Virus-like particle-mediated delivery of structure-selected neoantigens demonstrates immunogenicity and antitumoral activity in mice.

BACKGROUND: Neoantigens are patient- and tumor-specific peptides that arise from somatic mutations. They stand as promising targets for personalized therapeutic cancer vaccines. The identification process for neoantigens has evolved with the use of next-generation sequencing technologies and bioinformatic tools in tumor genomics. However, in-silico strategies for selecting immunogenic neoantigens still have very low accuracy rates, since they mainly focus on predicting peptide binding to Major Histocompatibility Complex (MHC) molecules, which is key but not the sole determinant for immunogenicity. Moreover, the therapeutic potential of neoantigen-based vaccines may be enhanced using an optimal delivery platform that elicits robust de novo immune responses. METHODS: We developed a novel neoantigen selection pipeline based on existing software combined with a novel prediction method, the Neoantigen Optimization Algorithm (NOAH), which takes into account structural features of the peptide/MHC-I interaction, as well as the interaction between the peptide/MHC-I complex and the TCR, in its prediction strategy. Moreover, to maximize neoantigens' therapeutic potential, neoantigen-based vaccines should be manufactured in an optimal delivery platform that elicits robust de novo immune responses and bypasses central and peripheral tolerance. RESULTS: We generated a highly immunogenic vaccine platform based on engineered HIV-1 Gag-based Virus-Like Particles (VLPs) expressing a high copy number of each in silico selected neoantigen. We tested different neoantigen-loaded VLPs (neoVLPs) in a B16-F10 melanoma mouse model to evaluate their capability to generate new immunogenic specificities. NeoVLPs were used in in vivo immunogenicity and tumor challenge experiments. CONCLUSIONS: Our results indicate the relevance of incorporating other immunogenic determinants beyond the binding of neoantigens to MHC-I. Thus, neoVLPs loaded with neoantigens enhancing the interaction with the TCR can promote the generation of de novo antitumor-specific immune responses, resulting in a delay in tumor growth. Vaccination with the neoVLP platform is a robust alternative to current therapeutic vaccine approaches and a promising candidate for future personalized immunotherapy.

Impact of Obefazimod on Viral Persistence, Inflammation, and Immune Activation in People With Human Immunodeficiency Virus on Suppressive Antiretroviral Therapy.

BACKGROUND: Persistence of viral reservoirs has been observed in people with human immunodeficiency virus (HIV), despite long-term antiretroviral therapy (ART), and likely contributes to chronic immune activation and inflammation. Obefazimod is a novel drug that inhibits human immunodeficiency virus type 1 (HIV-1) replication and reduces inflammation. Here we assess whether obefazimod is safe and might impact HIV-1 persistence, chronic immune activation, and inflammation in ART-suppressed people with HIV. METHODS: We evaluated obefazimod-related adverse events, changes in cell-associated HIV-1 DNA and RNA, residual viremia, immunophenotype, and inflammation biomarkers in blood and rectal tissue. We compared 24 ART-suppressed people with HIV who received daily doses of 50 mg obefazimod for 12 weeks (n = 13) or 150 mg for 4 weeks (n = 11) and 12 HIV-negative individuals who received 50 mg for 4 weeks. RESULTS: The 50- and 150-mg doses of obefazimod were safe, although the 150-mg dose showed inferior tolerability. The 150-mg dose reduced HIV-1 DNA (P = .008, median fold change = 0.6) and residual viremia in all individuals with detectable viremia at baseline. Furthermore, obefazimod upregulated miR-124 in all participants and reduced the activation markers CD38, HLA-DR, and PD-1 and several inflammation biomarkers. CONCLUSIONS: The effect of obefazimod by reducing chronic immune activation and inflammation suggests a potential role for the drug in virus remission strategies involving other compounds that can activate immune cells, such as latency-reversing agents.

Alanine-based spacers promote an efficient antigen processing and presentation in neoantigen polypeptide vaccines.

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-based 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-2 Kb-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.

Exploring FeLV-Gag-Based VLPs as a New Vaccine Platform-Analysis of Production and Immunogenicity.

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.

Altered T-cell subset distribution in the viral reservoir in HIV-1-infected individuals with extremely low proviral DNA (LoViReTs).

