Intra-Host Evolution Analyses in an Immunosuppressed Patient Supports SARS-CoV-2 Viral Reservoir Hypothesis.

Throughout the SARS-CoV-2 pandemic, several variants of concern (VOCs) have been identified, many of which share recurrent mutations in the spike glycoprotein's receptor-binding domain (RBD). This region coincides with known epitopes and can therefore have an impact on immune escape. Protracted infections in immunosuppressed patients have been hypothesized to lead to an enrichment of such mutations and therefore drive evolution towards VOCs. Here, we present the case of an immunosuppressed patient that developed distinct populations with immune escape mutations throughout the course of their infection. Notably, by investigating the co-occurrence of substitutions on individual sequencing reads in the RBD, we found quasispecies harboring mutations that confer resistance to known monoclonal antibodies (mAbs) such as S:E484K and S:E484A. These mutations were acquired without the patient being treated with mAbs nor convalescent sera and without them developing a detectable immune response to the virus. We also provide additional evidence for a viral reservoir based on intra-host phylogenetics, which led to a viral substrain that evolved elsewhere in the patient's body, colonizing their upper respiratory tract (URT). The presence of SARS-CoV-2 viral reservoirs can shed light on protracted infections interspersed with periods where the virus is undetectable, and potential explanations for long-COVID cases.

Enhanced detection of antigen-specific T cells by a multiplexed AIM assay.

Broadly applicable methods to identify and characterize antigen-specific CD4+ and CD8+ T cells are key to immunology research, including studies of vaccine responses and immunity to infectious diseases. We developed a multiplexed activation-induced marker (AIM) assay that presents several advantages compared to single pairs of AIMs. The simultaneous measurement of four AIMs (CD69, 4-1BB, OX40, and CD40L) creates six AIM pairs that define CD4+ T cell populations with partial and variable overlap. When combined in an AND/OR Boolean gating strategy for analysis, this approach enhances CD4+ T cell detection compared to any single AIM pair, while CD8+ T cells are dominated by CD69/4-1BB co-expression. Supervised and unsupervised clustering analyses show differential expression of the AIMs in defined T helper lineages and that multiplexing mitigates phenotypic biases. Paired and unpaired comparisons of responses to infections (HIV and cytomegalovirus [CMV]) and vaccination (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) validate the robustness and versatility of the method.

Gaining Biological Insights through Supervised Data Visualization.

Dimensionality reduction-based data visualization is pivotal in comprehending complex biological data. The most common methods, such as PHATE, t-SNE, and UMAP, are unsupervised and therefore reflect the dominant structure in the data, which may be independent of expert-provided labels. Here we introduce a supervised data visualization method called RF-PHATE, which integrates expert knowledge for further exploration of the data. RF-PHATE leverages random forests to capture intricate featurelabel relationships. Extracting information from the forest, RF-PHATE generates low-dimensional visualizations that highlight relevant data relationships while disregarding extraneous features. This approach scales to large datasets and applies to classification and regression. We illustrate RF-PHATE's prowess through three case studies. In a multiple sclerosis study using longitudinal clinical and imaging data, RF-PHATE unveils a sub-group of patients with non-benign relapsingremitting Multiple Sclerosis, demonstrating its aptitude for time-series data. In the context of Raman spectral data, RF-PHATE effectively showcases the impact of antioxidants on diesel exhaust-exposed lung cells, highlighting its proficiency in noisy environments. Furthermore, RF-PHATE aligns established geometric structures with COVID-19 patient outcomes, enriching interpretability in a hierarchical manner. RF-PHATE bridges expert insights and visualizations, promising knowledge generation. Its adaptability, scalability, and noise tolerance underscore its potential for widespread adoption.

Plasma Human Immunodeficiency Virus 1 Soluble Glycoprotein 120 Association With Correlates of Immune Dysfunction and Inflammation in Antiretroviral Therapy-Treated Individuals With Undetectable Viremia.

