Children develop robust and sustained cross-reactive spike-specific immune responses to SARS-CoV-2 infection.

SARS-CoV-2 infection is generally mild or asymptomatic in children but a biological basis for this outcome is unclear. Here we compare antibody and cellular immunity in children (aged 3-11 years) and adults. Antibody responses against spike protein were high in children and seroconversion boosted responses against seasonal Beta-coronaviruses through cross-recognition of the S2 domain. Neutralization of viral variants was comparable between children and adults. Spike-specific T cell responses were more than twice as high in children and were also detected in many seronegative children, indicating pre-existing cross-reactive responses to seasonal coronaviruses. Importantly, children retained antibody and cellular responses 6 months after infection, whereas relative waning occurred in adults. Spike-specific responses were also broadly stable beyond 12 months. Therefore, children generate robust, cross-reactive and sustained immune responses to SARS-CoV-2 with focused specificity for the spike protein. These findings provide insight into the relative clinical protection that occurs in most children and might help to guide the design of pediatric vaccination regimens.

Robust SARS-CoV-2-specific T cell immunity is maintained at 6 months following primary infection.

The immune response to SARS-CoV-2 is critical in controlling disease, but there is concern that waning immunity may predispose to reinfection. We analyzed the magnitude and phenotype of the SARS-CoV-2-specific T cell response in 100 donors at 6 months following infection. T cell responses were present by ELISPOT and/or intracellular cytokine staining analysis in all donors and characterized by predominant CD4+ T cell responses with strong interleukin (IL)-2 cytokine expression. Median T cell responses were 50% higher in donors who had experienced a symptomatic infection, indicating that the severity of primary infection establishes a 'set point' for cellular immunity. T cell responses to spike and nucleoprotein/membrane proteins were correlated with peak antibody levels. Furthermore, higher levels of nucleoprotein-specific T cells were associated with preservation of nucleoprotein-specific antibody level although no such correlation was observed in relation to spike-specific responses. In conclusion, our data are reassuring that functional SARS-CoV-2-specific T cell responses are retained at 6 months following infection.

Cytotoxic CD4+ T-cells specific for EBV capsid antigen BORF1 are maintained in long-term latently infected healthy donors.

Epstein Barr Virus (EBV) infects more than 95% of the population whereupon it establishes a latent infection of B-cells that persists for life under immune control. Primary EBV infection can cause infectious mononucleosis (IM) and long-term viral carriage is associated with several malignancies and certain autoimmune diseases. Current efforts developing EBV prophylactic vaccination have focussed on neutralising antibodies. An alternative strategy, that could enhance the efficacy of such vaccines or be used alone, is to generate T-cell responses capable of recognising and eliminating newly EBV-infected cells before the virus initiates its growth transformation program. T-cell responses against the EBV structural proteins, brought into the newly infected cell by the incoming virion, are prime candidates for such responses. Here we show the structural EBV capsid proteins BcLF1, BDLF1 and BORF1 are frequent targets of T-cell responses in EBV infected people, identify new CD8+ and CD4+ T-cell epitopes and map their HLA restricting alleles. Using T-cell clones we demonstrate that CD4+ but not CD8+ T-cell clones specific for the capsid proteins can recognise newly EBV-infected B-cells and control B-cell outgrowth via cytotoxicity. Using MHC-II tetramers we show a CD4+ T-cell response to an epitope within the BORF1 capsid protein epitope is present during acute EBV infection and in long-term viral carriage. In common with other EBV-specific CD4+ T-cell responses the BORF1-specific CD4+ T-cells in IM patients expressed perforin and granzyme-B. Unexpectedly, perforin and granzyme-B expression was sustained over time even when the donor had entered the long-term infected state. These data further our understanding of EBV structural proteins as targets of T-cell responses and how CD4+ T-cell responses to EBV change from acute disease into convalescence. They also identify new targets for prophylactic EBV vaccine development.

PD-1 is imprinted on cytomegalovirus-specific CD4+ T cells and attenuates Th1 cytokine production whilst maintaining cytotoxicity.

