Diagnosing Viral Infections Through T-Cell Receptor Sequencing of Activated CD8+ T Cells.

T-cell-based diagnostic tools identify pathogen exposure but lack differentiation between recent and historical exposures in acute infectious diseases. Here, T-cell receptor (TCR) RNA sequencing was performed on HLA-DR+/CD38+CD8+ T-cell subsets of hospitalized coronavirus disease 2019 (COVID-19) patients (n = 30) and healthy controls (n = 30; 10 of whom had previously been exposed to severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]). CDR3α and CDR3ß TCR regions were clustered separately before epitope specificity annotation using a database of SARS-CoV-2-associated CDR3α and CDR3ß sequences corresponding to >1000 SARS-CoV-2 epitopes. The depth of the SARS-CoV-2-associated CDR3α/ß sequences differentiated COVID-19 patients from the healthy controls with a receiver operating characteristic area under the curve of 0.84 ± 0.10. Hence, annotating TCR sequences of activated CD8+ T cells can be used to diagnose an acute viral infection and discriminate it from historical exposure. In essence, this work presents a new paradigm for applying the T-cell repertoire to accomplish TCR-based diagnostics.

Leveraging T-cell receptor - epitope recognition models to disentangle unique and cross-reactive T-cell response to SARS-CoV-2 during COVID-19 progression/resolution.

Despite the general agreement on the significance of T cells during SARS-CoV-2 infection, the clinical impact of specific and cross-reactive T-cell responses remains uncertain. Understanding this aspect could provide insights for adjusting vaccines and maintaining robust long-term protection against continuously emerging variants. To characterize CD8+ T-cell response to SARS-CoV-2 epitopes unique to the virus (SC2-unique) or shared with other coronaviruses (CoV-common), we trained a large number of T-cell receptor (TCR) - epitope recognition models for MHC-I-presented SARS-CoV-2 epitopes from publicly available data. These models were then applied to longitudinal CD8+ TCR repertoires from critical and non-critical COVID-19 patients. In spite of comparable initial CoV-common TCR repertoire depth and CD8+ T-cell depletion, the temporal dynamics of SC2-unique TCRs differed depending on the disease severity. Specifically, while non-critical patients demonstrated a large and diverse SC2-unique TCR repertoire by the second week of the disease, critical patients did not. Furthermore, only non-critical patients exhibited redundancy in the CD8+ T-cell response to both groups of epitopes, SC2-unique and CoV-common. These findings indicate a valuable contribution of the SC2-unique CD8+ TCR repertoires. Therefore, a combination of specific and cross-reactive CD8+ T-cell responses may offer a stronger clinical advantage. Besides tracking the specific and cross-reactive SARS-CoV-2 CD8+ T cells in any TCR repertoire, our analytical framework can be expanded to more epitopes and assist in the assessment and monitoring of CD8+ T-cell response to other infections.

Qualitative plasma viral load determination as a tool for screening of viral reservoir size in PWH.

OBJECTIVES: Suppression of viral replication in patients on antiretroviral therapy (ART) is determined by plasma viral load (pVL) measurement. Whenever pVL reaches values below the limit of quantification, the qualitative parameter 'target detected' or 'target not detected' is available but often not reported to the clinician. We investigated whether qualitative pVL measurements can be used to estimate the viral reservoir size. DESIGN: The study recruited 114 people with HIV (PWH) who are stable on ART between 2016 and 2018. The percentage of pVL measurements qualitatively reported as 'target detected' (PTD) within a 2-year period was calculated. METHODS: t-DNA and US-RNA were used to estimate viral reservoir size and were quantified on peripheral blood mononuclear cells (PBMCs) using droplet digital PCR. RESULTS: A median of 6.5 pVL measurements over a 2-year period was evaluated for each participant to calculate PTD. A positive correlation was found between t-DNA and PTD (r = 0.24; P = 0.011) but not between US-RNA and PTD (r = 0.1; P = 0.3). A significantly lower PTD was observed in PWH with a small viral reservoir, as estimated by t-DNA less than 66 copies/106 PBMCs and US-RNA less than 10 copies/106 PBMCs, compared with PWH with a larger viral reservoir (P = 0.001). We also show that t-DNA is detectable whenever PTD is higher than 56% and that ART regimen does not affect PTD. CONCLUSION: Our study shows that PTD provides an efficient parameter to preselect participants with a small viral reservoir based on already available pVL data for future HIV cure trials.

