Microbiology & Immunology - Research Publications

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    Development of virus-like particles with inbuilt immunostimulatory properties as vaccine candidates
    Collett, S ; Earnest, L ; Carrera Montoya, J ; Edeling, MAA ; Yap, A ; Wong, CY ; Christiansen, D ; Roberts, J ; Mumford, J ; Lecouturier, V ; Pavot, V ; Marco, S ; Loi, JK ; Simmons, C ; Gulab, SAA ; Mackenzie, JMM ; Elbourne, A ; Ramsland, PAA ; Cameron, G ; Hans, D ; Godfrey, DII ; Torresi, J (FRONTIERS MEDIA SA, 2023-06-07)
    The development of virus-like particle (VLP) based vaccines for human papillomavirus, hepatitis B and hepatitis E viruses represented a breakthrough in vaccine development. However, for dengue and COVID-19, technical complications, such as an incomplete understanding of the requirements for protective immunity, but also limitations in processes to manufacture VLP vaccines for enveloped viruses to large scale, have hampered VLP vaccine development. Selecting the right adjuvant is also an important consideration to ensure that a VLP vaccine induces protective antibody and T cell responses. For diseases like COVID-19 and dengue fever caused by RNA viruses that exist as families of viral variants with the potential to escape vaccine-induced immunity, the development of more efficacious vaccines is also necessary. Here, we describe the development and characterisation of novel VLP vaccine candidates using SARS-CoV-2 and dengue virus (DENV), containing the major viral structural proteins, as protypes for a novel approach to produce VLP vaccines. The VLPs were characterised by Western immunoblot, enzyme immunoassay, electron and atomic force microscopy, and in vitro and in vivo immunogenicity studies. Microscopy techniques showed proteins self-assemble to form VLPs authentic to native viruses. The inclusion of the glycolipid adjuvant, α-galactosylceramide (α-GalCer) in the vaccine formulation led to high levels of natural killer T (NKT) cell stimulation in vitro, and strong antibody and memory CD8+ T cell responses in vivo, demonstrated with SARS-CoV-2, hepatitis C virus (HCV) and DEN VLPs. This study shows our unique vaccine formulation presents a promising, and much needed, new vaccine platform in the fight against infections caused by enveloped RNA viruses.
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    Glycolipids from the gut symbiont Bacteroides fragilis are agonists for natural killer T cells and induce their regulatory differentiation
    Cameron, G ; Nguyen, T ; Ciula, M ; Williams, SJ ; Godfrey, DI (ROYAL SOC CHEMISTRY, 2023-07-26)
    Natural Killer T (NKT) cells are a lipid-antigen reactive T cell subset that is restricted to the antigen presenting molecule CD1d. They possess diverse functional properties that contribute to inflammatory and regulatory immune responses. The most studied lipid antigen target for these T cells is α-galactosylceramide (αGC). The commensal organism Bacteroides fragilis (B. fragilis) produces several forms of αGC, but conflicting information exists about the influence of these lipids on NKT cells. Herein, we report the total synthesis of a major form of αGC from B. fragilis (Bf αGC), and several analogues thereof. We confirm the T cell receptor (TCR)-mediated recognition of these glycolipids by mouse and human NKT cells. Despite the natural structure of Bf αGC containing lipid branching that limits potency, we demonstrate that Bf αGC drives mouse NKT cells to proliferate and differentiate into producers of the immunoregulatory cytokine, interleukin-10 (IL-10). These Bf αGC-experienced NKT cells display regulatory function by inhibiting the expansion of naïve NKT cells upon subsequent exposure to this antigen. Moreover, this regulatory activity impacts more than just NKT cells, as demonstrated by the NKT cell-mediated inhibition of antigen-stimulated mucosal-associated invariant T (MAIT) cells (a T cell subset restricted to a different antigen presenting molecule, MR1). These findings reveal that B. fragilis-derived NKT cell agonists may have broad immunoregulatory activity, providing insight into the mechanisms influencing immune tolerance to commensal bacteria and highlighting a potential means to manipulate NKT cell function for therapeutic benefit.
