Nationwide upsurge in invasive disease in the context of longitudinal surveillance of carriage and invasive Streptococcus pyogenes 2009-2023, the Netherlands: a molecular epidemiological study.

Since 2022, many countries have reported an upsurge in invasive group A streptococcal (iGAS) infections. We explored whether changes in Streptococcus pyogenes carriage rates or emergence of strains with potentially altered virulence, such as emm1 variants M1UK and M1DK, contributed to the 2022/2023 surge in the Netherlands. We determined emm (sub)type distribution for 2,698 invasive and 351 S. pyogenes carriage isolates collected between January 2009 and March 2023. Genetic evolution of emm1 was analyzed by whole-genome sequencing of 497 emm1 isolates. The nationwide iGAS upsurge coincided with a sharp increase of emm1.0 from 18% (18/100) of invasive isolates in Q1 2022 to 58% (388/670) in Q1 2023 (Fisher's exact test, P < 0.0001). M1UK became dominant among invasive emm1 isolates in 2016 and further expanded from 72% in Q1 2022 to 96% in Q1 2023. Phylogenetic comparison revealed evolution and clonal expansion of four new M1UK clades in 2022/2023. DNase Spd1 and superantigen SpeC were acquired in 9% (46/497) of emm1 isolates. S. pyogenes carriage rates and emm1 proportions in carriage isolates remained stable during this surge, and the expansion of M1UK in iGAS was not reflected in carriage isolates. During the 2022/2023 iGAS surge in the Netherlands, expansion of four new M1UK clades was observed among invasive isolates, but not carriage isolates, suggesting increased virulence and fitness of M1UK compared to contemporary M1 strains. The emergence of more virulent clades has important implications for public health strategies such as antibiotic prophylaxis for close contacts of iGAS patients.IMPORTANCEThis study describes the molecular epidemiology of invasive group A streptococcal (iGAS) infections in the Netherlands based on >3,000 Streptococcus pyogenes isolates from both asymptomatic carriers and iGAS patients collected before, during, and after the COVID-19 pandemic period (2009-2023) and is the first to assess whether changes in carriage rates or carried emm types contributed to the alarming post-COVID-19 upsurge in iGAS infections. We show that the 2022/2023 iGAS surge coincided with a sharp increase of emm1, particularly the toxicogenic M1UK variant, in invasive isolates, but not in carriage isolates. These findings suggest that increased virulence and fitness of M1UK likely contributes to an increased dissemination between hosts. The emergence of a more virulent and fit lineage has important implications for iGAS control interventions such as antibiotic prophylaxis for close contacts of iGAS patients and calls for a reappraisal of iGAS control interventions and guidelines.

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.

Maternal Pertussis Immunization and Immunoglobulin G Levels in Early- to Late-Term and Preterm Infants.

