Utility of accessible SARS-CoV-2 specific immunoassays in vaccinated adults with a history of advanced HIV infection.

Accessible SARS-CoV-2-specific immunoassays may inform clinical management in people with HIV, particularly in case of persisting immunodysfunction. We prospectively studied their application in vaccine recipients with HIV, purposely including participants with a history of advanced HIV infection. Participants received one (n = 250), two (n = 249) or three (n = 42) doses of the BNT162b2 vaccine. Adverse events were documented through questionnaires. Sample collection occurred pre-vaccination and a median of 4 weeks post-second dose and 14 weeks post-third dose. Anti-spike and anti-nucleocapsid antibodies were measured with the Roche Elecsys chemiluminescence immunoassays. Neutralising activity was evaluated using the GenScript cPass surrogate virus neutralisation test, following validation against a Plaque Reduction Neutralization Test. T-cell reactivity was assessed with the Roche SARS-CoV-2 IFNγ release assay. Primary vaccination (2 doses) was well tolerated and elicited measurable anti-spike antibodies in 202/206 (98.0%) participants. Anti-spike titres varied widely, influenced by previous SARS-CoV-2 exposure, ethnicity, intravenous drug use, CD4 counts and HIV viremia as independent predictors. A third vaccine dose significantly boosted anti-spike and neutralising responses, reducing variability. Anti-spike titres > 15 U/mL correlated with neutralising activity in 136/144 paired samples (94.4%). Three participants with detectable anti-S antibodies did not develop cPass neutralising responses post-third dose, yet displayed SARS-CoV-2 specific IFNγ responses. SARS-CoV-2 vaccination is well-tolerated and immunogenic in adults with HIV, with responses improving post-third dose. Anti-spike antibodies serve as a reliable indicator of neutralising activity. Discordances between anti-spike and neutralising responses were accompanied by detectable IFN-γ responses, underlining the complexity of the immune response in this population.

Advanced Cellular Models for Rare Disease Study: Exploring Neural, Muscle and Skeletal Organoids.

Organoids are self-organized, three-dimensional structures derived from stem cells that can mimic the structure and physiology of human organs. Patient-specific induced pluripotent stem cells (iPSCs) and 3D organoid model systems allow cells to be analyzed in a controlled environment to simulate the characteristics of a given disease by modeling the underlying pathophysiology. The recent development of 3D cell models has offered the scientific community an exceptionally valuable tool in the study of rare diseases, overcoming the limited availability of biological samples and the limitations of animal models. This review provides an overview of iPSC models and genetic engineering techniques used to develop organoids. In particular, some of the models applied to the study of rare neuronal, muscular and skeletal diseases are described. Furthermore, the limitations and potential of developing new therapeutic approaches are discussed.

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay.

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses. Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 °C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

Low-Cost Hyperspectral Imaging to Detect Drought Stress in High-Throughput Phenotyping.

Recent developments in low-cost imaging hyperspectral cameras have opened up new possibilities for high-throughput phenotyping (HTP), allowing for high-resolution spectral data to be obtained in the visible and near-infrared spectral range. This study presents, for the first time, the integration of a low-cost hyperspectral camera Senop HSC-2 into an HTP platform to evaluate the drought stress resistance and physiological response of four tomato genotypes (770P, 990P, Red Setter and Torremaggiore) during two cycles of well-watered and deficit irrigation. Over 120 gigabytes of hyperspectral data were collected, and an innovative segmentation method able to reduce the hyperspectral dataset by 85.5% was developed and applied. A hyperspectral index (H-index) based on the red-edge slope was selected, and its ability to discriminate stress conditions was compared with three optical indices (OIs) obtained by the HTP platform. The analysis of variance (ANOVA) applied to the OIs and H-index revealed the better capacity of the H-index to describe the dynamic of drought stress trend compared to OIs, especially in the first stress and recovery phases. Selected OIs were instead capable of describing structural changes during plant growth. Finally, the OIs and H-index results have revealed a higher susceptibility to drought stress in 770P and 990P than Red Setter and Torremaggiore genotypes.

