Publications by authors named "Brendon Y Chua"

34 Publications

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
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

The impact of influenza pulmonary infection and inflammation on vagal bronchopulmonary sensory neurons.

FASEB J 2021 Mar;35(3):e21320

Department of Anatomy and Neuroscience, The University of Melbourne, Parkville, VIC, Australia.

Influenza A virus (IAV) is rapidly detected in the airways by the immune system, with resident parenchymal cells and leukocytes orchestrating viral sensing and the induction of antiviral inflammatory responses. The airways are innervated by heterogeneous populations of vagal sensory neurons which also play an important role in pulmonary defense. How these neurons respond to IAV respiratory infection remains unclear. Here, we use a murine model to provide the first evidence that vagal sensory neurons undergo significant transcriptional changes following a respiratory IAV infection. RNA sequencing on vagal sensory ganglia showed that IAV infection induced the expression of many genes associated with an antiviral and pro-inflammatory response and this was accompanied by a significant increase in inflammatory cell recruitment into the vagal ganglia. Assessment of gene expression in single-vagal sensory neurons confirmed that IAV infection induced a neuronal inflammatory phenotype, which was most prominent in bronchopulmonary neurons, and also evident in some neurons innervating other organs. The altered transcriptome could be mimicked by intranasal treatment with cytokines and the lung homogenates of infected mice, in the absence of infectious virus. These data argue that IAV pulmonary infection and subsequent inflammation induces vagal sensory ganglia neuroinflammation and this may have important implications for IAV-induced morbidity.
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http://dx.doi.org/10.1096/fj.202001509RDOI Listing
March 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 Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, 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

Prophylactic intranasal administration of a TLR2/6 agonist reduces upper respiratory tract viral shedding in a SARS-CoV-2 challenge ferret model.

EBioMedicine 2021 Jan 3;63:103153. Epub 2020 Dec 3.

National Infection Service, Public Health England (PHE), Porton Down, Salisbury, Wiltshire, United Kingdom SP4 0JG; Nuffield Dept of Medicine, Oxford University, Oxford, UK. Electronic address:

Background: The novel human coronavirus SARS-CoV-2 is a major ongoing global threat with huge economic burden. Like all respiratory viruses, SARS-CoV-2 initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission.

Methods: SARS-CoV-2 Victoria/01/2020 was passaged in Vero/hSLAM cells and virus titre determined by plaque assay. Challenge virus was delivered by intranasal instillation to female ferrets at 5.0 × 10 pfu/ml. Treatment groups received intranasal INNA-051, developed by Ena Respiratory. SARS-CoV-2 RNA was detected using the 2019-nCoV CDC RUO Kit and QuantStudio™ 7 Flex Real-Time PCR System. Histopathological analysis was performed using cut tissues stained with haematoxylin and eosin (H&E).

Findings: We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. After 5 days post-exposure to SARS-CoV-2, INNA-051 significantly reduced virus in throat swabs (p=<0.0001) by up to a 24 fold (96% reduction) and in nasal wash (p=0.0107) up to a 15 fold (93% reduction) in comparison to untreated animals.

Interpretation: The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT, to reduce SARS-CoV-2 transmission and provide protection against COVID-19.

Funding: This work was funded by Ena Respiratory, Melbourne, Australia.
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http://dx.doi.org/10.1016/j.ebiom.2020.103153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711201PMC
January 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

Immune profiling of influenza-specific B- and T-cell responses in macaques using flow cytometry-based assays.

Immunol Cell Biol 2021 Jan 7;99(1):97-106. Epub 2020 Sep 7.

Department of Microbiology and Immunology, University of Melbourne, Peter Doherty Institute for Infection and Immunity, Parkville, VIC, 3010, Australia.

