Blood brain barrier disruption and glutamatergic excitotoxicity in post-acute sequelae of SARS COV-2 infection cognitive impairment: potential biomarkers and a window into pathogenesis.

Objective: To investigate the association between blood-brain barrier permeability, brain metabolites, microstructural integrity of the white matter, and cognitive impairment (CI) in post-acute sequelae of SARS-COV-2 infection (PASC). Methods: In this multimodal longitudinal MRI study 14 PASC participants with CI and 10 healthy controls were enrolled. All completed investigations at 3 months following acute infection (3 months ± 2 weeks SD), and 10 PASC participants completed at 12 months ± 2.22 SD weeks. The assessments included a standard neurological assessment, a cognitive screen using the brief CogState battery and multi-modal MRI derived metrics from Dynamic contrast enhanced (DCE) perfusion Imaging, Diffusion Tensor Imaging (DTI), and single voxel proton Magnetic Resonance Spectroscopy. These measures were compared between patients and controls and correlated with cognitive scores. Results: At baseline, and relative to controls, PASC participants had higher K-Trans and Myo-inositol, and lower levels of Glutamate/Glutamine in the frontal white matter (FWM) (p < 0.01) as well as in brain stem (p < 0.05), and higher FA and lower MD in the FWM (p < 0.05). In PASC participants, FA and MD decreased in the FWM at 12 months compared to baseline (p < 0.05). K-Trans and metabolite concentrations did not change significantly over time. Neurocognitive scores did not correlation with the increased permeability (K trans). Interpretation: PASC with CI is associated with BBB impairment, loss of WM integrity, and inflammation at 3 months which significantly but not uniformly improved at 12 months. The loss of WM integrity is possibly mediated by BBB impairment and associated glutamatergic excitotoxicity.

Improvement of immune dysregulation in individuals with long COVID at 24-months following SARS-CoV-2 infection.

This study investigates the humoral and cellular immune responses and health-related quality of life measures in individuals with mild to moderate long COVID (LC) compared to age and gender matched recovered COVID-19 controls (MC) over 24 months. LC participants show elevated nucleocapsid IgG levels at 3 months, and higher neutralizing capacity up to 8 months post-infection. Increased spike-specific and nucleocapsid-specific CD4+ T cells, PD-1, and TIM-3 expression on CD4+ and CD8+ T cells were observed at 3 and 8 months, but these differences do not persist at 24 months. Some LC participants had detectable IFN-γ and IFN-ß, that was attributed to reinfection and antigen re-exposure. Single-cell RNA sequencing at the 24 month timepoint shows similar immune cell proportions and reconstitution of naïve T and B cell subsets in LC and MC. No significant differences in exhaustion scores or antigen-specific T cell clones are observed. These findings suggest resolution of immune activation in LC and return to comparable immune responses between LC and MC over time. Improvement in self-reported health-related quality of life at 24 months was also evident in the majority of LC (62%). PTX3, CRP levels and platelet count are associated with improvements in health-related quality of life.

Advances in HIV Gene Therapy.

Early gene therapy studies held great promise for the cure of heritable diseases, but the occurrence of various genotoxic events led to a pause in clinical trials and a more guarded approach to progress. Recent advances in genetic engineering technologies have reignited interest, leading to the approval of the first gene therapy product targeting genetic mutations in 2017. Gene therapy (GT) can be delivered either in vivo or ex vivo. An ex vivo approach to gene therapy is advantageous, as it allows for the characterization of the gene-modified cells and the selection of desired properties before patient administration. Autologous cells can also be used during this process which eliminates the possibility of immune rejection. This review highlights the various stages of ex vivo gene therapy, current research developments that have increased the efficiency and safety of this process, and a comprehensive summary of Human Immunodeficiency Virus (HIV) gene therapy studies, the majority of which have employed the ex vivo approach.

Molecular epidemiology to aid virtual elimination of HIV transmission in Australia.

