Immunological imprinting: Understanding COVID-19.

Immunological imprinting generically refers to the effects prior exposures have on subsequent immune responses to, and eventually protection against, antigenically related viruses. Here, Koutsakos and Ellebedy explain different concepts and terms around imprinting and the fundamental immunological principles behind it. They also discuss the potential role imprinting may have in the context of COVID-19 vaccines.

SARS-CoV-2 breakthrough infection induces rapid memory and de novo T cell responses

While the protective role of neutralising antibodies against COVID-19 is well-established, questions remain about the relative importance of cellular immunity. Using 6 pMHC-multimers in a cohort with early and frequent sampling, we define the phenotype and kinetics of recalled and primary T cell responses following Delta or Omicron breakthrough infection in previously vaccinated individuals. Recall of spike-specific CD4+ T cells was rapid, with cellular proliferation and extensive activation evident as early as 1 day post-symptom onset. Similarly, spike-specific CD8+ T cells were rapidly activated but showed variable degrees of expansion. The frequency of activated SARS-CoV-2-specific CD8+ T cells at baseline and peak inversely correlated with peak SARS-CoV-2 RNA levels in nasal swabs and accelerated viral clearance. Our study demonstrates rapid and extensive recall of memory T cell populations occurs early after breakthrough infection and suggests that CD8+ T cells contribute to the control of viral replication in breakthrough SARS-CoV-2 infections. Graphical Our understanding of T cell responses to SARS-CoV-2 vaccination and breakthrough infection has lagged behind B cells and antibodies. Here, Koutsakos et al utilize longitudinal sampling to demonstrate rapid activation of SARS-CoV-2-specific CD4+ and CD8+ T cells during breakthrough infection. Furthermore, Spike-specific CD8+ T cell activation correlates with viral clearance.

SARS-CoV-2 breakthrough infection induces rapid memory and de novo T cell responses.

Although the protective role of neutralizing antibodies against COVID-19 is well established, questions remain about the relative importance of cellular immunity. Using 6 pMHC multimers in a cohort with early and frequent sampling, we define the phenotype and kinetics of recalled and primary T cell responses following Delta or Omicron breakthrough infection in previously vaccinated individuals. Recall of spike-specific CD4+ T cells was rapid, with cellular proliferation and extensive activation evident as early as 1 day post symptom onset. Similarly, spike-specific CD8+ T cells were rapidly activated but showed variable degrees of expansion. The frequency of activated SARS-CoV-2-specific CD8+ T cells at baseline and peak inversely correlated with peak SARS-CoV-2 RNA levels in nasal swabs and accelerated viral clearance. Our study demonstrates that a rapid and extensive recall of memory T cell populations occurs early after breakthrough infection and suggests that CD8+ T cells contribute to the control of viral replication in breakthrough SARS-CoV-2 infections.

Immunization with inactivated whole virus particle influenza virus vaccines improves the humoral response landscape in cynomolgus macaques.

Although antibody-inducing split virus vaccines (SV) are currently the most effective way to combat seasonal influenza, their efficacy can be modest, especially in immunologically-naïve individuals. We investigated immune responses towards inactivated whole influenza virus particle vaccine (WPV) formulations, predicated to be more immunogenic, in a non-human primate model, as an important step towards clinical testing in humans. Comprehensive analyses were used to capture 46 immune parameters to profile how WPV-induced responses differed to those elicited by antigenically-similar SV formulations. Naïve cynomolgus macaques vaccinated with either monovalent or quadrivalent WPV consistently induced stronger antibody responses and hemagglutination inhibition (HI) antibody titres against vaccine-matched viruses compared to SV formulations, while acute reactogenic effects were similar. Responses in WPV-primed animals were further increased by boosting with the same formulation, conversely to modest responses after priming and boosting with SV. 28-parameter multiplex bead array defined key antibody features and showed that while both WPV and SV induced elevated IgG responses against A/H1N1 nucleoprotein, only WPV increased IgG responses against A/H1N1 hemagglutinin (HA) and HA-Stem, and higher IgA responses to A/H1N1-HA after each vaccine dose. Antibodies to A/H1N1-HA and HA-Stem that could engage FcγR2a and FcγR3a were also present at higher levels after one dose of WPV compared to SV and remained elevated after the second dose. Furthermore, WPV-enhanced antibody responses were associated with higher frequencies of HA-specific B-cells and IFN-γ-producing CD4+ T-cell responses. Our data additionally demonstrate stronger boosting of HI titres by WPV following prior infection and support WPV administered as a priming dose irrespective of the follow up vaccine for the second dose. Our findings thus show that compared to SV vaccination, WPV-induced humoral responses are significantly increased in scope and magnitude, advocating WPV vaccination regimens for priming immunologically-naïve individuals and also in the event of a pandemic outbreak.