BACKGROUND: Chronic inflammation persists in some people living with human immunodeficiency virus (HIV) during antiretroviral therapy and is associated with premature aging. The glycoprotein 120 (gp120) subunit of HIV-1 envelope sheds and can be detected in plasma, showing immunomodulatory properties even in the absence of detectable viremia. We evaluated whether plasma soluble gp120 (sgp120) and a family of gp120-specific anti-cluster A antibodies, linked to CD4 depletion in vitro, contribute to chronic inflammation, immune dysfunction, and subclinical cardiovascular disease in participants of the Canadian HIV and Aging Cohort Study with undetectable viremia. METHODS: Cross-sectional assessment of sgp120 and anti-cluster A antibodies was performed in 386 individuals from the cohort. Their association with proinflammatory cytokines and subclinical coronary artery disease was assessed using linear regression models. RESULTS: High levels of sgp120 and anti-cluster A antibodies were inversely correlated with CD4+ T cell count and CD4/CD8 ratio. The presence of sgp120 was associated with increased levels of interleukin 6. In participants with detectable atherosclerotic plaque and detectable sgp120, anti-cluster A antibodies and their combination with sgp120 levels correlated positively with the total volume of atherosclerotic plaques. CONCLUSIONS: This study showed that sgp120 may act as a pan toxin causing immune dysfunction and sustained inflammation in a subset of people living with HIV, contributing to the development of premature comorbid conditions.

Plasma SARS-CoV-2 RNA elimination and RAGE kinetics distinguish COVID-19 severity.

Objectives: Identifying biomarkers causing differential SARS-CoV-2 infection kinetics associated with severe COVID-19 is fundamental for effective diagnostics and therapeutic planning. Methods: In this work, we applied mathematical modelling to investigate the relationships between patient characteristics, plasma SARS-CoV-2 RNA dynamics and COVID-19 severity. Using a straightforward mathematical model of within-host viral kinetics, we estimated key model parameters from serial plasma viral RNA (vRNA) samples from 256 hospitalised COVID-19+ patients. Results: Our model predicted that clearance rates distinguish key differences in plasma vRNA kinetics and severe COVID-19. Moreover, our analyses revealed a strong correlation between plasma vRNA kinetics and plasma receptor for advanced glycation end products (RAGE) concentrations (a plasma biomarker of lung damage), collected in parallel to plasma vRNA from patients in our cohort, suggesting that RAGE can substitute for viral plasma shedding dynamics to prospectively classify seriously ill patients. Conclusion: Overall, our study identifies factors of COVID-19 severity, supports interventions to accelerate viral clearance and underlines the importance of mathematical modelling to better understand COVID-19.

Humoral Responses Elicited after a Fifth Dose of SARS-CoV-2 mRNA Bivalent Vaccine.

While an important part of the world's population is vaccinated against SARS-CoV-2, new variants continue to emerge. We observe that even after a fifth dose of the mRNA bivalent vaccine, most vaccinated individuals have antibodies that poorly neutralize several Omicron subvariants, including BQ.1.1, XBB, XBB.1.5, FD.1.1, and CH.1.1. However, Fc-effector functions remain strong and stable over time against new variants, which may partially explain why vaccines continue to be effective. We also observe that donors who have been recently infected have stronger antibody functional activities, including neutralization and Fc-effector functions, supporting the observations that hybrid immunity leads to better humoral responses.

Spontaneous HIV expression during suppressive ART is associated with the magnitude and function of HIV-specific CD4+ and CD8+ T cells.

Spontaneous transcription and translation of HIV can persist during suppressive antiretroviral therapy (ART). The quantity, phenotype, and biological relevance of this spontaneously "active" reservoir remain unclear. Using multiplexed single-cell RNAflow-fluorescence in situ hybridization (FISH), we detect active HIV transcription in 14/18 people with HIV on suppressive ART, with a median of 28/million CD4+ T cells. While these cells predominantly exhibit abortive transcription, p24-expressing cells are evident in 39% of participants. Phenotypically diverse, active reservoirs are enriched in central memory T cells and CCR6- and activation-marker-expressing cells. The magnitude of the active reservoir positively correlates with total HIV-specific CD4+ and CD8+ T cell responses and with multiple HIV-specific T cell clusters identified by unsupervised analysis. These associations are particularly strong with p24-expressing active reservoir cells. Single-cell vDNA sequencing shows that active reservoirs are largely dominated by defective proviruses. Our data suggest that these reservoirs maintain HIV-specific CD4+ and CD8+ T responses during suppressive ART.

Post-COVID symptoms are associated with endotypes reflecting poor inflammatory and hemostatic modulation.

