Publications by authors named "Chinn Yi Wong"

17 Publications

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Systems serology detects functionally distinct coronavirus antibody features in children and elderly.

Nat Commun 2021 04 1;12(1):2037. Epub 2021 Apr 1.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, Australia.

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.
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http://dx.doi.org/10.1038/s41467-021-22236-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016934PMC
April 2021

Robust correlations across six SARS-CoV-2 serology assays detecting distinct antibody features.

Clin Transl Immunology 2021 28;10(3):e1258. Epub 2021 Feb 28.

Department of Microbiology and Immunology University of Melbourne, at the Peter Doherty Institute for Infection and Immunity Melbourne VIC Australia.

Objectives: As the world transitions into a new era of the COVID-19 pandemic in which vaccines become available, there is an increasing demand for rapid reliable serological testing to identify individuals with levels of immunity considered protective by infection or vaccination.

Methods: We used 34 SARS-CoV-2 samples to perform a rapid surrogate virus neutralisation test (sVNT), applicable to many laboratories as it circumvents the need for biosafety level-3 containment. We correlated results from the sVNT with five additional commonly used SARS-CoV-2 serology techniques: the microneutralisation test (MNT), in-house ELISAs, commercial Euroimmun- and Wantai-based ELISAs (RBD, spike and nucleoprotein; IgG, IgA and IgM), antigen-binding avidity, and high-throughput multiplex analyses to profile isotype, subclass and Fc effector binding potential. We correlated antibody levels with antibody-secreting cell (ASC) and circulatory T follicular helper (cTfh) cell numbers.

Results: Antibody data obtained with commercial ELISAs closely reflected results using in-house ELISAs against RBD and spike. A correlation matrix across ten measured ELISA parameters revealed positive correlations for all factors. The frequency of inhibition by rapid sVNT strongly correlated with spike-specific IgG and IgA titres detected by both commercial and in-house ELISAs, and MNT titres. Multiplex analyses revealed strongest correlations between IgG, IgG1, FcR and C1q specific to spike and RBD. Acute cTfh-type 1 cell numbers correlated with spike and RBD-specific IgG antibodies measured by ELISAs and sVNT.

Conclusion: Our comprehensive analyses provide important insights into SARS-CoV-2 humoral immunity across distinct serology assays and their applicability for specific research and/or diagnostic questions to assess SARS-CoV-2-specific humoral responses.
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http://dx.doi.org/10.1002/cti2.1258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916820PMC
February 2021

Integrated immune dynamics define correlates of COVID-19 severity and antibody responses.

Cell Rep Med 2021 Mar 5;2(3):100208. Epub 2021 Feb 5.

Department of Medicine, Central Clinical School, Monash University, Melbourne, VIC, Australia.

SARS-CoV-2 causes a spectrum of COVID-19 disease, the immunological basis of which remains ill defined. We analyzed 85 SARS-CoV-2-infected individuals at acute and/or convalescent time points, up to 102 days after symptom onset, quantifying 184 immunological parameters. Acute COVID-19 presented with high levels of IL-6, IL-18, and IL-10 and broad activation marked by the upregulation of CD38 on innate and adaptive lymphocytes and myeloid cells. Importantly, activated CXCR3cT1 cells in acute COVID-19 significantly correlate with and predict antibody levels and their avidity at convalescence as well as acute neutralization activity. Strikingly, intensive care unit (ICU) patients with severe COVID-19 display higher levels of soluble IL-6, IL-6R, and IL-18, and hyperactivation of innate, adaptive, and myeloid compartments than patients with moderate disease. Our analyses provide a comprehensive map of longitudinal immunological responses in COVID-19 patients and integrate key cellular pathways of complex immune networks underpinning severe COVID-19, providing important insights into potential biomarkers and immunotherapies.
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http://dx.doi.org/10.1016/j.xcrm.2021.100208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862905PMC
March 2021

TLR2-mediated activation of innate responses in the upper airways confers antiviral protection of the lungs.

JCI Insight 2021 Mar 8;6(5). Epub 2021 Mar 8.

