Robust and prototypical immune responses toward COVID-19 vaccine in First Nations peoples are impacted by comorbidities.

High-risk groups, including Indigenous people, are at risk of severe COVID-19. Here we found that Australian First Nations peoples elicit effective immune responses to COVID-19 BNT162b2 vaccination, including neutralizing antibodies, receptor-binding domain (RBD) antibodies, SARS-CoV-2 spike-specific B cells, and CD4+ and CD8+ T cells. In First Nations participants, RBD IgG antibody titers were correlated with body mass index and negatively correlated with age. Reduced RBD antibodies, spike-specific B cells and follicular helper T cells were found in vaccinated participants with chronic conditions (diabetes, renal disease) and were strongly associated with altered glycosylation of IgG and increased interleukin-18 levels in the plasma. These immune perturbations were also found in non-Indigenous people with comorbidities, indicating that they were related to comorbidities rather than ethnicity. However, our study is of a great importance to First Nations peoples who have disproportionate rates of chronic comorbidities and provides evidence of robust immune responses after COVID-19 vaccination in Indigenous people.

Robust SARS-CoV-2 T cell responses with common TCRαß motifs toward COVID-19 vaccines in patients with hematological malignancy impacting B cells.

Immunocompromised hematology patients are vulnerable to severe COVID-19 and respond poorly to vaccination. Relative deficits in immunity are, however, unclear, especially after 3 vaccine doses. We evaluated immune responses in hematology patients across three COVID-19 vaccination doses. Seropositivity was low after a first dose of BNT162b2 and ChAdOx1 (∼26%), increased to 59%-75% after a second dose, and increased to 85% after a third dose. While prototypical antibody-secreting cells (ASCs) and T follicular helper (Tfh) cell responses were elicited in healthy participants, hematology patients showed prolonged ASCs and skewed Tfh2/17 responses. Importantly, vaccine-induced expansions of spike-specific and peptide-HLA tetramer-specific CD4+/CD8+ T cells, together with their T cell receptor (TCR) repertoires, were robust in hematology patients, irrespective of B cell numbers, and comparable to healthy participants. Vaccinated patients with breakthrough infections developed higher antibody responses, while T cell responses were comparable to healthy groups. COVID-19 vaccination induces robust T cell immunity in hematology patients of varying diseases and treatments irrespective of B cell numbers and antibody response.

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.

Prospective comprehensive profiling of immune responses to COVID-19 vaccination in patients on zanubrutinib therapy.

Zanubrutinib-treated and treatment-naïve patients with chronic lymphocytic leukaemia (CLL) or Waldenstrom's macroglobulinaemia were recruited in this prospective study to comprehensively profile humoral and cellular immune responses to COVID-19 vaccination. Overall, 45 patients (median 72 years old) were recruited; the majority were male (71%), had CLL (76%) and were on zanubrutinib (78%). Seroconversion rates were 65% and 77% following two and three doses, respectively. CD4+ and CD8+ T-cell response rates increased with third dose. In zanubrutinib-treated patients, 86% developed either a humoral or cellular response. Patients on zanubrutinib developed substantial immune responses following two COVID-19 vaccine doses, which further improved following a third dose.

Circulating effector γδ T cell populations are associated with acute coronavirus disease 19 in unvaccinated individuals.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes severe coronavirus disease 2019 (COVID-19) in a small proportion of infected individuals. The immune system plays an important role in the defense against SARS-CoV-2, but our understanding of the cellular immune parameters that contribute to severe COVID-19 disease is incomplete. Here, we show that populations of effector γδ T cells are associated with COVID-19 in unvaccinated patients with acute disease. We found that circulating CD27neg CD45RA+ CX3CR1+ Vδ1effector cells expressing Granzymes (Gzms) were enriched in COVID-19 patients with acute disease. Moreover, higher frequencies of GzmB+ Vδ2+ T cells were observed in acute COVID-19 patients. SARS-CoV-2 infection did not alter the γδ T cell receptor repertoire of either Vδ1+ or Vδ2+ subsets. Our work demonstrates an association between effector populations of γδ T cells and acute COVID-19 in unvaccinated individuals.

