Search

Safe and effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have been crucial to fight against the coronavirus disease 2019 pandemic. Most vaccines are based on a mutated version of the Spike glycoprotein [K986P/V987P (S-2P)] with improved stability, yield and immunogenicity. However, S-2P is still produced at low levels. Here, we described a novel V987H mutation that increases by two-fold the production of the recombinant Spike and the exposure of the receptor binding domain (RBD). S-V987H immunogenicity was similar to S-2P in K18-hACE2 mice and golden Syrian hamsters, and superior to a monomeric RBD. Immunization with S-V987H, but not with S-2P or RBD, conferred full protection against severe disease in both animal models after SARS-CoV-2 challenge (D614G and B.1.351 variants). Furthermore, S-V987H immunized K18-hACE2 mice showed a faster tissue viral clearance than RBD- or S-2P-vaccinated animals. Thus, S-V987H protein provides an alternative to S-2P for future SARS-CoV-2 vaccines development.

We developed one-step reverse transcription (RT)-droplet digital PCR (ddPCR) protocols to detect SARS-CoV-2 RNA and compared them to the gold-standard RT-quantitative PCR (RT-qPCR) method. RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, while both techniques were equivalent in the mid- and high-viral-load ranges.

In the present study we report the functional and structural characterization of 17T2, a new highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody (mAb) isolated from a convalescent COVID-19 individual infected during the first wave of the COVID-19 pandemic. 17T2 is a class 1 VH1-58/κ3-20 antibody, derived from a receptor binding domain (RBD)-specific IgA memory B cell and developed as a human recombinant IgG1. Functional characterization revealed that 17T2 mAb has a high and exceptionally broad neutralizing activity against all SARS-CoV-2 spike variants tested, including BQ.1.1. Moreover, 17T2 mAb has in vivo prophylactic activity against Omicron BA.1.1 infection in K18-hACE2 transgenic mice. 3D reconstruction from cryogenic-electron microscopy (cryo-EM) showed that 17T2 binds the Omicron BA.1 spike protein with the RBD domains in up position and recognizes an epitope overlapping with the receptor binding motif, as it is the case for other structurally similar neutralizing mAbs, including S2E12. Yet, unlike S2E12, 17T2 retains its high neutralizing activity against all Omicron sublineages tested, probably due to a larger contact area with the RBD, which could confer a higher resilience to spike mutations. These results highlight the impact of small structural antibody changes on neutralizing performance and identify 17T2 mAb as a potential candidate for future therapeutic and prophylactic interventions., We acknowledge access to the cryo-EM CNB-CSIC facility in the context of the CRIOMECORR project (ESFRI-2019-01-CSIC-16) and we thank the staff of the Protein Technology Unity (CRG) for the help in protein production. This study was supported by the COVID-19 call grant from Generalitat de Catalunya, Department of Health (to GM), grant Miguel Servet research program (to GM), and partially funded by the crowdfunding initiative #joemcorono and the Fundació Glòria Soler (to JB). A.P-G. was supported by a predoctoral grant from Generalitat de Catalunya and Fons Social Europeu (2022 FI_B 00698).

Neoantigens are tumor-specific antigens that are mostly particular for each patient. Since the immune system is able to mount a specific immune response against these neoantigens, they are a promising tool for the development of therapeutic personalized cancer vaccines. Neoantigens must be presented to T cells by Antigen Presenting Cells (APC) in the context of MHC-I or MHC-II molecules. Therefore, the strategy of vaccine delivery may have a major impact on the magnitude and quality of T cell responses. Neoantigen vaccines are frequently administered as a pool of individual synthetic peptides that induce mainly CD4+ T cell responses. MHC-I-mediated presentation and the elicitation of CD8+ T cell responses may be improved using DNA or RNA sequences that code for a unique long polypeptide that concatenates the different neoantigens spaced by linker sequences. When administered this way, the selection of the spacer between neoantigens is of special interest, as it might influence the processing and presentation of the right peptides by APCs. Here, we evaluate the impact of such linker regions on the MHC-I-dependent antigen presentation using an in vitro assay that assesses the MHC-I presentation of SIINFEKL, a H-2Kb-restricted OVA peptide. Our results show that spacers used to generate epitope concatenates have a large impact on the efficiency of neoantigen processing and presentation by MHC-I molecules; in contrast, the peptide position and the flanking regions have a minimal impact. Moreover, linkers based on alanine residues promote a more efficient peptide presentation than the commonly used GGGS linker.

Feline leukemia virus (FeLV) is one of the most prevalent infectious diseases in domestic cats. Although different commercial vaccines are available, none of them provides full protection. Thus, efforts to design a more efficient vaccine are needed. Our group has successfully engineered HIV-1 Gag-based VLPs that induce a potent and functional immune response against the HIV-1 transmembrane protein gp41. Here, we propose to use this concept to generate FeLV-Gag-based VLPs as a novel vaccine strategy against this retrovirus. By analogy to our HIV-1 platform, a fragment of the FeLV transmembrane p15E protein was exposed on FeLV-Gag-based VLPs. After optimization of Gag sequences, the immunogenicity of the selected candidates was evaluated in C57BL/6 and BALB/c mice, showing strong cellular and humoral responses to Gag but failing to generate anti-p15E antibodies. Altogether, this study not only tests the versatility of the enveloped VLP-based vaccine platform but also sheds light on FeLV vaccine research.