Efficient Induction of Antigen-Specific CD8+ T-Cell Responses by Cationic Peptide-Based mRNA Nanoparticles.

A major determinant for the success of mRNA-based vaccines is the composition of the nanoparticles (NPs) used for formulation and delivery. Cationic peptides represent interesting candidate carriers for mRNA, since they have been shown to efficiently deliver nucleic acids to eukaryotic cells. mRNA NPs based on arginine-rich peptides have previously been demonstrated to induce potent antigen-specific CD8+ T-cell responses. We therefore compared the histidine-rich amphipathic peptide LAH4-L1 (KKALLAHALHLLALLALHLAHALKKA) to the fully substituted arginine variant (LAH4-L1R) for their capacity to formulate mRNA and transfect dendritic cells (DCs). Although both peptides encapsulated mRNA to the same extent, and showed excellent uptake in DCs, the gene expression level was significantly higher for LAH4-L1. The LAH4-L1-mRNA NPs also resulted in enhanced antigen presentation in the context of MHC I compared to LAH4-L1R in primary murine CD103+ DCs. Both peptides induced DC maturation and inflammasome activation. Subsequent ex vivo stimulation of OT-I splenocytes with transfected CD103+ DCs resulted in a high proportion of polyfunctional CD8+ T cells for both peptides. In addition, in vivo immunization with LAH4-L1 or LAH4-L1R-mRNA NPs resulted in proliferation of antigen-specific T cells. In conclusion, although LAH4-L1 outperformed LAH4-L1R in terms of transfection efficiency, the immune stimulation ex vivo and in vivo was equally efficient.

Reply to Davison, G. Comment on "Huijghebaert et al. Does Trypsin Oral Spray (Viruprotect®/ColdZyme®) Protect against COVID-19 and Common Colds or Induce Mutation? Caveats in Medical Device Regulations in the European Union. Int. J. Environ. Res. Public Health 2021, 18, 5066".

We have read the comment from Davison with great interest [...].

Does Trypsin Oral Spray (Viruprotect®/ColdZyme®) Protect against COVID-19 and Common Colds or Induce Mutation? Caveats in Medical Device Regulations in the European Union.

BACKGROUND: nasal or oral sprays are often marketed as medical devices (MDs) in the European Union to prevent common cold (CC), with ColdZyme®/Viruprotect® (trypsin/glycerol) mouth spray claiming to prevent colds and the COVID-19 virus from infecting host cells and to shorten/reduce CC symptoms as an example. We analyzed the published (pre)-clinical evidence. METHODS: preclinical: comparison of in vitro tests with validated host cell models to determine viral infectivity. Clinical: efficacy, proportion of users protected against virus (compared with non-users) and safety associated with trypsin/glycerol. RESULTS: preclinical data showed that exogenous trypsin enhances SARS-CoV-2 infectivity and syncytia formation in host models, while culture passages in trypsin presence induce spike protein mutants. The manufacturer claims >98% SARS-CoV-2 deactivation, although clinically irrelevant as based on a tryptic viral digest, inserting trypsin inactivation before host cells exposure. Efficacy and safety were not adequately addressed in clinical studies or leaflets (no COVID-19 data). Protection was obtained among 9-39% of users, comparable to or lower than placebo-treated or non-users. Several potential safety risks (tissue digestion, bronchoconstriction) were identified. CONCLUSIONS: the current European MD regulations may result in insufficient exploration of (pre)clinical proof of action. Exogenous trypsin exposure even raises concerns (higher SARS-CoV-2 infectivity, mutations), whereas its clinical protective performance against respiratory viruses as published remains poor and substandard.