PD-1 is expressed on exhausted T cells in cancer patients but its physiological role remains uncertain. We determined the phenotype, function and transcriptional correlates of PD-1 expression on cytomegalovirus-specific CD4+ T cells during latent infection. PD-1 expression ranged from 10-85% and remained stable over time within individual donors. This 'setpoint' was correlated with viral load at primary infection. PD-1+ CD4+ T cells display strong cytotoxic function but generate low levels of Th1 cytokines which is only partially reversed by PD-1 blockade. TCR clonotypes showed variable sharing between PD-1+ and PD-1- CMV-specific cells indicating that PD-1 status is defined either during T cell priming or subsequent clonal expansion. Physiological PD-1+ CD4+ T cells therefore display a unique 'high cytotoxicity-low cytokine' phenotype and may act to suppress viral reactivation whilst minimizing tissue inflammation. Improved understanding of the physiological role of PD-1 will help to delineate the mechanisms, and potential reversal, of PD-1+ CD4+ T cell exhaustion in patients with malignant disease.

Cytokines as effectors and predictors of responses in the treatment of bladder cancer by bacillus Calmette-Guérin.

The most effective intravesical treatment of non-muscle-invasive bladder cancer is instillation of live Mycobacterium bovis bacillus Calmette-Guérin (BCG). BCG stimulates the release of cytokines, contributing directly or indirectly to its effectiveness. However, the function of specific cytokines is not well understood. We have undertaken a nonsystematic review of primary evidence regarding cytokine detection, activation and response in BCG patients. Cytokines IL-2, IL-8 and TNF-α appear to be essential for effective BCG therapy and nonrecurrence, while IL-10 may have an inhibitory effect on BCG responses. IL-2, IL-8, TRAIL and TNF-α are potentially predictive of response to BCG. Alterations in genes encoding cytokines may also affect responses. There are significant data showing the association of certain cytokines with successful BCG treatment, and which may be useful predictive markers. Isolating those cytokines mediating efficacy may hold the key to ameliorating BCG's side effects and improving efficacy and patient compliance.

Current state of COVID-19 in children: 4 years on.

Children have been disproportionately affected by the COVID-19 pandemic. Despite evidence of a very low risk of severe disease, children were subjected to extensive lockdown, restriction and mitigation measures, including school closures, to control the rapid spread of SARS-CoV-2 in most parts of the world. In this review we summarise the UK experience of COVID-19 in children four years into the largest and longest pandemic of this century. We address the risks of SARS-CoV-2 infection, immunity, transmission, severity and outcomes in children. We also assess the implementation, uptake, effectiveness and impact of COVID-19 vaccination, as well as the emergence, evolution and near disappearance of PIMS-TS (paediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2) and current understanding of long COVID in children. This review consolidates current knowledge on childhood COVID-19 and emphasises the importance of continued research and the need for research-driven public health actions and policy decisions, especially in the context of new variants and future vaccines.

Nasal mucosal IgA levels against SARS-CoV-2 and seasonal coronaviruses are low in children but boosted by reinfection.

Repeated coronavirus infections in childhood drive progressive maturation of systemic immune responses into adulthood. Analyses of immune responses in children have focused primarily upon systemic assessment but the importance of mucosal immunity is increasingly recognised. We studied virus-specific antibody responses in contemporaneous nasal swabs and blood samples from 99 children (4-15 years) and 28 adults (22-56 years), all of whom had prior SARS-CoV-2 infection. Whilst mucosal IgA titres against Influenza and Respiratory Syncytial virus were comparable between children and adults, those against all coronaviruses, including SARS-CoV-2, were lower in children. Mucosal IgA antibodies demonstrated comparable relative neutralisation capacity in both groups and retained activity against recent omicron variants such as XBB.1 which are highly evasive of IgG neutralisation. SARS-CoV-2 reinfection preferentially enhanced mucosal IgA responses whilst the impact of vaccination was more modest. Nasal IgA levels against coronaviruses thus display a pattern of incremental response to reinfection which likely determines the natural history of reinfection. This highlights the particular significance of developing mucosal vaccines against coronaviruses in children.

Immunological imprinting of humoral immunity to SARS-CoV-2 in children.