Department of Microbiology and Immunology, the University of Melbourne, the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.

The impact of respiratory virus infections on global health is felt not just during a pandemic, but endemic seasonal infections pose an equal and ongoing risk of severe disease. Moreover, vaccines and antiviral drugs are not always effective or available for many respiratory viruses. We investigated how induction of effective and appropriate antigen-independent innate immunity in the upper airways can prevent the spread of respiratory virus infection to the vulnerable lower airways. Activation of TLR2, when restricted to the nasal turbinates, resulted in prompt induction of innate immune-driven antiviral responses through action of cytokines, chemokines, and cellular activity in the upper but not the lower airways. We have defined how nasal epithelial cells and recruitment of macrophages work in concert and play pivotal roles to limit progression of influenza virus to the lungs and sustain protection for up to 7 days. These results reveal underlying mechanisms of how control of viral infection in the upper airways can occur and support the implementation of strategies that can activate TLR2 in nasal passages to provide rapid protection, especially for at-risk populations, against severe respiratory infection when vaccines and antiviral drugs are not always effective or available.
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http://dx.doi.org/10.1172/jci.insight.140267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8021123PMC
March 2021

High antibody titres induced by protein subunit vaccines using antigens Hsp18 and MUL_3720 with a TLR-2 agonist fail to protect against Buruli ulcer in mice.

PeerJ 2020 7;8:e9659. Epub 2020 Aug 7.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia.

Background: is the causative agent of a debilitating skin and soft tissue infection known as Buruli ulcer (BU). There is no vaccine against BU. The purpose of this study was to investigate the vaccine potential of two previously described immunogenic proteins, MUL_3720 and Hsp18, using a mouse tail infection model of BU.

Methods: Recombinant versions of the two proteins were each electrostatically coupled with a previously described lipopeptide adjuvant. Seven C57BL/6 and seven BALB/c mice were vaccinated and boosted with each of the formulations. Vaccinated mice were then challenged with via subcutaneous tail inoculation. Vaccine performance was assessed by time-to-ulceration compared to unvaccinated mice.

Results: The MUL_3720 and Hsp18 vaccines induced high titres of antigen-specific antibodies that were predominately subtype IgG. However, all mice developed ulcers by day-40 post- challenge. No significant difference was observed in the time-to-onset of ulceration between the experimental vaccine groups and unvaccinated animals.

Conclusions: These data align with previous vaccine experiments using Hsp18 and MUL_3720 that indicated these proteins may not be appropriate vaccine antigens. This work highlights the need to explore alternative vaccine targets and different approaches to understand the role antibodies might play in controlling BU
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http://dx.doi.org/10.7717/peerj.9659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416718PMC
August 2020

Modular platforms for the assembly of self-adjuvanting lipopeptide-based vaccines for use in an out-bred population.

Vaccine 2020 01 15;38(3):597-607. Epub 2019 Nov 15.

Department of Microbiology and Immunology at the Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville 3010, Victoria, Australia.

To facilitate the preparation of synthetic epitope-based self-adjuvanting vaccines capable of eliciting antibody responses in an out-bred population, we have developed two modular approaches. In the first, the Toll-like receptor 2 agonist PamCys and the target antibody epitope are assembled as a module which is then coupled to a carrier protein as a source of antigens to stimulate T cell help. A vaccine candidate made in this way was shown to induce a specific immune response in four different strains of mice without the need for extraneous adjuvant. In the second approach, three vaccine components in the form of a target antibody epitope, a T helper cell epitope and PamCys, were prepared separately each carrying different chemical functional groups. By using pH-mediated chemo-selective ligations, the vaccine was assembled in a one-pot procedure. Using this approach, a number of vaccine constructs including a lipopeptide-protein conjugate were made and also shown to elicit immune responses in different strains of mice. These two modular approaches thus constitute a powerful platform for the assembly of self-adjuvanting lipopeptide-based vaccines that can potentially be used to induce robust antibody responses in an outbred population. Finally, our study of the impact of chemical linkages on immunogenicity of a lipopeptide vaccine shows that a stable covalent bond between Pam2Cys and a B cell epitope, rather than between Pam2Cys and T helper cell epitope is critical for the induction of antibody responses and biological efficacy, indicating that Pam2Cys functions not only as an adjuvant but also participates in processing and presentation of the immunogen.
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http://dx.doi.org/10.1016/j.vaccine.2019.10.055DOI Listing
January 2020

Geometry of a TLR2-Agonist-Based Adjuvant Can Affect the Resulting Antigen-Specific Immune Response.