Closing schools for SARS-CoV-2: a pragmatic rapid recommendation.

Background: In Belgium, schools closed during the first lockdown in March 2020, with a partial reopening in May. They fully reopened in September. During the summer, infections started to increase in the general population, speeding up in September. Some measures were taken to limit social contacts but those were insufficient to mitigate the exponential rise of infections in October. Children were still receiving all lessons at school at that time and it was questioned whether this position was tenable. We systematically compared the benefits and harms of closing primary and secondary schools and developed a recommendation. Methods: A multidisciplinary panel, including school pupils and teachers, educational experts, clinicians and researchers, produced this recommendation in compliance with the standards for trustworthy rapid guidelines. The recommendation is based on data collected through national surveillance or studies from Belgium, and supported by a rapid literature review. Results: Closing schools during the first lockdown probably resulted in a large learning delay and possibly led to more cases of child abuse. We are uncertain about the effect on the infection rate, hospitalisations, transmission rates, mental health of children, teachers and parents. The panel concluded that the balance of benefits and harms of closing schools clearly shifts against closing schools. Detrimental effects are even worse for vulnerable children. This recommendation is affected by the local virus circulation. Conclusion: The guideline panel issues a strong recommendation against closing schools when the virus circulation is low to moderate, and a weak recommendation against closing schools when the virus circulation is high. It does not apply when the school system cannot function due to lack of teachers, too many children who are at home or a shortage of support services. As the results of international studies are consistent with Belgian study results, this recommendation may also be relevant internationally.

Essentials in saline pharmacology for nasal or respiratory hygiene in times of COVID-19.

PURPOSE: Nasal irrigation or nebulizing aerosol of isotonic or hypertonic saline is a traditional method for respiratory or nasal care. A recent small study in outpatients with COVID-19 without acute respiratory distress syndrome suggests substantial symptom resolution. We therefore analyzed pharmacological/pharmacodynamic effects of isotonic or hypertonic saline, relevant to SARS-CoV-2 infection and respiratory care. METHODS: Mixed search method. RESULTS: Due to its wetting properties, saline achieves an improved spreading of alveolar lining fluid and has been shown to reduce bio-aerosols and viral load. Saline provides moisture to respiratory epithelia and gels mucus, promotes ciliary beating, and improves mucociliary clearance. Coronaviruses and SARS-CoV-2 damage ciliated epithelium in the nose and airways. Saline inhibits SARS-CoV-2 replication in Vero cells; possible interactions involve the viral ACE2-entry mechanism (chloride-dependent ACE2 configuration), furin and 3CLpro (inhibition by NaCl), and the sodium channel ENaC. Saline shifts myeloperoxidase activity in epithelial or phagocytic cells to produce hypochlorous acid. Clinically, nasal or respiratory airway care with saline reduces symptoms of seasonal coronaviruses and other common cold viruses. Its use as aerosol reduces hospitalization rates for bronchiolitis in children. Preliminary data suggest symptom reduction in symptomatic COVID-19 patients if saline is initiated within 48 h of symptom onset. CONCLUSIONS: Saline interacts at various levels relevant to nasal or respiratory hygiene (nasal irrigation, gargling or aerosol). If used from the onset of common cold symptoms, it may represent a useful add-on to first-line interventions for COVID-19. Formal evaluation in mild COVID-19 is desirable as to establish efficacy and optimal treatment regimens.

CD32+CD4+ memory T cells are enriched for total HIV-1 DNA in tissues from humanized mice.