Omicron variants of SARS-CoV-2 are globally dominant and infection rates are very high in children. We measure immune responses following Omicron BA.1/2 infection in children aged 6-14 years and relate this to prior and subsequent SARS-CoV-2 infection or vaccination. Primary Omicron infection elicits a weak antibody response with poor functional neutralizing antibodies. Subsequent Omicron reinfection or COVID-19 vaccination elicits increased antibody titres with broad neutralisation of Omicron subvariants. Prior pre-Omicron SARS-CoV-2 virus infection or vaccination primes for robust antibody responses following Omicron infection but these remain primarily focussed against ancestral variants. Primary Omicron infection thus elicits a weak antibody response in children which is boosted after reinfection or vaccination. Cellular responses are robust and broadly equivalent in all groups, providing protection against severe disease irrespective of SARS-CoV-2 variant. Immunological imprinting is likely to act as an important determinant of long-term humoral immunity, the future clinical importance of which is unknown.

Early evaluation of the safety, reactogenicity, and immune response after a single dose of modified vaccinia Ankara-Bavaria Nordic vaccine against mpox in children: a national outbreak response.

BACKGROUND: In response to a national mpox (formerly known as monkeypox) outbreak in England, children exposed to a confirmed mpox case were offered modified vaccinia Ankara-Bavaria Nordic (MVA-BN), a third-generation smallpox vaccine, for post-exposure prophylaxis. We aimed to assess the safety and reactogenicity and humoral and cellular immune response, following the first reported use of MVA-BN in children. METHODS: This is an assessment of children receiving MVA-BN for post-exposure prophylaxis in response to a national mpox outbreak in England. All children receiving MVA-BN were asked to complete a post-vaccination questionnaire online and provide a blood sample 1 month and 3 months after vaccination. Outcome measures for the questionnaire included reactogenicity and adverse events after vaccination. Blood samples were tested for humoural, cellular, and cytokine responses and compared with unvaccinated paediatric controls who had never been exposed to mpox. FINDINGS: Between June 1 and Nov 30, 2022, 87 children had one MVA-BN dose and none developed any serious adverse events or developed mpox disease after vaccination. Post-vaccination reactogenicity questionnaires were completed by 45 (52%) of 87 children. Their median age was 5 years (IQR 5-9), 25 (56%) of 45 were male, and 22 (49%) of 45 were White. 16 (36%) reported no symptoms, 18 (40%) reported local reaction only, and 11 (24%) reported systemic symptoms with or without local reactions. Seven (8%) of 87 children provided a first blood sample a median of 6 weeks (IQR 6·0-6·5) after vaccination and five (6%) provided a second blood sample at a median of 15 weeks (14-15). All children had poxvirus IgG antibodies with titres well above the assay cutoff of OD450nm 0·1926 with mean absorbances of 1·380 at six weeks and 0·9826 at 15 weeks post-vaccination. Assessment of reactivity to 27 recombinant vaccina virus and monkeypox virus proteins showed humoral antigen recognition, primarily to monkeypox virus antigens B6, B2, and vaccina virus antigen B5, with waning of humoral responses observed between the two timepoints. All children had a robust T-cell response to whole modified vaccinia Ankara virus and a select pool of conserved pan-Poxviridae peptides. A balanced CD4+ and CD8+ T-cell response was evident at 6 weeks, which was retained at 15 weeks after vaccination. INTERPRETATION: A single dose of MVA-BN for post-exposure prophylaxis was well-tolerated in children and induced robust antibody and cellular immune responses up to 15 weeks after vaccination. Larger studies are needed to fully assess the safety, immunogenicity, and effectiveness of MVA-BN in children. Our findings, however, support its on-going use to prevent mpox in children as part of an emergency public health response. FUNDING: UK Health Security Agency.

Long-term proliferation of functional human NK cells, with conversion of CD56(dim) NK cells to a CD56 (bright) phenotype, induced by carcinoma cells co-expressing 4-1BBL and IL-12.