The Global UNAIDS 95/95/95 targets aim to increase the percentage of persons who know their HIV status, receive antiretroviral therapy, and have achieved viral suppression. Achieving these targets requires efforts to improve the public health response to increase access to care for those living with HIV, identify those yet undiagnosed with HIV early, and increase access to prevention for those most at risk of HIV acquisition. HIV infections in Australia are among the lowest globally having recorded significant declines in new diagnoses in the last decade. However, the HIV epidemic has changed with an increasing proportion of newly diagnosed infections among those born outside Australia observed in the last five years. Thus, the current prevention efforts are not enough to achieve the UNAIDS targets and virtual elimination across all population groups. We believe both are possible by including molecular epidemiology in the public health response. Molecular epidemiology methods have been crucial in the field of HIV prevention, particularly in demonstrating the efficacy of treatment as prevention. Cluster detection using molecular epidemiology can provide opportunities for the real-time detection of new outbreaks before they grow, and cluster detection programs are now part of the public health response in the USA and Canada. Here, we review what molecular epidemiology has taught us about HIV evolution and spread. We summarize how we can use this knowledge to improve public health measures by presenting case studies from the USA and Canada. We discuss the successes and challenges of current public health programs in Australia, and how we could use cluster detection as an add-on to identify gaps in current prevention measures easier and respond quicker to growing clusters. Lastly, we raise important ethical and legal challenges that need to be addressed when HIV genotypic data is used in combination with personal data.

Does PET-CT Have a Role in the Evaluation of Tuberculosis Treatment in Phase 2 Clinical Trials?

Positron emission tomography-computed tomography (PET-CT) has the potential to revolutionize research in infectious diseases, as it has done with cancer. There is growing interest in it as a biomarker in the setting of early-phase tuberculosis clinical trials, particularly given the limitations of current biomarkers as adequate predictors of sterilizing cure for tuberculosis. PET-CT is a real-time tool that provides a 3-dimensional view of the spatial distribution of tuberculosis within the lung parenchyma and the nature of lesions with uptake (ie, whether nodular, consolidative, or cavitary). Its ability to provide functional data on changes in metabolism, drug penetration, and immune control of tuberculous lesions has the potential to facilitate drug development and regimen selection for advancement to phase 3 trials in tuberculosis. In this narrative review, we discuss the role that PET-CT may have in evaluating responses to drug therapy in active tuberculosis treatment and the challenges in taking PET-CT forward as predictive biomarker of relapse-free cure in the setting of phase 2 clinical trials.

Clinical and laboratory features of COVID-19 illness and outcomes in immunocompromised individuals during the first pandemic wave in Sydney, Australia.

People with immunocompromising conditions are at increased risk of SARS-CoV-2 infection and mortality, however early in the pandemic it was challenging to collate data on this heterogenous population. We conducted a registry study of immunocompromised individuals with polymerase chain reaction (PCR)-confirmed SARS-CoV-2 infection from March-October 2020 in Sydney, Australia to understand clinical and laboratory outcomes in this population prior to the emergence of the Delta variant. 27 participants were enrolled into the study including people with a haematologic oncologic conditions (n = 12), secondary immunosuppression (N = 8) and those with primary or acquired immunodeficiency (i.e. HIV; N = 7). All participants had symptomatic COVID-19 with the most common features being cough (64%), fever (52%) and headache (40%). Five patients demonstrated delayed SARS-CoV-2 clearance lasting three weeks to three months. The mortality rate in this study was 7% compared to 1.3% in the state of New South Wales Australia during the same period. This study provides data from the first eight months of the pandemic on COVID-19 outcomes in at-risk patient groups.

Immune response to COVID-19 vaccination in patients with Waldenström macroglobulinaemia who pause their BTKi therapy.

Patients with Waldenström macroglobulinaemia (WM) are at increased risk of severe COVID-19 infection and have poor immune responses to COVID-19 vaccination. This study assessed whether a closely monitored pause in Bruton's Tyrosine Kinase inhibitor (BTKi) therapy might result in an improved humoral response to a 3rd COVID-19 vaccine dose. Improved response was observed in WM patients who paused their BTKi, compared to a group who did not pause their BTKi. However, the response was attenuated after BTKi recommencement. This data contributes to our understanding of vaccination strategies in this patient group and may help inform consensus approaches in the future.

