Quantification of T-cell dynamics during latent cytomegalovirus infection in humans.

Cytomegalovirus (CMV) infection has a major impact on the T-cell pool, which is thought to be associated with ageing of the immune system. The effect on the T-cell pool has been interpreted as an effect of CMV on non-CMV specific T-cells. However, it remains unclear whether the effect of CMV could simply be explained by the presence of large, immunodominant, CMV-specific memory CD8+ T-cell populations. These have been suggested to establish through gradual accumulation of long-lived cells. However, little is known about their maintenance. We investigated the effect of CMV infection on T-cell dynamics in healthy older adults, and aimed to unravel the mechanisms of maintenance of large numbers of CMV-specific CD8+ T-cells. We studied the expression of senescence, proliferation, and apoptosis markers and quantified the in vivo dynamics of CMV-specific and other memory T-cell populations using in vivo deuterium labelling. Increased expression of late-stage differentiation markers by CD8+ T-cells of CMV+ versus CMV- individuals was not solely explained by the presence of large, immunodominant CMV-specific CD8+ T-cell populations. The lifespans of circulating CMV-specific CD8+ T-cells did not differ significantly from those of bulk memory CD8+ T-cells, and the lifespans of bulk memory CD8+ T-cells did not differ significantly between CMV- and CMV+ individuals. Memory CD4+ T-cells of CMV+ individuals showed increased expression of late-stage differentiation markers and decreased Ki-67 expression. Overall, the expression of senescence markers on T-cell populations correlated positively with their expected in vivo lifespan. Together, this work suggests that i) large, immunodominant CMV-specific CD8+ T-cell populations do not explain the phenotypical differences between CMV+ and CMV- individuals, ii) CMV infection hardly affects the dynamics of the T-cell pool, and iii) large numbers of CMV-specific CD8+ T-cells are not due to longer lifespans of these cells.

Vesicle-bound EBV-BART13-3p miRNA in circulation distinguishes nasopharyngeal from other head and neck cancer and asymptomatic EBV-infections.

Cell-free microRNA (miRNA) in biofluids released by tumors in either protein or vesicle-bound form, represent promising minimally-invasive cancer biomarkers. However, a highly abundant non-tumor background in human plasma and serum complicates the discovery and detection of tumor-selective circulating miRNAs. We performed small RNA sequencing on serum and plasma RNA from Nasopharyngeal Carcinoma (NPC) patients. Collectively, Epstein Barr virus-encoded miRNAs, more so than endogenous miRNAs, signify presence of NPC. However, RNAseq-based EBV miRNA profiles differ between NPC patients, suggesting inter-tumor heterogeneity or divergent secretory characteristics. We determined with sensitive qRT-PCR assays that EBV miRNAs BART7-3p, BART9-3p and BART13-3p are actively secreted by C666.1 NPC cells bound to extracellular vesicles (EVs) and soluble ribonucleoprotein complexes. Importantly, these miRNAs are expressed in all primary NPC tumor biopsies and readily detected in nasopharyngeal brushings from both early and late-stage NPC patients. Increased levels of BART7-3p, BART9-3p and particularly BART13-3p, distinguish NPC patient sera from healthy controls. Receiver operating characteristic curve analysis using sera from endemic NPC patients, other head and neck cancers and individuals with asymptomatic EBV-infections reveals a superior diagnostic performance of EBV miRNAs over anti-EBNA1 IgA serology and EBV-DNA load (AUC 0.87-0.96 vs 0.86 and 0.66 respectively). The high specificity of circulating EBV-BART13-3p (97%) for NPC detection is in agreement with active secretion from NPC tumor cells. We conclude EV-bound BART13-3p in circulation is a promising, NPC-selective, biomarker that should be considered as part of a screening strategy to identify NPC in endemic regions.

Pre-vaccination immunotypes reveal weak and robust antibody responders to influenza vaccination.

