The absence of preexisting neutralizing antibodies specific for the novel A (H7N9) influenza virus indicates a lack of prior human exposure. As influenza A virus–specific CD8 ⁺ T lymphocytes (CTLs) ...can be broadly cross-reactive, we tested whether immunogenic peptides derived from H7N9 might be recognized by memory CTLs established following infection with other influenza strains. Probing across multiple ethnicities, we identified 32 conserved epitopes derived from the nucleoprotein (NP) and matrix-1 (M1) proteins. These NP and M1 peptides are presented by HLAs prevalent in 16–57% of individuals. Remarkably, some HLA alleles (A*0201, A*0301, B*5701 , B*1801, and B*0801) elicit robust CTL responses against any human influenza A virus, including H7N9, whereas ethnicities where HLA-A*0101, A*6801, B*1501, and A*2402 are prominent, show limited CTL response profiles. By this criterion, some groups, especially the Alaskan and Australian Indigenous peoples, would be particularly vulnerable to H7N9 infection. This dissection of CTL-mediated immunity to H7N9 thus suggests strategies for both vaccine delivery and development.
The diversity of circulating human B cells is unknown. We use single-cell RNA sequencing (RNA-seq) to examine the diversity of both antigen-specific and total B cells in healthy subjects and ...malaria-exposed individuals. This reveals two B cell lineages: a classical lineage of activated and resting memory B cells and an alternative lineage, which includes previously described atypical B cells. Although atypical B cells have previously been associated with disease states, the alternative lineage is common in healthy controls, as well as malaria-exposed individuals. We further track Plasmodium-specific B cells after malaria vaccination in naive volunteers. We find that alternative lineage cells are primed after the initial immunization and respond to booster doses. However, alternative lineage cells develop an atypical phenotype with repeated boosts. The data highlight that atypical cells are part of a wider alternative lineage of B cells that are a normal component of healthy immune responses.
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•Single-cell RNA-seq reveals two distinct B cell lineages•An alternative lineage contains CXCR3+ and atypical B cells•Alternative B cells are primed after primary vaccination and respond to boosters•Alternative B cells adopt a more atypical phenotype following repeated antigen exposure
Using single-cell RNA sequencing, Sutton et al. show that a population of “atypical” B cells, normally associated with disease, are part of a wider landscape of alternative B cells that participate in normal responses to vaccination.
CD8
T cells recognizing antigenic peptides derived from conserved internal viral proteins confer broad protection against distinct influenza viruses. As memory CD8
T cells change throughout the human ...lifetime and across tissue compartments, we investigated how T cell receptor (TCR) composition and diversity relate to memory CD8
T cells across anatomical sites and immunological phases of human life. We used
peptide-HLA tetramer magnetic enrichment, single-cell multiplex RT-PCR for both the TCR-alpha (TCRα) and TCR-beta (TCRβ) chains, and new TCRdist and grouping of lymphocyte interactions by paratope hotspots (GLIPH) algorithms to compare TCRs directed against the most prominent human influenza epitope, HLA-A*02:01-M1
(A2
M1
). We dissected memory TCR repertoires directed toward A2
M1
CD8
T cells within human tissues and compared them to human peripheral blood of young and elderly adults. Furthermore, we compared these memory CD8
T cell repertoires to A2
M1
CD8
TCRs during acute influenza disease in patients hospitalized with avian A/H7N9 virus. Our study provides the first
comparative analysis of paired antigen-specific TCR-α/β clonotypes across different tissues and peripheral blood across different age groups. We show that human A2
M1
CD8
T cells can be readily detected in human lungs, spleens, and lymph nodes, and that tissue A2
M1
TCRαβ repertoires reflect A2
M1
TCRαβ clonotypes derived from peripheral blood in healthy adults and influenza-infected patients. A2
M1
TCRαβ repertoires displayed distinct features only in elderly adults, with large private TCRαβ clonotypes replacing the prominent and public TRBV19/TRAV27 TCRs. Our study provides novel findings on influenza-specific TCRαβ repertoires within human tissues, raises the question of how we can prevent the loss of optimal TCRαβ signatures with aging, and provides important insights into the rational design of T cell-mediated vaccines and immunotherapies.