The magnitude and timing of recalled immunity after breakthrough infection is shaped by SARS-CoV-2 variants.

Vaccination against SARS-CoV-2 protects from infection and improves clinical outcomes in breakthrough infections, likely reflecting residual vaccine-elicited immunity and recall of immunological memory. Here, we define the early kinetics of spike-specific humoral and cellular immunity after vaccination of seropositive individuals and after Delta or Omicron breakthrough infection in vaccinated individuals. Early longitudinal sampling revealed the timing and magnitude of recall, with the phenotypic activation of B cells preceding an increase in neutralizing antibody titers. While vaccination of seropositive individuals resulted in robust recall of humoral and T cell immunity, recall of vaccine-elicited responses was delayed and variable in magnitude during breakthrough infections and depended on the infecting variant of concern. While the delayed kinetics of immune recall provides a potential mechanism for the lack of early control of viral replication, the recall of antibodies coincided with viral clearance and likely underpins the protective effects of vaccination against severe COVID-19.

Establishment and recall of SARS-CoV-2 spike epitope-specific CD4+ T cell memory.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination elicit CD4+ T cell responses to the spike protein, including circulating follicular helper T (cTFH) cells that correlate with neutralizing antibodies. Using a novel HLA-DRB1*15:01/S751 tetramer to track spike-specific CD4+ T cells, we show that primary infection or vaccination induces robust S751-specific CXCR5- and cTFH cell memory responses. Secondary exposure induced recall of CD4+ T cells with a transitory CXCR3+ phenotype, and drove expansion of cTFH cells transiently expressing ICOS, CD38 and PD-1. In both contexts, cells exhibited a restricted T cell antigen receptor repertoire, including a highly public clonotype and considerable clonotypic overlap between CXCR5- and cTFH populations. Following a third vaccine dose, the rapid re-expansion of spike-specific CD4+ T cells contrasted with the comparatively delayed increase in antibody titers. Overall, we demonstrate that stable pools of cTFH and memory CD4+ T cells established by infection and/or vaccination are efficiently recalled upon antigen reexposure and may contribute to long-term protection against SARS-CoV-2.

Special Issue-Immunity to Influenza Viruses.

Influenza viruses remain a constant global threat with significant health and socioeconomic impact every year and have the potential to cause devastating pandemics [...].

Evaluation of Human Circulating T Follicular Helper Cells in Influenza- and SARS-CoV-2-Specific B Cell Immunity.

Generation of effective immune protection against viral infection and vaccination depends greatly on a successful engagement and stimulation of adaptive immune B cells and a specialized CD4+ T cell subset called T follicular helper cells (TFH cells). Since TFH cells primarily reside in lymphoid tissues, they can be challenging to study in human settings. However, a counterpart of these cells, circulating TFH (cTFH) cells, can be detected in peripheral blood. Assessment of cTFH cells serves as an informative marker of humoral responses following viral infection and vaccination and can be predictive of antibody titers. Here, we describe a comprehensive flow cytometry detection method for dissecting cTFH subsets and activation, together with the assessment of antibody-secreting cells (ASCs), from a small volume of human whole blood. This approach allows the investigation of cellular events that underpin successful immune responses following influenza and SARS-CoV-2 infection/vaccination in humans and is applicable to other viral disease settings.

T follicular helper cells in the humoral immune response to SARS-CoV-2 infection and vaccination.

Vaccination remains the most effective mechanism to reduce the impact of COVID-19. Induction of neutralizing antibodies is a strong correlate of protection from infection and severe disease. An understanding of the cellular events that underpin the generation of effective neutralizing antibodies is therefore key to the development of efficacious vaccines that target emerging variants of concern. Analysis of the immune response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and vaccination has identified circulating T follicular helper cells (cTFH ) as a robust correlate of the neutralizing antibody response. Here, we discuss the analysis of cTFH cells and their lymphoid counterparts in human humoral immune responses during COVID-19, and in response to vaccination with SARS-CoV-2 spike. We discuss the phenotypic heterogeneity of cTFH cells and the utility of cTFH subsets as informative biomarkers for development of humoral immunity. We posit that the analysis of the most effective cTFH will be critical to inducing durable immunity to new variants of SARS-CoV-2.