Effective vaccine-induced immune responses are particularly essential in older adults who face an increased risk of immunosenescence. However, the complexity and variability of the human immune system make predicting vaccine responsiveness challenging. To address this knowledge gap, our study aimed to characterize immune profiles that are predictive of vaccine responsiveness using "immunotypes" as an innovative approach. We analyzed an extensive set of innate and adaptive immune cell subsets in the whole blood of 307 individuals (aged 25-92) pre- and post-influenza vaccination which we associated with day 28 hemagglutination inhibition (HI) antibody titers. Building on our previous work that stratified individuals into nine immunotypes based on immune cell subsets, we identified two pre-vaccination immunotypes associated with weak and one showing robust day 28 antibody response. Notably, the weak responders demonstrated HLA-DR+ T-cell signatures, while the robust responders displayed a high naïve-to-memory T-cell ratio and percentage of nonclassical monocytes. These specific signatures deepen our understanding of the relationship between the baseline of the immune system and its functional potential. This approach could enhance our ability to identify individuals at risk of immunosenescence. Our findings highlight the potential of pre-vaccination immunotypes as an innovative tool for informing personalized vaccination strategies and improving health outcomes, particularly for aging populations.

Primary SARS-CoV-2 infection in children and adults results in similar Fc-mediated antibody effector function patterns.

Objectives: Increasing evidence suggests that Fc-mediated antibody effector functions have an important role in protection against respiratory viruses, including SARS-CoV-2. However, limited data are available on the potential differences in the development, heterogeneity and durability of these responses in children compared to adults. Methods: Here, we assessed the development of spike S1-specific serum antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD) and natural killer cell activation (ADNKA), alongside specific antibody binding concentrations (IgG, IgA and IgM) and IgG avidity in healthy adults (n = 38, 18-56 years) and children (n = 21, 5-16 years) following primary SARS-CoV-2 infection, with a 10-month longitudinal follow-up. Differences between groups were assessed using a nonparametric Kruskal-Wallis test with Dunn's multiple comparisons test. Results: We found similar (functional) antibody responses in children compared to adults, with a tendency for increased durability in children, which was statistically significant for ADCD (P < 0.05). While ADNKA was strongly reduced in both adults (P < 0.001) and children (P < 0.05) at the latest time point, ADCP remained relatively stable over time, possibly relating to an increase in avidity of the spike-specific antibodies (P < 0.001). Finally, the ADNKA capacity relative to antibody concentration appeared to decrease over time in both children and adults. Conclusion: In conclusion, our data provide novel insights into the development of SARS-CoV-2-specific antibody Fc-mediated effector functions in children and adults. An increased understanding of these characteristics in specific age populations is valuable for the future design of novel and improved vaccination strategies for respiratory viruses such as SARS-CoV-2.

Delineating immune variation between adult and children COVID-19 cases and associations with disease severity.

The SARS-CoV-2 pandemic has emphasized the need to explore how variations in the immune system relate to the severity of the disease. This study aimed to explore inter-individual variation in response to SARS-CoV-2 infection by comparing T cell, B cell, and innate cell immune subsets among primary infected children and adults (i.e., those who had never experienced SARS-CoV-2 infection nor received vaccination previously), with varying disease severity after infection. We also examined immune subset kinetics in convalescent individuals compared to those with persistent infection to identify possible markers of immune dysfunction. Distinct immune subset differences were observed between infected adults and children, as well as among adult cases with mild, moderate, and severe disease. IgM memory B cells were absent in moderate and severe cases whereas frequencies of B cells with a lack of surface immunoglobulin expression were significantly higher in severe cases. Interestingly, these immune subsets remained stable during recovery implying that these subsets could be associated with underlying baseline immune variation. Our results offer insights into the potential immune markers associated with severe COVID-19 and provide a foundation for future research in this area.

Distinct T cell responsiveness to different COVID-19 vaccines and cross-reactivity to SARS-CoV-2 variants with age and CMV status.