Novel siRNA therapeutics demonstrate multi-variant efficacy against SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a respiratory virus that causes COVID-19 disease, with an estimated global mortality of approximately 2%. While global response strategies, which are predominantly reliant on regular vaccinations, have shifted from zero COVID to living with COVID, there is a distinct lack of broad-spectrum direct acting antiviral therapies that maintain efficacy across evolving SARS-CoV-2 variants of concern. This is of most concern for immunocompromised and immunosuppressed individuals who lack robust immune responses following vaccination, and others at risk for severe COVID and long-COVID. RNA interference (RNAi) therapeutics induced by short interfering RNAs (siRNAs) offer a promising antiviral treatment option, with broad-spectrum antiviral capabilities unparalleled by current antiviral therapeutics and a high genetic barrier to antiviral escape. Here we describe novel siRNAs, targeting highly conserved regions of the SARS-CoV-1 and 2 genome of both human and animal species, with multi-variant antiviral potency against eight SARS-CoV-2 lineages - Ancestral VIC01, Alpha, Beta, Gamma, Delta, Zeta, Kappa and Omicron. Treatment with our siRNA resulted in significant protection against virus-mediated cell death in vitro, with >97% cell survival (P < 0.0001), and corresponding reductions of viral nucleocapsid RNA of up to 99.9% (P < 0.0001). When compared to antivirals; Sotrovimab and Remdesivir, the siRNAs demonstrated a more potent antiviral effect and similarly, when multiplexing siRNAs to target different viral regions simultaneously, an increased antiviral effect was observed compared to individual siRNA treatments (P < 0.0001). These results demonstrate the potential for a highly effective broad-spectrum direct acting antiviral against multiple SARS-CoV-2 variants, including variants resistant to antivirals and vaccine generated neutralizing antibodies.

The kynurenine pathway relates to post-acute COVID-19 objective cognitive impairment and PASC.

OBJECTIVE: To determine the prevalence and natural history of post-acute COVID-19 objective cognitive impairment and function, and their relationship to demographic, clinical factors, post-acute sequelae of COVID-19 (PASC), and biomarkers. METHODS: A total of 128 post-acute COVID-19 patients (age = 46 ± 15; 42% women, acute disease severity: not hospitalized: 38.6% mild: 0-1 symptoms, 52% 2+ symptoms; 9.4% hospitalized) completed standard cognition, olfaction, and mental health examinations 2-, 4-, and 12-month post diagnosis. Over the same time frame, WHO-defined PASC was determined. Blood cytokines, peripheral neurobiomarkers, and kynurenine pathway (KP) metabolites were measured. Objective cognitive function was demographically/practice corrected, and impairment prevalence was determined using the evidence-based Global Deficit Score method to detect at least mild cognitive impairment (GDS > 0.5). Linear mixed effect regression models with time effect (month post diagnosis) evaluated the relationships to cognition. RESULTS: Across the 12-month study period, mild to moderate cognitive impairment ranged from 16% to 26%, and 46.5% were impaired at least once. Impairment associated with poorer work capacity (p < 0.05), and 2-month objectively tested anosmia (p < 0.05). PASC with (p = 0.01) and without disability (p < 0.03) associated with acute COVID-19 severity. KP measures showed prolonged activation (2 to 8 months) (p < 0.0001) linked to IFN-beta in those with PASC. Of the blood analytes, only the KP metabolites (elevated quinolinic acid, 3-hydroxyanthranilic acid, kynurenine, the kynurenine/tryptophan ratio) associated (p < 0.001) with poorer cognitive performance and greater likelihood of impairment. PASC, independent of disability associated with abnormal kynurenine/tryptophan (p < 0.03). INTERPRETATION: The kynurenine pathway relates to post-acute COVID-19 objective cognitive impairment and PASC, thereby enabling biomarker and therapeutic possibilities.

The crossroads: divergent roles of virus-specific CD4+ T lymphocytes in determining the outcome for human papillomavirus infection.

Despite the widespread availability of effective prophylactic vaccines to prevent human papillomavirus (HPV) infection, HPV remains a major health burden. For health care systems in countries with the capacity for vaccine roll out, incomplete strategies result in citizens with naturally occurring infection, who are at an a posteriori risk of HPV-driven disease. Genital HPV infection is the most common sexually transmitted virus globally. Those classified as high-risk HPV strains are more likely to generate persistent disease. Within this group, HPV16 and 18 are the most prevalent and likely to induce persistent high-grade squamous intraepithelial neoplasia; neoplasia is a large step toward cancerous growth known as a squamous cell carcinoma which contribute to all cervical, 70% of oropharyngeal, 78% of vaginal and 88% of anal cancers. This review will illuminate the relevance of CD4+ T lymphocytes in determining the outcome of papillomavirus infection from the perspective of oropharyngeal and anogenital HPV-driven disease in the immune competent and immunocompromised. The focus is on recent investigations for this "silent" pandemic among current global health crises that should not be forgotten. Informing effective strategies that control viral infection through naturally acquired or induced immunity will identify aspects of scientific and clinical practice that may improve outcome.