SARS-CoV-2-specific CD8+ T-cell responses and TCR signatures in the context of a prominent HLA-A*24:02 allomorph.

In-depth understanding of human T-cell-mediated immunity in coronavirus disease 2019 (COVID-19) is needed if we are to optimize vaccine strategies and immunotherapies. Identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T-cell epitopes and generation of peptide-human leukocyte antigen (peptide-HLA) tetramers facilitate direct ex vivo analyses of SARS-CoV-2-specific T cells and their T-cell receptor (TCR) repertoires. We utilized a combination of peptide prediction and in vitro peptide stimulation to validate novel SARS-CoV-2 epitopes restricted by HLA-A*24:02, one of the most prominent HLA class I alleles, especially in Indigenous and Asian populations. Of the peptides screened, three spike-derived peptides generated CD8+ IFNγ+ responses above background, S1208-1216 (QYIKWPWYI), S448-456 (NYNYLYRLF) and S193-201 (VFKNIDGYF), with S1208 generating immunodominant CD8+ IFNγ+ responses. Using peptide-HLA-I tetramers, we performed direct ex vivo tetramer enrichment for HLA-A*24:02-restricted CD8+ T cells in COVID-19 patients and prepandemic controls. The precursor frequencies for HLA-A*24:02-restricted epitopes were within the range previously observed for other SARS-CoV-2 epitopes for both COVID-19 patients and prepandemic individuals. Naïve A24/SARS-CoV-2-specific CD8+ T cells increased nearly 7.5-fold above the average precursor frequency during COVID-19, gaining effector and memory phenotypes. Ex vivo single-cell analyses of TCRαß repertoires found that the A24/S448+ CD8+ T-cell TCRαß repertoire was driven by a common TCRß chain motif, whereas the A24/S1208+ CD8+ TCRαß repertoire was diverse across COVID-19 patients. Our study provides an in depth characterization and important insights into SARS-CoV-2-specific CD8+ T-cell responses associated with a prominent HLA-A*24:02 allomorph. This contributes to our knowledge on adaptive immune responses during primary COVID-19 and could be exploited in vaccine or immunotherapeutic approaches.

Systems serology detects functionally distinct coronavirus antibody features in children and elderly.

The hallmarks of COVID-19 are higher pathogenicity and mortality in the elderly compared to children. Examining baseline SARS-CoV-2 cross-reactive immunological responses, induced by circulating human coronaviruses (hCoVs), is needed to understand such divergent clinical outcomes. Here we show analysis of coronavirus antibody responses of pre-pandemic healthy children (n = 89), adults (n = 98), elderly (n = 57), and COVID-19 patients (n = 50) by systems serology. Moderate levels of cross-reactive, but non-neutralizing, SARS-CoV-2 antibodies are detected in pre-pandemic healthy individuals. SARS-CoV-2 antigen-specific Fcγ receptor binding accurately distinguishes COVID-19 patients from healthy individuals, suggesting that SARS-CoV-2 infection induces qualitative changes to antibody Fc, enhancing Fcγ receptor engagement. Higher cross-reactive SARS-CoV-2 IgA and IgG are observed in healthy elderly, while healthy children display elevated SARS-CoV-2 IgM, suggesting that children have fewer hCoV exposures, resulting in less-experienced but more polyreactive humoral immunity. Age-dependent analysis of COVID-19 patients, confirms elevated class-switched antibodies in elderly, while children have stronger Fc responses which we demonstrate are functionally different. These insights will inform COVID-19 vaccination strategies, improved serological diagnostics and therapeutics.

Robust correlations across six SARS-CoV-2 serology assays detecting distinct antibody features.