Mol Pharm 2019 05 12;16(5):2037-2047. Epub 2019 Apr 12.

Department of Microbiology and Immunology , The University of Melbourne, at The Peter Doherty Institute for Infection and Immunity , 792 Elizabeth Street , Melbourne , Victoria 3010 , Australia.

Targeted delivery of otherwise nonimmunogenic antigens to Toll-like receptors (TLRs) expressed on dendritic cells (DCs) has proven to be an effective means of improving immunogenicity. For this purpose, we have used a branched cationic lipopeptide, RPamCys, which is an agonist for TLR2 and enables electrostatic association with antigen for this purpose. Here, we compare the immunological properties of ovalbumin formulated with different geometrical configurations of RPamCys. Our results demonstrate that notwithstanding the presence of the same adjuvant, branched forms of RPamCys are more effective at inducing immune responses than are linear geometries. CD8 T-cell-mediated responses are particularly improved, resulting in significantly higher levels of antigen-specific cytokine secretion and cytolysis of antigen-bearing target cells in vivo. The results correlate with the ability of branched RPamCys conformations to encourage higher levels of DC maturation and facilitate superior antigen uptake, leading to increased production of proinflammatory cytokines. These differences are not attributable to particle size because both branched and linear lipopeptides associate with antigen-forming complexes of similar size, but rather the ability of branched lipopeptides to induce more efficient TLR2-mediated cell signaling. Branched lipopeptides are also more resistant to trypsin-mediated proteolysis, suggesting greater stability than their linear counterparts. The branched lipopeptide facilitates presentation of antigen more efficiently to CD8 T cells, resulting in rapid cell division and upregulation of early cell surface activation markers. These results as well as cognate recognition of PamCys by TLR2 indicate that the adjuvant's efficiency is also dependent on its geometry.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00026DOI Listing
May 2019

Human CD8 T cell cross-reactivity across influenza A, B and C viruses.

Nat Immunol 2019 05 18;20(5):613-625. Epub 2019 Feb 18.

Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Parkville, Victoria, Australia.

Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally and infect humans, with IAV and IBV causing the most severe disease. CD8 T cells confer cross-protection against IAV strains, however the responses of CD8 T cells to IBV and ICV are understudied. We investigated the breadth of CD8 T cell cross-recognition and provide evidence of CD8 T cell cross-reactivity across IAV, IBV and ICV. We identified immunodominant CD8 T cell epitopes from IBVs that were protective in mice and found memory CD8 T cells directed against universal and influenza-virus-type-specific epitopes in the blood and lungs of healthy humans. Lung-derived CD8 T cells displayed tissue-resident memory phenotypes. Notably, CD38Ki67CD8 effector T cells directed against novel epitopes were readily detected in IAV- or IBV-infected pediatric and adult subjects. Our study introduces a new paradigm whereby CD8 T cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for the design of universal vaccines.
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http://dx.doi.org/10.1038/s41590-019-0320-6DOI Listing
May 2019

Tear film inflammatory cytokine upregulation in contact lens discomfort.

Ocul Surf 2019 01 13;17(1):89-97. Epub 2018 Oct 13.

Department of Optometry and Vision Sciences, Australia. Electronic address:

Purpose: To investigate the ocular inflammatory response, using clinical and immunological techniques, in people experiencing contact lens (CL) discomfort.