4-1BB ligation co-stimulates T cell activation, and agonistic antibodies have entered clinical trials. Natural killer (NK) cells also express 4-1BB following activation and are implicated in the anti-tumour efficacy of 4-1BB stimulation in mice; however, the response of human NK cells to 4-1BB stimulation is not clearly defined. Stimulation of non-adherent PBMC with OVCAR-3 cells expressing 4-1BB ligand (4-1BBL) or IL-12 resulted in preferential expansion of the NK cell population, while the combination 4-1BBL + IL-12 was superior for the activation and proliferation of functional NK cells from healthy donors and patients with renal cell or ovarian carcinoma, supporting long-term (21 day) NK cell proliferation. The expanded NK cells are predominantly CD56(bright), and we show that isolated CD56(dim)CD16(+) NK cells can switch to a CD56(bright)CD16(-) phenotype and proliferate in response to 4-1BBL + IL-12. Whereas 4-1BB upregulation on NK cells in response to 4-1BBL required 'help' from other PBMC, it could be induced on isolated NK cells by IL-12, but only in the presence of target (OVCAR-3) cells. Following primary stimulation with OVCAR-3 cells expressing 4-1BBL + IL-12 and subsequent resting until day 21, NK cells remained predominantly CD56(bright) and retained both high cytotoxic capability against K562 targets and enhanced ability to produce IFNγ relative to NK cells in PBMC. These data support the concept that NK cells could contribute to anti-tumour activity of 4-1BB agonists in humans and suggest that combining 4-1BB-stimulation with IL-12 could be beneficial for ex vivo or in vivo expansion and activation of NK cells for cancer immunotherapy.

mRNA or ChAd0x1 COVID-19 Vaccination of Adolescents Induces Robust Antibody and Cellular Responses With Continued Recognition of Omicron Following mRNA-1273.

Children and adolescents generally experience mild COVID-19. However, those with underlying physical health conditions are at a significantly increased risk of severe disease. Here, we present a comprehensive analysis of antibody and cellular responses in adolescents with severe neuro-disabilities who received COVID-19 vaccination with either ChAdOx1 (n=6) or an mRNA vaccine (mRNA-1273, n=8, BNT162b2, n=1). Strong immune responses were observed after vaccination and antibody levels and neutralisation titres were both higher after two doses. Both measures were also higher after mRNA vaccination and were further enhanced by prior natural infection where one vaccine dose was sufficient to generate peak antibody response. Robust T-cell responses were generated after dual vaccination and were also higher following mRNA vaccination. Early T-cells were characterised by a dominant effector-memory CD4+ T-cell population with a type-1 cytokine signature with additional production of IL-10. Antibody levels were well-maintained for at least 3 months after vaccination and 3 of 4 donors showed measurable neutralisation titres against the Omicron variant. T-cell responses also remained robust, with generation of a central/stem cell memory pool and showed strong reactivity against Omicron spike. These data demonstrate that COVID-19 vaccines display strong immunogenicity in adolescents and that dual vaccination, or single vaccination following prior infection, generate higher immune responses than seen after natural infection and develop activity against Omicron. Initial evidence suggests that mRNA vaccination elicits stronger immune responses than adenoviral delivery, although the latter is also higher than seen in adult populations. COVID-19 vaccines are therefore highly immunogenic in high-risk adolescents and dual vaccination might be able to provide relative protection against the Omicron variant that is currently globally dominant.

Robust SARS-CoV-2-specific and heterologous immune responses in vaccine-naïve residents of long-term care facilities who survive natural infection.

We studied humoral and cellular immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 152 long-term care facility staff and 124 residents over a prospective 4-month period shortly after the first wave of infection in England. We show that residents of long-term care facilities developed high and stable levels of antibodies against spike protein and receptor-binding domain. Nucleocapsid-specific responses were also elevated but waned over time. Antibodies showed stable and equivalent levels of functional inhibition against spike-angiotensin-converting enzyme 2 binding in all age groups with comparable activity against viral variants of concern. SARS-CoV-2 seropositive donors showed high levels of antibodies to other beta-coronaviruses but serostatus did not impact humoral immunity to influenza or other respiratory syncytial viruses. SARS-CoV-2-specific cellular responses were similar across all ages but virus-specific populations showed elevated levels of activation in older donors. Thus, survivors of SARS-CoV-2 infection show a robust and stable immunity against the virus that does not negatively impact responses to other seasonal viruses.

PD-L2 Is Constitutively Expressed in Normal and Malignant Urothelium.