BACKGROUND: HIV cure strategies aim to eliminate viral reservoirs that persist despite successful antiretroviral therapy (ART). We have previously described that 9% of HIV-infected individuals who receive ART harbor low levels of provirus (LoViReTs). METHODS: We selected 22 LoViReTs matched with 22 controls ART suppressed for more than 3 years with fewer than 100 and more than 100 HIV-DNA copies/106  CD4+ T cells, respectively. We measured HIV reservoirs in blood and host genetic factors. Fourteen LoViReTs underwent leukapheresis to analyze replication-competent virus, and HIV-DNA in CD4+ T-cell subpopulations. Additionally, we measured HIV-DNA in rectum and/or lymph node biopsies from nine of them. RESULTS: We found that LoViReTs harbored not only lower levels of total HIV-DNA, but also significantly lower intact HIV-DNA, cell-associated HIV-RNA, and ultrasensitive viral load than controls. The proportion of intact versus total proviruses was similar in both groups. We found no differences in the percentage of host factors. In peripheral blood, 71% of LoViReTs had undetectable replication-competent virus. Minimum levels of total HIV-DNA were found in rectal and lymph node biopsies compared with HIV-infected individuals receiving ART. The main contributors to the reservoir were short-lived transitional memory and effector memory T cells (47% and 29%, respectively), indicating an altered distribution of the HIV reservoir in the peripheral T-cell subpopulations of LoViReTs. CONCLUSION: In conclusion, LoViReTs are characterized by low levels of viral reservoir in peripheral blood and secondary lymphoid tissues, which might be explained by an altered distribution of the proviral HIV-DNA towards more short-lived memory T cells. LoViReTs can be considered exceptional candidates for future interventions aimed at curing HIV.

Dynamics of the Decay of Human Immunodeficiency Virus (HIV) RNA and Distribution of Bictegravir in the Genital Tract and Rectum in Antiretroviral-naive Adults Living With HIV-1 Treated With Bictegravir/Emtricitabine/Tenofovir Alafenamide (Spanish HIV/AIDS Research Network, PreEC/RIS 58).

BACKGROUND: The 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. METHODS: We 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. RESULTS: The 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. CONCLUSIONS: BIC/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 REGISTRATION: EudraCT 2018-002310-12.

The Genome-wide Methylation Profile of CD4+ T Cells From Individuals With Human Immunodeficiency Virus (HIV) Identifies Distinct Patterns Associated With Disease Progression.

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, <50 HIV-1 RNA copies/mL), and 22 HIV-negative donors (HIVneg). We analyzed the methylation pattern of 485 577 CpG in DNA from peripheral CD4+ T lymphocytes. We selected the most differentially methylated gene (TNF) and analyzed its specific methylation, messenger RNA (mRNA) expression, and plasma protein levels in 5 individuals before and after initiation of cART. RESULTS: We observed 129 methylated CpG sites (associated with 43 gene promoters) for which statistically significant differences were recorded in viremic versus HIVneg, 162 CpG sites (55 gene promoters) in viremic versus cART, 441 CpG sites (163 gene promoters) in viremic versus EC, but none in EC versus HIVneg. The TNF promoter region was hypermethylated in viremic versus HIVneg, cART, and EC. Moreover, we observed greater plasma levels of TNF in viremic individuals than in EC, cART, and HIVneg. CONCLUSIONS: Our study shows that genome methylation patterns vary depending on HIV infection status and progression profile and that these variations might have an impact on controlling HIV infection in the absence of cART.

HIV-1 DNA decay dynamics in early treated individuals: practical considerations for clinical trial design.

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 < 0.001; R2 = 0.54, P = 0.012; R2 = -0.47, P = 0.031; and R2 = -0.59, P = 0.0046, respectively). Statistical modelling shows that after 2 years on cART the viral reservoir had reached a set point. 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.

Switching From a Protease Inhibitor-based Regimen to a Dolutegravir-based Regimen: A Randomized Clinical Trial to Determine the Effect on Peripheral Blood and Ileum Biopsies From Antiretroviral Therapy-suppressed Human Immunodeficiency Virus-infected Individuals.

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.

Impact of intensification with raltegravir on HIV-1-infected individuals receiving monotherapy with boosted PIs.

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.

Short-term Treatment With Interferon Alfa Diminishes Expression of HIV-1 and Reduces CD4+ T-Cell Activation in Patients Coinfected With HIV and Hepatitis C Virus and Receiving Antiretroviral Therapy.