Methods: This study involved 38 adults who were full-time, silicone-hydrogel CL wearers. Participants were categorized into groups based upon a validated CL dry-eye questionnaire (CLDEQ-8) (n = 17 'asymptomatic', CLDEQ-8 score <9; n = 21 'symptomatic', CLDEQ-8 score ≥13). Examinations were performed at two visits (one with, and one without, CL wear), separated by one-week. Testing included: tear osmolarity, ocular redness, tear stability, ocular surface staining, meibography, tear production and tear collection. Tear osmolarity was taken from the inferior-lateral and superior-lateral menisci. The 'Inferior-Superior Osmotic Difference', I-SOD, was the absolute osmolarity difference between these menisci. Concentrations of seven cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-gamma, TNF-alpha) were assayed from basal tears using multiplex cytometric bead array.

Results: At baseline, there was no significant difference in key clinical signs between asymptomatic and symptomatic CL wearers (p > 0.05). The I-SOD was greater in symptomatic than asymptomatic CL wearers (23.1 ± 2.6 versus 11.3 ± 1.4 mOsmol/L, p = 0.001). People experiencing CL discomfort had higher tear IL-17A (122.6 ± 23.7 versus 44.0 ± 10.0 pg/mL, p = 0.02) and reduced tear stability (6.3 ± 1.1 versus 10.4 ± 1.6 s, p = 0.03) after several hours of CL wear. Tear IL-17A levels correlated with both the I-SOD (r = 0.43, p = 0.01) and CLDEQ-8 score (r = 0.40, p = 0.01).

Conclusions: CL discomfort occurs in individuals having no clinical dry eye signs, and is associated with higher tear levels of the pro-inflammatory cytokine IL-17A. These findings support an association between the discomfort response and low-grade, ocular surface inflammation.
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http://dx.doi.org/10.1016/j.jtos.2018.10.004DOI Listing
January 2019

Modulating Contact Lens Discomfort With Anti-Inflammatory Approaches: A Randomized Controlled Trial.

Invest Ophthalmol Vis Sci 2018 07;59(8):3755-3766

Department of Optometry and Vision Sciences, The University of Melbourne, Parkville, Victoria, Australia.

Purpose: To assess the efficacy of anti-inflammatory approaches, comprising a topical corticosteroid and omega-3 supplements, for modulating the inflammatory overlay associated with contact lens discomfort (CLD).

Methods: This randomized controlled trial involved 72 adults with CLD, randomized (1:1:1:1) to one of the following: placebo (oral olive oil), oral fish oil (900 mg/d eicosapentaenoic acid [EPA] + 600 mg/d docosohexaenoic acid [DHA]), oral combined fish+flaxseed oils (900 mg/d EPA + 600 mg/d DHA + 900 mg/d alpha-linolenic acid), or omega-3 eye-drops (0.025% EPA + 0.0025% DHA four times per day [qid]) for 12 weeks, with visits at baseline, weeks 4 and 12. At week 12, participants who received placebo were assigned a low-potency corticosteroid (fluorometholone [FML] 0.1%, drops, three times per day [tid]) for 2 weeks (week 14).

Results: Sixty-five participants completed the primary endpoint. At week 12, contact lens dry-eye questionnaire (CLDEQ-8) score was reduced from baseline with oral fish oil (-7.3 ± 0.8 units, n = 17, P < 0.05), compared with placebo (-3.5 ± 0.9 units, n = 16). FML produced significant reductions in tear IL-17A (-71.1 ± 14.3%, n = 12) and IL-6 (-47.6 ± 17.5%, n = 12, P < 0.05) relative to its baseline (week 12). At week 12, tear IL-17A levels were reduced from baseline in the oral fish oil (-63.2 ± 12.8%, n = 12, P < 0.05) and topical omega-3 (-76.2 ± 10.8%, n = 10, P < 0.05) groups, compared with placebo (-3.8 ± 12.7%, n = 12). Tear IL-6 was reduced with all omega-3 interventions, relative to placebo (P < 0.05) at week 12.