BackgroundThe pharmacokinetics of bictegravir (BIC) and its association with the decay of human immunodeficiency virus (HIV)–1 RNA in genital fluids and the rectum have not yet been addressed.MethodsWe conducted a prospective, multicenter study of antiretroviral-naive people living with HIV-1 and initiating BIC/emtricitabine (FTC)/tenofovir alafenamide (TAF). HIV-1 RNA was measured (limit of quantification, 40 copies/mL) in blood plasma (BP), seminal plasma (SP), rectal fluid (RF), and cervicovaginal fluid (CVF) at baseline; Days 3, 7, 14, and 28; and Weeks 12 and 24. Total and protein-unbound BIC concentrations at 24 hours postdose (C24h) were quantified in BP, SP, CVF and rectal tissue (RT) on Day 28 and Week 12 using a validated liquid chromatography-tandem mass spectrometry assay.ResultsThe study population comprised 15 males and 8 females. In SP, RF, and CVF, the baseline HIV-1 RNA was >40 copies/mL in 12/15, 13/15, and 4/8 individuals, respectively, with medians of 3.54 (2.41–3.79), 4.19 (2.98–4.70), and 2.56 (1.61–3.56) log10 copies/mL, respectively. The initial decay slope was significantly lower in SP than in RF and BP. The time to undetectable HIV-1 RNA was significantly shorter in SP and RF than in BP. All women achieved undetectable HIV-1 RNA in CVF at Day 14. The median total BIC concentrations in SP, RT, and CVF were 65.5 (20.1–923) ng/mL, 74.1 (6.0–478.5) ng/g, and 61.6 (14.4–1760.2) ng/mL, respectively, representing 2.7%, 2.6%, and 2.8% of the BP concentration, respectively, while the protein-unbound fractions were 51.1%, 44.6%, and 42.6%, respectively.ConclusionsBIC/FTC/TAF led to rapid decay of HIV-1 RNA in genital and rectal fluids. Protein-unbound BIC concentrations in SP, RT, and CVF highly exceeded the half-maximal effective concentration (EC50) value (1.1 ng/mL).Clinical Trials RegistrationEudraCT 2018-002310-12.

ADN del VIH-1; Cura del VIH-1; Reservorio de VIH-1 HIV-1 DNA; HIV-1 cure; HIV-1 reservoir ADN del VIH-1; Cura del VIH-1; Reservori del VIH-1 Knowing the mechanisms that govern the persistence of infected CD4+ subpopulations could help us to design new therapies to cure HIV-1 infection. We evaluated the simultaneous distribution of the HIV-1 reservoir in 13 CD4+ subpopulations from 14 HIV-1-infected individuals on antiretroviral therapy to analyze its relationship with HIV-1 transcription, immune activation, and cell proliferation. A unique large blood donation was used to isolate CD4, CD4 resting (CD4r), CD4 activated (CD4a), T naive (TN), T stem cell memory (TSCM), T central memory (TCM), T transitional memory (TTM), T effector memory (TEM), circulating T follicular helper (cTFH), TCD20, TCD32, and resting memory TCD2high (rmTCD2high) cells. HIV-1 DNA measured by droplet digital PCR ranged from 3,636 copies/106 in TTM to 244 in peripheral blood mononuclear cells (PBMCs), with no subpopulation standing out for provirus enrichment. Importantly, all the subpopulations harbored intact provirus by intact provirus DNA assay (IPDA). TCD32, cTFH, and TTM had the highest levels of HIV-1 transcription measured by fluorescent in situ hybridization with flow cytometry (FISH/flow), but without reaching statistical differences. The subpopulations more enriched in provirus had a memory phenotype, were less activated (measured by CD38+/HLA-DR+), and expressed more programmed cell death 1 (PD-1). Conversely, subpopulations transcribing more HIV-1 RNA were not necessarily enriched in provirus and were more activated (measured by CD38+/HLA-DR+) and more proliferative (measured by Ki-67). In conclusion, the HIV reservoir is composed of a mosaic of subpopulations contributing to the HIV-1 persistence through different mechanisms such as susceptibility to infection, provirus intactness, or transcriptional status. The narrow range of reservoir differences between the different blood cell subsets tested suggests limited efficacy in targeting only specific cell subpopulations during HIV-1 cure strategies. IMPORTANCE The main barrier for HIV-1 cure is the presence of latently infected CD4+ T cells. Although various cell subpopulations have been identified as major HIV-1 reservoir cells, the relative contribution of infected CD4 subpopulations in the HIV-1 reservoir remains largely unknown. Here, we evaluated the simultaneous distribution of the HIV-1 reservoir in 13 CD4+ T-cell subpopulations in peripheral blood from HIV-1-infected individuals under suppressive antiretroviral therapy. We found that the HIV-1 reservoir is composed of a mosaic of cell subpopulations, with heterogeneous proviral DNA, HIV-1 transcription, and activation status. Hence, each cell subpopulation contributes to the HIV-1 persistence through different mechanisms such as susceptibility to infection, rates of intact provirus, transcriptional status or half-life. This research provides new insights into the composition of the HIV-1 reservoir, suggesting that, to be effective, eradication strategies must simultaneously target multiple cell subpopulations. This work was sponsored in part by Grifols and by Gilead (GLD17/00204). The funding organizations had no input in the design of the study or in the collection, analyses, and interpretation of the data. Similarly, they played no role in the writing of the manuscript or in the decision to submit the study for publication. J.M.-P. is supported by grant PID2019-109870RB-I00 from the Spanish Ministry of Science and Innovation. J.M.-P. and M.J.B. are supported by the RETIC RD16/0025/0041 and RD16/0025/0007 (cofunded by the European Regional Development Fund/European Social Fund). M.J.B. is supported by grant RTI2018-101082-B-I00 (MINECO/FEDER) and by the Miguel Servet program funded by the Spanish Health Institute Carlos III (CP17/00179). C.G. was supported by the Ph.D. fellowship of the Spanish Ministry of Education, Culture, and Sport (FPU15/03698).