Long-Term Benefits from Early Antiretroviral Therapy Initiation in HIV Infection.

BACKGROUND: For people with HIV and CD4+ counts >500 cells/mm3, early initiation of antiretroviral therapy (ART) reduces serious AIDS and serious non-AIDS (SNA) risk compared with deferral of treatment until CD4+ counts are <350 cells/mm3. Whether excess risk of AIDS and SNA persists once ART is initiated for those who defer treatment is uncertain. METHODS: The Strategic Timing of AntiRetroviral Treatment (START) trial, as previously reported, randomly assigned 4684 ART-naive HIV-positive adults with CD4+ counts .500 cells/mm3 to immediate treatment initiation after random assignment (n = 2325) or deferred treatment (n= 2359). In 2015, a 57% lower risk of the primary end point (AIDS, SNA, or death) for the immediate group was reported, and the deferred group was offered ART. This article reports the follow-up that continued to December 31, 2021. Cox proportional-hazards models were used to compare hazard ratios for the primary end point from randomization through December 31, 2015, versus January 1, 2016, through December 31, 2021. RESULTS: Through December 31, 2015, approximately 7 months after the cutoff date from the previous report, the median CD4+ count was 648 and 460 cells/mm3 in the immediate and deferred groups, respectively, at treatment initiation. The percentage of follow-up time spent taking ART was 95% and 36% for the immediate and deferred groups, respectively, and the time-averaged CD4+ difference was 199 cells/mm3. After January 1, 2016, the percentage of follow-up time on treatment was 97.2% and 94.1% for the immediate and deferred groups, respectively, and the CD4+ count difference was 155 cells/mm3. After January 1, 2016, a total of 89 immediate and 113 deferred group participants experienced a primary end point (hazard ratio of 0.79 [95% confidence interval, 0.60 to 1.04] versus hazard ratio of 0.47 [95% confidence interval, 0.34 to 0.65; P<0.001]) before 2016 (P=0.02 for hazard ratio difference). CONCLUSIONS: Among adults with CD4+ counts >500 cells/mm3, excess risk of AIDS and SNA associated with delaying treatment initiation was diminished after ART initiation, but persistent excess risk remained. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Emergence and antibody evasion of BQ, BA.2.75 and SARS-CoV-2 recombinant sub-lineages in the face of maturing antibody breadth at the population level.

BACKGROUND: The Omicron era of the COVID-19 pandemic commenced at the beginning of 2022 and whilst it started with primarily BA.1, it was latter dominated by BA.2 and the related sub-lineage BA.5. Following resolution of the global BA.5 wave, a diverse grouping of Omicron sub-lineages emerged derived from BA.2, BA.5 and recombinants thereof. Whilst emerging from distinct lineages, all shared similar changes in the Spike glycoprotein affording them an outgrowth advantage through evasion of neutralising antibodies. METHODS: Over the course of 2022, we monitored the potency and breadth of antibody neutralization responses to many emerging variants in the Australian community at three levels: (i) we tracked over 420,000 U.S. plasma donors over time through various vaccine booster roll outs and Omicron waves using sequentially collected IgG pools; (ii) we mapped the antibody response in individuals using blood from stringently curated vaccine and convalescent cohorts. (iii) finally we determine the in vitro efficacy of clinically approved therapies Evusheld and Sotrovimab. FINDINGS: In pooled IgG samples, we observed the maturation of neutralization breadth to Omicron variants over time through continuing vaccine and infection waves. Importantly, in many cases, we observed increased antibody breadth to variants that were yet to be in circulation. Determination of viral neutralization at the cohort level supported equivalent coverage across prior and emerging variants with isolates BQ.1.1, XBB.1, BR.2.1 and XBF the most evasive. Further, these emerging variants were resistant to Evusheld, whilst increasing neutralization resistance to Sotrovimab was restricted to BQ.1.1 and XBF. We conclude at this current point in time that dominant variants can evade antibodies at levels equivalent to their most evasive lineage counterparts but sustain an entry phenotype that continues to promote an additional outgrowth advantage. In Australia, BR.2.1 and XBF share this phenotype and, in contrast to global variants, are uniquely dominant in this region in the later months of 2022. INTERPRETATION: Whilst the appearance of a diverse range of omicron lineages has led to primary or partial resistance to clinically approved monoclonal antibodies, the maturation of the antibody response across both cohorts and a large donor pools importantly observes increasing breadth in the antibody neutralisation responses over time with a trajectory that covers both current and known emerging variants. FUNDING: This work was primarily supported by Australian Medical Foundation research grants MRF2005760 (SGT, GM & WDR), Medical Research Future Fund Antiviral Development Call grant (WDR), the New South Wales Health COVID-19 Research Grants Round 2 (SGT & FB) and the NSW Vaccine Infection and Immunology Collaborative (VIIM) (ALC). Variant modeling was supported by funding from SciLifeLab's Pandemic Laboratory Preparedness program to B.M. (VC-2022-0028) and by the European Union's Horizon 2020 research and innovation programme under grant agreement no. 101003653 (CoroNAb) to B.M.

