Microbiology & Immunology - Research Publications

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    Nice and slow make the germinal centers go: measured and escalating antigen delivery enhance durability and quality of humoral immune responses against HIV-1
    Tan, H-X ; Davenport, MP ; Kent, SJ ; Wheatley, AK (WILEY, 2022-11)
    A recently published article has confirmed that a novel immunization method of sustained and escalating antigen delivery augments the magnitude, quality and durability of humoral immune responses.
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    Cutting Edge: SARS-CoV-2 Infection Induces Robust Germinal Center Activity in the Human Tonsil
    Tan, H-X ; Wragg, KM ; Kelly, HG ; Esterbauer, R ; Dixon, BJ ; Lau, JSY ; Flanagan, KL ; van de Sandt, CE ; Kedzierska, K ; McMahon, JH ; Wheatley, AK ; Juno, JA ; Kent, SJ (AMER ASSOC IMMUNOLOGISTS, 2022-05-15)
    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.
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    Lung-resident memory B cells established after pulmonary influenza infection display distinct transcriptional and phenotypic profiles
    Tan, H-X ; Juno, JA ; Esterbauer, R ; Kelly, HG ; Wragg, KM ; Konstandopoulos, P ; Alcantara, S ; Alvarado, C ; Jones, R ; Starkey, G ; Wang, BZ ; Yoshino, O ; Tiang, T ; Grayson, ML ; Opdam, H ; D'Costa, R ; Vago, A ; Mackay, LK ; Gordon, CL ; Masopust, D ; Groom, JR ; Kent, SJ ; Wheatley, AK (AMER ASSOC ADVANCEMENT SCIENCE, 2022-01)
    Recent studies have established that memory B cells, largely thought to be circulatory in the blood, can take up long-term residency in inflamed tissues, analogous to widely described tissue-resident T cells. The dynamics of recruitment and retention of memory B cells to tissues and their immunological purpose remains unclear. Here, we characterized tissue-resident memory B cells (BRM) that are stably maintained in the lungs of mice after pulmonary influenza infection. Influenza-specific BRM were localized within inducible bronchus-associated lymphoid tissues (iBALTs) and displayed transcriptional signatures distinct from classical memory B cells in the blood or spleen while showing partial overlap with memory B cells in lung-draining lymph nodes. We identified lung-resident markers, including elevated expression of CXCR3, CCR6, and CD69, on hemagglutinin (HA)- and nucleoprotein (NP)-specific lung BRM. We found that CCR6 facilitates increased recruitment and/or retention of BRM in lungs and differentiation into antibody-secreting cells upon recall. Although expression of CXCR3 and CCR6 was comparable in total and influenza-specific memory B cells isolated across tissues of human donors, CD69 expression was higher in memory B cells from lung and draining lymph nodes of human organ donors relative to splenic and PBMC-derived populations, indicating that mechanisms underpinning BRM localization may be evolutionarily conserved. Last, we demonstrate that human memory B cells in lungs are transcriptionally distinct to populations in lung-draining lymph nodes or PBMCs. These data suggest that BRM may constitute a discrete component of B cell immunity, positioned at the lung mucosa for rapid humoral response against respiratory viral infections.
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    Immunological dysfunction persists for 8 months following initial mild-to-moderate SARS-CoV-2 infection
    Phetsouphanh, C ; Darley, D ; Wilson, DB ; Howe, A ; Munier, CML ; Patel, SK ; Juno, JA ; Burrell, LM ; Kent, SJ ; Dore, GJ ; Kelleher, AD ; Matthews, G (NATURE PORTFOLIO, 2022-02)
    A proportion of patients surviving acute coronavirus disease 2019 (COVID-19) infection develop post-acute COVID syndrome (long COVID (LC)) lasting longer than 12 weeks. Here, we studied individuals with LC compared to age- and gender-matched recovered individuals without LC, unexposed donors and individuals infected with other coronaviruses. Patients with LC had highly activated innate immune cells, lacked naive T and B cells and showed elevated expression of type I IFN (IFN-β) and type III IFN (IFN-λ1) that remained persistently high at 8 months after infection. Using a log-linear classification model, we defined an optimal set of analytes that had the strongest association with LC among the 28 analytes measured. Combinations of the inflammatory mediators IFN-β, PTX3, IFN-γ, IFN-λ2/3 and IL-6 associated with LC with 78.5-81.6% accuracy. This work defines immunological parameters associated with LC and suggests future opportunities for prevention and treatment.