Improved assays are critical to the successful implementation of novel HIV-1 cure strategies, given the limited ability of currently available assays to quantify true effects on the viral reservoir. As interventions based on immune clearance target infected cells producing viral antigens, irrespective of whether the viruses generated are infectious or not, we developed a novel assay to identify viral protein production at the single-cell level. The novel viral protein spot (VIP-SPOT) assay, based on the enzyme-linked ImmunoSpot (ELISpot) approach, quantifies the frequency of CD4

BackgroundUnderstanding the determinants of long-term immune responses to SARS-CoV-2 and the concurrent impact of vaccination and emerging variants of concern will guide optimal strategies to achieve global protection against the COVID-19 pandemic.MethodsA prospective cohort of 332 COVID-19 patients was followed beyond one year. Plasma neutralizing activity was evaluated using HIV-based reporter pseudoviruses expressing different SARS-CoV-2 spikes and was longitudinally analyzed using mixed-effects models.FindingsLong-term neutralizing activity was stable beyond one year after infection in mild/asymptomatic and hospitalized participants. However, longitudinal models suggest that hospitalized individuals generate both short- and long-lived memory B cells, while outpatient responses were dominated by long-lived B cells. In both groups, vaccination boosted responses to natural infection, although viral variants, mainly B.1.351, reduced the efficacy of neutralization. Importantly, despite showing higher neutralization titers, hospitalized patients showed lower cross-neutralization of B.1.351 variant compared to outpatients. Multivariate analysis identified severity of primary infection as the factor that independently determines both the magnitude and the inferior cross-neutralization activity of long-term neutralizing responses.ConclusionsNeutralizing response induced by SARS-CoV-2 is heterogeneous in magnitude but stable beyond one year after infection. Vaccination boosts these long-lasting natural neutralizing responses, counteracting the significant resistance to neutralization of new viral variants. Severity of primary infection determines higher magnitude but poorer quality of long-term neutralizing responses.

SARS-CoV-2 vaccination is the most effective strategy to protect individuals with haematologic malignancies against severe COVID-19, while eliciting limited vaccine responses. We characterized the humoral responses following 3 mo after mRNA-based vaccines in individuals at different plasma-cell disease stages: monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and multiple myeloma on first-line therapy (MM), compared with a healthy population. Plasma samples from uninfected MM patients showed lower SARS-CoV-2-specific antibody levels and neutralization capacity compared with MGUS, SMM, and healthy individuals. Importantly, COVID-19 recovered MM individuals presented significantly higher plasma neutralization capacity compared with their uninfected counterparts, highlighting that hybrid immunity elicit stronger immunity even in this immunocompromised population. No differences in the vaccine-induced humoral responses were observed between uninfected MGUS, SMM and healthy individuals. In conclusion, MGUS and SMM patients could be SARS-CoV-2 vaccinated following the vaccine recommendations for the general population, whereas a tailored monitoring of the vaccine-induced immune responses should be considered in uninfected MM patients. This work was partially funded by Grifols, the Departament de Salut of the Generalitat de Catalunya (grant DSL0016 to J Blanco and Grant DSL015 to J Carrillo), the Spanish Health Institute Carlos III (Grant PI17/01518 and PI20/00093 to J Blanco and PI18/01332 to J Carrillo), Fundació Gloria Soler, and the crowdfunding initiatives https://www.yomecorono.com, BonPreu/Esclat and Correos. M Trigueros was supported by a doctoral fellowship from the Departament de Salut from Generalitat de Catalunya (SLT017/20/000095). F Muñoz-Lopez is supported by a doctoral grant from Sorigué Foundation. E Pradenas was supported by a doctoral grant from ANID, Chile: Grant 72180406. M Massanella was granted with RYC2020-028934-I/AEI/10.13039/501100011033 from Spanish Ministry of Science and Innovation and State Research Agency, MCIN/AEI/10.13039/501100011033 and the European Social Fund “investing in your future.”

Improved assays are critical to the successful implementation of novel HIV-1 cure strategies, given the limited ability of currently available assays to quantify true effects on the viral reservoir. As interventions based on immune clearance target infected cells producing viral antigens, irrespective of whether the viruses generated are infectious or not, we developed a novel assay to identify viral protein production at the single-cell level. The novel viral protein spot (VIP-SPOT) assay, based on the enzyme-linked ImmunoSpot (ELISpot) approach, quantifies the frequency of CD4 T cells that produce HIV antigen upon stimulation. The performance of the VIP-SPOT assay was validated in samples from viremic (n= 18) and antiretroviral therapy (ART)-treated subjects (n=35), and the results were compared with total and intact proviral DNA and plasma viremia. The size of the functional reservoir, measured by VIP-SPOT, correlates with total HIV-1 DNA and, more strongly, with intact proviruses. However, the frequency of HIV antigen-producing cells is 100-fold lower than that of intact proviruses, thus suggesting that most latently infected cells harboring full-length proviruses are not prone to reactivation. Furthermore, VIP-SPOT was useful for evaluating the efficacy of latency reversing agents (LRAs) in primary cells. VIP-SPOT is a novel tool for measuring the size of the functional HIV-1 reservoir in a rapid, sensitive, and precise manner. It might benefit the evaluation of cure strategies based on immune clearance, as these will specifically target this minor fraction of the viral reservoir, and might assist in the identification of novel therapeutic candidates that modulate viral latency. IMPORTANCE Current efforts aimed at finding a definitive cure for HIV-1 infection are hampered mainly by the persistence of a viral reservoir in latently infected cells. While complete viral eradication from the body remains elusive, finding a functional cure to enable control of viremia without the need for continuous treatment is a key goal. As the lower reservoir size increases the likelihood of controlling viremia, new therapeutic strategies aim to reduce the size of this viral reservoir. Evaluating the efficacy of these strategies requires a robust assay to measure the viral reservoir. Currently available options are subject to overestimation or underestimation of the productive reservoir. In order to overcome this limitation, we have developed a novel assay, viral protein spot (VIP-SPOT), to precisely quantify the frequency of infected cells that retain the ability to reactivate and produce viral proteins.

To assess the potential impact of predominant circulating SARS-CoV-2 variants on neutralizing activity of infected and/or vaccinated individuals, we analyzed neutralization of pseudoviruses expressing the spike of the original Wuhan strain, the D614G and B.1.1.7 variants. Our data show that parameters of natural infection (time from infection and infecting variant) determined cross-neutralization. Importantly, upon vaccination, previously infected individuals developed equivalent B.1.1.7 and Wuhan neutralizing responses. In contrast, uninfected vaccinees showed reduced neutralization against B.1.1.7.FundingThis study was funded by Grifols, the Departament de Salut of the Generalitat de Catalunya, the Spanish Health Institute Carlos III, CERCA Programme/Generalitat de Catalunya, and the crowdfunding initiatives #joemcorono, BonPreu/Esclat and Correos.