Introduction: Accumulating evidence indicates the importance of T cell immunity in vaccination-induced protection against severe COVID-19 disease, especially against SARS-CoV-2 Variants-of-Concern (VOCs) that more readily escape from recognition by neutralizing antibodies. However, there is limited knowledge on the T cell responses across different age groups and the impact of CMV status after primary and booster vaccination with different vaccine combinations. Moreover, it remains unclear whether age has an effect on the ability of T cells to cross-react against VOCs. Methods: Therefore, we interrogated the Spike-specific T cell responses in healthy adults of the Dutch population across different ages, whom received different vaccine types for the primary series and/or booster vaccination, using IFNÉ£ ELISpot. Cells were stimulated with overlapping peptide pools of the ancestral Spike protein and different VOCs. Results: Robust Spike-specific T cell responses were detected in the vast majority of participants upon the primary vaccination series, regardless of the vaccine type (i.e. BNT162b2, mRNA-1273, ChAdOx1 nCoV-19, or Ad26.COV2.S). Clearly, in the 70+ age group, responses were overall lower and showed more variation compared to younger age groups. Only in CMV-seropositive older adults (>70y) there was a significant inverse relation of age with T cell responses. Although T cell responses increased in all age groups after booster vaccination, Spike-specific T cell frequencies remained lower in the 70+ age group. Regardless of age or CMV status, primary mRNA-1273 vaccination followed by BNT162b2 booster vaccination showed limited booster effect compared to the BNT162b2/BNT162b2 or BNT162b2/mRNA-1273 primary-booster regimen. A modest reduction in cross-reactivity to the Alpha, Delta and Omicron BA.1, but not the Beta or Gamma variant, was observed after primary vaccination. Discussion: Together, this study shows that age, CMV status, but also the primary-booster vaccination regimen influence the height of the vaccination-induced Spike-specific T cell response, but did not impact the VOC cross-reactivity.

Multiple vaccine comparison in the same adults reveals vaccine-specific and age-related humoral response patterns: an open phase IV trial.

Vaccine responsiveness is often reduced in older adults. Yet, our lack of understanding of low vaccine responsiveness hampers the development of effective vaccination strategies to reduce the impact of infectious diseases in the ageing population. Young-adult (25-49 y), middle-aged (50-64 y) and older-adult ( ≥ 65 y) participants of the VITAL clinical trials (n = 315, age-range: 28-98 y), were vaccinated with an annual (2019-2020) quadrivalent influenza (QIV) booster vaccine, followed by a primary 13-valent pneumococcal-conjugate (PCV13) vaccine (summer/autumn 2020) and a primary series of two SARS-CoV-2 mRNA-1273 vaccines (spring 2021). This unique setup allowed investigation of humoral responsiveness towards multiple vaccines within the same individuals over the adult age-range. Booster QIV vaccination induced comparable H3N2 hemagglutination inhibition (HI) titers in all age groups, whereas primary PCV13 and mRNA-1273 vaccination induced lower antibody concentrations in older as compared to younger adults (primary endpoint). The persistence of humoral responses, towards the 6 months timepoint, was shorter in older adults for all vaccines (secondary endpoint). Interestingly, highly variable vaccine responder profiles overarching multiple vaccines were observed. Yet, approximately 10% of participants, mainly comprising of older male adults, were classified as low responders to multiple vaccines. This study aids the identification of risk groups for low vaccine responsiveness and hence supports targeted vaccination strategies. Trial number: NL69701.041.19, EudraCT: 2019-000836-24.

Booster Immunization Improves Memory B Cell Responses in Older Adults Unresponsive to Primary SARS-CoV-2 Immunization.

The generation of a specific long-term immune response to SARS-CoV-2 is considered important for protection against COVID-19 infection and disease. Memory B cells, responsible for the generation of antibody-producing plasmablasts upon a new antigen encounter, play an important role in this process. Therefore, the induction of memory B cell responses after primary and booster SARS-CoV-2 immunizations was investigated in the general population with an emphasis on older adults. Participants, 20-99 years of age, due to receive the mRNA-1273 or BNT162b2 SARS-CoV-2 vaccine were included in the current study. Specific memory B cells were determined by ex vivo ELISpot assays. In a subset of participants, antibody levels, avidity, and virus neutralization capacity were compared to memory B cell responses. Memory B cells specific for both Spike S1 and receptor-binding domain (RBD) were detected in the majority of participants following the primary immunization series. However, a proportion of predominantly older adults showed low frequencies of specific memory B cells. Booster vaccination resulted in a large increase in the frequencies of S1- and RBD-specific memory B cells also for those in which low memory B cell frequencies were detected after the primary series. These data show that booster immunization is important for the generation of a memory B cell response, as a subset of older adults shows a suboptimal response to the primary SARS-CoV-2 immunization series. It is anticipated that these memory B cells will play a significant role in the immune response following viral re-exposure.

Contribution of SARS-CoV-2 infection preceding COVID-19 mRNA vaccination to generation of cellular and humoral immune responses in children.