Influenza remains a significant global public health burden, despite substantial annual vaccination efforts against circulating virus strains. As a result, novel vaccine approaches are needed to generate long-lasting and universal broadly cross-reactive immunity against distinct influenza virus strains and subtypes. Several new vaccine candidates are currently under development and/or in clinical trials. The successful development of new vaccines requires testing in animal models, other than mice, which capture the complexity of the human immune system. Importantly, following vaccination or challenge, the assessment of adaptive immunity at the antigen-specific level is particularly informative. In this study, using peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques, we describe detection methods and in-depth analyses of influenza virus-specific B cells by recombinant hemagglutinin probes and flow cytometry, as well as the detection of influenza virus-specific CD8 and CD4 T cells by stimulation with live influenza A virus and intracellular cytokine staining. We highlight the potential of these assays to be used with PBMCs from other macaque species, including rhesus macaques, pigtail macaques and African green monkeys. We also demonstrate the use of a human cytometric bead array kit in detecting inflammatory cytokines and chemokines from cynomolgus macaques to assess cytokine/chemokine milieu. Overall, the detection of influenza virus-specific B and T cells, together with inflammatory responses, as described in our study, provides useful insights for evaluating novel influenza vaccines. Our data deciphering immune responses toward influenza viruses can be also adapted to understanding immunity to other infections or vaccination approaches in macaque models.
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http://dx.doi.org/10.1111/imcb.12383DOI Listing
January 2021

Vaccine-Specific Immune Responses against Mycobacterium ulcerans Infection in a Low-Dose Murine Challenge Model.

Infect Immun 2020 02 20;88(3). Epub 2020 Feb 20.

Department of Microbiology and Immunology, Doherty Institute, University of Melbourne, Melbourne, Victoria, Australia

The neglected tropical disease Buruli ulcer (BU) is an infection of subcutaneous tissue with There is no effective vaccine. Here, we assessed an experimental prime-boost vaccine in a low-dose murine tail infection model. We used the enoyl reductase (ER) domain of the mycolactone polyketide synthases electrostatically coupled with a previously described Toll-like receptor 2 (TLR-2) agonist-based lipopeptide adjuvant, RPamCys. Mice were vaccinated and then challenged via tail inoculation with 14 to 20 CFU of a bioluminescent strain of Mice receiving either the experimental ER vaccine or bacillus Calmette-Guérin (BCG) were equally protected, with both groups faring significantly better than nonvaccinated animals (0.05). To explore potential correlates of protection, a suite of 29 immune parameters were assessed in the mice at the end of the experimental period. Multivariate statistical approaches were used to interrogate the immune response data to develop disease-prognostic models. High levels of interleukin 2 (IL-2) and low gamma interferon (IFN-γ) produced in the spleen best predicted control of infection across all vaccine groups. Univariate logistic regression revealed vaccine-specific profiles of protection. High titers of ER-specific IgG serum antibodies together with IL-2 and IL-4 in the draining lymph node (DLN) were associated with protection induced by the ER vaccine. In contrast, high titers of IL-6, tumor necrosis factor alpha (TNF-α), IFN-γ, and IL-10 in the DLN and low IFN-γ titers in the spleen were associated with protection following BCG vaccination. This study suggests that an effective BU vaccine must induce localized, tissue-specific immune profiles with controlled inflammatory responses at the site of infection.
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http://dx.doi.org/10.1128/IAI.00753-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035934PMC
February 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

In Vivo Imaging of Bioluminescent : A Tool to Refine the Murine Buruli Ulcer Tail Model.

Am J Trop Med Hyg 2019 12;101(6):1312-1321

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

Buruli ulcer (BU) is a neglected tropical disease caused by infection with . Unclear transmission, no available vaccine, and suboptimal treatment regimens hamper the control of this disease. Carefully designed preclinical research is needed to address these shortcomings. In vivo imaging (IVIS, Perkin Elmer, Waltham, MA) of infection is an emerging tool that permits monitoring of disease progression and reduces the need to using large numbers of mice at different time-points during the experiment, as individual mice can be imaged at multiple time-points. We aimed to further describe the use of in vivo imaging (IVIS) in BU. We studied the detection of in experimentally infected BALB/c mouse tails and the subsequent histopathology and immune response in this pilot study. IVIS-monitoring was performed weekly in ten infected BALB/c mice to measure light emitted as a proxy for bacterial load. Nine of 10 (90%) BALB/c mice infected subcutaneously with 3.3 × 10 JKD8049 (containing pMV306 hsp16+luxG13) exhibited light emission from the site of infection, indicating growth in vivo, whereas only five of 10 (50%) animals developed clinical signs of the disease. Specific antibody titers were detected within 2 weeks of the infection. Interferon (IFN)-γ and interleukin (IL)-10 were elevated in animals with pathology Histopathology revealed clusters of acid-fast bacilli in the subcutaneous tissue, with macrophage infiltration and granuloma formation resembling human BU. Our study successfully showed the utility of IVIS monitoring and lays a foundation for further research.
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http://dx.doi.org/10.4269/ajtmh.18-0959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896889PMC
December 2019

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

Opinion: Making Inactivated and Subunit-Based Vaccines Work.