Background Understanding mid-term kinetics of immunity to SARS-CoV-2 is the cornerstone for public health control of the pandemic and vaccine development. However, current evidence is rather based on limited measurements, losing sight of the temporal pattern of these changes. Methods We conducted a longitudinal analysis on a prospective cohort of COVID-19 patients followed up for >6 months. Neutralizing activity was evaluated using HIV reporter pseudoviruses expressing SARS-CoV-2 S protein. IgG antibody titer was evaluated by ELISA against the S2 subunit, the receptor binding domain (RBD), and the nucleoprotein (NP). Statistical analyses were carried out using mixed-effects models. Findings We found that individuals with mild or asymptomatic infection experienced an insignificant decay in neutralizing activity, which persisted 6 months after symptom onset or diagnosis. Hospitalized individuals showed higher neutralizing titers, which decreased following a 2-phase pattern, with an initial rapid decline that significantly slowed after day 80. Despite this initial decay, neutralizing activity at 6 months remained higher among hospitalized individuals compared to mild symptomatic. The slow decline in neutralizing activity at mid-term contrasted with the steep slope of anti-RBD, S2, or NP antibody titers, all of them showing a constant decline over the follow-up period. Conclusions Our results reinforce the hypothesis that the quality of the neutralizing immune response against SARS-CoV-2 evolves over the post-convalescent stage. Funding This study was funded by 10.13039/501100016387Grifols, the Departament de Salut of the Generalitat de Catalunya (grant nos. SLD016 to J.B. and SLD015 to J.C.), the Spanish Health Institute Carlos III (grant nos. PI17/01518 and PI18/01332 to J.C.), CERCA Programme/Generalitat de Catalunya2017 SGR 252, and the crowdfunding initiatives #joemcorono, BonPreu/Esclat, and Correos. The funders had no role in the study design, the data collection and analysis, the decision to publish, or the preparation of the manuscript. E.P. was supported by a doctoral grant from the National Agency for Research and Development of Chile (ANID; 72180406). C.A.-N. was supported by a doctoral grant from Generalitat de Catalunya and Fons Social Europeu (FI). S.P.-Y. was supported by 10.13039/501100011850Fundación Canaria Doctor Manuel Morales and 10.13039/100015528Universidad de La Laguna., Graphical Abstract, Context and significance Assessing the durability of neutralizing responses against SARS-CoV-2 is crucial to predict the level of protection in post-convalescent COVID-19 patients. We monitored for >6 months a cohort of 210 SARS-CoV-2-infected individuals with a wide range of symptoms (from asymptomatic infection to severe disease). Our results indicate that neutralizing antibodies are stable for at least 6 months after infection. However, individuals with mild or asymptomatic infection developed lower titers of neutralizing antibodies and could be at higher risk of reinfection. Despite the maintenance of neutralizing antibodies, total antibody titers slowly but gradually declined over time without apparent stabilization. This observation requires further analysis to evaluate the potential role of viral persistence or viral re-exposure in maintaining neutralization titers., Pradenas et al. describe the kinetics of neutralizing antibodies against SARS-CoV-2 and demonstrate their association with clinical severity and their stability for at least 6 months, despite constant decay of IgG titers. These findings help us to understand the mid-term immune response and the impact on herd immunity.

The protective effect of neutralizing antibodies in SARS-CoV-2 infected individuals is not yet well defined. To address this issue, we have analyzed the kinetics of neutralizing antibody responses and their association with disease severity. Between March and May 2020, the prospective KING study enrolled 72 COVID-19+ participants grouped according to disease severity. SARS-CoV-2 infection was diagnosed by serological and virological tests. Plasma neutralizing responses were assessed against replicative virus and pseudoviral particles. Multiple regression and non-parametric tests were used to analyze dependence of parameters. The magnitude of neutralizing titers significantly increased with disease severity. Hospitalized individuals developed higher titers compared to mild-symptomatic and asymptomatic individuals, which together showed titers below the detection limit in 50% of cases. Longitudinal analysis confirmed the strong differences in neutralizing titers between non-hospitalized and hospitalized participants and showed rapid kinetics of appearance of neutralizing antibodies (50% and 80% of maximal activity reached after 11 and 17 days after symptoms onset, respectively) in hospitalized patients. No significant impact of age, gender or treatment on the neutralizing titers was observed in this limited cohort. These data identify a clear association of humoral immunity with disease severity and point to immune mechanisms other than antibodies as relevant players in COVID-19 protection. This work was partially funded by Grifols, the Departament de Salut of the Generalitat de Catalunya (Grant DSL0016 to JB and Grant DSL015 to JC), the Spanish Health Institute Carlos III (Grant PI17/01518 and PI18/01332 to JC) and the crowdfunding initiatives #joemcorono, BonPreu/Esclat and Correos. The funders had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript. EP was supported by a doctoral grant from National Agency for Research and Development (ANID)—Formation of Advanced Human Capital Program, Becas Chile: Grant 72180406. We are deeply grateful to all participant and to the technical staff of IrsiCaixa for sample processing. We also thank Daniel Pérez-Zsolt and Jordana Muñoz-Basagoiti for assistance with ACE-2 expressing 293T cell culture. Peer Reviewed "Article signat per 26 autors/es: Benjamin Trinité, Ferran Tarrés-Freixas, Jordi Rodon, Edwards Pradenas, Víctor Urrea, Silvia Marfil, María Luisa Rodríguez de la Concepción, Carlos Ávila-Nieto, Carmen Aguilar-Gurrieri, Ana Barajas, Raquel Ortiz, Roger Paredes, Lourdes Mateu, Alfonso Valencia, Víctor Guallar, Lidia Ruiz, Eulàlia Grau, Marta Massanella, Jordi Puig, Anna Chamorro, Nuria Izquierdo-Useros, Joaquim Segalés, Bonaventura Clotet, Jorge Carrillo, Júlia Vergara-Alert & Julià Blanco"

