Microbiology & Immunology - Research Publications

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    Tear antibodies to SARS-CoV-2: implications for transmission
    Selva, K ; Davis, S ; Haycroft, E ; Lee, WS ; Lopez, E ; Reynaldi, A ; Davenport, M ; Kent, H ; Juno, J ; Chung, A ; Kent, S ( 2021-08-04)

    Objectives

    SARS-CoV-2 can be transmitted by aerosols and the ocular surface may be an important route of transmission. Little is known about protective antibody responses to SARS-CoV-2 in tears after infection or vaccination. We analysed SARS-CoV-2 specific IgG and IgA responses in human tears after either COVID-19 infection or vaccination.

    Methods

    We recruited 16 subjects with COVID-19 infection an average of 7 months previously and 15 subjects before and 2 weeks after Comirnaty (Pfizer-BioNtech) vaccination. Plasma, saliva and basal tears were collected. Pre-pandemic plasma, saliva and basal tears from 11 individuals were included as healthy controls. Antibody responses to 5 SARS-CoV-2 antigens were measured via multiplex.

    Results

    IgG antibodies to Spike and Nucleoprotein were detected in tears, saliva and plasma from subjects with prior SARS-CoV-2 infection in comparison to uninfected controls. While RBD-specific antibodies were detected in plasma, minimal RBD-specific antibodies were detected in tears and saliva. In contrast, high levels of IgG antibodies to Spike and RBD, but not Nucleoprotein, were induced in tears, saliva and plasma of subjects receiving 2 doses of the Comirnaty vaccine. Increased levels of IgA1 and IgA2 antibodies to SARS-CoV-2 antigens were detected in plasma following infection or vaccination, but were unchanged in tears and saliva.