Agreements, Behaviour, and Change: Sex Outside the Relationship in Male HIV-negative Partners in HIV Serodiscordant Relationships in Australia, Brazil, and Thailand.

Male HIV serodiscordant couples have diverse relationship agreements regarding sex outside the relationship. We examined the relationship agreements as described by 343 male HIV-negative partners in HIV serodiscordant relationships in Australia, Brazil and Thailand participating in a multi-year cohort study. At baseline, 125 (34.1%) HIV-negative partners reported no agreement, 115 (33.5%) had a monogamous agreement, and 103 (37.9%) had an open agreement allowing sex outside the relationship. Relationship agreements were largely stable over time, with 76% of HIV-negative men reporting the same agreement across follow up, while changes were predominantly towards having an open agreement. Behaviour largely matched relationship agreements, and the predictors of breaking an agreement by having condomless anal intercourse (CLAI) with an outside partner were CLAI within the relationship (OR = 3.17, 95%CI: 1.64-6.14, p < 0.001) and PrEP use in the last three months (OR = 3.42, 95%CI: 1.48-7.92, p = 0.004). When considering HIV transmission risk for HIV-negative men in serodiscordant relationships, greater focus needs to be placed on sex that is occurring outside the relationship and the agreements that facilitate this.

T follicular helper cell responses to SARS-CoV-2 vaccination among healthy and immunocompromised adults.

The worldwide rollout of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccinations in the last 2 years has produced a multitude of studies investigating T-cell responses in the peripheral blood and a limited number in secondary lymphoid tissues. As a key component to an effective immune response, vaccine-specific T follicular helper (Tfh) cells are localized in the draining lymph node (LN) and assist in the selection of highly specific B-cell clones for the production of neutralizing antibodies. While these cells have been noted in the blood as circulating Tfh (cTfh) cells, they are not often taken into consideration when examining effective CD4+ T-cell responses, particularly in immunocompromised groups. Furthermore, site-specific analyses in locations such as the LN have recently become an attractive area of investigation. This is mainly a result of improved sampling methods via ultrasound-guided fine-needle biopsy (FNB)/fine-needle aspiration (FNA), which are less invasive than LN excision and able to be performed longitudinally. While these studies have been undertaken in healthy individuals, data from immunocompromised groups are lacking. This review will focus on both Tfh and cTfh responses after SARS-CoV-2 vaccination in healthy and immunocompromised individuals. This area of investigation could identify key characteristics of a successful LN response required for the prevention of infection and viral clearance. This furthermore may highlight responses that could be fine-tuned to improve vaccine efficacy within immunocompromised groups that are at a risk of more severe disease.

Layer-by-Layer Particles Deliver Epigenetic Silencing siRNA to HIV-1 Latent Reservoir Cell Types.

