-existing and de novo antibody responses to SARS-CoV-2 will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.

immunology↗

bioRxiv2020

Extraction-free COVID-19 (SARS-CoV-2) diagnosis by RT-PCR to increase capacity for national testing programmes during a pandemic

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus disease 2019 (COVID-19), a respiratory tract infection. The standard molecular diagnostic test is a multistep process involving viral RNA extraction and real-time quantitative reverse transcriptase PCR (qRT-PCR). Laboratories across the globe face constraints on equipment and reagents during the COVID-19 pandemic. We have developed a simplified qRT-PCR assay that removes the need for an RNA extraction process and can be run on a real-time thermal cycler. The assay uses custom primers and probes, and maintains diagnostic sensitivity within 98.0% compared to the assay run on a high-throughput, random-access automated platform, the Panther Fusion (Hologic). This assay can be used to increase capacity for COVID-19 testing for national programmes worldwide.

molecular biology↗

bioRxiv2018

Bigger and Better? Representativeness of the Influenza A surveillance using one consolidated clinical microbiology laboratory data set as compared to the Belgian Sentinel Network of Laboratories

Infectious diseases remain a serious public health concern globally, while the need for reliable and representative surveillance systems remains as acute as ever. The public health surveillance of infectious diseases uses reported positive results from sentinel clinical laboratories or laboratory networks, to survey the presence of specific microbial agents known to constitute a threat to public health in a given population. This monitoring activity is commonly based on a representative fraction of the microbiology laboratories nationally reporting to a single central reference point. However in recent years a number of clinical microbiology laboratories (CML) have undergone a process of consolidation involving a shift towards laboratory amalgamation and closer real-time informational linkage. This report aims to investigate whether such merging activities might have a potential impact on infectious diseases surveillance. Influenza data was used from Belgian public health surveillance 2014-2017, to evaluate whether national infection trends could be estimated equally as effectively from only just one centralised CML serving the wider Brussels area (LHUB-ULB). The overall comparison reveals that there is a close correlation and representativeness of the LHUB-ULB data to the national and international data for the same time periods, both on epidemiological and molecular grounds. Notably, the effectiveness of the LHUB-ULB surveillance remains partially subject to local regional variations. These results illustrate that centralised CML-derived data are not only credible but also advantageous to use for future surveillance and prediction purposes, especially for automatic detection systems that might include multiple layers of information and timely implementation of control strategies.

epidemiology↗

bioRxiv2017

A High HIV-1 Strain Variability in London, UK, Revealed by Full-Genome Analysis: Results from the ICONIC Project

Background & MethodsThe ICONIC project has developed an automated high-throughput pipeline to generate HIV nearly full-length genomes (NFLG, i.e. from gag to nef) from next-generation sequencing (NGS) data. The pipeline was applied to 420 HIV samples collected at University College London Hospital and Barts Health NHS Trust (London) and sequenced using an Illumina MiSeq at the Wellcome Trust Sanger Institute (Cambridge). Consensus genomes were generated and subtyped using COMET, and unique recombinants were studied with jpHMM and SimPlot. Maximum-likelihood phylogenetic trees were constructed using RAxML to identify transmission networks using the Cluster Picker.\n\nResultsThe pipeline generated sequences of at least 1Kb of length (median=7.4Kb) for 375 out of the 420 samples (89%), with 174 (46.4%) being NFLG. A total of 365 sequences (169 of them NFLG) corresponded to unique subjects and were included in the down-stream analyses. The most frequent HIV subtypes were B (n=149, 40.8%) and C (n=77, 21.1%) and the circulating recombinant form CRF02_AG (n=32, 8.8%). We found 14 different CRFs (n=66, 18.1%) and multiple URFs (n=32, 8.8%) that involved recombination between 12 different subtypes/CRFs. The most frequent URFs were B/CRF01_AE (4 cases) and A1/D, B/C, and B/CRF02_AG (3 cases each). Most URFs (19/26, 73%) lacked breakpoints in the PR+RT pol region, rendering them undetectable if only that was sequenced. Twelve (37.5%) of the URFs could have emerged within the UK, whereas the rest were probably imported from sub-Saharan Africa, South East Asia and South America. For 2 URFs we found highly similar pol sequences circulating in the UK. We detected 31 phylogenetic clusters using the full dataset: 25 pairs (mostly subtypes B and C), 4 triplets and 2 quadruplets. Some of these were not consistent across different genes due to inter- and intra-subtype recombination. Clusters involved 70 sequences, 19.2% of the dataset.\n\nConclusionsThe initial analysis of genome sequences detected substantial hidden variability in the London HIV epidemic. Analysing full genome sequences, as opposed to only PR+RT, identified previously undetected recombinants. It provided a more reliable description of CRFs (that would be otherwise misclassified) and transmission clusters.

genomics↗