Altres ajuts: This work was partially funded by Grifols, the Departament de Salut of the Generalitat de Catalunya (grant SLD016 to J.B. and Grant SLD015 to J.C.), and the crowdfunding initiatives #joemcorono, BonPreu/Esclat and Correos. With the spread of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is a need to assess the protection conferred by both previous infections and current vaccination. Here we tested the neutralizing activity of infected and/or vaccinated individuals against pseudoviruses expressing the spike of the original SARS-CoV-2 isolate Wuhan-Hu-1 (WH1), the D614G mutant and the B.1.1.7 variant. Our data show that parameters of natural infection (time from infection and nature of the infecting variant) determined cross-neutralization. Uninfected vaccinees showed a small reduction in neutralization against the B.1.1.7 variant compared to both the WH1 strain and the D614G mutant. Interestingly, upon vaccination, previously infected individuals developed more robust neutralizing responses against B.1.1.7, suggesting that vaccines can boost the neutralization breadth conferred by natural infection.

Altres ajuts: Melinda Gates Foundation (OPP1068252) Primary HIV infection (PHI) and subsequent chronic infection alter B-cell compartment. However, longitudinal analysis defining the dynamics of B-cell alterations are still limited. We longitudinally studied B-cell subsets in individuals followed for 1 year after PHI (n = 40). Treated and untreated chronic HIV infected (n = 56) and HIV-uninfected individuals (n = 58) were recruited as reference groups at the Manhiça District in Mozambique. B cells were analyzed by multicolor flow-cytometry. Anti-HIV humoral response and plasma cytokines were assessed by ELISA or Luminex-based technology. A generalized activation of B cells induced by HIV occurs early after infection and is characterized by increases in Activated and Tissue-like memory cells, decreases in IgM-IgD- (switched) and IgM-only B cells. These alterations remain mostly stable until chronic infection and are reverted in part by ART. In contrast, other parameters followed particular dynamics: PD-1 expression in memory cells decreases progressively during the first year of infection, Transitional B cells expand at month 3-4 after infection, and Marginal zone-like B cells show a late depletion. Plasmablasts expand 2 months after infection linked to plasma viral load and anti-p24 IgG3 responses. Most of well-defined changes induced by HIV in B-cell activation and memory subsets are readily observed after PHI, lasting until ART initiation. However, subsequent changes occur after sustained viral infection. These data indicate that HIV infection impacts B cells in several waves over time, and highlight that early treatment would result in beneficial effects on the B-cell compartment.

Background Human genetic variation—mostly in the human leukocyte antigen (HLA) and C–C chemokine receptor type 5 (CCR5) regions—explains 25% of the variability in progression of human immunodeficiency virus (HIV) infection. However, it is also known that viral infections can modify cellular DNA methylation patterns. Therefore, changes in the methylation of cytosine-guanine (CpG) islands might modulate progression of HIV infection. Methods In total, 85 samples were analyzed: 21 elite controllers (EC), 21 subjects with HIV before combination antiretroviral therapy (cART) (viremic, 93 325 human immunodeficiency virus type 1 [HIV-1] RNA copies/mL) and under suppressive cART (cART, median of 17 months, This study shows that genome methylation patterns among nonhuman immunodeficiency virus (HIV)-infected and viremic, combination antiretroviral therapy (cART)-suppressed, and elite controller individuals with HIV vary depending on HIV viremia and progression profile, which might have an impact on control of HIV infection in the absence of cART.

Understanding mid-term kinetics of immunity to SARS-CoV-2 is the cornerstone for public health control of the pandemic and vaccine development. However, current evidence is rather based on limited measurements, thus losing sight of the temporal pattern of these changes1–6. In this longitudinal analysis, conducted on a prospective cohort of COVID-19 patients followed up to 242 days, we found that individuals with mild or asymptomatic infection experienced an insignificant decay in neutralizing activity that persisted six months after symptom onset or diagnosis. Hospitalized individuals showed higher neutralizing titers, which decreased following a two-phase pattern, with an initial rapid decline that significantly slowed after day 80. Despite this initial decay, neutralizing activity at six months remained higher among hospitalized individuals. The slow decline in neutralizing activity at mid-term contrasted with the steep slope of antibody titers change, reinforcing the hypothesis that the quality of immune response evolves over the post-convalescent stage4,5.

The identification of individuals with null alleles enables studying how the loss of gene function affects infection. We previously described a non‐functional variant in SIGLEC1, which encodes the myeloid‐cell receptor Siglec‐1/CD169 implicated in HIV‐1 cell‐to‐cell transmission. Here we report a significant association between the SIGLEC1 null variant and extrapulmonary dissemination of Mycobacterium tuberculosis (Mtb) in two clinical cohorts comprising 6,256 individuals. Local spread of bacteria within the lung is apparent in Mtb‐infected Siglec‐1 knockout mice which, despite having similar bacterial load, developed more extensive lesions compared to wild type mice. We find that Siglec‐1 is necessary to induce antigen presentation through extracellular vesicle uptake. We postulate that lack of Siglec‐1 delays the onset of protective immunity against Mtb by limiting antigen exchange via extracellular vesicles, allowing for an early local spread of mycobacteria that increases the risk for extrapulmonary dissemination.