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.

SARS-CoV-2-specific T cell memory with common TCRαß motifs is established in unvaccinated children who seroconvert after infection.

As the establishment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell memory in children remains largely unexplored, we recruited convalescent COVID-19 children and adults to define their circulating memory SARS-CoV-2-specific CD4+ and CD8+ T cells prior to vaccination. We analyzed epitope-specific T cells directly ex vivo using seven HLA class I and class II tetramers presenting SARS-CoV-2 epitopes, together with Spike-specific B cells. Unvaccinated children who seroconverted had comparable Spike-specific but lower ORF1a- and N-specific memory T cell responses compared with adults. This agreed with our TCR sequencing data showing reduced clonal expansion in children. A strong stem cell memory phenotype and common T cell receptor motifs were detected within tetramer-specific T cells in seroconverted children. Conversely, children who did not seroconvert had tetramer-specific T cells of predominantly naive phenotypes and diverse TCRαß repertoires. Our study demonstrates the generation of SARS-CoV-2-specific T cell memory with common TCRαß motifs in unvaccinated seroconverted children after their first virus encounter.

T Cells Targeting SARS-CoV-2: By Infection, Vaccination, and Against Future Variants.

T cell responses are a key cornerstone to viral immunity to drive high-quality antibody responses, establishing memory for recall and for viral clearance. Inefficient recruitment of T cell responses plays a role in the development of severe COVID-19 and is also represented by reduced cellular responses in men, children, and diversity compared with other epitope-specific subsets and available T cell receptor diversity. SARS-CoV-2-specific T cell responses are elicited by multiple vaccine formats and augmented by prior infection for hybrid immunity. Epitope conservation is relatively well-maintained leading to T cell crossreactivity for variants of concern that have diminished serological responses.

Anti-PEG Antibodies Boosted in Humans by SARS-CoV-2 Lipid Nanoparticle mRNA Vaccine.

Humans commonly have low level antibodies to poly(ethylene) glycol (PEG) due to environmental exposure. Lipid nanoparticle (LNP) mRNA vaccines for SARS-CoV-2 contain small amounts of PEG, but it is not known whether PEG antibodies are enhanced by vaccination and what their impact is on particle-immune cell interactions in human blood. We studied plasma from 130 adults receiving either the BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) mRNA vaccines or no SARS-CoV-2 vaccine for PEG-specific antibodies. Anti-PEG IgG was commonly detected prior to vaccination and was significantly boosted a mean of 13.1-fold (range 1.0-70.9) following mRNA-1273 vaccination and a mean of 1.78-fold (range 0.68-16.6) following BNT162b2 vaccination. Anti-PEG IgM increased 68.5-fold (range 0.9-377.1) and 2.64-fold (0.76-12.84) following mRNA-1273 and BNT162b2 vaccination, respectively. The rise in PEG-specific antibodies following mRNA-1273 vaccination was associated with a significant increase in the association of clinically relevant PEGylated LNPs with blood phagocytes ex vivo. PEG antibodies did not impact the SARS-CoV-2 specific neutralizing antibody response to vaccination. However, the elevated levels of vaccine-induced anti-PEG antibodies correlated with increased systemic reactogenicity following two doses of vaccination. We conclude that PEG-specific antibodies can be boosted by LNP mRNA vaccination and that the rise in PEG-specific antibodies is associated with systemic reactogenicity and an increase of PEG particle-leukocyte association in human blood. The longer-term clinical impact of the increase in PEG-specific antibodies induced by lipid nanoparticle mRNA vaccines should be monitored. It may be useful to identify suitable alternatives to PEG for developing next-generation LNP vaccines to overcome PEG immunogenicity in the future.

SARS-CoV-2 infection results in immune responses in the respiratory tract and peripheral blood that suggest mechanisms of disease severity.