Introduction: Persistent symptoms after COVID-19 infection ("long COVID") negatively affects almost half of COVID-19 survivors. Despite its prevalence, its pathophysiology is poorly understood, with multiple host systems likely affected. Here, we followed patients from hospital to discharge and used a systems-biology approach to identify mechanisms of long COVID. Methods: RNA-seq was performed on whole blood collected early in hospital and 4-12 weeks after discharge from 24 adult COVID-19 patients (10 reported post-COVID symptoms after discharge). Differential gene expression analysis, pathway enrichment, and machine learning methods were used to identify underlying mechanisms for post-COVID symptom development. Results: Compared to patients with post-COVID symptoms, patients without post-COVID symptoms had larger temporal gene expression changes associated with downregulation of inflammatory and coagulation genes over time. Patients could also be separated into three patient endotypes with differing mechanistic trajectories, which was validated in another published patient cohort. The "Resolved" endotype (lowest rate of post-COVID symptoms) had robust inflammatory and hemostatic responses in hospital that resolved after discharge. Conversely, the inflammatory/hemostatic responses of "Suppressive" and "Unresolved" endotypes (higher rates of patients with post-COVID symptoms) were persistently dampened and activated, respectively. These endotypes were accurately defined by specific blood gene expression signatures (6-7 genes) for potential clinical stratification. Discussion: This study allowed analysis of long COVID whole blood transcriptomics trajectories while accounting for the issue of patient heterogeneity. Two of the three identified and externally validated endotypes ("Unresolved" and "Suppressive") were associated with higher rates of post-COVID symptoms and either persistently activated or suppressed inflammation and coagulation processes. Gene biomarkers in blood could potentially be used clinically to stratify patients into different endotypes, paving the way for personalized long COVID treatment.

Plasmatic HIV-1 soluble gp120 is associated with immune dysfunction and inflammation in ART-treated individuals with undetectable viremia.

Background: Chronic inflammation persists in some people living with HIV (PLWH), even during antiretroviral therapy (ART) and is associated with premature aging. The gp120 subunit of the HIV-1 envelope glycoprotein can shed from viral and cellular membranes and can be detected in plasma and tissues, showing immunomodulatory properties even in the absence of detectable viremia. We evaluated whether plasmatic soluble gp120 (sgp120) and a family of gp120-specific anti-cluster A antibodies, which were previously linked to CD4 depletion in vitro , could contribute to chronic inflammation, immune dysfunction, and sub-clinical cardiovascular disease in participants of the Canadian HIV and Aging cohort (CHACS) with undetectable viremia. Methods: Cross-sectional assessment of plasmatic sgp120 and anti-cluster A antibodies was performed in 386 individuals from CHACS. Their association with pro-inflammatory cytokines, as well as subclinical coronary artery disease measured by computed tomography coronary angiography was assessed using linear regression models. Results: In individuals with high levels of sgp120, anti-cluster A antibodies inversely correlated with CD4 count (p=0.042) and CD4:CD8 ratio (p=0.004). The presence of sgp120 was associated with increased plasma levels of IL-6. In participants with detectable atherosclerotic plaque and detectable sgp120, sgp120 levels, anti-cluster A antibodies and their combination correlated positively with the total volume of atherosclerotic plaques (p=0.01, 0.018 and 0.006, respectively). Conclusion: Soluble gp120 may act as a pan toxin causing immune dysfunction and sustained inflammation in a subset of PLWH, contributing to the development of premature comorbidities. Whether drugs targeting sgp120 could mitigate HIV-associated comorbidities in PLWH with suppressed viremia warrants further studies. Key points: Soluble gp120 is detected in the plasma of people living with HIV-1 with undetectable viremia. The presence of soluble gp120 and anti-cluster A antibodies is associated with immune dysfunction, chronic inflammation, and sub-clinical cardiovascular disease.

A third SARS-CoV-2 mRNA vaccine dose in people receiving hemodialysis overcomes B cell defects but elicits a skewed CD4+ T cell profile.

Cellular immune defects associated with suboptimal responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccination in people receiving hemodialysis (HD) are poorly understood. We longitudinally analyze antibody, B cell, CD4+, and CD8+ T cell vaccine responses in 27 HD patients and 26 low-risk control individuals (CIs). The first two doses elicit weaker B cell and CD8+ T cell responses in HD than in CI, while CD4+ T cell responses are quantitatively similar. In HD, a third dose robustly boosts B cell responses, leads to convergent CD8+ T cell responses, and enhances comparatively more T helper (TH) immunity. Unsupervised clustering of single-cell features reveals phenotypic and functional shifts over time and between cohorts. The third dose attenuates some features of TH cells in HD (tumor necrosis factor alpha [TNFα]/interleukin [IL]-2 skewing), while others (CCR6, CXCR6, programmed cell death protein 1 [PD-1], and HLA-DR overexpression) persist. Therefore, a third vaccine dose is critical to achieving robust multifaceted immunity in hemodialysis patients, although some distinct TH characteristics endure.