CD32 has raised conflicting results as a putative marker of the HIV-1 reservoir. We measured CD32 expression in tissues from viremic and virally suppressed humanized mice treated relatively early or late after HIV-1 infection with combined antiretroviral therapy. CD32 was expressed in a small fraction of the memory CD4+ T-cell subsets from different tissues in viremic and aviremic mice, regardless of treatment initiation time. CD32+ memory CD4+ T cells were enriched in cell-associated (CA) HIV-1 DNA but not in CA HIV-1 RNA as compared to the CD32-CD4+ fraction. Using multidimensional reduction analysis, several memory CD4+CD32+ T-cell clusters were identified expressing HLA-DR, TIGIT, or PD-1. Importantly, although tissue-resident CD32+CD4+ memory cells were enriched with translation-competent reservoirs, most of it was detected in memory CD32-CD4+ T cells. Our findings support that CD32 labels highly activated/exhausted memory CD4+ T-cell subsets that contain only a small proportion of the translation-competent reservoir.

Off the beaten path: Novel mRNA-nanoformulations for therapeutic vaccination against HIV.

Over the last few years, immunotherapy for HIV in general and therapeutic vaccination in particular, has received a tremendous boost, both in preclinical research and in clinical applications. This interest is based on the evidence that the immune system plays a crucial role in controlling HIV infection, as shown for long-term non-progressors and elite controllers, and that immune responses can be manipulated towards targeting conserved epitopes. So far, the most successful approach has been vaccination with autologous dendritic cells (DCs) loaded ex vivo with antigens and activation signals. Although this approach offers much promise, it also comes with significant drawbacks such as the requirement of a specialized infrastructure and expertise, as well as major challenges for logistics and storage, making it extremely time consuming and costly. Therefore, methods are being developed to avoid the use of ex vivo generated, autologous DCs. One of these methods is based on mRNA for therapeutic vaccination. mRNA has proven to be a very promising vaccine platform, as the coding information for any desired protein, including antigens and activation signals, can be generated in a very short period of time, showing promise both as an off-the-shelf therapy and as a personalized approach. However, an important drawback of this approach is the short half-life of native mRNA, due to the presence of ambient RNases. In addition, proper immunization requires that the antigens are expressed, processed and presented at the right immunological site (e.g. the lymphoid tissues). An ambivalent aspect of mRNA as a vaccine is its capacity to induce type I interferons, which can have beneficial adjuvant effects, but also deleterious effects on mRNA stability and translation. Thus, proper formulation of the mRNA is crucially important. Many approaches for RNA formulation have already been tested, with mixed success. In this review we discuss the state-of-the-art and future trends for mRNA-nanoparticle formulations for HIV vaccination, both in the prophylactic and in the therapeutic setting.

COVID-19: Does the infectious inoculum dose-response relationship contribute to understanding heterogeneity in disease severity and transmission dynamics?

The variation in the speed and intensity of SARS-CoV-2 transmission and severity of the resulting COVID-19 disease are still imperfectly understood. We postulate a dose-response relationship in COVID-19, and that "the dose of virus in the initial inoculum" is an important missing link in understanding several incompletely explained observations in COVID-19 as a factor in transmission dynamics and severity of disease. We hypothesize that: (1) Viral dose in inoculum is related to severity of disease, (2) Severity of disease is related to transmission potential, and (3) In certain contexts, chains of severe cases can build up to severe local outbreaks, and large-scale intensive epidemics. Considerable evidence from other infectious diseases substantiates this hypothesis and recent evidence from COVID-19 points in the same direction. We suggest research avenues to validate the hypothesis. If proven, our hypothesis could strengthen the scientific basis for deciding priority containment measures in various contexts in particular the importance of avoiding super-spreading events and the benefits of mass masking.

A call for strengthened evidence on targeted, non-pharmaceutical interventions against COVID-19 for the protection of vulnerable individuals in sub-Saharan Africa.

Following the easing of lockdown measures in many sub-Saharan African countries, coronavirus disease 2019 (COVID-19) cases have been on the rise. As the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, may be difficult to stop in these settings, we propose that the existing COVID-19 prevention strategies aimed at reducing overall transmission are complemented with more targeted strategies to protect people at risk of severe COVID-19 disease. We suggest investigating the feasibility, acceptability, and efficacy of distributing COVID-19 prevention kits to households with persons at increased risk of severe COVID-19 disease.