Importance: Maternal tetanus, diphtheria, and acellular pertussis (Tdap) vaccination protects newborns against severe pertussis. Data on transplacental antibody transfer on Tdap vaccination before 24 weeks' gestation remain scarce and are particularly relevant for preterm infants to increase the time interval for maternal antibody transfer. Objective: To assess noninferiority of anti-pertussis toxin (anti-PT) immunoglobulin G (IgG) antibody levels at age 2 months in early- to late-term infants following Tdap vaccination between 20 0/7 and 24 0/7 weeks' gestation compared with 30 0/7 and 33 0/7 weeks' gestation and compared with preterm infants. Design, Setting, and Participants: This prospective, multicenter cohort study included pregnant women aged 18 years or older in birthing centers and hospitals in the Netherlands between August 2019 and November 2021 who received Tdap vaccination between 20 0/7 and 24 0/7 weeks' gestation. Women with imminent premature birth were recruited if they had received maternal Tdap vaccination between 20 and 24 weeks' gestation. Blood samples were collected from mothers at delivery, from the umbilical cord, and from infants at age 2 months. Data from infants' blood samples at age 2 months were compared with a reference cohort (recruited between January 2014 and February 2016) of early- to late-term infants of the same age whose mothers had received Tdap vaccination between 30 0/7 and 33 0/7 weeks' gestation. Exposure: Maternal Tdap vaccination between 20 0/7 and 24 0/7 weeks' gestation or 30 0/7 and 33 0/7 weeks' gestation. Main Outcomes and Measures: The primary outcome was the geometric mean concentration (GMC) of anti-PT IgG antibodies in early- to late-term infants (≥37 0/7 weeks' gestation) at age 2 months, comparing maternal Tdap vaccination between 20 0/7 and 24 0/7 weeks' vs 30 0/7 and 33 0/7 weeks' gestation (reference cohort). Anti-PT GMC in 2-month-old infants born preterm (<35 0/7 weeks' gestation) compared with early- to late-term infants after maternal Tdap vaccination between 20 and 24 weeks' gestation was a secondary outcome. Results: In total, 221 women who delivered 239 offspring were enrolled in the study; 66 early- to late-term infants (median gestational age [GA], 40.6 weeks [IQR, 39.8-41.0 weeks]; 38 [57.6%] male) and 73 preterm infants (median GA, 32.1 weeks [IQR, 29.5-33.0 weeks]; 42 [54.5%] female) had blood samples collected at 2 months of age. Anti-PT GMC was 14.7 IU/mL (95% CI, 10.6-20.4 IU/mL) in early- to late-term infants following maternal Tdap vaccination between 20 0/7 and 24 0/7 weeks' gestation compared with 27.3 IU/mL (95% CI, 20.1-37.1 IU/mL) in 55 infants in the reference group (median GA, 40.3 [IQR, 39.1-41.0]; 33 [60.0%] female). The mean anti-PT GMC in preterm infants in the study group was 11.2 IU/mL (95% CI, 8.1-15.3 IU/mL) (P = .23 compared with early- to late-term infants). Conclusions and Relevance: In this cohort study, 2-month-old preterm and early- to late-term infants showed significantly lower anti-PT antibody levels following maternal Tdap vaccination between 20 0/7 and 24 0/7 weeks' gestation compared with 30 0/7 and 33 0/7 weeks' gestation; preterm and early- to late-term infants had similar anti-PT antibody levels, but both groups showed significantly lower antibody levels compared with the reference group. Epidemiological research should investigate whether maternal Tdap vaccination before 24 weeks' gestation provides sufficient protection against clinical pertussis, particularly in preterm infants, as long as no correlate of protection is available.

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.

Quantity and Quality of Naturally Acquired Antibody Immunity to the Pneumococcal Proteome Throughout Life.

BACKGROUND: Young children and older adults are susceptible for invasive pneumococcal disease (IPD) caused by Streptococcus pneumoniae. Pneumococcal protein-specific antibodies play a protective role against IPD; however, not much is known about the pace of acquisition, maturation, and maintenance of these antibodies throughout life. METHODS: Immunoglobulin G (IgG) and IgA levels, avidity, and/or specificity to the pneumococcal proteome in serum and saliva from healthy young children, adults, and older adults, with known carriage status, were measured by enzyme-linked immunosorbent assay (ELISA) and 2-dimensional western blotting against ΔcpsTIGR4. RESULTS: Eleven-month-old children, the youngest age group tested, had the lowest pneumococcal proteome-specific IgG and IgA levels and avidity in serum and saliva, followed by 24-month-old children and were further elevated in adult groups. Among adult groups, the parents had the highest serum and saliva IgG and IgA antibody levels. In children, antibody levels and avidity correlated with daycare attendance and presence of siblings, posing as proxy for exposure and immunization. Immunodominance patterns slightly varied throughout life. CONCLUSIONS: Humoral immunity against the pneumococcal proteome is acquired through multiple episodes of pneumococcal exposure. Low-level and low-avidity antiproteome antibody profiles in young children may contribute to their IPD susceptibility, while in overall antiproteome antibody-proficient older adults other factors likely play a role.

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.

Prevalence of human respiratory pathogens and associated mucosal cytokine levels in young children and adults: a cross-sectional observational study in the Netherlands during the winter of 2012/2013.

