Inducible Bronchus-Associated Lymphoid Tissues (iBALT) Serve as Sites of B Cell Selection and Maturation Following Influenza Infection in Mice

Seasonally recurrent influenza virus infections are a significant cause of global morbidity and mortality. In murine models, primary influenza infection in the respiratory tract elicits potent humoral responses concentrated in the draining mediastinal lymph node and the spleen. In addition to immunity within secondary lymphoid organs (SLO), pulmonary infection is also associated with formation of ectopic inducible bronchus-associated tissues (iBALT) in the lung. These structures display a lymphoid organization, but their function and protective benefits remain unclear. Here we examined the phenotype, transcriptional profile and antigen specificity of B cell populations forming iBALT in influenza infected mice. We show that the cellular composition of iBALT was comparable to SLO, containing populations of follicular dendritic cells (FDC), T-follicular helper (Tfh) cells, and germinal center (GC)-like B cells with classical dark- and light-zone polarization. Transcriptional profiles of GC B cells in iBALT and SLO were conserved regardless of anatomical localization. The architecture of iBALT was pleiomorphic and less structurally defined than SLO. Nevertheless, we show that GC-like structures within iBALT serve as a distinct niche that independently support the maturation and selection of B cells primarily targeted against the influenza virus nucleoprotein. Our findings suggest that iBALT, which are positioned at the frontline of the lung mucosa, drive long-lived, and unique GC reactions that contribute to the diversity of the humoral response targeting influenza

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

Broad spectrum SARS‐CoV ‐2‐specific immunity in hospitalized First Nations peoples recovering from COVID ‐19

Indigenous peoples globally are at increased risk of COVID‐19‐associated morbidity and mortality. However, data that describe immune responses to SARS‐CoV‐2 infection in Indigenous populations are lacking. We evaluated immune responses in Australian First Nations peoples hospitalized with COVID‐19. Our work comprehensively mapped out inflammatory, humoral and adaptive immune responses following SARS‐CoV‐2 infection. Patients were recruited early following the lifting of strict public health measures in the Northern Territory, Australia, between November 2021 and May 2022. Australian First Nations peoples recovering from COVID‐19 showed increased levels of MCP‐1 and IL‐8 cytokines, IgG‐antibodies against Delta‐RBD and memory SARS‐CoV‐2‐specific T cell responses prior to hospital discharge in comparison with hospital admission, with resolution of hyperactivated HLA‐DR+CD38+ T cells. SARS‐CoV‐2 infection elicited coordinated ASC, Tfh and CD8+ T cell responses in concert with CD4+ T cell responses. Delta and Omicron RBD‐IgG, as well as Ancestral N‐IgG antibodies, strongly correlated with Ancestral RBD‐IgG antibodies and Spike‐specific memory B cells. We provide evidence of broad and robust immune responses following SARS‐CoV‐2 infection in Indigenous peoples, resembling those of non‐Indigenous COVID‐19 hospitalized patients

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

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

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

As establishment of 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 analysed 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 to 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 naïve phenotypes and diverse TCRαβ repertoires. Our study demonstrates generation of SARS-CoV-2-specific T cell memory with common TCRαβ motifs in unvaccinated seroconverted children after their first virus encounter

CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8+ T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8+ T cells directed toward subdominant epitopes (B7/N257, A2/S269, and A24/S1,208) CD8+ T cells specific for the immunodominant B7/N105 epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8+ T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαβ repertoires and promiscuous αβ-TCR pairing within B7/N105+CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαβ diversity within immunodominant B7/N105-specific CD8+ T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses

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 TCR?? 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

Recombinant HIV vaccines and mucosal immunity

© 2016 Dr. Hyon Xhi TanMore than 36 million people were infected with the human immunodeficiency virus (HIV)-1 in 2015, and 17 million of this population were females. Female HIV infection has wider implications on mother-to-child transmission of HIV and the survival of children in the event of their mother’s death from AIDS. This highlights a critical need for vaccines that prevent female acquisition of HIV infection. In female sexual transmission of HIV, infection of target CD4 T-cells is highly localised in the sub-mucosal cervico-vaginal tissues for several days before systemic dissemination, and displays little or no viral diversity. Cytotoxic CD8 T-lymphocyte (CTL) responses have been correlated with effective control of acute viraemia in HIV-1 infection of humans or simian immunodeficiency virus (SIV) infection in non-human primate model. Elimination of HIV infection by CTL responses is hypothesised to be most effective during this early phase of contained infection in the cervico-vaginal tissue. In this Ph.D. thesis, vaccination strategies for the induction of tissue-resident HIV-specific CTLs in the female reproductive mucosal tissues were investigated. This thesis demonstrated that influenza virus could be genetically engineered to induce intracellular expression of whole HIV-1 p24 capsid protein. The influenza vectors developed were immunogenic for HIV-specific CD8 T-cell responses when used in prime-boost regimens. We exploited the capacity of these recombinant influenza viruses to infect mucosal tissues in order to establish HIV-specific CTL responses in the vagina mucosa. The mucosal vaccination strategies proposed here provide novel approaches for the establishment of frontline vaginal-resident HIV- specific CTLs and may induce heightened local surveillance for protection against vaginal HIV-1 exposure