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    Newborn and child-like molecular signatures in older adults stem from TCR shifts across human lifespan
    van de Sandt, CE ; Nguyen, THO ; Gherardin, NA ; Crawford, JC ; Samir, J ; Minervina, AA ; Pogorelyy, MV ; Rizzetto, S ; Szeto, C ; Kaur, J ; Ranson, N ; Sonda, S ; Harper, A ; Redmond, SJ ; McQuilten, HA ; Menon, T ; Sant, S ; Jia, X ; Pedrina, K ; Karapanagiotidis, T ; Cain, N ; Nicholson, S ; Chen, Z ; Lim, R ; Clemens, EB ; Eltahla, A ; La Gruta, NL ; Crowe, J ; Lappas, M ; Rossjohn, J ; Godfrey, DI ; Thomas, PG ; Gras, S ; Flanagan, KL ; Luciani, F ; Kedzierska, K (NATURE PORTFOLIO, 2023-11)
    CD8+ T cells provide robust antiviral immunity, but how epitope-specific T cells evolve across the human lifespan is unclear. Here we defined CD8+ T cell immunity directed at the prominent influenza epitope HLA-A*02:01-M158-66 (A2/M158) across four age groups at phenotypic, transcriptomic, clonal and functional levels. We identify a linear differentiation trajectory from newborns to children then adults, followed by divergence and a clonal reset in older adults. Gene profiles in older adults closely resemble those of newborns and children, despite being clonally distinct. Only child-derived and adult-derived A2/M158+CD8+ T cells had the potential to differentiate into highly cytotoxic epitope-specific CD8+ T cells, which was linked to highly functional public T cell receptor (TCR)αβ signatures. Suboptimal TCRαβ signatures in older adults led to less proliferation, polyfunctionality, avidity and recognition of peptide mutants, although displayed no signs of exhaustion. These data suggest that priming T cells at different stages of life might greatly affect CD8+ T cell responses toward viral infections.
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    Antibody Fc-binding profiles and ACE2 affinity to SARS-CoV-2 RBD variants
    Haycroft, ER ; Davis, SK ; Ramanathan, P ; Lopez, E ; Purcell, RA ; Tan, LL ; Pymm, P ; Wines, BD ; Hogarth, PM ; Wheatley, AK ; Juno, JA ; Redmond, SJ ; Gherardin, NA ; Godfrey, DI ; Tham, W-H ; Selva, KJ ; Kent, SJ ; Chung, AW (SPRINGER, 2023-08)
    Emerging SARS-CoV-2 variants, notably Omicron, continue to remain a formidable challenge to worldwide public health. The SARS-CoV-2 receptor-binding domain (RBD) is a hotspot for mutations, reflecting its critical role at the ACE2 interface during viral entry. Here, we comprehensively investigated the impact of RBD mutations, including 5 variants of concern (VOC) or interest-including Omicron (BA.2)-and 33 common point mutations, both on IgG recognition and ACE2-binding inhibition, as well as FcγRIIa- and FcγRIIIa-binding antibodies, in plasma from two-dose BNT162b2-vaccine recipients and mild-COVID-19 convalescent subjects obtained during the first wave using a custom-designed bead-based 39-plex array. IgG-recognition and FcγR-binding antibodies were decreased against the RBD of Beta and Omicron, as well as point mutation G446S, found in several Omicron sub-variants as compared to wild type. Notably, while there was a profound decrease in ACE2 inhibition against Omicron, FcγR-binding antibodies were less affected, suggesting that Fc functional antibody responses may be better retained against the RBD of Omicron in comparison to neutralization. Furthermore, while measurement of RBD-ACE2-binding affinity via biolayer interferometry showed that all VOC RBDs have enhanced affinity to human ACE2, we demonstrate that human ACE2 polymorphisms, E35K (rs1348114695) has reduced affinity to VOCs, while K26R (rs4646116) and S19P (rs73635825) have increased binding kinetics to the RBD of VOCs, potentially affecting virus-host interaction and, thereby, host susceptibility. Collectively, our findings provide in-depth coverage of the impact of RBD mutations on key facets of host-virus interactions.
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    MAIT and Vδ2 unconventional T cells are supported by a diverse intestinal microbiome and correlate with favorable patient outcome after allogeneic HCT.