The COVID-19 pandemic: diverse contexts; different epidemics-how and why?

It is very exceptional that a new disease becomes a true pandemic. Since its emergence in Wuhan, China, in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, has spread to nearly all countries of the world in only a few months. However, in different countries, the COVID-19 epidemic takes variable shapes and forms in how it affects communities. Until now, the insights gained on COVID-19 have been largely dominated by the COVID-19 epidemics and the lockdowns in China, Europe and the USA. But this variety of global trajectories is little described, analysed or understood. In only a few months, an enormous amount of scientific evidence on SARS-CoV-2 and COVID-19 has been uncovered (knowns). But important knowledge gaps remain (unknowns). Learning from the variety of ways the COVID-19 epidemic is unfolding across the globe can potentially contribute to solving the COVID-19 puzzle. This paper tries to make sense of this variability-by exploring the important role that context plays in these different COVID-19 epidemics; by comparing COVID-19 epidemics with other respiratory diseases, including other coronaviruses that circulate continuously; and by highlighting the critical unknowns and uncertainties that remain. These unknowns and uncertainties require a deeper understanding of the variable trajectories of COVID-19. Unravelling them will be important for discerning potential future scenarios, such as the first wave in virgin territories still untouched by COVID-19 and for future waves elsewhere.

Rapid viral rebound after analytical treatment interruption in patients with very small HIV reservoir and minimal on-going viral transcription.

INTRODUCTION: Viral remission after analytical treatment interruption (ATI), termed post-treatment control, has been described in a small proportion of HIV-positive patients. This phenomenon has been separately associated to both low levels of HIV-1 proviral DNA as well as cell-associated RNA. We investigated whether the combination of both parameters could help predict delayed viral rebound after treatment interruption (TI). METHODS: We conducted an open single-arm ATI study in four Belgian HIV reference centres from January 2016 to July 2018. Eligible participants were adults who had fewer than 50 HIV-1 RNA copies/mL for more than two years, more than 500 CD4 cells/µL for more than three months, and were in general good health. Consenting participants who had fewer than 66 copies total HIV-1 DNA (t-DNA) and fewer than 10 copies cell-associated HIV-1 unspliced RNA (US-RNA) per million peripheral blood mononuclear cells (PBMCs), interrupted therapy and were monitored closely. Antiretroviral therapy (ART) was resumed after two consecutive viral loads exceeding 1000 copies or one exceeding 10,000 copies/mL. The primary outcome was the proportion of participants with fewer than 50 HIV-1 RNA copies/mL 48 weeks after TI. Secondary outcomes were time to viral rebound, the frequency of serious adverse events (AEs) and evolution of t-DNA and US-RNA after TI. RESULTS: All 16 consenting participants who interrupted therapy experienced rapid viral rebound two to eight weeks after TI. No serious AEs were observed. Levels of t-DNA and US-RNA increased after TI but returned to pre-ATI levels after treatment restart. None of the studied demographic, clinical and biological parameters were predictive of time of viral rebound. CONCLUSIONS: The combination of low levels of t-DNA and US-RNA in PBMCs, corresponding respectively to a small and transcriptionally silent viral reservoir, is not predictive of viral remission after TI in patients on ART.

Cytotoxic CD8+ T Cells Expressing CXCR5 Are Detectable in HIV-1 Elite Controllers After Prolonged In Vitro Peptide Stimulation.