Long-term treatment with interferon (IFN) alfa plus ribavirin decreases the proviral human immunodeficiency virus type 1 (HIV) DNA level. However, the short-term impact of IFN alfa on persistent HIV and its effects on immune activation after antiretroviral therapy remain unknown. Our study showed that the cell-associated HIV RNA level and CD4(+) T-cell activation decreased in the IFN group (n = 10). No changes were detected in levels of residual plasma viremia, replication-competent reservoirs, proviral DNA, or 2-long-terminal repeat circles, although APOBEC3G, TRIM5α, BST2, and TRIM22 were upregulated in the IFN group. These data suggest that short-term treatment with IFN alfa combined with RBV decreases HIV expression, in part through inhibition of HIV transcription by TRIM22 and decrease in T-cell activation.

Production and Immunogenicity of FeLV Gag-Based VLPs Exposing a Stabilized FeLV Envelope Glycoprotein.

The envelope glycoprotein (Env) of retroviruses, such as the Feline leukemia virus (FeLV), is the main target of neutralizing humoral response, and therefore, a promising vaccine candidate, despite its reported poor immunogenicity. The incorporation of mutations that stabilize analogous proteins from other viruses in their prefusion conformation (e.g., HIV Env, SARS-CoV-2 S, or RSV F glycoproteins) has improved their capability to induce neutralizing protective immune responses. Therefore, we have stabilized the FeLV Env protein following a strategy based on the incorporation of a disulfide bond and an Ile/Pro mutation (SOSIP) previously used to generate soluble HIV Env trimers. We have characterized this SOSIP-FeLV Env in its soluble form and as a transmembrane protein present at high density on the surface of FeLV Gag-based VLPs. Furthermore, we have tested its immunogenicity in DNA-immunization assays in C57BL/6 mice. Low anti-FeLV Env responses were detected in SOSIP-FeLV soluble protein-immunized animals; however, unexpectedly no responses were detected in the animals immunized with SOSIP-FeLV Gag-based VLPs. In contrast, high humoral response against FeLV Gag was observed in the animals immunized with control Gag VLPs lacking SOSIP-FeLV Env, while this response was significantly impaired when the VLPs incorporated SOSIP-FeLV Env. Our data suggest that FeLV Env can be stabilized as a soluble protein and can be expressed in high-density VLPs. However, when formulated as a DNA vaccine, SOSIP-FeLV Env remains poorly immunogenic, a limitation that must be overcome to develop an effective FeLV vaccine.

VLPs generated by the fusion of RSV-F or hMPV-F glycoprotein to HIV-Gag show improved immunogenicity and neutralizing response in mice.

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) vaccines have been long overdue. Structure-based vaccine design created a new momentum in the last decade, and the first RSV vaccines have finally been approved in older adults and pregnant individuals. These vaccines are based on recombinant stabilized pre-fusion F glycoproteins administered as soluble proteins. Multimeric antigenic display could markedly improve immunogenicity and should be evaluated in the next generations of vaccines. Here we tested a new virus like particles-based vaccine platform which utilizes the direct fusion of an immunogen of interest to the structural human immunodeficient virus (HIV) protein Gag to increase its surface density and immunogenicity. We compared, in mice, the immunogenicity of RSV-F or hMPV-F based immunogens delivered either as soluble proteins or displayed on the surface of our VLPs. VLP associated F-proteins showed better immunogenicity and induced superior neutralizing responses. Moreover, when combining both VLP associated and soluble immunogens in a heterologous regimen, VLP-associated immunogens provided added benefits when administered as the prime immunization.

A monoclonal antibody targeting a large surface of the receptor binding motif shows pan-neutralizing SARS-CoV-2 activity.

Here we report the characterization of 17T2, a SARS-CoV-2 pan-neutralizing human monoclonal antibody isolated from a COVID-19 convalescent individual infected during the first pandemic wave. 17T2 is a class 1 VH1-58/κ3-20 antibody, derived from a receptor binding domain (RBD)-specific IgA+ memory B cell, with a broad neutralizing activity against former and new SARS-CoV-2 variants, including XBB.1.16 and BA.2.86 Omicron subvariants. Consistently, 17T2 demonstrates in vivo prophylactic and therapeutic activity against Omicron BA.1.1 infection in K18-hACE2 mice. Cryo-electron microscopy reconstruction shows that 17T2 binds the BA.1 spike with the RBD in "up" position and blocks the receptor binding motif, as other structurally similar antibodies do, including S2E12. Yet, unlike S2E12, 17T2 retains its neutralizing activity against all variants tested, probably due to a larger RBD contact area. These results highlight the impact of small structural antibody changes on neutralizing performance and identify 17T2 as a potential candidate for future clinical interventions.

Immunization with V987H-stabilized Spike glycoprotein protects K18-hACE2 mice and golden Syrian hamsters upon SARS-CoV-2 infection.