Viral Immunol 2018 03 25;31(2):150-158. Epub 2018 Jan 25.

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

Empirically derived vaccines have in the past relied on the isolation and growth of disease-causing microorganisms that are then inactivated or attenuated before being administered. This is often done without prior knowledge of the mechanisms involved in conferring protective immunity. Recent advances in scientific technologies and in our knowledge of how protective immune responses are induced enable us to rationally design novel and safer vaccination strategies. Such advances have accelerated the development of inactivated whole-organism- and subunit-based vaccines. In this review, we discuss ideal attributes and criteria that need to be considered for the development of vaccines and some existing vaccine platforms. We focus on inactivated vaccines against influenza virus and ways by which vaccine efficacy can be improved with the use of adjuvants and Toll-like receptor-2 signaling.
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http://dx.doi.org/10.1089/vim.2017.0146DOI Listing
March 2018

Structure-function relationships of protein-lipopeptide complexes and influence on immunogenicity.

Amino Acids 2017 10 17;49(10):1691-1704. Epub 2017 Jul 17.

Department of Microbiology and Immunology, The University of Melbourne, at The Peter Doherty Institute for Infection and Immunity, 792 Elizabeth St, Melbourne, VIC, 3000, Australia.

The lipopeptide, RPamCys, associates electrostatically with soluble protein antigens and significantly enhances their ability to induce protective humoral and cell-mediated responses. We demonstrate that antibody titers elicited by the antigen ovalbumin (OVA) associated with RPamCys are higher than those elicited by OVA in the presence of alum and comparable to those elicited by OVA formulated with complete Freund's adjuvant (CFA). The hierarchy of anti-OVA antibody avidities was CFA > RPamCys = alum. Each of the three adjuvants facilitated IgG class-switching with significantly more IgG1 elicited by OVA when formulated with RPamCys. The effects of substituting naturally occurring L-stereoisomers of the cationic residues within RPamCys with D-stereoisomers revealed that substitution did not affect the ability of RPamCys to stimulate dendritic cell maturation or its ability to elicit antibody production when used as an adjuvant. Minor detrimental effects were, however, observed in the ensuing CD8 T cell responses suggesting that the use of D-amino acids affects antigen processing and presentation pathways involved in generation of cell-mediated immunity at least when facilitated through TLR2. Both D- and L-forms were found to be resistant to digestion by trypsin, indicating resistance of the branched structure to protease activity.
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http://dx.doi.org/10.1007/s00726-017-2466-6DOI Listing
October 2017

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

Competition within the virus-specific CD4 T-cell pool limits the T follicular helper response after influenza infection.

Immunol Cell Biol 2016 09 22;94(8):729-40. Epub 2016 Apr 22.

Department of Microbiology and Immunology, The Peter Doherty Institute, The University of Melbourne, Parkville, Victoria, Australia.

CD4 T follicular helper cells (TFH) are critical in the generation of potent and long-lived B-cell responses after viral infection. However, the factors that dictate the generation and maintenance of these cells are not fully understood. Here we use adoptive transfer of OTII T-cell receptor transgenic CD4 T cells, followed by infection with recombinant influenza A virus (IAV), as a means of identifying and tracking virus-specific CD4(+) T-cell responses. We show that T-cell competition within the virus-specific CD4 T-cell pool induced by IAV infection limits the proliferation and differentiation of IAV-specific CD4(+) TFH responses. In particular, increased T-cell competition for antigen results in a diminished IAV-specific TFH CD4 T-cell responses, particularly germinal center TFH responses. Strikingly, competition in the form of preexisting cellular immunity generated by heterosubtypic IAV immunization limits de novo CD4 T-cell responses in secondary lymphoid tissue. Taken together, these data show a profound linkage between antigen availability and promotion of TFH CD4(+) T-cell responses in response to infection. These data suggest that competition within the CD4 T-cell pool limits TFH responses and may be an important regulatory mechanism for controlling immunity.
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http://dx.doi.org/10.1038/icb.2016.42DOI 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

Establishment of functional influenza virus-specific CD8(+) T cell memory pools after intramuscular immunization.