On-demand, hospital-based, severe acute respiratory coronavirus virus 2 (SARS-CoV-2) genomic epidemiology to support nosocomial outbreak investigations: A prospective molecular epidemiology study.

Objectives: We evaluated the added value of infection control-guided, on demand, and locally performed severe acute respiratory coronavirus virus 2 (SARS-CoV-2) genomic sequencing to support outbreak investigation and control in acute-care settings. Design and setting: This 18-month prospective molecular epidemiology study was conducted at a tertiary-care hospital in Montreal, Canada. When nosocomial transmission was suspected by local infection control, viral genomic sequencing was performed locally for all putative outbreak cases. Molecular and conventional epidemiology data were correlated on a just-in-time basis to improve understanding of coronavirus disease 2019 (COVID-19) transmission and reinforce or adapt control measures. Results: Between April 2020 and October 2021, 6 outbreaks including 59 nosocomial infections (per the epidemiological definition) were investigated. Genomic data supported 7 distinct transmission clusters involving 6 patients and 26 healthcare workers. We identified multiple distinct modes of transmission, which led to reinforcement and adaptation of infection control measures. Molecular epidemiology data also refuted (n = 14) suspected transmission events in favor of community acquired but institutionally clustered cases. Conclusion: SARS-CoV-2 genomic sequencing can refute or strengthen transmission hypotheses from conventional nosocomial epidemiological investigations, and guide implementation of setting-specific control strategies. Our study represents a template for prospective, on site, outbreak-focused SARS-CoV-2 sequencing. This approach may become increasingly relevant in a COVID-19 endemic state where systematic sequencing within centralized surveillance programs is not available. Trial registration: clinicaltrials.gov identifier: NCT05411562.

Temsavir blocks the immunomodulatory activities of HIV-1 soluble gp120.

While HIV-1-mediated CD4 downregulation protects infected cells from antibody-dependent cellular cytotoxicity (ADCC), shed gp120 binds to CD4 on uninfected bystander CD4+ T cells, sensitizing them to ADCC mediated by HIV+ plasma. Soluble gp120-CD4 interaction on multiple immune cells also triggers a cytokine burst. The small molecule temsavir acts as an HIV-1 attachment inhibitor by preventing envelope glycoprotein (Env)-CD4 interaction and alters the overall antigenicity of Env by affecting its processing and glycosylation. Here we show that temsavir also blocks the immunomodulatory activities of shed gp120. Temsavir prevents shed gp120 from interacting with uninfected bystander CD4+ cells, protecting them from ADCC responses and preventing a cytokine burst. Mechanistically, this depends on temsavir's capacity to prevent soluble gp120-CD4 interaction, to reduce gp120 shedding, and to alter gp120 antigenicity. This suggests that the clinical benefits provided by temsavir could extend beyond blocking viral entry.

Humoral Responses against BQ.1.1 Elicited after Breakthrough Infection and SARS-CoV-2 mRNA Vaccination.

The Omicron BQ.1.1 variant is now the major SARS-CoV-2 circulating strain in many countries. Because of the many mutations present in its Spike glycoprotein, this variant is resistant to humoral responses elicited by monovalent mRNA vaccines. With the goal to improve immune responses against Omicron subvariants, bivalent mRNA vaccines have recently been approved in several countries. In this study, we measure the capacity of plasma from vaccinated individuals, before and after a fourth dose of mono- or bivalent mRNA vaccine, to recognize and neutralize the ancestral (D614G) and the BQ.1.1 Spikes. Before and after the fourth dose, we observe a significantly better recognition and neutralization of the ancestral Spike. We also observe that fourth-dose vaccinated individuals who have been recently infected better recognize and neutralize the BQ.1.1 Spike, independently of the mRNA vaccine used, than donors who have never been infected or have an older infection. Our study supports that hybrid immunity, generated by vaccination and a recent infection, induces higher humoral responses than vaccination alone, independently of the mRNA vaccine used.

Spike recognition and neutralization of SARS-CoV-2 Omicron subvariants elicited after the third dose of mRNA vaccine.

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants have recently emerged, becoming the dominant circulating strains in many countries. These variants contain a large number of mutations in their spike glycoprotein, raising concerns about vaccine efficacy. In this study, we evaluate the ability of plasma from a cohort of individuals that received three doses of mRNA vaccine to recognize and neutralize these Omicron subvariant spikes. We observed that BA.4/5 and BQ.1.1 spikes are markedly less recognized and neutralized compared with the D614G and other Omicron subvariant spikes tested. Also, individuals who have been infected before or after vaccination present better humoral responses than SARS-CoV-2-naive vaccinated individuals, thus indicating that hybrid immunity generates better humoral responses against these subvariants.