The use of immune checkpoint blockade, in particular PD-1 and PD-L1 inhibitors, is now commonplace in many clinical settings including the treatment of muscle-invasive bladder cancer (MIBC). Notwithstanding, little information exists regarding the expression of the alternative PD-1 ligand, PD-L2 in urothelial bladder cancer (UBC). We therefore set out to characterise the expression of PD-L2 in comparison to PD-L1. Firstly, we assessed PD-L2 expression by immunohistochemistry and found widespread expression of PD-L2 in UBC, albeit with reduced expression in MIBC. We further investigated these findings using RNA-seq data from a cohort of 575 patients demonstrating that PDCD1LG2 (PD-L2) is widely expressed in UBC and correlated with CD274 (PD-L1). However, in contrast to our immunohistochemistry findings, expression was significantly increased in advanced disease. We have also provided detailed evidence of constitutive PD-L2 expression in normal urothelium and propose a mechanism by which PD-L2 is cleaved from the cell surface in MIBC. These data provide a comprehensive assessment of PD-L2 in UBC, showing PD-L2 is abundant in UBC and, importantly, constitutively present in normal urothelium. These data have implications for future development of immune checkpoint blockade, and also the understanding of the function of the immune system in the normal urinary bladder.

BCL-W is dispensable for the sustained survival of select Burkitt lymphoma and diffuse large B-cell lymphoma cell lines.

Dysregulated expression of BCL-2 family proteins allows cancer cells to escape apoptosis. To counter this, BH3-mimetic drugs that target and inhibit select BCL-2 prosurvival proteins to induce apoptosis have been developed for cancer therapy. Venetoclax, which targets BCL-2, has been effective as therapy for patients with chronic lymphocytic leukemia, and MCL-1-targeting BH3-mimetic drugs have been extensively evaluated in preclinical studies for a range of blood cancers. Recently, BCL-W, a relatively understudied prosurvival member of the BCL-2 protein family, has been reported to be abnormally upregulated in Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), and Hodgkin lymphoma patient samples. Therefore, to determine if BCL-W would be a promising therapeutic target for B-cell lymphomas, we have examined the role of BCL-W in the sustained growth of human BL- and DLBCL-derived cell lines. We found that CRISPR/CAS9-mediated loss or short hairpin RNA-mediated knockdown of BCL-W expression in selected BL and DLBCL cell lines did not lead to spontaneous apoptosis and had no effect on their sensitivity to a range of BH3-mimetic drugs targeting other BCL-2 prosurvival proteins. Our results suggest that BCL-W is not universally required for the sustained growth and survival of human BL and DLBCL cell lines. Thus, targeting BCL-W in this subset of B-cell lymphomas may not be of broad therapeutic benefit.

Association of Spike-Specific T Cells With Relative Protection From Subsequent SARS-CoV-2 Omicron Infection in Young Children.

This cohort study investigates the risk of SARS-CoV-2 reinfection among young children with and without spike-specific T-cell responses.

Interleukin-17-positive mast cells influence outcomes from BCG for patients with CIS: Data from a comprehensive characterisation of the immune microenvironment of urothelial bladder cancer.

The tumour immune microenvironment is considered to influence cancer behaviour and outcome. Using a panel of markers for innate and adaptive immune cells we set out to characterise and understand the bladder tumour microenvironment of 114 patients from a prospective multicentre cohort of newly-diagnosed bladder cancer patients, followed-up for 4.33±1.71 years. We found IL-17-positive cells were significantly increased in primary and concomitant carcinoma in situ (CIS), p<0.0001, a highly malignant lesion which is the most significant single risk factor for disease progression. Further characterisation of the tumour immunophenotype identified IL-17+ cells as predominantly mast cells rather than T-cells, in contrast to most other tumour types. Expression of the IL-17-receptor in bladder tumours, and functional effects and gene expression changes induced by IL-17 in bladder tumour cells in vitro suggest a role in tumour behaviour. Finally, we assessed the effects of IL-17 in the context of patient outcome, following intravesical BCG immunotherapy which is the standard of care; higher numbers of IL-17+ cells were associated with improved event-free survival (p = 0.0449, HR 0.2918, 95% CI 0.08762-0.9721) in patients with primary and concomitant CIS (n = 41), we propose a model of IL-17+ Mast cells mechanism of action. Thus, in the context of bladder CIS, IL-17+ mast cells predict favourable outcome following BCG immunotherapy indicative of a novel mechanism of BCG immunotherapy in UBC and could form the basis of a stratified approach to treatment.