Subjects who developed SARS-CoV-2 specific IgM after vaccination show a longer humoral immunity and a lower frequency of infection.

BACKGROUND: We have previously shown that eliciting SARS-CoV-2-specific IgM after vaccination is associated with higher levels of SARS-CoV-2 neutralizing IgG. This study aims to assess whether IgM development is also associated with longer-lasting immunity. METHODS: We analysed anti-SARS-CoV-2 spike protein IgG and IgM (IgG-S, IgM-S), and anti-nucleocapsid IgG (IgG-N) in 1872 vaccinees at different time points: before the first dose (D1; w0), before the second dose (D2; w3) at three (w6) and 23 weeks (w29) after D2; moreover, 109 subjects were further tested at the booster dose (D3, w44), at 3 weeks (w47) and 6 months (w70) after D3. Two-level linear regression models were used to evaluate the differences in IgG-S levels. FINDINGS: In subjects who had no evidence of a previous infection at D1 (non-infected, NI), IgM-S development after D1 and D2 was associated with higher IgG-S levels at short (w6, p < 0.0001) and long (w29, p < 0.001) follow-up. Similar IgG-S levels were observed after D3. The majority (28/33, 85%) of the NI subjects who had developed IgM-S in response to vaccination did not experience infection. INTERPRETATION: The development of anti-SARS-CoV-2 IgM-S following D1 and D2 is associated with higher IgG-S levels. Most individuals who developed IgM-S never became infected, suggesting that IgM elicitation may be associated with a lower risk of infection. FUNDING: "Fondi Ricerca Corrente" and "Progetto Ricerca Finalizzata" COVID-2020 (Italian Ministry of Health); FUR 2020 Department of Excellence 2018-2022 (MIUR, Italy); the Brain Research Foundation Verona.

A Quantitative Method for the Study of HIV-1 and Mycobacterium tuberculosis Coinfection.

Mycobacterium tuberculosis and human immunodeficiency virus-1 (HIV-1) syndemic interactions are a major global health concern. Despite the clinical significance of coinfection, our understanding of the cellular pathophysiology and the therapeutic pharmacodynamic impact of coinfection is limited. Here, we use single-round infectious HIV-1 pseudotyped viral particles expressing green fluorescent protein alongside M. tuberculosis expressing mCherry to study pathogenesis and treatment. We report that HIV-1 infection inhibited intracellular replication of M. tuberculosis and demonstrate the therapeutic activity of antiviral treatment (efavirenz) and antimicrobial treatment (rifampicin). The described method could be applied for detailed mechanistic studies to inform the development of novel treatment strategies.

HIV and SARS-CoV-2 Co-Infection: From Population Study Evidence to In Vitro Studies.

Human immunodeficiency virus type 1 (HIV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have caused two major viral outbreaks during the last century. Two major aspects of HIV-1 and SARS-CoV-2 co-infection have been extensively investigated and deserve attention. First, the impact of the co-infection on the progression of disease caused by HIV-1 or SARS-CoV-2. Second, the impact of the HIV-1 anti-retroviral treatment on SARS-CoV-2 infection. In this review, we aim to summarize and discuss the works produced since the beginning of the SARS-CoV-2 pandemic ranging from clinical studies to in vitro experiments in the context of co-infection and drug development.

Transcriptomic and splicing changes underlying tomato responses to combined water and nutrient stress.