Safe and effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are 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 describe the 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 is similar to S-2P in mice and golden Syrian hamsters (GSH), and superior to a monomeric RBD. S-V987H immunization confer full protection against severe disease in K18-hACE2 mice and GSH upon SARS-CoV-2 challenge (D614G or B.1.351 variants). Furthermore, S-V987H immunized K18-hACE2 mice show a faster tissue viral clearance than RBD- or S-2P-vaccinated animals challenged with D614G, B.1.351 or Omicron BQ1.1 variants. Thus, S-V987H protein might be considered for future SARS-CoV-2 vaccines development.

Dynamics of virological and immunological markers of HIV persistence after allogeneic haematopoietic stem-cell transplantation in the IciStem cohort: a prospective observational cohort study.

BACKGROUND: Allogeneic haematopoietic stem-cell transplantation (allo-HSCT) markedly reduces HIV reservoirs, but the mechanisms by which this occurs are only partly understood. In this study, we aimed to describe the dynamics of virological and immunological markers of HIV persistence after allo-HSCT. METHODS: In this prospective observational cohort study, we analysed the viral reservoir and serological dynamics in IciStem cohort participants with HIV who had undergone allo-HSCT and were receiving antiretroviral therapy, ten of whom had received cells from donors with the CCR5Δ32 mutation. Participants from Belgium, Canada, Germany, Italy, the Netherlands, Spain, Switzerland, and the UK were included in the cohort both prospectively and retrospectively between June 1, 2014 and April 30, 2019. In the first 6 months after allo-HSCT, participants had monthly assessments, with annual assessments thereafter, with the protocol tailored to accommodate for the individual health status of each participant. HIV reservoirs were measured in blood and tissues and HIV-specific antibodies were measured in plasma. We used the Wilcoxon signed-rank test to compare data collected before and after allo-HSCT in participants for whom longitudinal data were available. When the paired test was not possible, we used the Mann-Whitney U test. We developed a mathematical model to study the factors influencing HIV reservoir reduction in people with HIV after allo-HSCT. FINDINGS: We included 30 people with HIV with haematological malignancies who received a transplant between Sept 1, 2009 and April 30, 2019 and were enrolled within the IciStem cohort and included in this analysis. HIV reservoirs in peripheral blood were reduced immediately after full donor chimerism was achieved, generally accompanied by undetectable HIV-DNA in bone marrow, ileum, lymph nodes, and cerebrospinal fluid, regardless of donor CCR5 genotype. HIV-specific antibody levels and functionality values declined more slowly than direct HIV reservoir values, decaying significantly only months after full donor chimerism. Mathematical modelling suggests that allogeneic immunity mediated by donor cells is the main viral reservoir depletion mechanism after massive reservoir reduction during conditioning chemotherapy before allo-HSCT (half-life of latently infected replication-competent cells decreased from 44 months to 1·5 months). INTERPRETATION: Our work provides, for the first time, data on the effects of allo-HSCT in the context of HIV infection. Additionally, we raise the question of which marker can serve as the last reporter of the residual viraemia, postulating that the absence of T-cell immune responses might be a more reliable marker than antibody decline after allo-HSCT. FUNDING: amfAR (American Foundation for AIDS Research; ARCHE Program), National Institutes of Health, National Institute of Allergy and Infectious Diseases, and Dutch Aidsfonds.

An efficient algorithm to perform multiple testing in epistasis screening.

BACKGROUND: Research in epistasis or gene-gene interaction detection for human complex traits has grown over the last few years. It has been marked by promising methodological developments, improved translation efforts of statistical epistasis to biological epistasis and attempts to integrate different omics information sources into the epistasis screening to enhance power. The quest for gene-gene interactions poses severe multiple-testing problems. In this context, the maxT algorithm is one technique to control the false-positive rate. However, the memory needed by this algorithm rises linearly with the amount of hypothesis tests. Gene-gene interaction studies will require a memory proportional to the squared number of SNPs. A genome-wide epistasis search would therefore require terabytes of memory. Hence, cache problems are likely to occur, increasing the computation time. In this work we present a new version of maxT, requiring an amount of memory independent from the number of genetic effects to be investigated. This algorithm was implemented in C++ in our epistasis screening software MBMDR-3.0.3. We evaluate the new implementation in terms of memory efficiency and speed using simulated data. The software is illustrated on real-life data for Crohn's disease. RESULTS: In the case of a binary (affected/unaffected) trait, the parallel workflow of MBMDR-3.0.3 analyzes all gene-gene interactions with a dataset of 100,000 SNPs typed on 1000 individuals within 4 days and 9 hours, using 999 permutations of the trait to assess statistical significance, on a cluster composed of 10 blades, containing each four Quad-Core AMD Opteron(tm) Processor 2352 2.1 GHz. In the case of a continuous trait, a similar run takes 9 days. Our program found 14 SNP-SNP interactions with a multiple-testing corrected p-value of less than 0.05 on real-life Crohn's disease (CD) data. CONCLUSIONS: Our software is the first implementation of the MB-MDR methodology able to solve large-scale SNP-SNP interactions problems within a few days, without using much memory, while adequately controlling the type I error rates. A new implementation to reach genome-wide epistasis screening is under construction. In the context of Crohn's disease, MBMDR-3.0.3 could identify epistasis involving regions that are well known in the field and could be explained from a biological point of view. This demonstrates the power of our software to find relevant phenotype-genotype higher-order associations.