Conclusions: CLD was attenuated by oral long-chain omega-3 supplementation for 12 weeks. Acute (2 week) topical corticosteroids and longer-term (12 week) omega-3 supplementation reduced tear levels of the proinflammatory cytokines IL-17A and IL-6, demonstrating parallels in modulating ocular inflammation with these approaches.
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http://dx.doi.org/10.1167/iovs.18-24758DOI Listing
July 2018

PEGylation of a TLR2-agonist-based vaccine delivery system improves antigen trafficking and the magnitude of ensuing antibody and CD8 T cell responses.

Biomaterials 2017 Aug 11;137:61-72. Epub 2017 May 11.

Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, 3000, Australia; Research Center for Zoonosis Control, Hokkaido University, Sapporo, 001-0020, Japan; Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo, 001-0020, Japan. Electronic address:

The lipopeptide RPamCys is an agonist for toll-like receptor-2 (TLR2), a key pathogen-associated molecular pattern receptor expressed on many antigen-presenting cells such as dendritic cells (DCs). Electrostatic association of RPamCys with soluble protein antigens significantly enhances their immunogenicity and there is evidence to suggest that reducing the size of suitably adjuvanted-antigen complexes in solution may further improve their immunostimulatory capabilities. In this study, we investigated how incorporation of polyethylene glycol (PEG) into RPamCys affects the size, activity and efficacy of formed antigen-lipopeptide complexes. The presence of PEG was shown to increase solubility with a concomitant reduction in the particle size of vaccine formulations that was dependent on the length of PEG used. When compared to non-PEGylated RPamCys, vaccination of animals with antigen-complexed PEGylated RPamCys resulted not only in improvements in antibody production but significantly higher antigen-specific CD8 T cell responses. Both lipopeptides exhibited similar in vitro capabilities to induce DC maturation, facilitate antigen uptake and presentation to T cells. Moreover, analyses of the transcriptomes obtained from DCs treated with either lipopeptide revealed a large number of commonly induced genes with similar transcript expression levels, suggesting that common signalling pathways and processes were engaged following activation by either lipopeptide. In vivo analysis however revealed that vaccination with antigen-complexed PEGylated RPamCys resulted in improved antigen presentation to T cells. These heightened responses were not attributed to prolonged antigen persistence but rather due to more rapid transportation of antigen from the injection site into the draining lymph nodes over a short period of time. Our results indicate that reducing the size of formed antigen-TLR2-agonist complexes by PEGylation does not compromise the activity of the agonist but in fact enhances its trafficking in vivo ultimately leading to improved humoral and cell-mediated immune responses.
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http://dx.doi.org/10.1016/j.biomaterials.2017.05.018DOI Listing
August 2017

A Randomized, Double-Masked, Placebo-Controlled Clinical Trial of Two Forms of Omega-3 Supplements for Treating Dry Eye Disease.

Ophthalmology 2017 01 3;124(1):43-52. Epub 2016 Nov 3.

Department of Optometry and Vision Sciences, The University of Melbourne, Parkville, Australia. Electronic address:

Purpose: To assess the efficacy of 2 forms of oral long-chain omega-3 (ω-3) essential fatty acid (EFA) supplements, phospholipid (krill oil) and triacylglyceride (fish oil), for treating dry eye disease (DED).

Design: Randomized, double-masked, placebo-controlled clinical trial.

Participants: This study was conducted at a single site and involved 60 participants with mild to moderate DED who were randomized (1:1:1) to 1 of 3 groups: placebo (olive oil), krill oil, or fish oil supplements.

Methods: Participants received 1 of the 3 interventions: placebo (olive oil 1500 mg/day), krill oil (945 mg/day eicosapentaenoic acid [EPA], + 510 mg/day docosahexaenoic acid [DHA]), or fish oil (1000 mg/day EPA + 500 mg/day DHA) for 90 days, with monthly study visits.

Main Outcome Measures: Primary outcome measures were mean change in (1) tear osmolarity and (2) DED symptoms (Ocular Surface Disease Index [OSDI] score) between days 1 and 90. Secondary outcomes included mean change in key clinical signs (tear stability, tear production, ocular surface staining, bulbar and limbal redness, tear volume, anterior blepharitis, meibomian gland capping) and tear inflammatory cytokine levels.