Tomato is a horticultural crop of high economic and nutritional value. Suboptimal environmental conditions, such as limited water and nutrient availability, cause severe yield reductions. Thus, selection of genotypes requiring lower inputs is a goal for the tomato breeding sector. We screened 10 tomato varieties exposed to water deficit, low nitrate or a combination of both. Biometric, physiological and molecular analyses revealed different stress responses among genotypes, identifying T270 as severely affected, and T250 as tolerant to the stresses applied. Investigation of transcriptome changes caused by combined stress in roots and leaves of these two genotypes yielded a low number of differentially expressed genes (DEGs) in T250 compared to T270, suggesting that T250 tailors changes in gene expression to efficiently respond to combined stress. By contrast, the susceptible tomato activated approximately one thousand and two thousand genes in leaves and roots respectively, indicating a more generalized stress response in this genotype. In particular, developmental and stress-related genes were differentially expressed, such as hormone responsive factors and transcription factors. Analysis of differential alternative splicing (DAS) events showed that combined stress greatly affects the splicing landscape in both genotypes, highlighting the important role of AS in stress response mechanisms. In particular, several stress and growth-related genes as well as transcription and splicing factors were differentially spliced in both tissues. Taken together, these results reveal important insights into the transcriptional and post-transcriptional mechanisms regulating tomato adaptation to growth under reduced water and nitrogen inputs.

Increased Prevalence of Unstable HLA-C Variants in HIV-1 Rapid-Progressor Patients.

HIV-1 infection in the absence of treatment results in progression toward AIDS. Host genetic factors play a role in HIV-1 pathogenesis, but complete knowledge is not yet available. Since less-expressed HLA-C variants are associated with poor HIV-1 control and unstable HLA-C variants are associated with higher HIV-1 infectivity, we investigated whether there was a correlation between the different stages of HIV-1 progression and the presence of specific HLA-C allotypes. HLA-C genotyping was performed using allele-specific PCR by analyzing a treatment-naïve cohort of 96 HIV-1-infected patients from multicentric cohorts in the USA, Canada, and Brazil. HIV-1-positive subjects were classified according to their different disease progression status as progressors (Ps, n = 48), long-term non-progressors (LTNPs, n = 37), and elite controllers (ECs, n = 11). HLA-C variants were classified as stable or unstable according to their binding stability to ß2-microglobulin/peptide complex. Our results showed a significant correlation between rapid progression to AIDS and the presence of two or one unstable HLA-C variants (p-value: 0.0078, p-value: 0.0143, respectively). These findings strongly suggest a link between unstable HLA-C variants both at genotype and at allele levels and rapid progression to AIDS. This work provides further insights into the impact of host genetic factors on AIDS progression.

Atlas of phenotypic, genotypic and geographical diversity present in the European traditional tomato.

The Mediterranean basin countries are considered secondary centres of tomato diversification. However, information on phenotypic and allelic variation of local tomato materials is still limited. Here we report on the evaluation of the largest traditional tomato collection, which includes 1499 accessions from Southern Europe. Analyses of 70 traits revealed a broad range of phenotypic variability with different distributions among countries, with the culinary end use within each country being the main driver of tomato diversification. Furthermore, eight main tomato types (phenoclusters) were defined by integrating phenotypic data, country of origin, and end use. Genome-wide association study (GWAS) meta-analyses identified associations in 211 loci, 159 of which were novel. The multidimensional integration of phenoclusters and the GWAS meta-analysis identified the molecular signatures for each traditional tomato type and indicated that signatures originated from differential combinations of loci, which in some cases converged in the same tomato phenotype. Our results provide a roadmap for studying and exploiting this untapped tomato diversity.

Perinatally Human Immunodeficiency Virus-Infected Adolescents and Young Adults Demonstrate Distinct BNT162b2 Messenger RNA Coronavirus Disease 2019 Vaccine Immunogenicity.