Letter to the editor: Stability of Random Forest importance measures.

The goal of this article (letter to the editor) is to emphasize the value of exploring ranking stability when using the importance measures, mean decrease accuracy (MDA) and mean decrease Gini (MDG), provided by Random Forest. We illustrate with a real and a simulated example that ranks based on the MDA are unstable to small perturbations of the dataset and ranks based on the MDG provide more robust results.

Impact of chemotherapy and/or immunotherapy on neutralizing antibody response to SARS-CoV-2 mRNA-1237 vaccine in patients with solid tumors.

Patients with solid tumors have been a risk group since the beginning of the SARS-CoV-2 pandemic due to more significant complications, hospitalizations or deaths. The immunosuppressive state of cancer treatments or the tumor itself could influence the development of post-vaccination antibodies. This study prospectively analyzed 89 patients under chemotherapy and/or immunotherapy, who received two doses of the mRNA-1237 vaccine, and were compared with a group of 26 non-cancer individuals. Information on adverse events and neutralizing antibodies against the ancestral strain of SARS-CoV-2 (WH1) have been analyzed. Local reactions accounted for 65%, while systemic reactions accounted for 46% of oncologic individuals/cancer patients. Regarding the response to vaccination, 6.7% of cancer patients developed low neutralizing antibody levels. Lower levels of neutralizing antibodies between cancer and non-cancer groups were significant in individuals without previous SARS-CoV-2 infection, but not in previously infected individuals. We also observed that patients receiving chemotherapy or chemoimmunotherapy have significantly lower levels of neutralizing antibodies than non-cancer individuals. In conclusion, our study confirms the importance of prioritizing cancer patients receiving anticancer treatment in SARS-CoV-2 vaccination programs.

Comparison of Reverse Transcription (RT)-Quantitative PCR and RT-Droplet Digital PCR for Detection of Genomic and Subgenomic SARS-CoV-2 RNA.

Most individuals acutely infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibit mild symptoms. However, 10 to 20% of those infected develop long-term symptoms, referred to as post-coronavirus disease 2019 (COVID-19) condition (PCC). One hypothesis is that PCC might be exacerbated by viral persistence in tissue sanctuaries. Therefore, the accurate detection and quantification of SARS-CoV-2 are not only necessary for viral load monitoring but also crucial for detecting long-term viral persistence and determining whether viral replication is occurring in tissue reservoirs. In this study, the sensitivity and robustness of reverse transcription (RT)-droplet digital PCR (ddPCR) and RT-quantitative PCR (qPCR) techniques have been compared for the detection and quantification of SARS-CoV-2 genomic and subgenomic RNAs from oropharyngeal swabs taken from confirmed SARS-CoV-2-positive, SARS-CoV-2-exposed, and nonexposed individuals as well as from samples from mice infected with SARS-CoV-2. Our data demonstrated that both techniques presented equivalent results in the mid- and high-viral-load ranges. Additionally, RT-ddPCR was more sensitive than RT-qPCR in the low-viral-load range, allowing the accurate detection of positive results in individuals exposed to the virus. Overall, these data suggest that RT-ddPCR might be an alternative to RT-qPCR for detecting low viral loads in samples and for assessing viral persistence in samples from individuals with PCC. IMPORTANCE 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. Overall, these results suggest that RT-ddPCR might be a viable alternative to RT-qPCR when it comes to detecting low viral loads in samples, which is a highly relevant issue for determining viral persistence in as-yet-unknown tissue reservoirs in individuals suffering from post-COVID conditions or long COVID.