Primary COVID-19 vaccination for children, 5-17 years of age, was offered in the Netherlands at a time when a substantial part of this population had already experienced a SARS-CoV-2 infection. While vaccination has been shown effective, underlying immune responses have not been extensively studied. We studied immune responsiveness to one and/or two doses of primary BNT162b2 mRNA vaccination and compared the humoral and cellular immune response in children with and without a preceding infection. Antibodies targeting the original SARS-CoV-2 Spike or Omicron Spike were measured by multiplex immunoassay. B-cell and T-cell responses were investigated using enzyme-linked immunosorbent spot (ELISpot) assays. The activation of CD4+ and CD8+ T cells was studied by flowcytometry. Primary vaccination induced both a humoral and cellular adaptive response in naive children. These responses were stronger in those with a history of infection prior to vaccination. A second vaccine dose did not further boost antibody levels in those who previously experienced an infection. Infection-induced responsiveness prior to vaccination was mainly detected in CD8+ T cells, while vaccine-induced T-cell responses were mostly by CD4+ T cells. Thus, SARS-CoV-2 infection prior to vaccination enhances adaptive cellular and humoral immune responses to primary COVID-19 vaccination in children. As most children are now expected to contract infection before the age of five, the impact of infection-induced immunity in children is of high relevance. Therefore, considering natural infection as a priming immunogen that enhances subsequent vaccine-responsiveness may help decision-making on the number and timing of vaccine doses.

Identification of aging-associated immunotypes and immune stability as indicators of post-vaccination immune activation.

Immunosenescence describes immune dysfunction observed in older individuals. To identify individuals at-risk for immune dysfunction, it is crucial to understand the diverse immune phenotypes and their intrinsic functional capabilities. We investigated immune cell subsets and variation in the aging population. We observed that inter-individual immune variation was associated with age and cytomegalovirus seropositivity. Based on the similarities of immune subset composition among individuals, we identified nine immunotypes that displayed different aging-associated immune signatures, which explained inter-individual variation better than age. Additionally, we correlated the immune subset composition of individuals over approximately a year as a measure of stability of immune parameters. Immune stability was significantly lower in immunotypes that contained aging-associated immune subsets and correlated with a circulating CD38 + CD4+ T follicular helper cell increase 7 days after influenza vaccination. In conclusion, immune stability is a feature of immunotypes and could be a potential indicator of post-vaccination cellular kinetics.

Corrigendum: Children and Adults With Mild COVID-19: Dynamics of the Memory T Cell Response Up to 10 Months.

[This corrects the article DOI: 10.3389/fimmu.2022.817876.].

Children and Adults With Mild COVID-19: Dynamics of the Memory T Cell Response up to 10 Months.

Background: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to considerable morbidity/mortality worldwide, but most infections, especially among children, have a mild course. However, it remains largely unknown whether infected children develop cellular immune memory. Methods: To determine whether a memory T cell response is being developed, we performed a longitudinal assessment of the SARS-CoV-2-specific T cell response by IFN-γ ELISPOT and activation marker analyses of peripheral blood samples from unvaccinated children and adults with mild-to-moderate COVID-19. Results: Upon stimulation of PBMCs with heat-inactivated SARS-CoV-2 or overlapping peptides of spike (S-SARS-CoV-2) and nucleocapsid proteins, we found S-SARS-CoV-2-specific IFN-γ T cell responses in infected children (83%) and adults (100%) that were absent in unexposed controls. Frequencies of SARS-CoV-2-specific T cells were higher in infected adults, especially several cases with moderate symptoms, compared to infected children. The S-SARS-CoV-2 IFN-γ T cell response correlated with S1-SARS-CoV-2-specific serum antibody concentrations. Predominantly, effector memory CD4+ T cells of a Th1 phenotype were activated upon exposure to SARS-CoV-2 antigens. Frequencies of SARS-CoV-2-specific T cells were significantly reduced at 10 months after symptom onset, while S1-SARS-CoV-2-specific IgG concentrations were still detectable in 90% of all children and adults. Conclusions: Our data indicate that an antigen-specific T cell and antibody response is developed after mild SARS-CoV-2 infection in children and adults. It remains to be elucidated to what extent this SARS-CoV-2-specific response can contribute to an effective recall response after reinfection.

Reduced Antibody Acquisition with Increasing Age following Vaccination with BNT162b2: Results from Two Longitudinal Cohort Studies in The Netherlands.