    Andrlová, H ; Miltiadous, O ; Kousa, AI ; Dai, A ; DeWolf, S ; Violante, S ; Park, H-Y ; Janaki-Raman, S ; Gardner, R ; El Daker, S ; Slingerland, J ; Giardina, P ; Clurman, A ; Gomes, ALC ; Nguyen, C ; da Silva, MB ; Armijo, GK ; Lee, N ; Zappasodi, R ; Chaligne, R ; Masilionis, I ; Fontana, E ; Ponce, D ; Cho, C ; Bush, A ; Hill, L ; Chao, N ; Sung, AD ; Giralt, S ; Vidal, EH ; Hosszu, KK ; Devlin, SM ; Peled, JU ; Cross, JR ; Perales, M-A ; Godfrey, DI ; van den Brink, MRM ; Markey, KA (American Association for the Advancement of Science (AAAS), 2022-05-25)
    Microbial diversity is associated with improved outcomes in recipients of allogeneic hematopoietic cell transplantation (allo-HCT), but the mechanism underlying this observation is unclear. In a cohort of 174 patients who underwent allo-HCT, we demonstrate that a diverse intestinal microbiome early after allo-HCT is associated with an increased number of innate-like mucosal-associated invariant T (MAIT) cells, which are in turn associated with improved overall survival and less acute graft-versus-host disease (aGVHD). Immune profiling of conventional and unconventional immune cell subsets revealed that the prevalence of Vδ2 cells, the major circulating subpopulation of γδ T cells, closely correlated with the frequency of MAIT cells and was associated with less aGVHD. Analysis of these populations using both single-cell transcriptomics and flow cytometry suggested a shift toward activated phenotypes and a gain of cytotoxic and effector functions after transplantation. A diverse intestinal microbiome with the capacity to produce activating ligands for MAIT and Vδ2 cells appeared to be necessary for the maintenance of these populations after allo-HCT. These data suggest an immunological link between intestinal microbial diversity, microbe-derived ligands, and maintenance of unconventional T cells.
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    Differential antigen requirements by diverse MR1-restricted T cells (vol 100, pg 112, 2022)
    Seneviratna, R ; Redmond, SJ ; McWilliam, HEG ; Reantragoon, R ; Villadangos, JA ; McCluskey, J ; Godfrey, D ; Gherardin, NA (WILEY, 2022-03)
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    Are NKT cells a useful predictor of COVID-19 severity?
    Koay, H-F ; Gherardin, NA ; Nguyen, THO ; Zhang, W ; Habel, JR ; Seneviratna, R ; James, F ; Holmes, NE ; Smibert, OC ; Gordon, CL ; Trubiano, JA ; Kedzierska, K ; Godfrey, DI (CELL PRESS, 2022-02-08)
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    Identification of a novel role for the immunomodulator ILRUN in the development of several T cell subsets in mice.
    Tribolet, L ; Brice, AM ; Fulford, TS ; Layton, DS ; Godfrey, DI ; Bean, AGD ; Stewart, CR (Elsevier BV, 2023-05)
    Inflammation and lipid regulator with UBA-like and NBR1-like domains (ILRUN) is a protein-encoding gene associated with innate immune signaling, lipid metabolism and cancer. In the context of innate immunity, ILRUN inhibits IRF3-mediated transcription of antimicrobial and proinflammatory cytokines by inducing degradation of the transcriptional coactivators CBP and p300. There remains a paucity of information, however, regarding the innate immune roles of ILRUN beyond in vitro analyses. To address this, we utilize a knockout mouse model to investigate the effect of ILRUN on cytokine expression in splenocytes and on the development of immune cell populations in the spleen and thymus. We show elevated production of tumor necrosis factor and interleukin-6 cytokines in ILRUN-deficient splenocytes following stimulation with the innate immune ligands polyinosinic:polycytidylic acid or lipopolysaccharide. Differences were also observed in the populations of several T cell subsets, including regulatory, mucosal-associated invariant and natural killer. These data identify novel functions for ILRUN in the development of certain immune cell populations and support previous in vitro findings that ILRUN negatively regulates the synthesis of pathogen-stimulated cytokines. This establishes the ILRUN knockout mouse model as a valuable resource for further study of the functions of ILRUN in health and disease.
