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Cytokine polyfunctionality is a well-established concept in immune cells, especially T cells, and their ability to concurrently produce multiple cytokines has been associated with better immunological disease control and subsequent effectiveness during infection and disease. To date, only little is known about the secretion dynamics of those cells, masked by the widespread deployment of mainly time-integrated endpoint measurement techniques that do not easily differentiate between concurrent and sequential secretion. Here, we employed a single-cell microfluidic platform capable of resolving the secretion dynamics of individual PBMCs. To study the dynamics of poly-cytokine secretion, as well as the dynamics of concurrent and sequential polyfunctionality, we analyzed the response at different time points after ex vivo activation. First, we observed the simultaneous secretion of cytokines over the measurement time for most stimulants in a subpopulation of cells only. Second, polyfunctionality generally decreased with prolonged stimulation times and revealed no correlation with the concentration of secreted cytokines in response to stimulation. However, we observed a general trend towards higher cytokine secretion in polyfunctional cells, with their secretion dynamics being distinctly different from mono-cytokine-secreting cells. This study provided insights into the distinct secretion behavior of heterogenous cell populations after stimulation with well-described agents and such a system could provide a better understanding of various immune dynamics in therapy and disease.

Human peripheral blood mononuclear cells (hPBMCs) are widely used in fundamental research and clinical applications as studying their responses to activation is an effective way to uncover functional alterations and disease associated phenotypes. However, the availability of samples in large numbers at a specific time and location remains challenging, hence they often might preferably be collected and cryopreserved for later analysis. While the effect of cryopreservation on viability and cell surface expression is well established, changes in activity and cytokine secretion still lead to conflicting results as it is often measured in bulk or within the cells. Here, we used our platform for dynamic single-cell multiplexed cytokine secretion measurement and compared it to a traditional intracellular cytokine staining to quantify the effect of cryopreservation on cytokine secretion and expression of individual hPBMCs. Following stimulation with LPS or anti-CD3/CD28 antibodies for up to 36 or 72 h incubation, we observed distinct alterations in cytokine responses due to cryopreservation when comparing to fresh samples, but also remarkable consistencies for some cytokines and parameters. In short, the frequencies of cytokine-secreting cells in cryopreserved samples were lower for IL-6 (LPS), IL1-β (CD3/CD28) and IFN-γ (CD3/CD28), while the frequency and dynamics of IL-8 secretion were strongly impacted in all cases. We observed a large disconnect between cytokine expression and secretion for TNF-α, where the expression dramatically increased after cryopreservation, but actual secretion was, in comparison, remarkably stable. The polyfunctionality of single cells was altered by cryopreservation in specific co-secreting populations led by the effects on IL-6 or IL-8 secretion. Among immune cells, cryopreservation seemed to affect lymphocytes and monocytes differently as effects appeared early on in lymphocytes while generally observed in later time points in monocytes. Together, this study offers an in-depth quantitative insight into the biological behavior of immune cells in response to cryopreservation and stimulation, further providing some insights into conflicting results in the literature as well as guidelines for researchers planning to assess cytokine-secreting from frozen hPBMCs in immunological research or clinical applications.

The extent to which immune responses to natural infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and immunization with vaccines protect against variants of concern (VOC) is of increasing importance. Accordingly, here we analyse antibodies and T cells of a recently vaccinated, UK cohort, alongside those recovering from natural infection in early 2020. We show that neutralization of the VOC compared to a reference isolate of the original circulating lineage, B, is reduced: more profoundly against B.1.351 than for B.1.1.7, and in responses to infection or a single dose of vaccine than to a second dose of vaccine. Importantly, high magnitude T cell responses are generated after two vaccine doses, with the majority of the T cell response directed against epitopes that are conserved between the prototype isolate B and the VOC. Vaccination is required to generate high potency immune responses to protect against these and other emergent variants. Understanding the effect of vaccination on emerging SARS-CoV-2 variants of concern is of increasing importance. Here, James et al. report that two doses of vaccination with the Pfizer-BioNTech vaccine induce more robust immune responses to the B.1.1.7 and B.1.351 SARS-CoV-2 lineages than does natural infection.

While cellular metabolism was proposed to be a driving factor of the activation and differentiation of B cells and the function of the resulting antibody-secreting cells (ASCs), the study of correlations between cellular metabolism and functionalities has been difficult due to the absence of technologies enabling the parallel measurement. Herein, we performed single-cell transcriptomics and introduced a direct concurrent functional and metabolic flux quantitation of individual murine B cells. Our transcriptomic data identified lactate metabolism as dynamic in ASCs, but antibody secretion did not correlate with lactate secretion rates (LSRs). Instead, our study of all splenic B cells during an immune response linked increased lactate metabolism with acidic intracellular pH and the upregulation of apoptosis. T cell-dependent responses increased LSRs, and added TLR4 agonists affected the magnitude and boosted LSR high B cells in vivo, while resulting in only a few immunoglobulin-G secreting cells (IgG-SCs). Therefore, our observations indicated that LSR high cells were not differentiating into IgG-SCs, and were rather removed due to apoptosis.