Vaccine-induced protection against severe COVID-19, hospitalization, and death is of the utmost importance, especially in the elderly. However, limited data are available on humoral immune responses following COVID-19 vaccination in the general population across a broad age range. We performed an integrated analysis of the effect of age, sex, and prior SARS-CoV-2 infection on Spike S1-specific (S1) IgG concentrations up to three months post-BNT162b2 (Pfizer/BioNTech; Comirnaty) vaccination. In total, 1735 persons, eligible for COVID-19 vaccination through the national program, were recruited from the general population (12 to 92 years old). Sixty percent were female, and the median vaccination interval was 35 days (interquartile range, IQR: 35−35). All participants had seroconverted to S1 one month after two vaccine doses. S1 IgG was higher in participants with a history of SARS-CoV-2 infection (median: 4535 BAU/mL, IQR: 2341−7205) compared to infection-naive persons (1842 BAU/mL, 1019−3116), p < 0.001. In infection-naive persons, linear mixed effects regression showed a strong negative association between age and S1 IgG (p < 0.001) across the entire age range. Females had higher S1 IgG than males (p < 0.001). In persons with an infection history, age nor sex was associated with S1 IgG concentrations. The lower magnitude of S1 antibodies in older persons following COVID-19 vaccination will affect long-term protection.

Reduced Bordetella pertussis-specific CD4+ T-Cell Responses at Older Age.

Pertussis, a human-specific respiratory infectious disease caused by the Gram-negative bacterium Bordetella pertussis (Bp), remains endemic with epidemic years despite high vaccination coverage. Whereas pertussis vaccines and natural infection with Bp confer immune protection, the duration of protection varies and is not lifelong. Recent evidence indicates a considerable underestimation of the pertussis burden among older adults. Whereas the impact of increasing age on Bp-specific humoral immunity has been demonstrated, little is known on immunosenescence of CD4+ T-cell responses in the context of Bp. Here, we aimed to address whether increasing age impacts responsiveness of the Bp-specific CD4+ T-cells in the memory pool following a clinically symptomatic pertussis infection in whole cell vaccine-primed pediatric and adult cases. Cytokine and proliferative responses and phenotypical profiles of CD4+ T cells specific for Bp antigens at an early and late convalescent timepoint were compared. Responses of various Th cytokines, including IFNγ, were significantly lower in older adults at early and late timepoints post diagnosis. In addition, we found lower frequencies of Bp-specific proliferated CD4+ T cells in older adults, in the absence of differences in replication profile. Phenotyping of Bp-specific CD4+ T cells suggested reduced expression of activation markers rather than increased expression of co-inhibitory markers. Altogether, our findings show that the magnitude and functionality of the Bp-specific memory CD4+ T-cell pool decrease at older age. Declined CD4+ T-cell responsiveness to Bp is suggested to contribute to the burden of pertussis in older adults.

BRUCELLA PINNIPEDIALIS IN GREY SEALS ( HALICHOERUS GRYPUS) AND HARBOR SEALS ( PHOCA VITULINA) IN THE NETHERLANDS.

Brucellosis is a zoonotic disease with terrestrial or marine wildlife animals as potential reservoirs for the disease in livestock and human populations. The primary aim of this study was to assess the presence of Brucella pinnipedialis in marine mammals living along the Dutch coast and to observe a possible correlation between the presence of B. pinnipedialis and accompanying pathology found in infected animals. The overall prevalence of Brucella spp. antibodies in sera from healthy wild grey seals ( Halichoerus grypus; n=11) and harbor seals ( Phoca vitulina; n=40), collected between 2007 and 2013 ranged from 25% to 43%. Additionally, tissue samples of harbor seals collected along the Dutch shores between 2009 and 2012, were tested for the presence of Brucella spp. In total, 77% (30/39) seals were found to be positive for Brucella by IS 711 real-time PCR in one or more tissue samples, including pulmonary nematodes. Viable Brucella was cultured from 40% (12/30) real-time PCR-positive seals, and was isolated from liver, lung, pulmonary lymph node, pulmonary nematode, or spleen, but not from any PCR-negative seals. Tissue samples from lung and pulmonary lymph nodes were the main source of viable Brucella bacteria. All isolates were typed as B. pinnipedialis by multiple-locus variable number of tandem repeats analysis-16 clustering and matrix-assisted laser desorption ionization-time of flight mass spectrometry, and of sequence type ST25 by multilocus sequence typing analysis. No correlation was observed between Brucella infection and pathology. This report displays the isolation and identification of B. pinnipedialis in marine mammals in the Dutch part of the Atlantic Ocean.