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    Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine
    Deliyannis, G ; Gherardin, NA ; Wong, CY ; Grimley, SL ; Cooney, JP ; Redmond, SJ ; Ellenberg, P ; Davidson, KC ; Mordant, FL ; Smith, T ; Gillard, M ; Lopez, E ; McAuley, J ; Tan, CW ; Wang, JJ ; Zeng, W ; Littlejohn, M ; Zhou, R ; Chan, JF-W ; Chen, Z-W ; Hartwig, AE ; Bowen, R ; Mackenzie, JM ; Vincan, E ; Torresi, J ; Kedzierska, K ; Pouton, CW ; Gordon, TP ; Wang, L-F ; Kent, SJ ; Wheatley, AK ; Lewin, SR ; Subbarao, K ; Chung, AW ; Pellegrini, M ; Munro, T ; Nolan, T ; Rockman, S ; Jackson, DC ; Purcell, DFJ ; Godfrey, DI (ELSEVIER, 2023-06)
    BACKGROUND: The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines. METHODS: We report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid α-Galactosylceramide, or MF59® squalene oil-in-water adjuvant, using mice, rats and hamsters. We also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the immuno-evasive beta variant (N501Y, E484K, K417N). These vaccines were also tested as a heterologous third dose booster in mice, following priming with whole spike vaccine. FINDINGS: Each formulation of the RBD-Fc vaccines drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. The 'beta variant' RBD vaccine, combined with MF59® adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a heterologous third dose booster, the RBD-Fc vaccines combined with MF59® increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1, BA.2 and BA.5. INTERPRETATION: These results demonstrated that an RBD-Fc protein subunit/MF59® adjuvanted vaccine can induce high levels of broadly reactive nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial. FUNDING: This work was supported by grants from the Medical Research Future Fund (MRFF) (2005846), The Jack Ma Foundation, National Health and Medical Research Council of Australia (NHMRC; 1113293) and Singapore National Medical Research Council (MOH-COVID19RF-003). Individual researchers were supported by an NHMRC Senior Principal Research Fellowship (1117766), NHMRC Investigator Awards (2008913 and 1173871), Australian Research Council Discovery Early Career Research Award (ARC DECRA; DE210100705) and philanthropic awards from IFM investors and the A2 Milk Company.
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    Immune profiling of SARS-CoV-2 infection during pregnancy reveals NK cell and ?? T cell perturbations
    Habel, JR ; Chua, BY ; Kedzierski, L ; Selva, KJ ; Damelang, T ; Haycroft, ER ; Nguyen, THO ; Koay, H-F ; Nicholson, S ; McQuilten, HA ; Jia, X ; Allen, LF ; Hensen, L ; Zhang, W ; Sandt, CEVD ; Neil, JA ; Pragastis, K ; Lau, JSY ; Jumarang, J ; Allen, EK ; Amanant, F ; Krammer, F ; Wragg, KM ; Juno, JA ; Wheatley, AK ; Tan, H-X ; Pell, G ; Walker, S ; Audsley, J ; Reynaldi, A ; Thevarajan, I ; Denholm, JT ; Subbarao, K ; Davenport, MP ; Hogarth, PM ; Godfrey, DI ; Cheng, AC ; Tong, SYC ; Bond, K ; Williamson, DA ; McMahon, JH ; Thomas, PG ; Pannaraj, PS ; James, F ; Holmes, NE ; Smibert, OC ; Trubiano, JA ; Gordon, CL ; Chung, AW ; Whitehead, CL ; Kent, SJ ; Lappas, M ; Rowntree, LC ; Kedzierska, K (AMER SOC CLINICAL INVESTIGATION INC, 2023-03-22)
    Pregnancy poses a greater risk for severe COVID-19; however, underlying immunological changes associated with SARS-CoV-2 during pregnancy are poorly understood. We defined immune responses to SARS-CoV-2 in unvaccinated pregnant and nonpregnant women with acute and convalescent COVID-19, quantifying 217 immunological parameters. Humoral responses to SARS-CoV-2 were similar in pregnant and nonpregnant women, although our systems serology approach revealed distinct antibody and FcγR profiles between pregnant and nonpregnant women. Cellular analyses demonstrated marked differences in NK cell and unconventional T cell activation dynamics in pregnant women. Healthy pregnant women displayed preactivated NK cells and γδ T cells when compared with healthy nonpregnant women, which remained unchanged during acute and convalescent COVID-19. Conversely, nonpregnant women had prototypical activation of NK and γδ T cells. Activation of CD4+ and CD8+ T cells and T follicular helper cells was similar in SARS-CoV-2-infected pregnant and nonpregnant women, while antibody-secreting B cells were increased in pregnant women during acute COVID-19. Elevated levels of IL-8, IL-10, and IL-18 were found in pregnant women in their healthy state, and these cytokine levels remained elevated during acute and convalescent COVID-19. Collectively, we demonstrate perturbations in NK cell and γδ T cell activation in unvaccinated pregnant women with COVID-19, which may impact disease progression and severity during pregnancy.