For over two decades, nanomaterials have been employed to facilitate intracellular delivery of small interfering RNA (siRNA), both in vitro and in vivo, to induce post-transcriptional gene silencing (PTGS) via RNA interference. Besides PTGS, siRNAs are also capable of transcriptional gene silencing (TGS) or epigenetic silencing, which targets the gene promoter in the nucleus and prevents transcription via repressive epigenetic modifications. However, silencing efficiency is hampered by poor intracellular and nuclear delivery. Here, polyarginine-terminated multilayered particles are reported as a versatile system for the delivery of TGS-inducing siRNA to potently suppress virus transcription in HIV-infected cells. siRNA is complexed with multilayered particles formed by layer-by-layer assembly of poly(styrenesulfonate) and poly(arginine) and incubated with HIV-infected cell types, including primary cells. Using deconvolution microscopy, uptake of fluorescently labeled siRNA is observed in the nuclei of HIV-1 infected cells. Viral RNA and protein are measured to confirm functional virus silencing from siRNA delivered using particles 16 days post-treatment. This work extends conventional particle-enabled PTGS siRNA delivery to the TGS pathway and paves the way for future studies on particle-delivered siRNA for efficient TGS of various diseases and infections, including HIV.

Viral competition assay to assess the role of HIV-1 proteins in immune evasion.

CD8+ T lymphocytes can recognize and eliminate cells infected by viruses. However, the human immunodeficiency virus (HIV-1) has developed mechanisms to evade CD8+ T-cell-mediated clearance. Here, we describe a protocol to assess the role of the HIV-1 protein Nef in immune evasion. The viral competition assay reveals the preferential killing of HIV-1-infected cells unable to express Nef. This methodology can be extended to study HIV-1 proteins involved in immune evasion and viral variants encoding cytotoxic T lymphocyte escape mutations. For complete details on the use and execution of this protocol, please refer to Duette et al. (2022).1.

Broadly neutralizing SARS-CoV-2 antibodies through epitope-based selection from convalescent patients.

Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells of convalescent patients using SARS-CoV-2 receptor binding domains carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (S309) by orders of magnitude. They also provide prophylactic and therapeutic in vivo protection of female hACE2 mice against viral challenge. Our results indicate that exposure to SARS-CoV-2 induces antibodies that maintain broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.

Tracking the clonal dynamics of SARS-CoV-2-specific T cells in children and adults with mild/asymptomatic COVID-19.

Children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop less severe coronavirus disease 2019 (COVID-19) than adults. The mechanisms for the age-specific differences and the implications for infection-induced immunity are beginning to be uncovered. We show by longitudinal multimodal analysis that SARS-CoV-2 leaves a small footprint in the circulating T cell compartment in children with mild/asymptomatic COVID-19 compared to adult household contacts with the same disease severity who had more evidence of systemic T cell interferon activation, cytotoxicity and exhaustion. Children harbored diverse polyclonal SARS-CoV-2-specific naïve T cells whereas adults harbored clonally expanded SARS-CoV-2-specific memory T cells. A novel population of naïve interferon-activated T cells is expanded in acute COVID-19 and is recruited into the memory compartment during convalescence in adults but not children. This was associated with the development of robust CD4+ memory T cell responses in adults but not children. These data suggest that rapid clearance of SARS-CoV-2 in children may compromise their cellular immunity and ability to resist reinfection.

Parallel analysis of multiple human memory CD4+ T-cell subsets within antigen-specific responses using cell proliferation dyes.

Activation induced marker (AIM) assays are being used increasingly to measure antigen-specific T-cell responses, but this activation can alter cell lineage defining phenotypic markers. We aimed to extend the utility of AIM assays to enable pre-activation defined cell populations to be tracked and quantified within T-cell memory responses. We sorted three ex vivo CD4+ T-cell populations prior to any activation using well defined ex vivo lineage surface marker combinations. These populations were memory non-Tregs, CD39+ Tregs and CD39neg Tregs, although any three memory CD4+ T-cell populations able to be isolated by cell surface markers could potentially be tracked. These cells were labeled with three distinct fluorescent cell proliferation dyes (CFSE, CellTrace Violet and Cell Proliferation Dye eF670) and then all autologous PBMCs were reconstituted maintaining ex vivo cell ratios and CD25/OX40 AIM assays performed with CMV and HSV antigens. This approach enabled tracking of pre-defined cell populations within antigen stimulated responses using both activation marker and cell proliferation readouts. We confirmed that although CD39+ Tregs comprise a substantial proportion of AIM assay responses, they do not make substantial contributions to the proliferative response. This extends the utility of AIM assays to enable parallel analysis of the relative contribution of several CD4+ memory T-cell subsets to recall responses.