Altres ajuts: This research was sponsored in part by Grifols and by Merck Sharp & Dohme España, S.A. (IISP 54925). The funding organizations had no input in the design of the study; in the collection, analyses, or interpretation of the data; writing of the manuscript; or in the decision to submit the study for publication. NH received a post-doctoral grant from the Jaqueline Beytout Foundation. FG received the support of "José María Segovia de Arana" contracts (2019) and MMT from the NIH (R01AI143457). Small viral reservoirs are found predominantly in HIV-1 controllers and individuals treated during acute/early HIV-1 infection. However, other HIV + individuals could naturally also harbour low viral reservoirs. We screened 451 HIV-1-infected treated-individuals with suppressed plasma viremia for at least 3 years and stored cryopreserved peripheral blood mononuclear cells (PBMCs). Total HIV-DNA was analysed in PBMCs with ddPCR. Individuals with 50 HIV-DNA copies/10 6 PBMCs) to analyse total HIV-DNA, T-cell and NK-cell populations, HIV-1 specific antibodies, and plasma inflammation markers. We found that 9.3% of the individuals screened had

This is an openaccessarticledistributedunderthe termsof theCreativeCommonsAttributionLicense,whichpermitsunrestricted use, distribution, andreproductionin any medium,providedthe originalauthorand sourceare credited. Objective Optimization of antiretroviral therapy and anti-inflammatory treatments, such as statins, are among the strategies aimed at reducing metabolic disorders, inflammation and immune activation in people living with HIV (PLWH). We evaluated the potential benefit of combining both strategies. Design Forty-two PLWH aged =40 years receiving a protease inhibitor (PI)-based regimen were randomized (1:1) to switch from PI to Raltegravir (n = 20), or to remain on PI (n = 22). After 24 weeks, all patients received atorvastatin 20mg/day for 48 weeks. Methods We analyzed plasma inflammatory as well as T-cell maturation, activation, exhaustion and senescence markers at baseline, 24 and 72 weeks, Results Plasma inflammatory markers remained unchanged. Furthermore, no major changes on T-cell maturation subsets, immunoactivation, exhaustion or immunosenescence markers in both CD4 and CD8 T cell compartments were observed. Only a modest decrease in the frequency of CD38+ CD8 T cells and an increase in the frequency of CD28-CD57+ in both CD4 and CD8 T-cell compartments were noticed in the Raltegravir-switched group. Conclusions The study combined antiretroviral switch to Raltegravir and Statin-based anti-inflammatory strategies to reduce inflammation and chronic immune activation in PLWH. Although this combination was safe and well tolerated, it had minimal impact on inflammatory and immunological markers. Clinical Peer Reviewed Ativle signat per 10 autors/es: Eugènia Negredo, Montse Jiménez, Jordi Puig, Cora Loste, Núria Pérez-Álvarez, Victor Urrea, Patricia Echeverría, Anna Bonjoch, Bonaventura Clotet, Julià Blanco

Background Initiation of combination antiretroviral therapy (cART) soon after HIV-1 infection limits the establishment of viral reservoirs. Thus, early treated individuals are preferred candidates to evaluate novel viral remission strategies. However, their cART-dependent HIV-1 DNA decay dynamics are still poorly defined. This can hamper the design and interpretation of results from clinical trials intended to further reduce viral reservoirs. Objectives To clarify the duration of cART needed for the HIV-1 reservoir to be stabilized in early treated individuals. Methods We characterized the longitudinal decline of total HIV-1 DNA levels by droplet digital PCR in 21 individuals initiating cART within 6 months after estimated HIV-1 acquisition. Measurements were taken at cART initiation, after 6 months and annually until Year 4. Correlations between virological and clinical parameters were statistically analysed. Statistical modelling was performed applying a mixed-effects model. Results Total HIV-1 DNA experienced a median overall decrease of 1.43 log10 units (IQR = 1.17–1.69) throughout the 4 years of follow-up. Baseline levels for total HIV-1 DNA, viral load, absolute CD4+ T cell count and CD4+/CD8+ ratio correlate with final HIV-1 DNA measurements (R2 = 0.68, P Conclusions A waiting period of 2 years on cART should be considered when designing interventions aiming to impact latent HIV-1 reservoir levels and viral rebound kinetics after cART discontinuation, in order to facilitate interpretation of results and enhance the chance of viral control.

Background Monotherapy with ritonavir-boosted PIs (PI/r) has been used to simplify treatment of HIV-1-infected patients. In previous studies raltegravir intensification evidenced ongoing viral replication and reduced T cell activation, preferentially in subjects receiving PI-based triple ART. However, data about low-level viral replication and its consequences in patients receiving PI/r monotherapy are scarce. Methods We evaluated the impact of 24 weeks of intensification with raltegravir on markers of viral persistence, cellular immune activation and inflammation biomarkers in 33 patients receiving maintenance PI/r monotherapy with darunavir or lopinavir boosted with ritonavir. ClinicalTrials.gov identifier: NCT01480713. Results The addition of raltegravir to PI/r monotherapy resulted in a transient increase in 2-LTR (long-terminal repeat) circles in a significant proportion of participants, along with decreases in CD8+ T cell activation levels and a temporary increase in the expression of the exhaustion marker CTLA-4 in peripheral T lymphocytes. Intensification with raltegravir also reduced the number of samples with intermediate levels of residual viraemia (10-60 HIV-1 RNA copies/mL) compared with samples taken during PI/r monotherapy. However, there were no changes in cell-associated HIV-1 DNA in peripheral CD4+ T cells or soluble inflammatory biomarkers (CD14, IP-10, IL-6, C-reactive protein and D-dimer). Conclusions Intensification of PI/r monotherapy with raltegravir revealed persistent low-level viral replication and reduced residual viraemia in some patients during long-term PI/r monotherapy. The concomitant change in T cell phenotype suggests an association between active viral production and T cell activation. These results contribute to understanding the lower efficacy rates of PI/r monotherapies compared with triple therapies in clinical trials.