Vaccine 2015 Sep 13;33(39):5148-54. Epub 2015 Aug 13.

Department of Microbiology and Immunology at The Peter Doherty Institute for Infection & Immunity, The University of Melbourne, Royal Parade, Parkville, Victoria, 3010, Australia. Electronic address:

The emergence of the avian-origin influenza H7N9 virus and its pandemic potential has highlighted the ever-present need to develop vaccination approaches to induce cross-protective immunity. In this study, we examined the establishment of cross-reactive CD8(+) T cell immunity in mice following immunization with live A/Puerto Rico/8/1934 (PR8; H1N1) influenza virus via two non-productive inoculation routes. We found that immunization via the intramuscular (IM) route established functional influenza-virus specific memory CD8(+) T cell pools capable of cross-reactive recall responses. Epitope-specific primary, memory and recall CD8(+) T-cell responses induced by the IM route, highly relevant to human influenza immunisations, were of comparable magnitude and quality to those elicited by the intraperitoneal (IP) priming, commonly used in mice. Furthermore, IM immunisation resulted in lower lung viral titres following heterologous challenge with A/Aichi/68 (X31; H3N2) compared to the IP route. Examining the ability of DCs from lymphoid organs to present viral antigen revealed that immune induction following IM immunization occurred in draining lymph nodes, while immunization via the IP route resulted in the priming of responses in distal lymphoid organs, indicative of a systemic distribution of antigen. No major differences in the pulmonary cytokine environment of immunized animals following X31 challenge were observed that could account for the improved heterologous protection induced by the IM route. However, while both routes induced similar levels of PR8-specific antibodies, higher levels of cross-reactive antibodies against X31 were induced following IM inoculation. Our data demonstrate how non-replicative routes of infection can induce efficient cross-reactive CD8(+) T cell responses and strong strain-specific antibody responses, with the additional benefit from IM priming of enhanced heterosubtypic antibody production.
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http://dx.doi.org/10.1016/j.vaccine.2015.08.001DOI Listing
September 2015

Reducing the impact of influenza-associated secondary pneumococcal infections.

Immunol Cell Biol 2016 Jan 21;94(1):101-8. Epub 2015 Jul 21.

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

When administered prophylactically, we show that the Toll-like receptor-2 (TLR-2) agonist PEG-Pam2Cys (pegylated-S-(2,3-bis(palmitoyloxy)propyl)cysteine) not only mediates potent anti-viral activity against influenza virus but also reduces the impact of secondary infections with Streptococcus pneumoniae (the pneumococcus) by reducing (i) pulmonary viral and bacterial burdens, (ii) the levels of proinflammatory cytokines that normally accompany influenza and S. pneumoniae secondary infections and (iii) the vascular permeability of the pulmonary tract that can allow bacterial invasion of the blood in mice. We also show that an inactivated detergent-disrupted influenza virus vaccine formulated with the Pam2Cys-based adjuvant R4-Pam2Cys provides the host with both immediate and long-term protection against secondary pneumococcal infections following influenza virus infection through innate and specific immune mechanisms, respectively. Vaccinated animals generated influenza virus-specific immune responses that provided the host with long-term protection against influenza virus and its sequelae. This vaccine, which generates an immediate response, provides an additional countermeasure, which is ideal for use even in the midst of an influenza outbreak.
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http://dx.doi.org/10.1038/icb.2015.71DOI Listing
January 2016

Oseltamivir Prophylaxis Reduces Inflammation and Facilitates Establishment of Cross-Strain Protective T Cell Memory to Influenza Viruses.

PLoS One 2015 18;10(6):e0129768. Epub 2015 Jun 18.