Antiretroviral therapy (ART) is not curative as HIV-1 persists in long-lived viral reservoirs. Consequently, patients are dependent on life-long drug adherence with possible side effects. To overcome these limitations strategies of a functional cure aim at ART free viral remission. In this study, we sought to identify detailed subsets of anti-viral CD8+ T cell immunity linked to natural long-term control of HIV-1 infection. Here, we analyzed HIV controllers and ART suppressed progressors for in vitro viral suppressive capacity (VSC) at baseline and after peptide stimulation. Functional properties and phenotypes of CD8+ T cells were assessed by IFN-γ ELISPOT and 18 color flow cytometry. HIV controllers showed significantly increased suppression at baseline as well as after peptide stimulation. IFN-γ secretion and the proliferation marker Ki67 positively correlated with VSC. Moreover, the detailed phenotype of three distinct multifunctional memory CD8+ T cell subsets were specific traits of HIV controllers of which two correlated convincingly with VSC. Our results underline the importance of multifunctional CD8+ T cell responses during natural control. Especially the role of CXCR5 expressing cytotoxic subsets emphasizes potential surveillance in sites of reservoir persistence and demand further study.

Correction to: iHIVARNA phase IIa, a randomized, placebo-controlled, double-blinded trial to evaluate the safety and immunogenicity of iHIVARNA-01 in chronically HIV-infected patients under stable combined antiretroviral therapy.

Following publication of the original article [1], we have been notified that end note would need to be adjusted.

Therapeutic Vaccine in Chronically HIV-1-Infected Patients: A Randomized, Double-Blind, Placebo-Controlled Phase IIa Trial with HTI-TriMix.

Therapeutic vaccinations aim to re-educate human immunodeficiency virus (HIV)-1-specific immune responses to achieve durable control of HIV-1 replication in virally suppressed infected individuals after antiretroviral therapy (ART) is interrupted. In a double blinded, placebo-controlled phase IIa multicenter study, we investigated the safety and immunogenicity of intranodal administration of the HIVACAT T cell Immunogen (HTI)-TriMix vaccine. It consists of naked mRNA based on cytotoxic T lymphocyte (CTL) targets of subdominant and conserved HIV-1 regions (HTI), in combination with mRNAs encoding constitutively active TLR4, the ligand for CD40 and CD70 as adjuvants (TriMix). We recruited HIV-1-infected individuals under stable ART. Study-arms HTI-TriMix, TriMix or Water for Injection were assigned in an 8:3:3 ratio. Participants received three vaccinations at weeks 0, 2, and 4 in an inguinal lymph node. Two weeks after the last vaccination, immunogenicity was evaluated using ELISpot assay. ART was interrupted at week 6 to study the effect of the vaccine on viral rebound. The vaccine was considered safe and well tolerated. Eighteen percent (n = 37) of the AEs were considered definitely related to the study product (grade 1 or 2). Three SAEs occurred: two were unrelated to the study product, and one was possibly related to ART interruption (ATI). ELISpot assays to detect T cell responses using peptides covering the HTI sequence showed no significant differences in immunogenicity between groups. There were no significant differences in viral load rebound dynamics after ATI between groups. The vaccine was safe and well tolerated. We were not able to demonstrate immunogenic effects of the vaccine.

Viral Inhibitory Activity of CD8+ T Cells in HIV Infection.

Cytotoxic T lymphocytes, or CD8+ T cells, play an important role in the control of replication of HIV. Inducing effective and durable HIV-specific CD8+ T cell responses are, therefore, a major objective in prophylactic and curative strategies for HIV infection. To evaluate such strategies, reliable immunological assays are needed that measure the capacity of CD8+ T cells to exert their effector functions and control viremia. Classical immunological assays such as interferon-γ (IFN-γ) enzyme-linked immunospot or intracellular cytokine staining measure the production of one or several effector molecules but do not actually show suppression of viral replication. Perhaps unsurprisingly, these assays do not correlate with either prevention of infection or lower viral set-points after infection. Therefore, more relevant assays are needed which directly measure the viral inhibitory activity (VIA) of CD8+ T cells and are more likely to predict success or failure of different immune interventions. The present review discusses the methodology of the VIA in detail as well as the practical implications of the several variations that have been described. We then go onto discuss existent literature on the relationship between VIA and HIV control, give an overview of examples where VIA has been induced or boosted in vivo or in vitro, and finally discuss observed associations between VIA and other immunological parameters. We conclude that while VIA is complex and laborious, it provides functional information about CD8+ T cells that no other assay can deliver.