Plasma samples taken at different time points from donors who received either AstraZeneca (Vaxzevria) or Pfizer (Comirnaty) or Moderna (Spikevax) coronavirus disease-19 (COVID-19) vaccine were ...assessed in virus neutralization assays against Delta and Omicron variants of concern and a reference isolate (VIC31). With the Pfizer vaccine there was 6-8-fold reduction in 50% neutralizing antibody titres (NT
) against Delta and VIC31 at 6 months compared to 2 weeks after the second dose; followed by 25-fold increase at 2 weeks after the third dose. Neutralisation of Omicron was only consistently observed 2 weeks after the third dose, with most samples having titres below the limit of detection at earlier timepoints. Moderna results were similar to Pfizer at 2 weeks after the second dose, while the titres for AstraZeneca samples derived from older donors were 7-fold lower against VIC31 and below the limit of detection against Delta and Omicron. Age and gender were not found to significantly impact our results. These findings indicate that vaccine matching may be needed, and that at least a third dose of these vaccines is necessary to generate sufficient neutralising antibodies against emerging variants of concern, especially Omicron, amidst the challenges of ensuring vaccine equity worldwide.
Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that recognize small molecule metabolites presented by major histocompatibility complex class I related protein 1 ...(MR1), via an αβ T cell receptor (TCR). MAIT TCRs feature an essentially invariant TCR α-chain, which is highly conserved between mammals. Similarly, MR1 is the most highly conserved major histocompatibility complex-I–like molecule. This extreme conservation, including the mode of interaction between the MAIT TCR and MR1, has been shown to allow for species-mismatched reactivities unique in T cell biology, thereby allowing the use of selected species-mismatched MR1–antigen (MR1–Ag) tetramers in comparative immunology studies. However, the pattern of cross-reactivity of species-mismatched MR1–Ag tetramers in identifying MAIT cells in diverse species has not been formally assessed. We developed novel cattle and pig MR1–Ag tetramers and utilized these alongside previously developed human, mouse, and pig-tailed macaque MR1–Ag tetramers to characterize cross-species tetramer reactivities. MR1–Ag tetramers from each species identified T cell populations in distantly related species with specificity that was comparable to species-matched MR1–Ag tetramers. However, there were subtle differences in staining characteristics with practical implications for the accurate identification of MAIT cells. Pig MR1 is sufficiently conserved across species that pig MR1–Ag tetramers identified MAIT cells from the other species. However, MAIT cells in pigs were at the limits of phenotypic detection. In the absence of sheep MR1–Ag tetramers, a MAIT cell population in sheep blood was identified phenotypically, utilizing species-mismatched MR1–Ag tetramers. Collectively, our results validate the use and define the limitations of species-mismatched MR1–Ag tetramers in comparative immunology studies.
Understanding the generation of immunity to SARS-CoV-2 in lymphoid tissues draining the site of infection has implications for immunity to SARS-CoV-2. We performed tonsil biopsies under local ...anesthesia in 19 subjects who had recovered from SARS-CoV-2 infection 24-225 d previously. The biopsies yielded >3 million cells for flow cytometric analysis in 17 subjects. Total and SARS-CoV-2 spike-specific germinal center B cells, and T follicular helper cells, were readily detectable in human tonsils early after SARS-CoV-2 infection, as assessed by flow cytometry. Responses were higher in samples within 2 mo of infection but still detectable in some subjects out to 7 mo following infection. We conclude the tonsils are a secondary lymphoid organ that develop germinal center responses to SARS-CoV-2 infection and could play a role in the long-term development of immunity.