Results: In total, 54 participants completed the study. At day 90, tear osmolarity was reduced from baseline with both krill oil (mean ± standard error of the mean: -18.6±4.5 mOsmol/l; n = 18; P < 0.001) and fish oil (-19.8±3.9 mOsmol/l; n = 19; P < 0.001) supplements, compared with placebo (-1.5±4.4 mOsmol/l; n = 17). OSDI score was significantly reduced at day 90 relative to baseline in the krill oil group only, compared with placebo (-18.6±2.4 vs. -10.5±3.3; P = 0.02). At day 90, there were also relative improvements in tear breakup time and ocular bulbar redness, compared with placebo, for both forms of ω-3 EFAs. Basal tear levels of the proinflammatory cytokine interleukin 17A were significantly reduced in the krill oil group, compared with placebo, at day 90 (-27.1±10.9 vs. 46.5±30.4 pg/ml; P = 0.02).

Conclusions: A moderate daily dose of both forms of long-chain ω-3 EFAs, for 3 months, resulted in reduced tear osmolarity and increased tear stability in people with DED. Omega-3 EFAs in a predominantly phospholipid form (krill oil) may confer additional therapeutic benefit, with improvements in DED symptoms and lower basal tear levels of interleukin 17A, relative to placebo.
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http://dx.doi.org/10.1016/j.ophtha.2016.09.023DOI Listing
January 2017

Tear Interferon-Gamma as a Biomarker for Evaporative Dry Eye Disease.

Invest Ophthalmol Vis Sci 2016 09;57(11):4824-4830

Department of Optometry and Vision Sciences, The University of Melbourne, Parkville, Victoria, Australia

Purpose: To assess whether tear hyperosmolarity, being diagnostic of dry eye disease (DED), is associated with specific alterations to the cytokine content of human tears that may provide a biomarker for DED.

Methods: In this prospective, cross-sectional, clinical study, participants (n = 77) were recruited from a single clinical site and categorized into groups based upon tear osmolarity status (n = 62 hyperosmolar, n = 15 normo-osmolar). Comprehensive anterior eye clinical assessments were undertaken. Concentrations of seven cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α) in basal tears were assayed using multiplex cytometric bead array. The main outcome measure was difference in cytokine concentration between groups. Group comparisons were undertaken using 2-tailed t-tests. Cohen's effect size was calculated for each finding. Spearman correlations between cytokine concentrations, clinical symptoms, and clinical parameters of DED were calculated.

Results: Tear hyperosmolarity was specifically associated with increased tear IFN-γ levels (13.3 ± 2.0 vs. 4.4 ± 1.4 pg/mL, P = 0.03). Cohen's effect size was large (0.8) for changes to tear IFN-γ levels. Significant correlations were observed between IFN-γ concentration and each of: tear osmolarity (r = 0.34; P = 0.007), total ocular surface staining (r = 0.56, P < 0.0001), and Schirmer test score (r = -0.33, P = 0.003).

Conclusions: Tear hyperosmolarity is specifically associated with higher levels of the proinflammatory cytokine IFN-γ, which correlate with key clinical parameters of DED. The calculated effect size (0.8) suggests that this assay has diagnostic power as a biomarker for evaporative DED.
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http://dx.doi.org/10.1167/iovs.16-19757DOI Listing
September 2016

Inactivated Influenza Vaccine That Provides Rapid, Innate-Immune-System-Mediated Protection and Subsequent Long-Term Adaptive Immunity.

mBio 2015 Oct 27;6(6):e01024-15. Epub 2015 Oct 27.

Department of Microbiology and Immunology, The University of Melbourne, at The Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo, Japan.