BACKGROUND: During acute HIV infection (AHI) HIV actively replicates but seroconversion has not yet occurred. Primary HIV infection (PHI) is characterized by a transient non-specific febrile illness, a massive inflammatory response and the progressive appearance of anti-HIV specific antibodies. In this study we have identified patterns of inflammatory biomarkers associated with the innate immunological reaction prior to completion of a full humoral response. METHODS: A symptom-based screening was used to identify AHI in the Manhica District Hospital in Mozambique. Plasma levels of biomarkers were determined by luminex and ELISA. Anti-HIV antibodies were analysed by flow-cytometry and Western blot. Statistical analyses used Random Forest and logistic regression models. RESULTS: Of 3116 rapid test seronegative or indeterminate individuals, 85 (2.7%) had positive plasma HIV viral load and were enrolled as PHI, of which n=45 (52.9%), n=8 (9.4%), n=12 (14.1%) and n=20 (23.5%) were classified as Fiebig I-III, IV, V and VI stages, respectively by Western-blot. Comparison of individuals at early (Fiebig I-IV) and late (Fiebig V-VI) immune stages identified significant differences in the expression level of plasma BAFF, MCP-1, sCD163 and MIG. This cytokine signature classified patients in the pre-seroconversion phase with a sensitivity of 92.5% and a specificity of 81.2% CONCLUSIONS:: Identification of a cytokine signature specific for the pre-seroconversion stage of PHI may help to understand the earliest HIV pathogenic events and identify new potential targets for immunotherapy aimed at modulating the cytokine response to HIV infection.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

HIV-1 infection remains incurable despite the efficient combined antiretroviral therapy (cART) due to the formation of long-lived viral reservoirs that are mostly settled in CD4+T cells and maintained by homeostatic proliferation. The use of cytostatic drugs such as tyrosine kinase inhibitors (TKIs) as adjuvants to cART could be helpful to avoid the reservoir establishment and replenishment. We determined previously that TKI dasatinib, which is successfully used for treating chronic myeloid leukemia (CML), shows antiviral effect against HIV-1 infection of CD4+ T cells in vitro. HIV-infected subjects that developed CML may safely combine long-term treatment with TKIs and cART but there is no information about the effect of dasatinib on HIV-1 reservoir in vivo. Therefore, we analyzed the ability of dasatinib to protect NSG mice engrafted with human CD34+ hematopoietic stem cells from HIV-1 infection. Mice were randomly assigned to two groups that received dasatinib or placebo daily by oral gavage. After five days, all mice were infected intraperitoneally with HIV-1 and followed up for 21 days in the absence of cART. Daily administration of dasatinib decreased viral and proviral load in all treated mice, showing in 40% of these mice undetectable viral RNA or DNA in blood. Proviral HIV-1 DNA in gut-associated lymphoid tissue (GALT) was also reduced in all dasatinib-treated mice and under the limit of detection in one of these mice. Finally, treatment with dasatinib modified the distribution of CD4+ and CD8+ T-cell subpopulations, delaying their differentiation into memory T-cell subsets that are a major component of the viral reservoir. In conclusion, dasatinib afforded protection of NSG mice from HIV-1 intraperitoneal infection in the absence of cART. Funding for this research was provided by: Ministerio de Economía y Competitividad (SAF2013-44677-R, SAF2016-78480-R) Ministerio de Ciencia, Innovación y Universidades (FIS PI16CIII/00034-ISCIII-FEDER) European Regional Development Fund Bristol-Myers Squibb (BMS AI471-041) Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ANRS 2014-2) Spanish Ministry of Education, Culture and Sport (FPU15/03698). Sí

In untreated HIV-1-infected individuals, viremia is positively associated with disease progression. However, some viremic non progressors (VNPs) individuals show paradoxical high CD4+ T cell counts. HIV-1 envelope glycoprotein complex (Env) is a major cytopathic determinant in viral replication; therefore, we have deeply characterized Env function in this rare clinical phenotype. Full-length Env clones isolated from individuals with Viral Load (VL) > 10,000 copies/mL classified as VNPs (n = 15) or rapid progressors (RPs, n = 17) were geno- and phenotypically analyzed by determining diversity, expression, CD4 binding/signaling, fusogenicity, infectivity and autophagy induction. Selected Env clones from VNPs and RPs (n = 32) showed similar expression, fusion and infection abilities. Env clones from both groups showed similar affinity for CD4 during cell-to-cell transmission and consistently induced similar levels of CD4 signaling, measured by α-tubulin acetylation. Moreover, we demonstrate for the first time that primary Env clones from VNP and RP induce autophagy in uninfected cells and that this feature correlated with fusogenic capacity but was unrelated to disease progression. In conclusion, our data suggest that Env clones from VNP individuals are fully functional. Therefore, the paradoxical CD4+ T cell count stability coexisting with high levels of viral replication is unrelated to Env function. This work is supported by Spanish AIDS network “Red Temática Cooperativa de Investigación en SIDA” RD12/0017/0002, RD12/0017/0028, RD12/0017/0034, RD16/0025/0011, RD16/0025/00XX and RD16/0025/0041 as part of the Plan Nacional R + D+ I and cofunded by Spanish “Instituto de Salud Carlos III (ISCIII)-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER)”. Work in JB’s lab is supported by the Spanish Ministry of Health Project Number EC11-043 (to JB and AV-F), by the CNRS PICS program (to MB-P), J.B. and C.C. are researchers from Fundació Institut de Recerca en Ciències de la Salut Germans Trias i Pujol supported by the Health Department of the Catalan Government/Generalitat de Catalunya and ISCIII grant numbers PI14/01307 (to JB) and PI15/01053 (to CC). Work in CL-G’s lab was supported by grants SAF (2010-17226) and (2016-77894-R) from MINECO (Spain) and FIS (PI 13/02269, ISCIII). A.V-F’s Lab is supported by the European Regional Development Fund (ERDF), SAF2015-64118-R (MINECO, Spain) “Fundación CajaCanarias” BIO29, UNLL10-3E-783 (ERDF and “Fundación CajaCanarias”). D.M-A and S.M-H are funded by Fundación CajaCanarias BIO29, SAF2011-24671-FPI, “TESIS2017010116- and TESIS2015-010038-Programa Predoctoral de Formación del Personal Investigador, Agencia Canaria de Investigación, Innovación y Sociedad de la Información” and European Social Fund-fellowships. R.C-R is funded by RIS-RETIC RD16/0025/0011 and J.E-H is funded by the Cabildo Tenerife “Agustin de Betancourt” 2017 Program. Sí