    Conclusion

    Both infection and vaccination induce SARS-CoV-2-specific IgG antibodies in tears. RBD-specific IgG antibodies in tears were induced by vaccination but were not present 7 months post-infection. This suggests neutralising antibodies may be low in the tears late following infection.
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    Integrated immune networks in SARS-CoV-2 infected pregnant women reveal differential NK cell and unconventional T cell activation
    Kedzierska, K ; Habel, J ; Chua, B ; Kedzierski, L ; Selva, K ; Damelang, T ; Haycroft, E ; Nguyen, T ; Koay, H-F ; Nicholson, S ; McQuilten, H ; Jia, X ; Allen, L ; Hensen, L ; Zhang, W ; de Sandt, CV ; Neil, J ; Amanat, F ; Krammer, F ; Wragg, K ; Juno, J ; Wheatley, A ; Tan, H-X ; Pell, G ; Audsley, J ; Thevarajan, I ; Denholm, J ; Subbarao, K ; Godfrey, D ; Cheng, A ; Tong, S ; Bond, K ; Williamson, D ; James, F ; Holmes, N ; Smibert, O ; Trubiona, J ; Gordon, C ; Chung, A ; Whitehead, C ; Kent, S ; Lappas, M ; Rowntree, L ( 2021-10-04)
    Although pregnancy poses a greater risk for severe COVID-19, the underlying immunological changes associated with SARS-CoV-2 infection during pregnancy are poorly understood. We defined immune responses to SARS-CoV-2 in pregnant and non-pregnant women during acute and convalescent COVID-19 up to 258 days post symptom onset, quantifying 217 immunological parameters. Additionally, matched maternal and cord blood were collected from COVID-19 convalescent pregnancies. Although serological responses to SARS-CoV-2 were similar in pregnant and non-pregnant women, cellular immune analyses revealed marked differences in key NK cell and unconventional T cell responses during COVID-19 in pregnant women. While NK, γδ T cells and MAIT cells displayed pre-activated phenotypes in healthy pregnant women when compared to non-pregnant age-matched women, activation profiles of these pre-activated NK and unconventional T cells remained unchanged at acute and convalescent COVID-19 in pregnancy. Conversely, activation dynamics of NK and unconventional T cells were prototypical in non-pregnant women in COVID-19. In contrast, activation of αβ CD4 + and CD8 + T cells, T follicular helper cells and antibody-secreting cells was similar in pregnant and non-pregnant women with COVID-19. Elevated levels of IL-1β, IFN-γ, IL-8, IL-18 and IL-33 were also found in pregnant women in their healthy state, and these cytokine levels remained elevated during acute and convalescent COVID-19. Collectively, our study provides the first comprehensive map of longitudinal immunological responses to SARS-CoV-2 infection in pregnant women, providing insights into patient management and education during COVID-19 pregnancy.
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    Impaired Th17 immunity in recurrent C. difficile infection is ameliorated by fecal microbial transplantation
    Cook, L ; Rees, WD ; Wong, MQ ; Wang, X ; Peters, H ; Oliveira, L ; Lau, T ; Mah, R ; Bressler, B ; Gomez, R ; Chow, I-T ; James, EA ; Kwok, WW ; Levings, MK ; Steiner, TS ( 2020-06-10)
    Background & Aims: Clostridioides difficile is a leading cause of infectious diarrhea and an urgent antimicrobial resistant threat. Symptoms are caused by its toxins, TcdA and TcdB, with many patients developing recurrent C. difficile infection (CDI), requiring fecal microbiota transplant (FMT). Antibody levels have not been useful in predicting patient outcomes, which is an unmet need. We aimed to characterize T cell-mediated immunity to C. difficile toxins and assess how these responses were affected by FMT. Methods: We obtained blood samples from patients with newly acquired CDI, recurrent CDI (with a subset receiving FMT), inflammatory bowel disease with no history of CDI, and healthy individuals (controls). Toxin-specific CD4+ T cell responses were analysed using a whole blood flow cytometry antigen-induced marker assay. Serum antibodies were measured by ELISA. Tetramer guided mapping was used to identify HLA-II-restricted TcdB epitopes and DNA was extracted from TcdB-specific CD4+ T cells for TCR repertoire analysis by Sanger sequencing. Results: CD4+ T cell responses to C. difficile toxins were functionally diverse. Compared to controls, individuals with CDI, or inflammatory bowel disease had significantly higher frequencies of TcdB-specific CD4+ T cells. Subjects with recurrent CDI had reduced proportions of TcdB-specific CD4+ Th17 cells, FMT reversed this deficit and increased toxin-specific antibody production. Conclusions: These data suggest that effective T cell immunity to C. difficile requires the development of Th17 cells. In addition, they show that an unknown aspect of the therapeutic effect of FMT may be enhanced T and B cell-mediated immunity to TcdB.
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    An inulin-type fructan enriched exclusive enteral nutrition formula suppresses colitis through gut microbiome modulation and promoting expansion of anti-inflammatory T cell subsets
    Healey, GR ; Tsai, K ; Lisko, DJ ; Cook, L ; Vallance, BA ; Jacobson, K ( 2021-02-02)
    Background & Aims: Exclusive enteral nutrition (EEN) is used to treat pediatric Crohn’s disease (CD), but therapeutic benefits are not long lasting. Due to reported lower efficacy EEN is not routinely used to treat pediatric ulcerative colitis (UC). Inulin-type fructans (IN) beneficially modulate the gut microbiome and promote expansion of anti-inflammatory immune cells. We hypothesized that enriching EEN with IN (EENIN) would enhance treatment efficacy. To test this, we examined the effects of EEN-IN on colitis development, the gut microbiome and CD4+ T cells using an adoptive T cell transfer model of colitis. Methods: TCR-ß deficient mice were randomized to one of four groups: 1) Control, 2) Chow, 3) EEN and 4) EEN-IN, and naïve CD4+ T cells were adoptively transferred into groups 2-4, after which mice were monitored for 5-weeks prior to experimental endpoint. Results: Mice fed EEN-IN showed greater colitis protection, with colonic shortening, goblet cell and crypt density loss reduced over that of EEN fed mice and reduced disease activity and immune cell infiltration compared to chow fed mice, and less crypt hyperplasia and higher survival compared to both groups. EENIN mice maintained colonic mucus layer thickness and had increased levels of Foxp3+IL-10+ and Rorγt+IL- 22+ and reduced levels of Tbet+
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    Lasting changes to circulating leukocytes in people with mild SARS-CoV-2 infections
    Kennedy, AE ; Cook, L ; Breznik, JA ; Cowbrough, B ; Wallace, JG ; Huynh, A ; Smith, JW ; Son, K ; Stacey, H ; Ang, J ; McGeer, A ; Coleman, BL ; Larché, M ; Larché, M ; Hambly, N ; Nair, P ; Ask, K ; Miller, MS ; Bramson, J ; Levings, MK ; Nazy, I ; Svenningsen, S ; Mukherjee, M ; Bowdish, DME ( 2021-09-27)
    Abstract: Survivors of severe SARS-CoV-2 infections frequently suffer from a range of post-infection sequelae. Whether survivors of mild or asymptomatic infections can expect any long-term health consequences is not yet known. Herein we investigated lasting changes to soluble inflammatory factors and cellular immune phenotype and function in individuals who had recovered from mild SARS-CoV-2 infections (n=22) compared to those that had recovered from other mild respiratory infections (n=11). Individuals who had mild SARS-CoV-2 infections had elevated levels of C-reactive protein 1-3 months after symptom onset, and changes in phenotype and function of circulating T cells that were not apparent in individuals 6-9 months post-symptom onset. Markers of monocyte activation and expression of adherence and chemokine receptors indicative of altered migratory capacity were also higher at 1-3 months post-infection in individuals who had mild SARS-CoV-2, but these were no longer elevated by 6-9 months post-infection. Perhaps most surprisingly, polyclonal activation of T cells was higher in individuals who had recently experienced a mild SARS-CoV-2 infection compared to individuals with other recent respiratory infections. These data are indicative of prolonged immune activation and systemic inflammation that persists for up to three months after mild or asymptomatic SARS-CoV-2 infections.
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    Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance
    Fareh, M ; Zhao, W ; Hu, W ; Casan, JML ; Kumar, A ; Symons, J ; Voskoboinik, I ; Ekert, P ; Rudraraju, R ; Lewin, S ; Trapani, J ( 2020-11-19)