Unlabelled: The continual threat to global health posed by influenza has led to increased efforts to improve the effectiveness of influenza vaccines for use in epidemics and pandemics. We show in this study that formulation of a low dose of inactivated detergent-split influenza vaccine with a Toll-like receptor 2 (TLR2) agonist-based lipopeptide adjuvant (R4Pam2Cys) provides (i) immediate, antigen-independent immunity mediated by the innate immune system and (ii) significant enhancement of antigen-dependent immunity which exhibits an increased breadth of effector function. Intranasal administration of mice with vaccine formulated with R4Pam2Cys but not vaccine alone provides protection against both homologous and serologically distinct (heterologous) viral strains within a day of administration. Vaccination in the presence of R4Pam2Cys subsequently also induces high levels of systemic IgM, IgG1, and IgG2b antibodies and pulmonary IgA antibodies that inhibit hemagglutination (HA) and neuraminidase (NA) activities of homologous but not heterologous virus. Improved primary virus nucleoprotein (NP)-specific CD8(+) T cell responses are also induced by the use of R4Pam2Cys and are associated with robust recall responses to provide heterologous protection. These protective effects are demonstrated in wild-type and antibody-deficient animals but not in those depleted of CD8(+) T cells. Using a contact-dependent virus transmission model, we also found that heterologous virus transmission from vaccinated mice to naive mice is significantly reduced. These results demonstrate the potential of adding a TLR2 agonist to an existing seasonal influenza vaccine to improve its utility by inducing immediate short-term nonspecific antiviral protection and also antigen-specific responses to provide homologous and heterologous immunity.

Importance: The innate and adaptive immune systems differ in mechanisms, specificities, and times at which they take effect. The innate immune system responds within hours of exposure to infectious agents, while adaptive immunity takes several days to become effective. Here we show, by using a simple lipopeptide-based TLR2 agonist, that an influenza detergent-split vaccine can be made to simultaneously stimulate and amplify both systems to provide immediate antiviral protection while giving the adaptive immune system time to implement long-term immunity. Both types of immunity induced by this approach protect against vaccine-matched as well as unrelated virus strains and potentially even against strains yet to be encountered. Conferring dual functionality to influenza vaccines is beneficial for improving community protection, particularly during periods between the onset of an outbreak and the time when a vaccine becomes available or in scenarios in which mass vaccination with a strain to which the population is immunologically naive is imperative.
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http://dx.doi.org/10.1128/mBio.01024-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4626850PMC
October 2015

A totally synthetic lipopeptide-based self-adjuvanting vaccine induces neutralizing antibodies against heat-stable enterotoxin from enterotoxigenic Escherichia coli.

Vaccine 2012 Jul 23;30(32):4800-6. Epub 2012 May 23.

Department of Microbiology & Immunology, The University of Melbourne, Parkville 3010, Victoria, Australia.

ST-based lipopeptide vaccine candidates were constructed in which ST was chemically synthesized and folded into the correct conformation prior to ligation to a module containing a T-helper cell epitope (T(H)) and the Toll-like receptor 2 (TLR2) agonist, S-[2,3-bis(palmitoyloxy)propyl]cysteine (P2C). Two different chemistries, thioether-based and oxime-based, were then used to ligate ST to the lipidated T(H) epitope. The enterotoxic activity of synthetic ST and the ST-based lipopeptide vaccines was determined in mice followed by an evaluation of immunological efficacy. The importance of the fine detail in chemical composition used in vaccine design was demonstrated by the findings that (i) the oxime-based vaccine exhibited little or no toxicity but the thioether-based vaccine, exhibited residual toxicity in suckling mice, (ii) although each of the synthetic vaccines generated specific anti-ST antibodies, it was the low titer antibodies induced by the oxime-based vaccine that demonstrated better neutralizing activity suggesting that the chemical linkage also affects the specificity of antibodies, (iii) the geometric arrangement of ST within a vaccine can profoundly affect the specificity and biological function of the antibodies that are elicited, and (iv) the lipopeptide-based ST vaccine candidate assembled using oxime chemistry induced a better neutralizing antibody response to ST when administered by the mucosal (intranasal) route.
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http://dx.doi.org/10.1016/j.vaccine.2012.05.017DOI Listing
July 2012

A modular approach to assembly of totally synthetic self-adjuvanting lipopeptide-based vaccines allows conformational epitope building.