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

CD8(+) T cells directed against conserved viral regions elicit broad immunity against distinct influenza viruses, promote rapid virus elimination and enhanced host recovery. The influenza neuraminidase inhibitor, oseltamivir, is prescribed for therapy and prophylaxis, although it remains unclear how the drug impacts disease severity and establishment of effector and memory CD8(+) T cell immunity. We dissected the effects of oseltamivir on viral replication, inflammation, acute CD8(+) T cell responses and the establishment of immunological CD8(+) T cell memory. In mice, ferrets and humans, the effect of osteltamivir on viral titre was relatively modest. However, prophylactic oseltamivir treatment in mice markedly reduced morbidity, innate responses, inflammation and, ultimately, the magnitude of effector CD8(+) T cell responses. Importantly, functional memory CD8(+) T cells established during the drug-reduced effector phase were capable of mounting robust recall responses. Moreover, influenza-specific memory CD4(+) T cells could be also recalled after the secondary challenge, while the antibody levels were unaffected. This provides evidence that long-term memory T cells can be generated during an oseltamivir-interrupted infection. The anti-inflammatory effect of oseltamivir was verified in H1N1-infected patients. Thus, in the case of an unpredicted influenza pandemic, while prophylactic oseltamivir treatment can reduce disease severity, the capacity to generate memory CD8(+) T cells specific for the newly emerged virus is uncompromised. This could prove especially important for any new influenza pandemic which often occurs in separate waves.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0129768PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4473273PMC
April 2016

A single dose biodegradable vaccine depot that induces persistently high levels of antibody over a year.

Biomaterials 2015 Jun 9;53:50-7. Epub 2015 Mar 9.

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

In this study, we describe a biodegradable vaccine depot which persists in vivo for at least 4-months, provides synergistic adjuvant effects and also allows dose sparing of both antigen and adjuvant. A single administration results in immediate release of a priming dose of vaccine, by a process of syneresis, which is then followed by release of remaining vaccine which maintains robust antibody levels that last for more than a year. The platform technology comprises two aqueous components; one contains chitosan and hydroxyapatite, in which the vaccine is incorporated, and the other consists of a crosslinking agent, tripolyphosphate (TPP) and chondroitin sulphate. When co-injected into tissue, they spontaneously crosslink forming a firm yet compliant vaccine-containing depot. Whole body imaging of animals inoculated with the material show that the depot persists in situ for up to 19 weeks. Vaccination of mice with depot formulations containing ovalbumin (OVA) emulsified in Montanide ISA 61 adjuvant results in the induction of robust antibody responses using doses of adjuvant 40-fold less than those recommended by the manufacturer. Dose sparing effects were also apparent with antigen when delivered in the depot. Similar dose sparing effects were observed with Montanide ISA 50, complete and incomplete Freund's adjuvants but not with aluminium hydroxide nor Quil A. Antibody titres, induced by a single dose of antigen/adjuvant formulation incorporated in the depot, persisted at high levels for at least 55 weeks following a single dose of vaccine.
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http://dx.doi.org/10.1016/j.biomaterials.2015.02.066DOI Listing
June 2015

Considerations for the rapid deployment of vaccines against H7N9 influenza.

Expert Rev Vaccines 2014 Nov 12;13(11):1327-37. Epub 2014 Jul 12.

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

The threat of an outbreak of avian-origin influenza H7N9 and the devastating consequences that a pandemic could have on global population health and economies has mobilized programs of constant surveillance and the implementation of preemptive plans. Central to these plans is the production of prepandemic vaccines that can be rapidly deployed to minimize disease severity and deaths resulting from such an occurrence. In this article, we review current H7N9 vaccine strategies in place and the available technologies and options that can help accelerate vaccine production and increase dose-sparing capabilities to provide enough vaccines to cover the population. We also present possible means of reducing disease impact during the critical period after an outbreak occurs before a strain matched vaccine becomes available and consider the use of existing stockpiles and seed strains of phylogenetically related subtypes, alternate vaccination regimes and vaccine forms that induce cross-reactive immunity.
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http://dx.doi.org/10.1586/14760584.2014.938641DOI Listing
November 2014

The use of a TLR2 agonist-based adjuvant for enhancing effector and memory CD8 T-cell responses.