BACKGROUND: Immunization of vulnerable populations with distinct immunity often results in suboptimal immunogenicity, durability, and efficacy. METHODS: Safety and immunogenicity profiles of BNT162b2 messenger RNA coronavirus disease 2019 (COVID-19) vaccine, among people living with human immunodeficiency virus (HIV), were evaluated in 28 perinatally HIV-infected patients under antiretroviral therapy (ART) and 65 healthy controls (HCs) with no previous history of COVID-19. Thus, we measured severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific humoral and CD4+ T cell responses. Samples were collected before vaccination (baseline, day [D] 0), at the second dose (D21), and at 4 weeks (D28) and 6 months (D180) after D0. Proteomic profiles at D0 and D28 were assessed with a multiplexed proximity extension assay (Olink) on plasma samples. RESULTS: All HIV-infected patients mounted similar anti-SARS-CoV-2 humoral responses to those of HCs, albeit with lower titers of anti-trimeric S at D28 (P = .01). Only peripheral blood mononuclear cells of HIV-infected patients demonstrated at D28 an impaired ability to expand their specific (CD40L+) CD4+ T-cell populations. Similar humoral titers were maintained between the 2 groups at 6-months follow-up. We additionally correlated baseline protein levels to either humoral or cellular responses, identifying clusters of molecules involved in immune response regulation with inverse profiles between the 2 study groups. CONCLUSIONS: Responses of ART-treated HIV-infected patients, compared to those of HCs, were characterized by distinct features especially within the proteomic compartment, supporting their eligibility to an additional dose, similarly to the HC schedule.

Clinical, Virological and Immunological Subphenotypes in a Cohort of Early Treated HIV-Infected Children.

Background: Identifying subphenotypes within heterogeneous diseases may have an impact in terms of therapeutic options. In this study, we aim to assess different subphenotypes in children living with human immunodeficiency virus (HIV-1), according to the clinical, virological, and immunological characteristics. Methods: We collected clinical and sociodemographic data, baseline viral load (VL), CD4 and CD8 count and percentage, age at initiation of ART, HIV DNA reservoir size in peripheral blood mononuclear cells (PBMCs), cell-associated RNA (CA-RNA), ultrasensitive VL, CD4 subsets (T effector CD25+, activated memory cells, Treg cells), humoral-specific HIV response (T-bet B cells), innate response (CD56dim natural killer (NK) cells, NKp46+, perforin), exhaustion markers (PD-1, PD-L1, DNAM), CD8 senescence, and biomarkers for T-lymphocyte thymic output (TREC) and endothelial activation (VCAM). The most informative variables were selected using an unsupervised lasso-type penalty selection for sparse clustering. Hierarchical clustering was performed using Pearson correlation as the distance metric and WARD.D2 as the clustering method. Internal validation was applied to select the best number of clusters. To compare the characteristics among clusters, boxplot and Kruskal Wallis test were assessed. Results: Three subphenotypes were discovered (cluster1: n=18, 45%; cluster2: n=11, 27.5%; cluster3: n=11, 27.5%). Patients in cluster1 were treated earlier, had higher baseline %CD4, low HIV reservoir size, low western blot score, higher TREC values, and lower VCAM values than the patients in the other clusters. In contrast, cluster3 was the less favorable. Patients were treated later and presented poorer outcomes with lower %CD4, and higher reservoir size, along with a higher percentage of CD8 immunosenescent cells, lower TREC, higher VCAM cytokine, and a higher %CD4 PD-1. Cluster2 was intermediate. Patients were like those of cluster1, but had lower levels of t-bet expression and higher HIV DNA reservoir size. Conclusions: Three HIV pediatric subphenotypes with different virological and immunological features were identified. The most favorable cluster was characterized by a higher rate of immune reconstitution and a slower disease progression, and the less favorable with more senescence and high reservoir size. In the near future therapeutic interventions for a path of a cure might be guided or supported by the different subphenotypes.

Determinants of B-Cell Compartment Hyperactivation in European Adolescents Living With Perinatally Acquired HIV-1 After Over 10 Years of Suppressive Therapy.