OBJECTIVES: As the world transitions into a new era of the COVID-19 pandemic in which vaccines become available, there is an increasing demand for rapid reliable serological testing to identify individuals with levels of immunity considered protective by infection or vaccination. METHODS: We used 34 SARS-CoV-2 samples to perform a rapid surrogate virus neutralisation test (sVNT), applicable to many laboratories as it circumvents the need for biosafety level-3 containment. We correlated results from the sVNT with five additional commonly used SARS-CoV-2 serology techniques: the microneutralisation test (MNT), in-house ELISAs, commercial Euroimmun- and Wantai-based ELISAs (RBD, spike and nucleoprotein; IgG, IgA and IgM), antigen-binding avidity, and high-throughput multiplex analyses to profile isotype, subclass and Fc effector binding potential. We correlated antibody levels with antibody-secreting cell (ASC) and circulatory T follicular helper (cTfh) cell numbers. RESULTS: Antibody data obtained with commercial ELISAs closely reflected results using in-house ELISAs against RBD and spike. A correlation matrix across ten measured ELISA parameters revealed positive correlations for all factors. The frequency of inhibition by rapid sVNT strongly correlated with spike-specific IgG and IgA titres detected by both commercial and in-house ELISAs, and MNT titres. Multiplex analyses revealed strongest correlations between IgG, IgG1, FcR and C1q specific to spike and RBD. Acute cTfh-type 1 cell numbers correlated with spike and RBD-specific IgG antibodies measured by ELISAs and sVNT. CONCLUSION: Our comprehensive analyses provide important insights into SARS-CoV-2 humoral immunity across distinct serology assays and their applicability for specific research and/or diagnostic questions to assess SARS-CoV-2-specific humoral responses.

Integrated immune dynamics define correlates of COVID-19 severity and antibody responses.

SARS-CoV-2 causes a spectrum of COVID-19 disease, the immunological basis of which remains ill defined. We analyzed 85 SARS-CoV-2-infected individuals at acute and/or convalescent time points, up to 102 days after symptom onset, quantifying 184 immunological parameters. Acute COVID-19 presented with high levels of IL-6, IL-18, and IL-10 and broad activation marked by the upregulation of CD38 on innate and adaptive lymphocytes and myeloid cells. Importantly, activated CXCR3+cTFH1 cells in acute COVID-19 significantly correlate with and predict antibody levels and their avidity at convalescence as well as acute neutralization activity. Strikingly, intensive care unit (ICU) patients with severe COVID-19 display higher levels of soluble IL-6, IL-6R, and IL-18, and hyperactivation of innate, adaptive, and myeloid compartments than patients with moderate disease. Our analyses provide a comprehensive map of longitudinal immunological responses in COVID-19 patients and integrate key cellular pathways of complex immune networks underpinning severe COVID-19, providing important insights into potential biomarkers and immunotherapies.

Suboptimal SARS-CoV-2-specific CD8+ T cell response associated with the prominent HLA-A*02:01 phenotype.

An improved understanding of human T cell-mediated immunity in COVID-19 is important for optimizing therapeutic and vaccine strategies. Experience with influenza shows that infection primes CD8+ T cell memory to peptides presented by common HLA types like HLA-A2, which enhances recovery and diminishes clinical severity upon reinfection. Stimulating peripheral blood mononuclear cells from COVID-19 convalescent patients with overlapping peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the clonal expansion of SARS-CoV-2-specific CD8+ and CD4+ T cells in vitro, with CD4+ T cells being robust. We identified two HLA-A*02:01-restricted SARS-CoV-2-specfic CD8+ T cell epitopes, A2/S269-277 and A2/Orf1ab3183-3191 Using peptide-HLA tetramer enrichment, direct ex vivo assessment of A2/S269+CD8+ and A2/Orf1ab3183+CD8+ populations indicated that A2/S269+CD8+ T cells were detected at comparable frequencies (∼1.3 × 10-5) in acute and convalescent HLA-A*02:01+ patients. These frequencies were higher than those found in uninfected HLA-A*02:01+ donors (∼2.5 × 10-6), but low when compared to frequencies for influenza-specific (A2/M158) and Epstein-Barr virus (EBV)-specific (A2/BMLF1280) (∼1.38 × 10-4) populations. Phenotyping A2/S269+CD8+ T cells from COVID-19 convalescents ex vivo showed that A2/S269+CD8+ T cells were predominantly negative for CD38, HLA-DR, PD-1, and CD71 activation markers, although the majority of total CD8+ T cells expressed granzymes and/or perforin. Furthermore, the bias toward naïve, stem cell memory and central memory A2/S269+CD8+ T cells rather than effector memory populations suggests that SARS-CoV-2 infection may be compromising CD8+ T cell activation. Priming with appropriate vaccines may thus be beneficial for optimizing CD8+ T cell immunity in COVID-19.