    ABSTRACT

    Mutation-driven evolution of SARS coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape routes from host immunity and antiviral therapeutics. Here, we employed genome-wide computational prediction and singlenucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus free-models. Further, optimized and multiplexed gRNAs suppressed viral replication by up to 90% in mammalian cells infected with replication-competent SARS-CoV-2. Unexpectedly, the comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches do not impair the capacity of a potent single gRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 in infected mammalian cells, including the highly infectious and globally disseminated Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint of antiviral therapeutics to simultaneously suppress a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.
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    Cross-reactive antibodies after SARS-CoV-2 infection and vaccination
    Grobben, M ; van der Straten, K ; Brouwer, PJM ; Brinkkemper, M ; Maisonnasse, P ; Dereuddre-Bosquet, N ; Burger, J ; Poniman, M ; Oomen, M ; Eggink, D ; Bijl, TPL ; van Willigen, HDG ; Wynberg, E ; Verkaik, B ; Figaroa, OJA ; de Vries, P ; Boertien, T ; Grand, RL ; de Jong, M ; Prins, M ; Chung, A ; de Bree, G ; Sanders, R ; van Gils, M ( 2021-06-02)
    Current SARS-CoV-2 vaccines are losing efficacy against emerging variants and may not protect against future novel coronavirus outbreaks, emphasizing the need for more broadly protective vaccines. To inform the development of a pan-coronavirus vaccine, we investigated the presence and specificity of cross-reactive antibodies against the spike (S) proteins of human coronaviruses (hCoV) after SARS-CoV-2 infection and vaccination. We found an 11 to 123-fold increase in antibodies binding to SARS-CoV and MERS-CoV as well as a 2 to 4-fold difference in antibodies binding to seasonal hCoVs in COVID-19 convalescent sera compared to pre-pandemic healthy donors, with the S2 subdomain of the S protein being the main target for cross-reactivity. In addition, we detected cross-reactive antibodies to all hCoV S proteins after SARS-CoV-2 S protein immunization in macaques, with higher responses for hCoV more closely related to SARS-CoV-2. These findings support the feasibility of and provide guidance for development of a pan-coronavirus vaccine.
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    Simultaneous evaluation of antibodies that inhibit SARS-CoV-2 RBD variants with a novel competitive multiplex assay
    Lopez, E ; Haycroft, E ; Adair, A ; Mordant, F ; O’Neill, M ; Pymm, P ; Redmond, S ; Gherardin, N ; Wheatley, A ; Juno, J ; Selva, K ; Davis, S ; Harty, L ; Purcell, DFJ ; Subbarao, K ; Godfrey, D ; Kent, S ; Tham, W-H ; Chung, A ( 2021-03-26)