Respiratory pathogens can cause severe disease and even death, especially in the very young and very old. Studies investigating their prevalence often focus on individuals presenting to healthcare providers with symptoms. However, the design of prevention strategies, e.g. which target groups to vaccinate, will benefit from knowledge on the prevalence of, risk factors for and host response to these pathogens in the general population. In this study, upper respiratory samples (n = 1311) were collected cross-sectionally during winter from 11- and 24-month old children, their parents, and adults ≥60 years of age that were recruited irrespective of seeking medical care. Almost all children, approximately two-thirds of parents and a quarter of older adults tested positive for at least one pathogen, often in the absence of symptoms. Viral interference was evident for the combination of rhinovirus and respiratory syncytial virus. Attending childcare facilities and having siblings associated with increased pathogen counts in children. On average, children showed increased levels of mucosal cytokines compared to parents and especially proinflammatory molecules associated with the presence of symptoms. These findings may guide further research into transmission patterns of respiratory pathogens and assist in determining the most appropriate strategies for the prediction and prevention of disease.

Serological profiling of pneumococcal proteins reveals unique patterns of acquisition, maintenance and waning of antibodies throughout life.

Streptococcus pneumoniae is a leading cause of morbidity and mortality in children and older adults. Yet knowledge on the development of pneumococcal protein-specific antibody responses throughout life is limited. To investigate this, we measured serum IgG levels to 55 pneumococcal proteins in 11-month old infants (n=73), 24-month old children (n=101), parents (n=99), adults without children <6 years of age (n= 99) and older adults aged >60 years (n=100). Our findings revealed low IgG levels in infancy, with distinct development patterns peaking in adults. A decrease in levels was observed for 27 antigens towards older age. Adults and older adults had increased IgG levels during pneumococcal carriage and at increased exposure risk to S. pneumoniae. Carriage was a stronger predictor than exposure or age for antibody responses. These findings highlight the dynamic nature of naturally acquired humoral immunity to pneumococcal proteins throughout life, offering insights for age-targeted interventions.

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.

Impact of age on pneumococcal colonization of the nasopharynx and oral cavity: an ecological perspective.

Pneumococcal carriage studies have suggested that pneumococcal colonization in adults is largely limited to the oral cavity and oropharynx. In this study, we used total abundance-based ß-diversity (dissimilarity) and ß-diversity components to characterize age-related differences in pneumococcal serotype composition of respiratory samples. quantitative PCR (qPCR) was applied to detect pneumococcal serotypes in nasopharyngeal samples collected from 946 toddlers and 602 adults, saliva samples collected from a subset of 653 toddlers, and saliva and oropharyngeal samples collected from a subset of 318 adults. Bacterial culture rates from nasopharyngeal samples were used to characterize age-related differences in rates of colonizing bacteria. Dissimilarity in pneumococcal serotype composition was low among saliva and nasopharyngeal samples from children. In contrast, respiratory samples from adults exhibited high serotype dissimilarity, which predominantly consisted of abundance gradients and was associated with reduced nasopharyngeal colonization. Age-related serotype dissimilarity was high among nasopharyngeal samples and relatively low for saliva samples. Reduced nasopharyngeal colonization by pneumococcal serotypes coincided with significantly reduced Moraxella catarrhalis and Haemophilus influenzae and increased Staphylococcus aureus nasopharyngeal colonization rates among adults. Findings from this study suggest that within-host environmental conditions, utilized in the upper airways by pneumococcus and other bacteria, undergo age-related changes. It may result in a host-driven ecological succession of bacterial species colonizing the nasopharynx and lead to competitive exclusion of pneumococcus from the nasopharynx but not from the oral habitat. This explains the poor performance of nasopharyngeal samples for pneumococcal carriage among adults and indicates that in adults saliva more accurately represents the epidemiology of pneumococcal carriage than nasopharyngeal samples.

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.

Saliva as an alternative sample type for detection of pneumococcal carriage in young children.