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 TCRαβ repertoires found that the A24/S448+CD8+ T‐cell TCRαβ repertoire was driven by a common TCRβ chain motif, whereas the A24/S1208+CD8+ TCRαβ 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.
Analysis of unmanipulated SARS‐CoV‐2‐specific CD8+ T cells was performed by direct ex vivo tetramer staining in COVID‐19 patients. We investigated in‐depth SARS‐CoV‐2‐specific CD8+ T‐cell responses and their T‐cell receptor (TCR) signatures restricted by a prominent HLA‐A*24:02 allomorph.
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.
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•Breakthrough infection elicits a rapid recall of CD4+ and CD8+ spike-specific T cells•CD4+ T cell recall is robust and coordinated, peaking by day 5 post symptoms•CD8+ T cells become activated but variably expand following infection•Spike-specific CD8+ T cell activation inversely correlates with the rate of viral decay
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 a 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.
Memory CD8⁺ T lymphocytes (CTLs) specific for antigenic peptides derived from internal viral proteins confer broad protection against distinct strains of influenza A virus (IAV). However, immune ...efficacy can be undermined by the emergence of escape mutants. To determine how T-cell receptor (TCR) composition relates to IAV epitope variability, we used ex vivo peptide–HLA tetramer enrichment and single-cell multiplex analysis to compare TCRs targeted to the largely conserved HLA-A*0201-M158 and the hypervariable HLA-B*3501-NP418 antigens. The TCRαβs for HLA-B*3501-NP418⁺ CTLs varied among individuals and across IAV strains, indicating that a range of mutated peptides will prime different NP418-specific CTL sets. Conversely, a dominant public TRAV27/TRBV19⁺ TCRαβ was selected in HLA-A*0201⁺ donors responding to M158. This public TCR cross-recognized naturally occurring M158 variants complexed with HLA-A*0201. Ternary structures showed that induced-fit molecular mimicry underpins TRAV27/TRBV19⁺ TCR specificity for the WT and mutant M158 peptides, suggesting the possibility of universal CTL immunity in HLA-A*0201⁺ individuals. Combined with the high population frequency of HLA-A*0201, these data potentially explain the relative conservation of M158. Moreover, our results suggest that vaccination strategies aimed at generating broad protection should incorporate variant peptides to elicit cross-reactive responses against other specificities, especially those that may be relatively infrequent among IAV-primed memory CTLs.
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.
Given the scale of the ongoing COVID-19 pandemic, the development of vaccines based on different platforms is essential, particularly in light of emerging viral variants, the absence of information ...on vaccine-induced immune durability, and potential paediatric use. We aimed to assess the safety and immunogenicity of an MF59-adjuvanted subunit vaccine for COVID-19 based on recombinant SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a novel molecular clamp (spike glycoprotein-clamp sclamp).
We did a phase 1, double-blind, placebo-controlled, block-randomised trial of the sclamp subunit vaccine in a single clinical trial site in Brisbane, QLD, Australia. Healthy adults (aged ≥18 to ≤55 years) who had tested negative for SARS-CoV-2, reported no close contact with anyone with active or previous SARS-CoV-2 infection, and tested negative for pre-existing SARS-CoV-2 immunity were included. Participants were randomly assigned to one of five treatment groups and received two doses via intramuscular injection 28 days apart of either placebo, sclamp vaccine at 5 μg, 15 μg, or 45 μg, or one dose of sclamp vaccine at 45 μg followed by placebo. Participants and study personnel, except the dose administration personnel, were masked to treatment. The primary safety endpoints included solicited local and systemic adverse events in the 7 days after each dose and unsolicited adverse events up to 12 months after dosing. Here, data are reported up until day 57. Primary immunogenicity endpoints were antigen-specific IgG ELISA and SARS-CoV-2 microneutralisation assays assessed at 28 days after each dose. The study is ongoing and registered with ClinicalTrials.gov, NCT04495933.