J Biol Chem 2011 Apr 14;286(15):12944-51. Epub 2011 Feb 14.

Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria 3010, Australia.

The technology described here allows the chemical synthesis of vaccines requiring correctly folded epitopes and that contain difficult or long peptide sequences. The final self-adjuvanting product promotes strong humoral and/or cell-mediated immunity. A module containing common components of the vaccine (T helper cell epitope and the adjuvanting lipid moiety S-[2,3-bis(palmitoyloxy)propyl]cysteine) was assembled to enable a plug and play approach to vaccine assembly. The inclusion within the module of a chemical group with chemical properties complementary and orthogonal to a chemical group present in the target epitope allowed chemoselective ligation of the two vaccine components. The heat-stable enterotoxin of enterotoxigenic Escherichia coli that requires strict conformational integrity for biological activity and the reproductive hormone luteinizing hormone-releasing hormone were used as the target epitopes for the antibody vaccines. An epitope from the acid polymerase of influenza virus was used to assemble a CD8(+) T cell vaccine. Evaluation of each vaccine candidate in animals demonstrated the feasibility of the approach and that the type of immune response required, viz. antibody or cytotoxic T lymphocyte, dictates the nature of the chemical linkage between the module and target epitope. The use of a thioether bond between the module and target epitope had little or no adverse effect on antibody responses, whereas the use of a disulfide bond between the module and target epitope almost completely abrogated the antibody response. In contrast, better cytotoxic T lymphocyte responses were obtained when a disulfide bond was used.
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http://dx.doi.org/10.1074/jbc.M111.227744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075641PMC
April 2011

Investigating the function of the putative mycolic acid methyltransferase UmaA: divergence between the Mycobacterium smegmatis and Mycobacterium tuberculosis proteins.

J Biol Chem 2008 Jan 15;283(3):1419-1427. Epub 2007 Nov 15.

Institut de Pharmacologie et Biologie Structurale (IPBS), Université Paul Sabatier (Toulouse III), 205 route de Narbonne, 31077 Toulouse Cedex, France; Department Mécanismes Moléculaires des Infections Mycobactériennes, Institut de Pharmacologie et Biologie Structurale (IPBS), Centre National de la Recherche Scientifique (Unité Mixte de Recherche 5089), 205 route de Narbonne, 31077 Toulouse Cedex, France. Electronic address:

Mycolic acids are major and specific lipid components of the cell envelope of mycobacteria that include the causative agents of tuberculosis and leprosy, Mycobacterium tuberculosis and Mycobacterium leprae, respectively. Subtle structural variations that are known to be crucial for both their virulence and the permeability of their cell envelope occur in mycolic acids. Among these are the introduction of cyclopropyl groups and methyl branches by mycolic acid S-adenosylmethionine-dependent methyltransferases (MA-MTs). While the functions of seven of the M. tuberculosis MA-MTs have been either established or strongly presumed nothing is known of the roles of the remaining umaA gene product and those of M. smegmatis MA-MTs. Mutants of the M. tuberculosis umaA gene and its putative M. smegmatis orthologue, MSMEG0913, were created. The lipid extracts of the resulting mutants were analyzed in detail using a combination of analytical techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proton nuclear magnetic resonance spectroscopy, and chemical degradation methods. The M. smegmatis mutants no longer synthesized subtypes of mycolates containing a methyl branch adjacent to either trans cyclopropyl group or trans double bond at the "proximal" position of both alpha- and epoxy-mycolates. Complementation with MSMEG0913, but not with umaA, fully restored the wild-type phenotype in M. smegmatis. Consistently, no modification was observed in the structures of mycolic acids produced by the M. tuberculosis umaA mutant. These data proved that despite their synteny and high similarity umaA and MSMEG0913 are not functionally orthologous.
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http://dx.doi.org/10.1074/jbc.M708859200DOI Listing
January 2008
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