For children, the gold standard for the detection of pneumococcal carriage is conventional culture of a nasopharyngeal swab. Saliva, however, has a history as one of the most sensitive methods for surveillance of pneumococcal colonization and has recently been shown to improve carriage detection in older age groups. Here, we compared the sensitivity of paired nasopharyngeal and saliva samples from PCV7-vaccinated 24-month-old children for pneumococcal carriage detection using conventional and molecular detection methods. Nasopharyngeal and saliva samples were collected from 288 24-month-old children during the autumn/winter, 2012/2013. All samples were first processed by conventional diagnostic culture. Next, DNA extracted from all plate growth was tested by qPCR for the presence of the pneumococcal genes piaB and lytA and a subset of serotypes. By culture, 161/288 (60 %) nasopharyngeal swabs tested positive for pneumococcus, but detection was not possible from saliva due to abundant polymicrobial growth on culture plates. By qPCR, 155/288 (54 %) culture-enriched saliva samples and 187/288 (65 %) nasopharyngeal swabs tested positive. Altogether, 219/288 (76 %) infants tested positive for pneumococcus, with qPCR-based carriage detection of culture-enriched nasopharyngeal swabs detecting significantly more carriers compared to either conventional culture (P<0.001) or qPCR detection of saliva (P=0.002). However, 32/219 (15 %) carriers were only positive in saliva, contributing significantly to the overall number of carriers detected (P=0.002). While testing nasopharyngeal swabs by qPCR proved most sensitive for pneumococcal detection in infants, saliva sampling could be considered as complementary to provide additional information on carriage and serotypes that may not be detected in the nasopharynx and may be particularly useful in longitudinal studies, requiring repeated sampling of study participants.

Difference in respiratory syncytial virus-specific Fc-mediated antibody effector functions between children and adults.

Respiratory syncytial virus (RSV) infections are a major cause of bronchiolitis and pneumonia in infants and older adults, for which there is no known correlate of protection. Increasing evidence suggests that Fc-mediated antibody effector functions have an important role, but little is known about the development, heterogeneity, and durability of these functional responses. In light of future vaccine strategies, a clear view of the immunological background and differences between various target populations is of crucial importance. In this study, we have assessed both quantitative and qualitative aspects of RSV-specific serum antibodies, including IgG/IgA levels, IgG subclasses, antibody-dependent complement deposition, cellular phagocytosis, and NK cell activation (ADNKA). Samples were collected cross-sectionally in different age groups (11-, 24-, and 46-month-old children, adults, and older adults; n = 31-35 per group) and longitudinally following natural RSV infection in (older) adults (2-36 months post-infection; n = 10). We found that serum of 24-month-old children induces significantly lower ADNKA than the serum of adults (P < 0.01), which is not explained by antibody levels. Furthermore, in (older) adults we observed boosting of antibody levels and functionality at 2-3 months after RSV infection, except for ADNKA. The strongest decrease was subsequently observed within the first 9 months, after which levels remained relatively stable up to three years post-infection. Together, these data provide a comprehensive overview of the functional landscape of RSV-specific serum antibodies in the human population, highlighting that while antibodies reach adult levels already at a young age, ADNKA requires more time to fully develop.

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.

Fourth mRNA COVID-19 vaccination in immunocompromised patients with haematological malignancies (COBRA KAI): a cohort study.

Background: Patients with haematological malignancies have impaired antibody responses to SARS-CoV-2 vaccination. We aimed to investigate whether a fourth mRNA COVID-19 vaccination improved antibody quantity and quality. Methods: In this cohort study, conducted at 5 sites in the Netherlands, we compared antibody concentrations 28 days after 4 mRNA vaccinations (3-dose primary series plus 1 booster vaccination) in SARS-CoV-2 naive, immunocompromised patients with haematological malignancies to those obtained by age-matched, healthy individuals who had received the standard primary 2-dose mRNA vaccination schedule followed by a first booster mRNA vaccination. Prior to and 4 weeks after each vaccination, peripheral blood samples and data on demographic parameters and medical history were collected. Concentrations of antibodies that bind spike 1 (S1) and nucleocapsid (N) protein of SARS-CoV-2 were quantified in binding antibody units (BAU) per mL according to the WHO International Standard for COVID-19 serological tests. Seroconversion was defined as an S1 IgG concentration >10 BAU/mL and a previous SARS-CoV-2 infection as N IgG >14.3 BAU/mL. Antibody neutralising activity was tested using lentiviral-based pseudoviruses expressing spike protein of SARS-CoV-2 wild-type (D614G), Omicron BA.1, and Omicron BA.4/5 variants. This study is registered with EudraCT, number 2021-001072-41. Findings: Between March 24, 2021 and May 4, 2021, 723 patients with haematological diseases were enrolled, of which 414 fulfilled the inclusion criteria for the current analysis. Although S1 IgG concentrations in patients significantly improved after the fourth dose, they remained significantly lower compared to those obtained by 58 age-matched healthy individuals after their first booster (third) vaccination. The rise in neutralising antibody concentration was most prominent in patients with a recovering B cell compartment, although potent responses were also observed in patients with persistent immunodeficiencies. 19% of patients never seroconverted, despite 4 vaccinations. Patients who received their first 2 vaccinations when they were B cell depleted and the third and fourth vaccination during B cell recovery demonstrated similar antibody induction dynamics as patients with normal B cell numbers during the first 2 vaccinations. However, the neutralising capacity of these antibodies was significantly better than that of patients with normal B cell numbers after two vaccinations. Interpretation: A fourth mRNA COVID-19 vaccination improved S1 IgG concentrations in the majority of patients with a haematological malignancy. Vaccination during B cell depletion may pave the way for better quality of antibody responses after B cell reconstitution. Funding: The Netherlands Organisation for Health Research and Development and Amsterdam UMC.