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    Disentangling the relative importance of T cell responses in COVID-19: leading actors or supporting cast?
    Kent, SJ ; Khoury, DS ; Reynold, A ; Juno, JA ; Wheatley, AK ; Stadler, E ; Wherry, EJ ; Triccas, J ; Sasson, SC ; Cromer, D ; Davenport, MP (NATURE PORTFOLIO, 2022-06)
    The rapid development of multiple vaccines providing strong protection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been a major achievement. There is now compelling evidence for the role of neutralizing antibodies in protective immunity. T cells may play a role in resolution of primary SARS-CoV-2 infection, and there is a widely expressed view that T cell-mediated immunity also plays an important role in vaccine-mediated protection. Here we discuss the role of vaccine-induced T cells in two distinct stages of infection: firstly, in protection from acquisition of symptomatic SARS-CoV-2 infection following exposure; secondly, if infection does occur, the potential for T cells to reduce the risk of developing severe COVID-19. We describe several lines of evidence that argue against a direct impact of vaccine-induced memory T cells in preventing symptomatic SARS-CoV-2 infection. However, the contribution of T cell immunity in reducing the severity of infection, particularly in infection with SARS-CoV-2 variants, remains to be determined. A detailed understanding of the role of T cells in COVID-19 is critical for next-generation vaccine design and development. Here we discuss the challenges in determining a causal relationship between vaccine-induced T cell immunity and protection from COVID-19 and propose an approach to gather the necessary evidence to clarify any role for vaccine-induced T cell memory in protection from severe COVID-19.
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    Establishment and recall of SARS-CoV-2 spike epitope-specific CD4+ T cell memory
    Wragg, KM ; Lee, WS ; Koutsakos, M ; Tan, H-X ; Amarasena, T ; Reynaldi, A ; Gare, G ; Konstandopoulos, P ; Field, KR ; Esterbauer, R ; Kent, HE ; Davenport, MP ; Wheatley, AK ; Kent, SJ ; Juno, JA (NATURE PORTFOLIO, 2022-05)
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination elicit CD4+ T cell responses to the spike protein, including circulating follicular helper T (cTFH) cells that correlate with neutralizing antibodies. Using a novel HLA-DRB1*15:01/S751 tetramer to track spike-specific CD4+ T cells, we show that primary infection or vaccination induces robust S751-specific CXCR5- and cTFH cell memory responses. Secondary exposure induced recall of CD4+ T cells with a transitory CXCR3+ phenotype, and drove expansion of cTFH cells transiently expressing ICOS, CD38 and PD-1. In both contexts, cells exhibited a restricted T cell antigen receptor repertoire, including a highly public clonotype and considerable clonotypic overlap between CXCR5- and cTFH populations. Following a third vaccine dose, the rapid re-expansion of spike-specific CD4+ T cells contrasted with the comparatively delayed increase in antibody titers. Overall, we demonstrate that stable pools of cTFH and memory CD4+ T cells established by infection and/or vaccination are efficiently recalled upon antigen reexposure and may contribute to long-term protection against SARS-CoV-2.
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    Immunization with inactivated whole virus particle influenza virus vaccines improves the humoral response landscape in cynomolgus macaques
    Chua, BY ; Sekiya, T ; Koutsakos, M ; Nomura, N ; Rowntree, LC ; Nguyen, THO ; McQuilten, HA ; Ohno, M ; Ohara, Y ; Nishimura, T ; Endo, M ; Itoh, Y ; Habel, JR ; Selva, KJ ; Wheatley, AK ; Wines, BD ; Hogarth, PM ; Kent, SJ ; Chung, AW ; Jackson, DC ; Brown, LE ; Shingai, M ; Kedzierska, K ; Kida, H ; Klein, SL (PUBLIC LIBRARY SCIENCE, 2022-10)
    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.