Between June 23, 2020, and Aug 17, 2020, of 314 healthy volunteers screened, 120 were randomly assigned (n=24 per group), and 114 (95%) completed the study up to day 57 (mean age 32·5 years SD 10·4, 65 54% male, 55 46% female). Severe solicited reactions were infrequent and occurred at similar rates in participants receiving placebo (two 8% of 24) and the SARS-CoV-2 sclamp vaccine at any dose (three 3% of 96). Both solicited reactions and unsolicited adverse events occurred at a similar frequency in participants receiving placebo and the SARS-CoV-2 sclamp vaccine. Solicited reactions occurred in 19 (79%) of 24 participants receiving placebo and 86 (90%) of 96 receiving the SARS-CoV-2 sclamp vaccine at any dose. Unsolicited adverse events occurred in seven (29%) of 24 participants receiving placebo and 35 (36%) of 96 participants receiving the SARS-CoV-2 sclamp vaccine at any dose. Vaccination with SARS-CoV-2 sclamp elicited a similar antigen-specific response irrespective of dose: 4 weeks after the initial dose (day 29) with 5 μg dose (geometric mean titre GMT 6400, 95% CI 3683–11 122), with 15 μg dose (7492, 4959–11 319), and the two 45 μg dose cohorts (8770, 5526–13 920 in the two-dose 45 μg cohort; 8793, 5570–13 881 in the single-dose 45 μg cohort); 4 weeks after the second dose (day 57) with two 5 μg doses (102 400, 64 857–161 676), with two 15 μg doses (74 725, 51 300–108 847), with two 45 μg doses (79 586, 55 430–114 268), only a single 45 μg dose (4795, 2858–8043). At day 57, 67 (99%) of 68 participants who received two doses of sclamp vaccine at any concentration produced a neutralising immune response, compared with six (25%) of 24 who received a single 45 μg dose and none of 22 who received placebo. Participants receiving two doses of sclamp vaccine elicited similar neutralisation titres, irrespective of dose: two 5 μg doses (GMT 228, 95% CI 146–356), two 15 μg doses (230, 170–312), and two 45 μg doses (239, 187–307).
This first-in-human trial shows that a subunit vaccine comprising mammalian cell culture-derived, MF59-adjuvanted, molecular clamp-stabilised recombinant spike protein elicits strong immune responses with a promising safety profile. However, the glycoprotein 41 peptide present in the clamp created HIV diagnostic assay interference, a possible barrier to widespread use highlighting the criticality of potential non-spike directed immunogenicity during vaccine development. Studies are ongoing with alternative molecular clamp trimerisation domains to ameliorate this response.
Coalition for Epidemic Preparedness Innovations, National Health and Medical Research Council, Queensland Government, and further philanthropic sources listed in the acknowledgments.
Severe influenza A virus (IAV) infection is associated with immune dysfunction. Here, we show circulating CD8
T-cell profiles from patients hospitalized with avian H7N9, seasonal IAV, and influenza ...vaccinees. Patient survival reflects an early, transient prevalence of highly activated CD38
HLA-DR
PD-1
CD8
T cells, whereas the prolonged persistence of this set is found in ultimately fatal cases. Single-cell T cell receptor (TCR)-αβ analyses of activated CD38
HLA-DR
CD8
T cells show similar TCRαβ diversity but differential clonal expansion kinetics in surviving and fatal H7N9 patients. Delayed clonal expansion associated with an early dichotomy at a transcriptome level (as detected by single-cell RNAseq) is found in CD38
HLA-DR
CD8
T cells from patients who succumbed to the disease, suggesting a divergent differentiation pathway of CD38
HLA-DR
CD8
T cells from the outset during fatal disease. Our study proposes that effective expansion of cross-reactive influenza-specific TCRαβ clonotypes with appropriate transcriptome signatures is needed for early protection against severe influenza disease.
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