Background: Despite a successful antiretroviral therapy (ART), adolescents living with perinatally acquired HIV (PHIV) experience signs of B-cell hyperactivation with expansion of 'namely' atypical B-cell phenotypes, including double negative (CD27-IgD-) and termed age associated (ABCs) B-cells (T-bet+CD11c+), which may result in reduced cell functionality, including loss of vaccine-induced immunological memory and higher risk of developing B-cells associated tumors. In this context, perinatally HIV infected children (PHIV) deserve particular attention, given their life-long exposure to chronic immune activation. Methods: We studied 40 PHIV who started treatment by the 2nd year of life and maintained 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-disciplinary 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. Results: Phenotypic signs of B cell hyperactivation were elevated in subjects starting ART later (%DN T-bet+CD11c+ p=0.03; %AM T-bet+CD11c+ p=0.02) and were associated with detectable cell-associated HIV-1 RNA (%AM T-bet+CD11c+ p=0.0003) and transient elevation of the plasma viral load (spike). Furthermore, B-cell hyperactivation appeared to be present in individuals with higher frequency of exhausted T-cells, in particular: %CD4 TIGIT+ were associated with %DN (p=0.008), %DN T-bet+CD11c+ (p=0.0002) and %AM T-bet+CD11c+ (p=0.002) and %CD4 PD-1 were associated with %DN (p=0.048), %DN T-bet+CD11c+ (p=0.039) and %AM T-bet+CD11c+ (p=0.006). The proteomic analysis revealed that subjects with expansion of these atypical B-cells and exhausted T-cells had enrichment of proteins involved in immune inflammation and complement activation pathways. Furthermore, we observed that higher levels of ABCs were associated a reduced capacity to maintain vaccine-induced antibody immunity against measles (%B-cells CD19+CD10- T-bet+, p=0.035). Conclusion: We identified that the levels of hyperactivated B cell subsets were strongly affected by time of ART start and associated with clinical, viral, cellular and plasma soluble markers. Furthermore, the expansion of ABCs also had a direct impact on the capacity to develop antibodies response following routine vaccination.

The HCV Envelope Glycoprotein Down-Modulates NF-κB Signalling and Associates With Stimulation of the Host Endoplasmic Reticulum Stress Pathway.

Following acute HCV infection, the virus establishes a chronic disease in the majority of patients whilst few individuals clear the infection spontaneously. The precise mechanisms that determine chronic HCV infection or spontaneous clearance are not completely understood but are proposed to be driven by host and viral genetic factors as well as HCV encoded immunomodulatory proteins. Using the HIV-1 LTR as a tool to measure NF-κB activity, we identified that the HCV E1E2 glycoproteins and more so the E2 protein down-modulates HIV-1 LTR activation in 293T, TZM-bl and the more physiologically relevant Huh7 liver derived cell line. We demonstrate this effect is specifically mediated through inhibiting NF-κB binding to the LTR and show that this effect was conserved for all HCV genotypes tested. Transcriptomic analysis of 293T cells expressing the HCV glycoproteins identified E1E2 mediated stimulation of the endoplasmic reticulum (ER) stress response pathway and upregulation of stress response genes such as ATF3. Through shRNA mediated inhibition of ATF3, one of the components, we observed that E1E2 mediated inhibitory effects on HIV-1 LTR activity was alleviated. Our in vitro studies demonstrate that HCV Env glycoprotein activates host ER Stress Pathways known to inhibit NF-κB activity. This has potential implications for understanding HCV induced immune activation as well as oncogenesis.

European traditional tomatoes galore: a result of farmers' selection of a few diversity-rich loci.

A comprehensive collection of 1254 tomato accessions, corresponding to European traditional and modern varieties, early domesticated varieties, and wild relatives, was analyzed by genotyping by sequencing. A continuous genetic gradient between the traditional and modern varieties was observed. European traditional tomatoes displayed very low genetic diversity, with only 298 polymorphic loci (95% threshold) out of 64 943 total variants. European traditional tomatoes could be classified into several genetic groups. Two main clusters consisting of Spanish and Italian accessions showed higher genetic diversity than the remaining varieties, suggesting that these regions might be independent secondary centers of diversity with a different history. Other varieties seem to be the result of a more recent complex pattern of migrations and hybridizations among the European regions. Several polymorphic loci were associated in a genome-wide association study with fruit morphological traits in the European traditional collection. The corresponding alleles were found to contribute to the distinctive phenotypic characteristic of the genetic varietal groups. The few highly polymorphic loci associated with morphological traits in an otherwise a low-diversity population suggests a history of balancing selection, in which tomato farmers likely maintained the morphological variation by inadvertently applying a high selective pressure within different varietal types.