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    Heterologous SARS-CoV-2 IgA neutralising antibody responses in convalescent plasma
    Davis, SK ; Selva, KJ ; Lopez, E ; Haycroft, ER ; Lee, WS ; Wheatley, AK ; Juno, JA ; Adair, A ; Pymm, P ; Redmond, SJ ; Gherardin, NA ; Godfrey, D ; Tham, W-H ; Kent, SJ ; Chung, AW (WILEY, 2022)
    OBJECTIVES: Following infection with SARS-CoV-2, virus-specific antibodies are generated, which can both neutralise virions and clear infection via Fc effector functions. The importance of IgG antibodies for protection and control of SARS-CoV-2 has been extensively reported. By comparison, other antibody isotypes including IgA have been poorly characterised. METHODS: Here, we characterised plasma IgA from 41 early convalescent COVID-19 subjects for neutralisation and Fc effector functions. RESULTS: Convalescent plasma IgA from > 60% of the cohort had the capacity to inhibit the interaction between wild-type RBD and ACE2. Furthermore, a third of the cohort induced stronger IgA-mediated ACE2 inhibition than matched IgG when tested at equivalent concentrations. Plasma IgA and IgG from this cohort broadly recognised similar RBD epitopes and had similar capacities to inhibit ACE2 from binding to 22 of the 23 prevalent RBD mutations assessed. However, plasma IgA was largely incapable of mediating antibody-dependent phagocytosis in comparison with plasma IgG. CONCLUSION: Overall, convalescent plasma IgA contributed to the neutralising antibody response of wild-type SARS-CoV-2 RBD and various RBD mutations. However, this response displayed large heterogeneity and was less potent than IgG.
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    Biparatopic nanobodies targeting the receptor binding domain efficiently neutralize SARS-CoV-2
    Pymm, P ; Redmond, SJ ; Dolezal, O ; Mordant, F ; Lopez, E ; Cooney, JP ; Davidson, KC ; Haycroft, ER ; Tan, CW ; Seneviratna, R ; Grimley, SL ; Purcell, DFJ ; Kent, SJ ; Wheatley, AK ; Wang, L-F ; Leis, A ; Glukhova, A ; Pellegrini, M ; Chung, AW ; Subbarao, K ; Uldrich, AP ; Tham, W-H ; Godfrey, DI ; Gherardin, NA (CELL PRESS, 2022-11-18)
    The development of therapeutics to prevent or treat COVID-19 remains an area of intense focus. Protein biologics, including monoclonal antibodies and nanobodies that neutralize virus, have potential for the treatment of active disease. Here, we have used yeast display of a synthetic nanobody library to isolate nanobodies that bind the receptor-binding domain (RBD) of SARS-CoV-2 and neutralize the virus. We show that combining two clones with distinct binding epitopes within the RBD into a single protein construct to generate biparatopic reagents dramatically enhances their neutralizing capacity. Furthermore, the biparatopic nanobodies exhibit enhanced control over clinically relevant RBD variants that escaped recognition by the individual nanobodies. Structural analysis of biparatopic binding to spike (S) protein revealed a unique binding mode whereby the two nanobody paratopes bridge RBDs encoded by distinct S trimers. Accordingly, biparatopic nanobodies offer a way to rapidly generate powerful viral neutralizers with enhanced ability to control viral escape mutants.
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    The magnitude and timing of recalled immunity after breakthrough infection is shaped by SARS-CoV-2 variants
    Koutsakos, M ; Lee, WS ; Reynaldi, A ; Tan, H-X ; Gare, G ; Kinsella, P ; Liew, KC ; Taiaroa, G ; Williamson, DA ; Kent, HE ; Stadler, E ; Cromer, D ; Khoury, DS ; Wheatley, AK ; Juno, JA ; Davenport, MP ; Kent, SJ (CELL PRESS, 2022-07-12)
    Vaccination against SARS-CoV-2 protects from infection and improves clinical outcomes in breakthrough infections, likely reflecting residual vaccine-elicited immunity and recall of immunological memory. Here, we define the early kinetics of spike-specific humoral and cellular immunity after vaccination of seropositive individuals and after Delta or Omicron breakthrough infection in vaccinated individuals. Early longitudinal sampling revealed the timing and magnitude of recall, with the phenotypic activation of B cells preceding an increase in neutralizing antibody titers. While vaccination of seropositive individuals resulted in robust recall of humoral and T cell immunity, recall of vaccine-elicited responses was delayed and variable in magnitude during breakthrough infections and depended on the infecting variant of concern. While the delayed kinetics of immune recall provides a potential mechanism for the lack of early control of viral replication, the recall of antibodies coincided with viral clearance and likely underpins the protective effects of vaccination against severe COVID-19.