Natural killer (NK) cells are important anti-viral effector cells. The function and phenotype of the NK cells that constitute an individual's NK cell repertoire can be influenced by ongoing or ...previous viral infections. Indeed, infection with human cytomegalovirus (HCMV) drives the expansion of a highly differentiated NK cell population characterized by expression of CD57 and the activating NKG2C receptor. This NK cell population has also been noted to occur in HIV-1-infected individuals. We evaluated the NK cells of HIV-1-infected and HIV-1-uninfected individuals to determine the relative frequency of highly differentiated CD57+NKG2C+ NK cells and characterize these cells for their receptor expression and responsiveness to diverse stimuli. Highly differentiated CD57+NKG2C+ NK cells occurred at higher frequencies in HCMV-infected donors relative to HCMV-uninfected donors and were dramatically expanded in HIV-1/HCMV co-infected donors. The expanded CD57+NKG2C+ NK cell population in HIV-1-infected donors remained stable following antiretroviral therapy. CD57+NKG2C+ NK cells derived from HIV-1-infected individuals were robustly activated by antibody-dependent stimuli that contained anti-HIV-1 antibodies or therapeutic anti-CD20 antibody, and these NK cells mediated cytolysis through NKG2C. Lastly, CD57+NKG2C+ NK cells from HIV-1-infected donors were characterized by reduced expression of the inhibitory NKG2A receptor. The abundance of highly functional CD57+NKG2C+ NK cells in HIV-1-infected individuals raises the possibility that these NK cells could play a role in HIV-1 pathogenesis or serve as effector cells for therapeutic/cure strategies.
Protective antibody (Ab) responses induced by natural infection or vaccination play a central role in defense against invasive pathogens. Germinal centers (GCs) are the sites of Ab affinity ...maturation and T follicular helper (Tfh) cells are a critical factor for driving GC formation and B cell selection. Therefore characterization of antigen (Ag)-specific Tfh cells is increasingly essential to define the mechanistic basis of protective antibody responses. However, since Tfh are weak producers of cytokines it is difficult to detect Ag-specific Tfh cells using conventional intracellular cytokine staining (ICS). Here, we report an assay identifying mouse Ag-specific Tfh cells by assessing the upregulation of surface activation-induced markers (AIM). Murine lymph node (LN)-derived Tfh cells largely retained CXCR5 and PD-1 expression following 18-hour cell culture. After influenza infection or influenza hemagglutinin (HA) protein vaccination of mice, stimulation of lymph node cell suspensions with peptide pools or whole protein drove upregulation of CD25, OX40 (CD134), ICOS (CD278) and CD154 on Tfh cells. Upregulation of either CD154 or CD25/OX40 proved a sensitive method for delineating HA-specific Tfh cells. This assay provides the opportunity to quantify antigen-specific Tfh cells in mice without the need for transgenic models or MHC-II tetramer reagents restricted to specific epitopes.
•Tfh cells are poor cytokine producers in vitro.•Activation induced marker assays identify antigen-specific T cells.•Murine antigen-specific Tfh cells upregulate CD25, OX40 and CD154 after stimulation.•Peptides or proteins are both effective in stimulating murine Tfh cells.
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.
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.
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.
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.
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.
Pre-existing HIV infection increases tuberculosis (TB) risk in children. Antiretroviral therapy (ART) reduces, but does not abolish, this risk in children with HIV. The immunologic mechanisms ...involved in TB progression in both HIV-naive and HIV-infected children have not been explored. Much of our current understanding is based on human studies in adults and adult animal models. In this study, we sought to model childhood HIV/Mycobacterium tuberculosis (Mtb) coinfection in the setting of ART and characterize T cells during TB progression. Macaques equivalent to 4 to 8 year-old children were intravenously infected with SIVmac239M, treated with ART 3 months later, and coinfected with Mtb 3 months after initiating ART. SIV-naive macaques were similarly infected with Mtb alone. TB pathology and total Mtb burden did not differ between SIV-infected, ART-treated and SIV-naive macaques, although lung Mtb burden was lower in SIV-infected, ART-treated macaques. No major differences in frequencies of CD4
and CD8
T cells and unconventional T cell subsets (Vγ9+ γδ T cells, MAIT cells, and NKT cells) in airways were observed between SIV-infected, ART-treated and SIV-naive macaques over the course of Mtb infection, with the exception of CCR5+ CD4
and CD8
T cells which were slightly lower. CD4
and CD8
T cell frequencies did not differ in the lung granulomas. Immune checkpoint marker levels were similar, although ki-67 levels in CD8
T cells were elevated. Thus, ART treatment of juvenile macaques, 3 months after SIV infection, resulted in similar progression of Mtb and T cell responses compared to Mtb in SIV-naive macaques.