Novel emm4 lineage associated with an upsurge in invasive group A streptococcal disease in the Netherlands, 2022.

Invasive group A streptococcal (iGAS) disease cases increased in the first half of 2022 in the Netherlands, with a remarkably high proportion of emm4 isolates. Whole-genome sequence analysis of 66 emm4 isolates, 40 isolates from the pre-coronavirus disease 2019 (COVID-19) pandemic period 2009-2019 and 26 contemporary isolates from 2022, identified a novel Streptococcus pyogenes lineage (M4NL22), which accounted for 85 % of emm4 iGAS cases in 2022. Surprisingly, we detected few isolates of the emm4 hypervirulent clone, which has replaced nearly all other emm4 in the USA and the UK. M4NL22 displayed genetic differences compared to other emm4 strains, although these were of unclear biological significance. In publicly available data, we identified a single Norwegian isolate belonging to M4NL22, which was sampled after the isolates from this study, possibly suggesting export of M4NL22 to Norway. In conclusion, our study identified a novel S. pyogenes emm4 lineage underlying an increase of iGAS disease in early 2022 in the Netherlands and the results have been promptly communicated to public health officials.

A spitting image: molecular diagnostics applied to saliva enhance detection of Streptococcus pneumoniae and pneumococcal serotype carriage.

Background: Despite strong historical records on the accuracy of saliva testing, oral fluids are considered poorly suited for pneumococcal carriage detection. We evaluated an approach for carriage surveillance and vaccine studies that increases the sensitivity and specificity of pneumococcus and pneumococcal serotype detection in saliva samples. Methods: Quantitative PCR (qPCR)-based methods were applied to detect pneumococcus and pneumococcal serotypes in 971 saliva samples collected from 653 toddlers and 318 adults. Results were compared with culture-based and qPCR-based detection in nasopharyngeal samples collected from children and in nasopharyngeal and oropharyngeal samples collected from adults. Optimal C q cut-offs for positivity in qPCRs were determined via receiver operating characteristic curve analysis and accuracy of different approaches was assessed using a composite reference for pneumococcal and for serotype carriage based on isolation of live pneumococcus from the person or positivity of saliva samples determined with qPCR. To evaluate the inter-laboratory reproducibility of the method, 229 culture-enriched samples were tested independently in the second center. Results: In total, 51.5% of saliva samples from children and 31.8% of saliva samples from adults were positive for pneumococcus. Detection of pneumococcus by qPCR in culture-enriched saliva exhibited enhanced sensitivity and higher agreement with a composite reference compared to diagnostic culture of nasopharyngeal samples in children (Cohen's κ: 0.69-0.79 vs. 0.61-0.73) and in adults (κ: 0.84-0.95 vs. 0.04-0.33) and culture of oropharyngeal samples in adults (κ: 0.84-0.95 vs. -0.12-0.19). Similarly, detection of serotypes with qPCR in culture-enriched saliva exhibited enhanced sensitivity and higher agreement with a composite reference compared to nasopharyngeal culture in children (κ: 0.73-0.82 vs. 0.61-0.73) and adults (κ: 0.90-0.96 vs. 0.00-0.30) and oropharyngeal culture in adults (κ: 0.90-0.96 vs. -0.13 to 0.30). However, results of qPCRs targeting serotype 4, 5, and 17F and serogroups 9, 12, and 35 were excluded due to assays' lack of specificity. We observed excellent quantitative agreement for qPCR-based detection of pneumococcus between laboratories. After exclusion of serotype/serogroup-specific assays with insufficient specificity, moderate agreement (κ 0.68, 95% CI 0.58-0.77) was observed. Conclusion: Molecular testing of culture-enriched saliva samples improves the sensitivity of overall surveillance of pneumococcal carriage in children and adults, but limitations of qPCR-based approaches for pneumococcal serotypes carriage detection should be considered.