Several Ebola viruses cause outbreaks of lethal haemorrhagic fever in humans, but developing therapies tackle only Zaire Ebola virus. Dendritic cells (DCs) are targets of this infection in vivo. Here, we found that Ebola virus entry into activated DCs requires the sialic acid-binding Ig-like lectin 1 (Siglec-1/CD169), which recognizes sialylated gangliosides anchored to viral membranes. Blockage of the Siglec-1 receptor by anti-Siglec-1 monoclonal antibodies halted Ebola viral uptake and cytoplasmic entry, offering cross-protection against other ganglioside-containing viruses such as human immunodeficiency virus type 1., J.M.-P. and N.I.-U. are supported by the Spanish Secretariat of State of Research, Development and Innovation through grant No. SAF2016-80033-R. J.M.-P. is supported by the Spanish AIDS network Red Temática Cooperativa de Investigación en SIDA. D.P.-Z. is supported by the Spanish Ministry of Science, Innovation and Universities and the European Regional Development Fund under agreement No. BES-2014-069931. J.C. is supported by Catalan Department of Health, PERIS fellowship (No. SLT006/17/00214). S.B. is supported by the Rio Hortega programme funded by the Spanish Health Institute CarlosIII (No. CM17/00242). X.M.-T. is supported by the Spanish Ministry of Science, Innovation and Universities and the European Regional Development Fund under agreement No. BES-2017-082900. This research was sponsored in part by Grifols.

Background In rhesus macaques, simian immunodeficiency virus infection is followed by expansion of enteric viruses but has a limited impact on the gut bacteriome. To understand the longitudinal effects of HIV-1 infection on the human gut microbiota, we prospectively followed 49 Mozambican subjects diagnosed with recent HIV-1 infection (RHI) and 54 HIV-1-negative controls for 9–18 months and compared them with 98 chronically HIV-1-infected subjects treated with antiretrovirals (n = 27) or not (n = 71). Results We show that RHI is followed by increased fecal adenovirus shedding, which persists during chronic HIV-1 infection and does not resolve with ART. Recent HIV-1 infection is also followed by transient non-HIV-specific changes in the gut bacterial richness and composition. Despite early resilience to change, an HIV-1-specific signature in the gut bacteriome—featuring depletion of Akkermansia, Anaerovibrio, Bifidobacterium, and Clostridium—previously associated with chronic inflammation, CD8+ T cell anergy, and metabolic disorders, can be eventually identified in chronically HIV-1-infected subjects. Conclusions Recent HIV-1 infection is associated with increased fecal shedding of eukaryotic viruses, transient loss of bacterial taxonomic richness, and long-term reductions in microbial gene richness. An HIV-1-associated microbiome signature only becomes evident in chronically HIV-1-infected subjects. Electronic supplementary material The online version of this article (10.1186/s40168-019-0687-5) contains supplementary material, which is available to authorized users.

Intravesical Bacille Calmette-Guerin (BCG) remains the most effective treatment for high-risk non-muscle-invasive bladder cancer (NMIBC), unfortunately there is no validated biomarker to predict clinical outcome. Here we tried to explore the possibility that a combination of the density of peritumoral infiltrating cells (Th1, Th2 and PD-L1) and the composition of peripheral immune cells (neutrophil and lymphocyte counts) could generate a more reliable prognostic biomarker. Twenty-two patients with high-risk NMIBC treated with BCG (10 BCG nonresponders and 12 BCG responders) were selected. BCG responders had significantly lower level of peritumoral T-bet(+) cells with an associated higher GATA-3(+)/T-bet(+) ratio (p = 0.04, p = 0.02, respectively). Furthermore, the immune polarization in tissue (GATA-3(+)/T-bet(+) ratio) adjusted for the systemic inflammation (neutrophil-to-lymphocyte ratio) showed a significantly higher association with the BCG response (p = 0.004). A survival analysis demonstrated prolonged recurrence-free survival (RFS) in patients with a lower T-bet(+)/Lymphocyte ratio and higher GTR/NLR (p = 0.01). No association was observed between peritumoral PD-L1(+) expression and the BCG response. In conclusion, alterations in overall immune function, both local and systemic, may influence the therapeutic response to BCG, therefore a combined analysis of tumoral (Th2/Th1 ratio) and peripheral (NLR) immune composition prior to treatment may be a promising approach to predict the BCG response in high-risk NMIBC patients.

Background Optimization of combination antiretroviral therapy (cART) can impact the human immunodeficiency virus (HIV) reservoir. We evaluated the effect on the HIV reservoir in peripheral blood and ileum biopsies in patients switching from boosted protease inhibitor (PI/r)–based therapy to dolutegravir (DTG)–based therapy. Methods Impact of Integrase-inhibitor DOlutegravir On the viral Reservoir (INDOOR) is a phase 4 open-label clinical trial that randomly included 42 HIV type 1–infected individuals on effective cART: 20 who switched from PI/r-based to DTG-based cART (switch group), and 22 who remained in PI/r-based regimens (control group). We analyzed blood and ileum biopsies to quantify episomal, total, and integrated HIV DNA, cell-associated HIV RNA, residual plasma viremia, T-cell subsets, cell activation, and inflammation markers. Results There were no related adverse events or treatment discontinuations due to drug intolerance. The HIV reservoir was consistently larger in ileal than in peripheral CD4+ T cells in both groups (P < .01). Residual viremia in plasma decreased in the switch group (P = .03). However, we did not observe significant longitudinal changes in low-level viral replication, total and integrated HIV reservoir, HIV transcription, T-cell maturation subsets, immunoactivation markers, inflammatory soluble proteins, or cellular markers of latently infected cells. Conclusions The INDOOR study is the first evaluation of changes in HIV reservoir size in ileum biopsies and in peripheral blood in individuals switched from PI/r- to DTG-based cART. Although this switch was safe and well tolerated, it had no impact on a large array of immunological and inflammatory markers or on HIV reservoir markers in peripheral or in ileal CD4+ T cells. Clinical Trials Registration EudraCT 2014-004331-39., INDOOR evaluated the effect of switching from boosted protease inhibitor– to dolutegravir-based antiretroviral therapy on the HIV reservoir in peripheral blood and ileum. Residual plasma viremia and soluble CD14 levels decreased, but low-level HIV replication, the HIV reservoir, and immune activation remained unaffected.