Immunol Cell Biol 2014 Apr 7;92(4):377-83. Epub 2014 Jan 7.

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

We have previously shown that the immunogenicity of protein antigens can be significantly enhanced if electrostatically associated with the Toll-like receptor-2 agonist-based lipopeptide R4Pam2Cys. The precise mechanisms and effectiveness of the cytotoxic T-lymphocyte (CTL)-mediated response facilitated by this agonist, however, have not been studied. Here we show that priming by dendritic cells (DCs) in the draining lymph nodes of animals vaccinated with antigen delivered using R4Pam2Cys results in significantly improved T-cell proliferation and induces their differentiation into polyfunctional effector CTLs characterised by granzyme B expression and the ability to secrete interferon-γ, interleukin-2 and tumor necrosis factor-α 7 days after vaccination. After 30 days, frequencies of antigen-specific CD62(low)CD127(high) (effector memory), CD62(high)CD127(high) (central memory) and CD43(low)CD27(high) CD8(+) T cells, a phenotype associated with strong recall responses against respiratory infections, are also increased compared with responses obtained with antigens formulated in the adjuvants Alum (alhydrogel) and CFA (complete Freund's adjuvant). The phenotypic changes observed in these mice vaccinated using R4Pam2Cys further correlated with their ability to recall specific T cells into the lung to mediate the reduction of pulmonary viral titres following challenge with a chimeric influenza virus containing the K(b)OVA257-264 epitope compared with animals vaccinated using Alum or CFA. The findings from this study not only demonstrate that better T-cell responses can be elicited using R4Pam2Cys compared with classically utilised adjuvants but also highlight the potential effectiveness of this lipopeptide-based adjuvant particularly against viral infections that require resolution through cell-mediated immunity.
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http://dx.doi.org/10.1038/icb.2013.102DOI Listing
April 2014

Hepatitis C VLPs delivered to dendritic cells by a TLR2 targeting lipopeptide results in enhanced antibody and cell-mediated responses.

PLoS One 2012 16;7(10):e47492. Epub 2012 Oct 16.

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

Although many studies provide strong evidence supporting the development of HCV virus-like particle (VLP)-based vaccines, the fact that heterologous viral vectors and/or multiple dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity. In this study, we have evaluated the use of an anionic self-adjuvanting lipopeptide containing the TLR2 agonist Pam(2)Cys (E(8)Pam(2)Cys) to enhance the immunogenicity of VLPs containing the HCV structural proteins (core, E1 and E2) of genotype 1a. While co-formulation of this lipopeptide with VLPs only resulted in marginal improvements in dendritic cell (DC) uptake, its ability to concomitantly induce DC maturation at very small doses is a feature not observed using VLPs alone or in the presence of an aluminium hydroxide-based adjuvant (Alum). Dramatically improved VLP and E2-specific antibody responses were observed in VLP+E(8)Pam(2)Cys vaccinated mice where up to 3 doses of non-adjuvanted or traditionally alum-adjuvanted VLPs was required to match the antibody titres obtained with a single dose of VLPs formulated with this lipopeptide. This result also correlated with significantly higher numbers of specific antibody secreting cells that was detected in the spleens of VLP+E(8)Pam(2)Cys vaccinated mice and greater ability of sera from these mice to neutralise the binding and uptake of VLPs by Huh7 cells. Moreover, vaccination of HLA-A2 transgenic mice with this formulation also induced better VLP-specific IFN-γ-mediated responses compared to non-adjuvanted VLPs but comparable levels to that achieved when coadministered with complete freund's adjuvant. These results suggest overall that the immunogenicity of HCV VLPs can be significantly improved by the addition of this novel adjuvant by targeting their delivery to DCs and could therefore constitute a viable vaccine strategy for the treatment of HCV.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047492PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3472981PMC
March 2013

Control of size dispersity of chitosan biopolymer microparticles and nanoparticles to influence vaccine trafficking and cell uptake.

J Biomed Mater Res A 2012 Jul 4;100(7):1859-67. Epub 2012 Apr 4.