The trajectories of acquired immunity to severe acute respiratory syndrome coronavirus 2 infection are not fully understood. We present a detailed longitudinal cohort study of UK healthcare workers prior to vaccination, presenting April-June 2020 with asymptomatic or symptomatic infection. Here we show a highly variable range of responses, some of which (T cell interferon-gamma ELISpot, N-specific antibody) wane over time, while others (spike-specific antibody, B cell memory ELISpot) are stable. We use integrative analysis and a machine-learning approach (SIMON - Sequential Iterative Modeling OverNight) to explore this heterogeneity. We identify a subgroup of participants with higher antibody responses and interferon-gamma ELISpot T cell responses, and a robust trajectory for longer term immunity associates with higher levels of neutralising antibodies against the infecting (Victoria) strain and also against variants B.1.1.7 (alpha) and B.1.351 (beta). These variable trajectories following early priming may define subsequent protection from severe disease from novel variants. The engagement of immunological memory is a key component to the protective anti-SARS-CoV-2 B and T cell responses. Here the authors assess the B and T cells of a cohort of UK healthcare workers in response to infection and longitudinally track the compartment showing distinct trajectories following early priming.

Background Disease caused by the capsular group B meningococcus (MenB) is the leading cause of infectious death in UK infants. A novel adenovirus-based vaccine encoding the MenB factor H binding protein (fHbp) with an N-terminal dual signal sequence induces high titres of protective antibody after a single dose in mice. A panel of N-terminal signal sequence variants were created to assess the contribution of components of this sequence to transgene expression kinetics of the encoded antigen from mammalian cells and the resultant effect on immunogenicity of fHbp. Results The full-length signal sequence (FL SS) resulted in superior early antigen expression compared with the panel of variants, as measured by flow cytometry and confocal imaging, and supported higher bactericidal antibody levels against the expressed antigen in mouse sera < 6 weeks post-immunisation than the licensed four component MenB vaccine. The FL SS also significantly increased antigen-specific T cell responses against other adenovirus-encoded bacterial antigens in mice. Conclusions These findings demonstrate that the FL SS enhances immunogenicity of the encoded antigen, supporting its inclusion in other viral vectored bacterial antigen transgenes.

Neisseria meningitidis outer membrane vesicles (OMV) are a component of the capsular group B meningococcal vaccine 4CMenB (Bexsero) and have been shown to induce 30% efficacy against gonococcal infection. They are composed of multiple antigens and are considered an interesting delivery platform for vaccines against several bacterial diseases. Neisseria meningitidis outer membrane vesicle (OMV) vaccines are safe and provide strain-specific protection against invasive meningococcal disease (IMD) primarily by inducing serum bactericidal antibodies against the outer membrane proteins (OMP). To design broader coverage vaccines, knowledge of the immunogenicity of all the antigens contained in OMVs is needed. In a Phase I clinical trial, an investigational meningococcal OMV vaccine, MenPF1, made from a meningococcus genetically modified to constitutively express the iron-regulated FetA induced bactericidal responses to both the PorA and the FetA antigen present in the OMP. Using peripheral blood mononuclear cells collected from this trial, we analyzed the kinetics of and relationships between IgG, IgA, and IgM B cell responses against recombinant PorA and FetA, including (i) antibody-secreting cells, (ii) memory B cells, and (iii) functional antibody responses (opsonophagocytic and bactericidal activities). Following MenPF1vaccination, PorA-specific IgG secreting cell responses were detected in up to 77% of participants and FetA-specific responses in up to 36%. Memory B cell responses to the vaccine were low or absent and mainly detected in participants who had evidence of preexisting immunity ( P = 0.0069). Similarly, FetA-specific antibody titers and bactericidal activity increased in participants with preexisting immunity and is consistent with the idea that immune responses are elicited to minor antigens during asymptomatic Neisseria carriage, which can be boosted by OMV vaccines. IMPORTANCE Neisseria meningitidis outer membrane vesicles (OMV) are a component of the capsular group B meningococcal vaccine 4CMenB (Bexsero) and have been shown to induce 30% efficacy against gonococcal infection. They are composed of multiple antigens and are considered an interesting delivery platform for vaccines against several bacterial diseases. However, the protective antibody response after two or three doses of OMV-based meningococcal vaccines appears short-lived. We explored the B cell response induced to a dominant and a subdominant antigen in a meningococcal OMV vaccine in a clinical trial and showed that immune responses are elicited to minor antigens. However, memory B cell responses to the OMV were low or absent and mainly detected in participants who had evidence of preexisting immunity against the antigens. Failure to induce a strong B cell response may be linked with the low persistence of protective responses.