Respiratory tract infection with SARS-CoV-2 results in varying immunopathology underlying COVID-19. We examine cellular, humoral and cytokine responses covering 382 immune components in longitudinal blood and respiratory samples from hospitalized COVID-19 patients. SARS-CoV-2-specific IgM, IgG, IgA are detected in respiratory tract and blood, however, receptor-binding domain (RBD)-specific IgM and IgG seroconversion is enhanced in respiratory specimens. SARS-CoV-2 neutralization activity in respiratory samples correlates with RBD-specific IgM and IgG levels. Cytokines/chemokines vary between respiratory samples and plasma, indicating that inflammation should be assessed in respiratory specimens to understand immunopathology. IFN-α2 and IL-12p70 in endotracheal aspirate and neutralization in sputum negatively correlate with duration of hospital stay. Diverse immune subsets are detected in respiratory samples, dominated by neutrophils. Importantly, dexamethasone treatment does not affect humoral responses in blood of COVID-19 patients. Our study unveils differential immune responses between respiratory samples and blood, and shows how drug therapy affects immune responses during COVID-19.

T Cell Epitope Discovery in the Context of Distinct and Unique Indigenous HLA Profiles.

CD8+ T cells are a pivotal part of the immune response to viruses, playing a key role in disease outcome and providing long-lasting immunity to conserved pathogen epitopes. Understanding CD8+ T cell immunity in humans is complex due to CD8+ T cell restriction by highly polymorphic Human Leukocyte Antigen (HLA) proteins, requiring T cell epitopes to be defined for different HLA allotypes across different ethnicities. Here we evaluate strategies that have been developed to facilitate epitope identification and study immunogenic T cell responses. We describe an immunopeptidomics approach to sequence HLA-bound peptides presented on virus-infected cells by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Using antigen presenting cell lines that stably express the HLA alleles characteristic of Indigenous Australians, this approach has been successfully used to comprehensively identify influenza-specific CD8+ T cell epitopes restricted by HLA allotypes predominant in Indigenous Australians, including HLA-A*24:02 and HLA-A*11:01. This is an essential step in ensuring high vaccine coverage and efficacy in Indigenous populations globally, known to be at high risk from influenza disease and other respiratory infections.

SARS-CoV-2 mRNA vaccination elicits a robust and persistent T follicular helper cell response in humans.

SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine-needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we evaluated the T cell receptor sequences and phenotype of lymph node TFH. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLA-DPB1∗04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.

A point-of-care lateral flow assay for neutralising antibodies against SARS-CoV-2.

BACKGROUND: As vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus, whether from infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure antibody-based protection to SARS-CoV-2, ideally with rapid turnaround and without the need for laboratory-based testing. METHODS: We have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibody (NAb) from whole blood, with a result that can be determined by eye or quantitatively on a small instrument. We compared our lateral flow test with the gold-standard microneutralisation assay, using samples from convalescent and vaccinated donors, as well as immunised macaques. FINDINGS: We show a high correlation between our lateral flow test with conventional neutralisation and that this test is applicable with animal samples. We also show that this assay is readily adaptable to test for protection to newly emerging SARS-CoV-2 variants, including the beta variant which revealed a marked reduction in NAb activity. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (specificity 100% and sensitivity 96% at a microneutralisation cutoff of 1:40) and that fingerprick whole blood samples are sufficient for this test. INTERPRETATION: Taken together, the COVID-19 NAb-testTM device described here provides a rapid readout of NAb based protection to SARS-CoV-2 at the point of care. FUNDING: Support was received from the Victorian Operational Infrastructure Support Program and the Australian Government Department of Health. This work was supported by grants from the Department of Health and Human Services of the Victorian State Government; the ARC (CE140100011, CE140100036), the NHMRC (1113293, 2002317 and 1116530), and Medical Research Future Fund Awards (2005544, 2002073, 2002132). Individual researchers were supported by an NHMRC Emerging Leadership Level 1 Investigator Grants (1194036), NHMRC APPRISE Research Fellowship (1116530), NHMRC Leadership Investigator Grant (1173871), NHMRC Principal Research Fellowship (1137285), NHMRC Investigator Grants (1177174 and 1174555) and NHMRC Senior Principal Research Fellowships (1117766 and 1136322). Grateful support was also received from the A2 Milk Company and the Jack Ma Foundation.