Antibody and T-Cell Responses 6 Months After Coronavirus Disease 2019 Messenger RNA-1273 Vaccination in Patients With Chronic Kidney Disease, on Dialysis, or Living With a Kidney Transplant.

BACKGROUND: The immune response to COVID-19 vaccination is inferior in kidney transplant recipients (KTRs) and to a lesser extent in patients on dialysis or with chronic kidney disease (CKD). We assessed the immune response 6 months after mRNA-1273 vaccination in kidney patients and compared this to controls. METHODS: A total of 152 participants with CKD stages G4/5 (eGFR <30 mL/min/1.73 m2), 145 participants on dialysis, 267 KTRs, and 181 controls were included. SARS-CoV-2 Spike S1 specific IgG antibodies were measured using fluorescent bead-based multiplex-immunoassay, neutralizing antibodies to ancestral, Delta, and Omicron (BA.1) variants by plaque reduction, and T-cell responses by interferon-γ release assay. RESULTS: At 6 months after vaccination, S1-specific antibodies were detected in 100% of controls, 98.7% of CKD G4/5 patients, 95.1% of dialysis patients, and 56.6% of KTRs. These figures were comparable to the response rates at 28 days, but antibody levels waned significantly. Neutralization of the ancestral and Delta variants was detected in most participants, whereas neutralization of Omicron was mostly absent. S-specific T-cell responses were detected at 6 months in 75.0% of controls, 69.4% of CKD G4/5 patients, 52.6% of dialysis patients, and 12.9% of KTRs. T-cell responses at 6 months were significantly lower than responses at 28 days. CONCLUSIONS: Although seropositivity rates at 6 months were comparable to rates at 28 days after vaccination, significantly decreased antibody levels and T-cell responses were observed. The combination of low antibody levels, reduced T-cell responses, and absent neutralization of the newly emerging variants indicates the need for additional boosts or alternative vaccination strategies in KTRs. CLINICAL TRIALS REGISTRATION: NCT04741386.

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.

Omicron BA.1 Mutations in SARS-CoV-2 Spike Lead to Reduced T-Cell Response in Vaccinated and Convalescent Individuals.

Omicron BA.1 variant can readily infect people with vaccine-induced or naturally acquired SARS-CoV-2 immunity facilitated by escape from neutralizing antibodies. In contrast, T-cell reactivity against the Omicron BA.1 variant seems relatively well preserved. Here, we studied the preexisting T cells elicited by either vaccination with the mRNA-based BNT162b2 vaccine or by natural infection with ancestral SARS-CoV-2 for their cross-reactive potential to 20 selected CD4+ T-cell epitopes of spike-protein-harboring Omicron BA.1 mutations. Although the overall memory CD4+ T-cell responses primed by the ancestral spike protein was still preserved generally, we show here that there is also a clear loss of memory CD4+ T-cell cross-reactivity to immunodominant epitopes across the spike protein due to Omicron BA.1 mutations. Complete or partial loss of preexisting T-cell responsiveness was observed against 60% of 20 nonconserved CD4+ T-cell epitopes predicted to be presented by a broad set of common HLA class II alleles. Monitoring such mutations in circulating strains helps predict which virus variants may escape previously induced cellular immunity and could be of concern.