SARS-CoV-2 vaccination elicits unconventional IgM specific responses in naïve and previously COVID-19-infected individuals.

BACKGROUND: Currently, evaluation of the IgG antibodies specific for the SARS-CoV-2 Spike protein following vaccination is used worldwide to estimate vaccine response. Limited data are available on vaccine-elicited IgM antibodies and their potential implication in immunity to SARS-CoV-2. METHODS: We performed a longitudinal study to quantify anti-S SARS-CoV-2 IgG and IgM (IgG-S and IgM-S) in health care worker (HCW) recipients of the BNT162b2 vaccine. Samples were collected before administration (T0), at the second dose (T1) and three weeks after T1 (T2). The cohort included 1584 immunologically naïve to SARS-CoV-2 (IN) and 289 with history of previous infection (PI). FINDINGS: IN showed three patterns of responses: (a) IgG positive/IgM negative (36.1%), (b) coordinated IgM-S/IgG-S responses appearing at T1 (37.4%) and (c) IgM appearing after IgG (26.3%). Coordinated IgM-S/IgG-S responses were associated with higher IgG titres. In IgM-S positive PI, 64.5% were IgM-S positive before vaccination, whereas 32% and 3.5% developed IgM-S after the first and second vaccine dose, respectively. IgM-S positive sera had higher pseudovirus neutralization titres compared to the IgM-S negative. INTERPRETATION: Coordinated expression of IgG-S and IgM-S after vaccination was associated with a significantly more efficient response in both antibody levels and virus-neutralizing activity. The unconventional IgG-S positive/IgM-S negative responses may suggest a recruitment of cross coronaviruses immunity by vaccination, warranting further investigation. FUNDING: Italian Ministry of Health under "Fondi Ricerca Corrente"- L1P5 and "Progetto Ricerca Finalizzata COVID-2020-12371675"; FUR 2020 Department of Excellence 2018-2022, MIUR, Italy; The Brain Research Foundation Verona.

Different decay of antibody response and VOC sensitivity in naïve and previously infected subjects at 15 weeks following vaccination with BNT162b2.

BACKGROUND: COVID-19 vaccines have demonstrated effectiveness in reducing SARS-CoV-2 mild and severe outcomes. In vaccinated subjects with SARS-CoV-2 history, RBD-specific IgG and pseudovirus neutralization titers were rapidly recalled by a single BTN162b2 vaccine dose to higher levels than those in naïve recipients after the second dose, irrespective of waning immunity. In this study, we inspected the long-term kinetic and neutralizing responses of S-specific IgG induced by two administrations of BTN162b2 vaccine in infection-naïve subjects and in subjects previously infected with SARS-CoV-2. METHODS: Twenty-six naïve and 9 previously SARS-CoV-2 infected subjects during the second wave of the pandemic in Italy were enrolled for this study. The two groups had comparable demographic and clinical characteristics. By means of ELISA and pseudotyped-neutralization assays, we investigated the kinetics of developed IgG-RBD and their neutralizing activity against both the ancestral D614G and the SARS-CoV-2 variants of concern emerged later, respectively. The Wilcoxon matched pair signed rank test and the Kruskal-Wallis test with Dunn's correction for multiple comparison were applied when needed. RESULTS: Although after 15 weeks from vaccination IgG-RBD dropped in all participants, naïve subjects experienced a more dramatic decline than those with previous SARS-CoV-2 infection. Neutralizing antibodies remained higher in subjects with SARS-CoV-2 history and conferred broad-spectrum protection. CONCLUSIONS: These data suggest that hybrid immunity to SARS-CoV-2 has a relevant impact on the development of IgG-RBD upon vaccination. However, the rapid decay of vaccination-elicited antibodies highlights that the administration of a third dose is expected to boost the response and acquire high levels of cross-neutralizing antibodies.