Predicting severity in COVID-19 disease using sepsis blood gene expression signatures.

Severely-afflicted COVID-19 patients can exhibit disease manifestations representative of sepsis, including acute respiratory distress syndrome and multiple organ failure. We hypothesized that diagnostic tools used in managing all-cause sepsis, such as clinical criteria, biomarkers, and gene expression signatures, should extend to COVID-19 patients. Here we analyzed the whole blood transcriptome of 124 early (1-5 days post-hospital admission) and late (6-20 days post-admission) sampled patients with confirmed COVID-19 infections from hospitals in Quebec, Canada. Mechanisms associated with COVID-19 severity were identified between severity groups (ranging from mild disease to the requirement for mechanical ventilation and mortality), and established sepsis signatures were assessed for dysregulation. Specifically, gene expression signatures representing pathophysiological events, namely cellular reprogramming, organ dysfunction, and mortality, were significantly enriched and predictive of severity and lethality in COVID-19 patients. Mechanistic endotypes reflective of distinct sepsis aetiologies and therapeutic opportunities were also identified in subsets of patients, enabling prediction of potentially-effective repurposed drugs. The expression of sepsis gene expression signatures in severely-afflicted COVID-19 patients indicates that these patients should be classified as having severe sepsis. Accordingly, in severe COVID-19 patients, these signatures should be strongly considered for the mechanistic characterization, diagnosis, and guidance of treatment using repurposed drugs.

An extended SARS-CoV-2 mRNA vaccine prime-boost interval enhances B cell immunity with limited impact on T cells.

Spacing the first two doses of SARS-CoV-2 mRNA vaccines beyond 3-4 weeks raised initial concerns about vaccine efficacy. While studies have since shown that long-interval regimens induce robust antibody responses, their impact on B and T cell immunity is poorly known. Here, we compare SARS-CoV-2 naive donors B and T cell responses to two mRNA vaccine doses administered 3-4 versus 16 weeks apart. After boost, the longer interval results in a higher magnitude and a more mature phenotype of RBD-specific B cells. While the two geographically distinct cohorts present quantitative and qualitative differences in T cell responses at baseline and after priming, the second dose led to convergent features with overall similar magnitude, phenotype, and function of CD4+ and CD8+ T cell responses at post-boost memory time points. Therefore, compared to standard regimens, a 16-week interval has a favorable impact on the B cell compartment but minimally affects T cell immunity.

Excess BAFF May Impact HIV-1-Specific Antibodies and May Promote Polyclonal Responses Including Those from First-Line Marginal Zone B-Cell Populations.

We have previously shown that blood levels of B-cell Activating Factor (BAFF) rise relatively to disease progression status in the context of HIV-1 infection. Excess BAFF was concomitant with hyperglobulinemia and the deregulation of blood B-cell populations, notably with increased frequencies of a population sharing characteristics of transitional immature and marginal zone (MZ) B-cells, which we defined as marginal zone precursor-like" (MZp). In HIV-uninfected individuals, MZp present a B-cell regulatory (Breg) profile and function, which are lost in classic-progressors. Moreover, RNASeq analyses of blood MZp from classic-progressors depict a hyperactive state and signs of exhaustion, as well as an interferon signature similar to that observed in autoimmune disorders such as Systemic Lupus Erythematosus (SLE) and Sjögren Syndrome (SS), in which excess BAFF and deregulated MZ populations have also been documented. Based on the above, we hypothesize that excess BAFF may preclude the generation of HIV-1-specific IgG responses and drive polyclonal responses, including those from MZ populations, endowed with polyreactivity/autoreactivity. As such, we show that the quantity of HIV-1-specific IgG varies with disease progression status. In vitro, excess BAFF promotes polyclonal IgM and IgG responses, including those from MZp. RNASeq analyses reveal that blood MZp from classic-progressors are prone to Ig production and preferentially make usage of IGHV genes associated with some HIV broadly neutralizing antibodies (bNAbs), but also with autoantibodies, and whose impact in the battle against HIV-1 has yet to be determined.

Innate Immunity and SARS-CoV-2 Vaccine Response in Hemodialysis Patients.

Patients receiving hemodialysis (HD) have more inflammatory monocytes and less plasmacytoid dendritic cells (DCs) compared with healthy controls.Patients on HD who have a poor antibody response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine had fewer monocyte-derived DCs and conventional DCs compared with good responders.The defects in antigen presentation might be possible therapeutic targets to increase vaccine efficacy in HD patients.