Defective Severe Acute Respiratory Syndrome Coronavirus 2 Immune Responses in an Immunocompromised Individual With Prolonged Viral Replication.

We describe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immune responses in a patient with lymphoma and recent programmed death 1 (PD-1) inhibitor therapy with late onset of severe coronavirus disease 2019 disease and prolonged SARS-CoV-2 replication, in comparison to age-matched and immunocompromised controls. High levels of HLA-DR+/CD38+ activation, interleukin 6, and interleukin 18 in the absence of B cells and PD-1 expression was observed. SARS-CoV-2-specific antibody responses were absent and SARS-CoV-2-specific T cells were minimally detected. This case highlights challenges in managing immunocompromised hosts who may fail to mount effective virus-specific immune responses.

Structural basis of biased T cell receptor recognition of an immunodominant HLA-A2 epitope of the SARS-CoV-2 spike protein.

CD8+ T cells play an important role in vaccination and immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although numerous SARS-CoV-2 CD8+ T cell epitopes have been identified, the molecular basis underpinning T cell receptor (TCR) recognition of SARS-CoV-2-specific T cells remains unknown. The T cell response directed toward SARS-CoV-2 spike protein-derived S269-277 peptide presented by the human leukocyte antigen (HLA)-A∗02:01 allomorph (hereafter the HLA-A2S269-277 epitope) is, to date, the most immunodominant SARS-CoV-2 epitope found in individuals bearing this allele. As HLA-A2S269-277-specific CD8+ T cells utilize biased TRAV12 gene usage within the TCR α-chain, we sought to understand the molecular basis underpinning this TRAV12 dominance. We expressed four TRAV12+ TCRs which bound the HLA-A2S269-277 complex with low micromolar affinity and determined the crystal structure of the HLA-A2S269-277 binary complex, and subsequently a ternary structure of the TRAV12+ TCR complexed to HLA-A2S269-277. We found that the TCR made extensive contacts along the entire length of the S269-277 peptide, suggesting that the TRAV12+ TCRs would be sensitive to sequence variation within this epitope. To examine this, we investigated cross-reactivity toward analogous peptides from existing SARS-CoV-2 variants and closely related coronaviruses. We show via surface plasmon resonance and tetramer studies that the TRAV12+ T cell repertoire cross-reacts poorly with these analogous epitopes. Overall, we defined the structural basis underpinning biased TCR recognition of CD8+ T cells directed at an immunodominant epitope and provide a framework for understanding TCR cross-reactivity toward viral variants within the S269-277 peptide.

Altered microRNA expression in COVID-19 patients enables identification of SARS-CoV-2 infection.

The host response to SARS-CoV-2 infection provide insights into both viral pathogenesis and patient management. The host-encoded microRNA (miRNA) response to SARS-CoV-2 infection, however, remains poorly defined. Here we profiled circulating miRNAs from ten COVID-19 patients sampled longitudinally and ten age and gender matched healthy donors. We observed 55 miRNAs that were altered in COVID-19 patients during early-stage disease, with the inflammatory miR-31-5p the most strongly upregulated. Supervised machine learning analysis revealed that a three-miRNA signature (miR-423-5p, miR-23a-3p and miR-195-5p) independently classified COVID-19 cases with an accuracy of 99.9%. In a ferret COVID-19 model, the three-miRNA signature again detected SARS-CoV-2 infection with 99.7% accuracy, and distinguished SARS-CoV-2 infection from influenza A (H1N1) infection and healthy controls with 95% accuracy. Distinct miRNA profiles were also observed in COVID-19 patients requiring oxygenation. This study demonstrates that SARS-CoV-2 infection induces a robust host miRNA response that could improve COVID-19 detection and patient management.

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.