Measuring Proviral HIV-1 DNA: Hurdles and Improvements to an Assay Monitoring Integration Events Utilising Human Alu Repeat Sequences.

Integrated HIV-1 DNA persists despite antiretroviral therapy and can fuel viral rebound following treatment interruption. Hence, methods to specifically measure the integrated HIV-1 DNA portion only are important to monitor the reservoir in eradication trials. Here, we provide an up-to-date overview of the literature on the different approaches used to measure integrated HIV-1 DNA. Further, we propose an implemented standard-curve free assay to quantify integrated HIV-1 DNA, so-called Alu-5LTR PCR, which utilises novel primer combinations. We tested the Alu-5LTR PCR in 20 individuals on suppressive ART for a median of nine years; the results were compared to those produced with the standard-free Alu-gag assay. The numbers of median integrated HIV-1 DNA copies were 5 (range: 1-12) and 14 (5-26) with the Alu-gag and Alu-5LTR, respectively. The ratios between Alu-gag vs Alu-5LTR results were distributed within the cohort as follows: most patients (12/20, 60%) provided ratios between 2-5, with 3/20 (15%) and 5/20 (25%) being below or above this range, respectively. Alu-5LTR assay sensitivity was also determined using an "integrated standard"; the data confirmed the increased sensitivity of the assay, i.e., equal to 0.25 proviruses in 10,000 genomes. This work represents an improvement in the field of measuring proviral HIV-1 DNA that could be employed in future HIV-1 persistence and eradication studies.

Size of HIV-1 reservoir is associated with telomere shortening and immunosenescence in early-treated European children with perinatally acquired HIV-1.

INTRODUCTION: Persistence of HIV-1, causing chronic immune activation, is a key determinant of premature senescence. Early antiretroviral therapy (ART) has been associated with a reduced HIV-1 reservoir in children with perinatally acquired HIV-1 (PHIV), but its impact on the senescence process is an open question. We investigated the association between HIV-1 reservoir and biological and immune ageing profile in PHIV enrolled in the multicentre cross-sectional study CARMA (Child and Adolescent Reservoir Measurements on early suppressive ART) conducted within the EPIICAL (Early treated Perinatally HIV Infected individuals: Improving Children's Actual Life) consortium. METHODS: Between September 2017 and June 2018, CARMA enrolled 40 PHIV who started ART before 2 years of age and had undetectable viremia for at least 5 years before sampling date. Samples from 37 children with a median age of 13.8 years were available for this study. HIV-1 DNA copies on CD4 cells, relative telomere length (marker of cellular senescence) and levels of T-cell receptor rearrangement excision circle (TREC, marker of thymic output) on CD4 and CD8 cells were quantified by qPCR. Immunological profile was assessed by flow cytometry. Associations between molecular and phenotypic markers, HIV-1 reservoir and age at ART initiation were explored using a multivariable Poisson regression. RESULTS: Higher HIV-1 reservoir was associated (p<0.001) with telomere shortening (incidence rate ratio [IRR] = 0.15 [0.13-0.17]), immunosenescence (CD28- CD57+ , IRR = 1.23 [1.21-1.26]) and immunoactivation (CD38+ HLADR+ , IRR = 7.29 [6.58-8.09]) of CD4 cells. Late ART initiation (after 6 months of age) correlated with higher HIV-1 reservoir levels (552 [303-1001] vs. 89 [56-365] copies/106 CD4 cells, p = 0.003) and percentage of CD4 senescent cells (2.89 [1.95-6.31] vs. 1.02 [0.45-2.69, p = 0.047). TREC levels in CD8 cells were inversely associated with HIV-1 reservoir (IRR = 0.77 [0.76-0.79]) and were significantly lower in late treated PHIV (1128 [486-1671] vs. 2278 [1425-3314], p = 0.042). CONCLUSIONS: Later ART initiation is associated with higher HIV-1 reservoir size, which correlates with increased telomere shortening and senescence of CD4 cells. Timing of ART initiation in infancy has long-term consequences on the immune and biological ageing profile of children with perinatally acquired HIV-1.