Despite remarkable successes of immunization in protecting public health, safe and effective vaccines against a number of life‐threatening pathogens such as HIV, ebola, influenza, and SARS‐CoV‐2 ...remain urgently needed. Subunit vaccines can avoid potential toxicity associated with traditional whole virion‐inactivated and live‐attenuated vaccines; however, the immunogenicity of subunit vaccines is often poor. A facile method is here reported to produce lipid nanoparticle subunit vaccines that exhibit high immunogenicity and elicit protection against influenza virus. Influenza hemagglutinin (HA) immunogens are functionalized on the surface of liposomes via stable metal chelation chemistry, using a scalable advanced microfluidic mixing technology (NanoAssemblr). Immunization of mice with HA‐liposomes elicits increased serum antibody titers and superior protection against highly pathogenic virus challenge compared with free HA protein. HA‐liposomal vaccines display enhanced antigen deposition into germinal centers within the draining lymph nodes, driving increased HA‐specific B cell, and follicular helper T cell responses. This work provides mechanistic insights into highly protective HA‐liposome vaccines and informs the rational design and rapid production of next generation nanoparticle subunit vaccines.
Liposomes attached with hemagglutinin (HA)—a key antigen on influenza viral surface are prepared. HA‐liposome immunization elicits higher levels of HA‐specific antibodies in mouse serum compared to soluble HA vaccination. Increased deposition of HA‐liposomes in germinal centers of draining lymph nodes leads to enhanced formation of HA‐specific germinal center B cells, which is responsible for improved humoral immune responses.
Objectives
Although antiretroviral therapy (ART) efficiently suppresses HIV viral load, immune dysregulation and dysfunction persist in people living with HIV (PLWH). γδ T cells are functionally ...impaired during untreated HIV infection, but the extent to which they are reconstituted upon ART is currently unclear.
Methods
Utilising a cohort of ART‐treated PLWH, we assessed the frequency and phenotype, characterised in vitro functional responses and defined the impact of immune checkpoint marker expression on effector functions of both Vδ1 and Vδ2 T cells. We additionally explore the in vitro expansion of Vδ2 T cells from PLWH on ART and the mechanisms by which such expanded cells may sense and kill HIV‐infected targets.
Results
A matured NK cell‐like phenotype was observed for Vδ1 T cells among 25 ART‐treated individuals (PLWH/ART) studied compared to 17 HIV‐uninfected controls, with heightened expression of 2B4, CD160, TIGIT and Tim‐3. Despite persistent phenotypic perturbations, Vδ1 T cells from PLWH/ART exhibited strong CD16‐mediated activation and degranulation, which were suppressed upon Tim‐3 and TIGIT crosslinking. Vδ2 T cell degranulation responses to the phosphoantigen (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl pyrophosphate at concentrations up to 2 ng mL−1 were significantly impaired in an immune checkpoint‐independent manner among ART‐treated participants. Nonetheless, expanded Vδ2 T cells from PLWH/ART retained potent anti‐HIV effector functions, with the NKG2D receptor contributing substantially to the elimination of infected cells.
Conclusion
Our findings highlight that although significant perturbations remain within the γδ T cell compartment throughout ART‐treated HIV, both subsets retain the capacity for robust anti‐HIV effector functions.
We assessed the phenotype and function of Vδ1 and Vδ2 T cells in the context of antiretroviral therapy (ART) treated HIV‐1 infection. Despite exhibiting elevated expression of immune checkpoint molecules in comparison to uninfected controls, both γδ T cell subsets remained capable of robust anti‐HIV effector functions in ART‐treated people living with HIV.
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