Neisseria gonorrhoeae causes 82 million global cases of gonorrhoea annually. Multidrug-resistant gonococci threaten to make gonorrhoea untreatable. Meningococcal vaccines MeNZB and 4CMenB (Bexsero), containing Neisseria meningitidis group B detergent-extracted outer membrane vesicles (dOMV), cross-protect against gonorrhoea with 31–59% effectiveness. We hypothesised that gonococcal OMV-based vaccines would have greater efficacy against gonorrhoea than meningococcal vaccines. We developed native OMV (nOMV) candidate Neisseria vaccines from gonococcal strains GC_0817560 and FA1090, and meningococcal B strain NZ98/254. lpxL1 and rmp genes were deleted to reduce reactogenicity and minimise induction of unprotective or blocking antibodies. nOMV were characterised and formulated with aluminium hydroxide. Deletion of lpxL1 markedly reduced nOMV-induced IL-6 release from peripheral blood mononuclear cells. nOMV derived from GC_0817560 lpxL1 − rmp − and FA1090 lpxL1 − rmp − induced greater quantities of gonococcal-specific serum IgG and accelerated clearance of FA1090 from oestradiol-treated BALB/c mice significantly faster than 4CMenB and NZ98/254 lpxL1 − rmp − nOMV ( P  < 0.0001). Gonococcal nOMV-based vaccines represent promising candidates for further development.

Cytokine polyfunctionality is a well-established concept in immune cells, especially T cells, and their ability to concurrently produce multiple cytokines has been associated with better immunological disease control and subsequent effectiveness during infection and disease. To date, only little is known about the secretion dynamics of those cells, masked by the widespread deployment of mainly time-integrated endpoint measurement techniques that do not easily differentiate between concurrent and sequential secretion. Here, we employed a single-cell microfluidic platform capable of resolving the secretion dynamics of individual PBMCs. To study the dynamics of poly-cytokine secretion, as well as the dynamics of concurrent and sequential polyfunctionality, we analyzed the response at different time points after ex vivo activation. First, we observed the simultaneous secretion of cytokines over the measurement time for most stimulants in a subpopulation of cells only. Second, polyfunctionality generally decreased with prolonged stimulation times and revealed no correlation with the concentration of secreted cytokines in response to stimulation. However, we observed a general trend towards higher cytokine secretion in polyfunctional cells, with their secretion dynamics being distinctly different from mono-cytokine-secreting cells. This study provided insights into the distinct secretion behavior of heterogenous cell populations after stimulation with well-described agents and such a system could provide a better understanding of various immune dynamics in therapy and disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic in 2020. Testing is crucial for mitigating public health and economic effects. Serology is considered key to population-level surveillance and potentially individual-level risk assessment. However, immunoassay performance has not been compared on large, identical sample sets. We aimed to investigate the performance of four high-throughput commercial SARS-CoV-2 antibody immunoassays and a novel 384-well ELISA. We did a head-to-head assessment of SARS-CoV-2 IgG assay (Abbott, Chicago, IL, USA), LIAISON SARS-CoV-2 S1/S2 IgG assay (DiaSorin, Saluggia, Italy), Elecsys Anti-SARS-CoV-2 assay (Roche, Basel, Switzerland), SARS-CoV-2 Total assay (Siemens, Munich, Germany), and a novel 384-well ELISA (the Oxford immunoassay). We derived sensitivity and specificity from 976 pre-pandemic blood samples (collected between Sept 4, 2014, and Oct 4, 2016) and 536 blood samples from patients with laboratory-confirmed SARS-CoV-2 infection, collected at least 20 days post symptom onset (collected between Feb 1, 2020, and May 31, 2020). Receiver operating characteristic (ROC) curves were used to assess assay thresholds. At the manufacturers' thresholds, for the Abbott assay sensitivity was 92·7% (95% CI 90·2–94·8) and specificity was 99·9% (99·4–100%); for the DiaSorin assay sensitivity was 96·2% (94·2–97·7) and specificity was 98·9% (98·0–99·4); for the Oxford immunoassay sensitivity was 99·1% (97·8–99·7) and specificity was 99·0% (98·1–99·5); for the Roche assay sensitivity was 97·2% (95·4–98·4) and specificity was 99·8% (99·3–100); and for the Siemens assay sensitivity was 98·1% (96·6–99·1) and specificity was 99·9% (99·4–100%). All assays achieved a sensitivity of at least 98% with thresholds optimised to achieve a specificity of at least 98% on samples taken 30 days or more post symptom onset. Four commercial, widely available assays and a scalable 384-well ELISA can be used for SARS-CoV-2 serological testing to achieve sensitivity and specificity of at least 98%. The Siemens assay and Oxford immunoassay achieved these metrics without further optimisation. This benchmark study in immunoassay assessment should enable refinements of testing strategies and the best use of serological testing resource to benefit individuals and population health. Public Health England and UK National Institute for Health Research.