    ABSTRACT

    The SARS-CoV-2 Receptor Binding Domain (RBD) is both the principal target of neutralizing antibodies, and one of the most rapidly evolving domains, which can result in the emergence of immune escape mutations limiting the effectiveness of vaccines and antibody therapeutics. To facilitate surveillance, we developed a rapid, high-throughput, multiplex assay able to assess the inhibitory response of antibodies to 24 RBD natural variants simultaneously. We demonstrate that immune escape can occur through two mechanisms, antibodies that fail to recognize mutations, along with antibodies that have reduced inhibitory capacity due to enhanced variant RBD-ACE2 affinity. A competitive approach where antibodies simultaneously compete with ACE2 for binding to the RBD may therefore more accurately reflect the physiological dynamics of infection. We describe the enhanced affinity of RBD variants N439K, S477N, Q493L, S494P and N501Y to the ACE2 receptor, and demonstrate the ability of this assay to bridge a major gap for SARS-CoV-2 research; informing selection of complementary monoclonal antibody candidates and the rapid identification of immune escape to emerging RBD variants following vaccination or natural infection.
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    Evaluation of serological tests for SARS-CoV-2: Implications for serology testing in a low-prevalence setting
    Bond, K ; Nicholson, S ; Ming Lim, S ; Karapanagiotidis, T ; Williams, E ; Johnson, D ; Hoang, T ; Sia, C ; Purcell, D ; R Lewin, S ; Catton, M ; P Howden, B ; A Williamson, D ( 2020-06-03)

    Background

    Robust serological assays are essential for long-term control of the COVID-19 pandemic. Many recently released point-of-care (PoCT) serological assays have been distributed with little pre-market validation.

    Methods

    Performance characteristics for five PoCT lateral flow devices approved for use in Australia were compared to a commercial enzyme immunoassay (ELISA) and a recently described novel surrogate virus neutralisation test (sVNT).

    Results

    Sensitivities for PoCT ranged from 51.8% (95% CI 43.1 to 60.4%) to 67.9% (95% CI 59.4–75.6%), and specificities from 95.6% (95% CI 89.2–98.8%) to 100.0% (95% CI 96.1–100.0%). Overall ELISA sensitivity for either IgA or IgG detection was 67.9% (95% CI 59.4–75.6), increasing to 93.8% (95% CI 85.0–98.3%) for samples > 14 days post symptom onset. Overall, sVNT sensitivity was 60.9% (95% CI 53.2–68.4%), rising to 91.2%% (95% CI 81.8–96.7%) for samples collected > 14 days post-symptom onset, with a specificity 94.4% (95% CI 89.2–97.5%),

    Conclusion

    Performance characteristics for COVID-19 serological assays were generally lower than those reported by manufacturers. Timing of specimen collection relative to onset of illness or infection is crucial in the reporting of performance characteristics for COVID-19 serological assays. The optimal algorithm for implementing serological testing for COVID-19 remains to be determined, particularly in low-prevalence settings.
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    Immune responses to SARS-CoV-2 in children of parents with symptomatic COVID-19
    Tosif, S ; Neeland, M ; Sutton, P ; Licciardi, P ; Sarkar, S ; Selva, K ; Do, LAH ; Donato, C ; Toh, ZQ ; Higgins, R ; de Sandt, CV ; Lemke, M ; Lee, C ; Shoffner, S ; Flanagan, K ; Arnold, K ; Mordant, F ; Mulholland, K ; Bines, J ; Dohle, K ; Pellicci, D ; Curtis, N ; McNab, S ; Steer, A ; Saffery, R ; Subbarao, K ; Chung, A ; Kedzierska, K ; Burgner, D ; Crawford, N ( 2020-07-28)
    Compared to adults, children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have mild or asymptomatic infection, but the underlying immunological differences remain unclear. We describe clinical features, virology, longitudinal cellular and cytokine immune profile, SARS-CoV-2-specific serology and salivary antibody responses in a family of two parents with PCR-confirmed symptomatic SARS-CoV-2 infection and their three children, who were repeatedly SARS-CoV-2 PCR negative. Cellular immune profiles and cytokine responses of all children were similar to their parents at all timepoints. All family members had salivary anti-SARS-CoV-2 antibodies detected, predominantly IgA, that coincided with symptom resolution in 3 of 4 symptomatic members. Plasma from both parents and one child had IgG antibody detected against the S1 protein and virus neutralising activity ranging from just detectable to robust titers. Using a systems serology approach, we show that all family members demonstrated higher levels of SARS-CoV-2-specific antibody features than healthy controls. These data indicate that children can mount an immune response to SARS-CoV-2 without virological evidence of infection. This raises the possibility that despite chronic exposure, immunity in children prevents establishment of SARS-CoV-2 infection. Relying on routine virological and serological testing may therefore not identify exposed children, with implications for epidemiological and clinical studies across the life-span.