School of Applied Sciences, Royal Melbourne Institute of Technology, Melbourne 3000, Australia.

Structurally related surfactant molecules were exploited to generate chitosan emulsions to provide systematic variation in micelle radii of curvature and size. These compositions provide precise control of chitosan particle dispersity, that is, size distribution according to three quantitative distribution parameters as well as shape distribution. This resulted in a suite of particle size distributions spanning 71 nm to 3.7 μm and a very high degree of particle sphericity, allowing the influence of particle size to be isolated in two in vivo studies relating biopolymer particle size to cellular uptake and trafficking to lymph nodes. Flow cytometry and fluorescence microscopy indicated that the three cell lines examined preferentially internalized chitosan microparticles to a greater extent than nanoparticles over a 24 h period. In an in vivo mouse model, nanoparticles initially trafficked rapidly to lymph nodes draining the site of inoculation followed by further slower uptake. Microparticles trafficked to the lymph nodes with a similar pattern except that the initial discharge was ∼50-fold less than that observed with nanoparticles indicating a profound difference in the physiological transport properties of the two particle types.
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http://dx.doi.org/10.1002/jbm.a.34153DOI Listing
July 2012

Chitosan microparticles and nanoparticles as biocompatible delivery vehicles for peptide and protein-based immunocontraceptive vaccines.

Mol Pharm 2012 Jan 16;9(1):81-90. Epub 2011 Dec 16.

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

It has become increasingly recognized that polymer particle size can have a profound effect on the interactions of particle-based vaccines with antigen presenting cells (APCs) thereby influencing and modulating ensuing immune responses. With the aim of developing chitosan particle-based immunocontraceptive vaccines, we have compared the use of chitosan-based nanoparticles and chitosan-based microparticles as vaccine delivery vehicles for vaccine candidates based on luteinizing hormone-releasing hormone (LHRH). Particles, functionalized with chloroacetyl groups, which allows the covalent attachment of thiol-containing antigens, were able to adsorb ~60-70% of their weight of peptide-based antigen and 10-20% of their weight of protein-based antigen. Quantitation by amino acid analysis of antigen associated with particles demonstrated a correlation between associated antigen and the degree of chloracetylation of particles. Visualization of fluorescently labeled antigen-loaded particles by confocal microscopy indicated that the majority of antigen was localized at the particle surface with a smaller amount located in the interior. We also found that uptake of both fluoresceinated nanoparticles and microparticles by dendritic cells occurred in a manner dependent on particle concentration. Nanoparticles trafficked from the injection site to draining lymph nodes faster than microparticles; high numbers of nanoparticle-bearing cells appeared in draining lymph nodes on day 3 and microparticles on day 4. This difference in trafficking rate did not, however, appear to have any significant impact on the ensuing immune response because inoculation with both peptide-conjugated and protein-conjugated particles induced high levels of LHRH-specific antibodies. In the case of protein-conjugated particles, the levels of antibodies elicited were similar to those elicited following inoculation with antigen emulsified with complete Freund's adjuvant. The approach to vaccine design that we have described here could represent another useful method for inducing immune responses against microbial, viral and tumorigenic protein antigens.
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http://dx.doi.org/10.1021/mp200264mDOI Listing
January 2012

Soluble proteins induce strong CD8+ T cell and antibody responses through electrostatic association with simple cationic or anionic lipopeptides that target TLR2.

J Immunol 2011 Aug 8;187(4):1692-701. Epub 2011 Jul 8.

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

The low immunogenicity exhibited by most soluble proteins is generally due to the absence of molecular signatures that are recognized by the immune system as dangerous. In this study, we show that electrostatic binding of synthetic branched cationic or anionic lipopeptides that contain the TLR-2 agonist Pam(2)Cys markedly enhance a protein's immunogenicity. Binding of a charged lipopeptide to oppositely charged protein Ags resulted in the formation of stable complexes and occurs at physiologic pH and salt concentrations. The induction of cell-mediated responses is dependent on the electrostatic binding of lipopeptide to the protein, with no CD8(+) T cells being elicited when protein and lipopeptide possessed the same electrical charge. The CD8(+) T cells elicited after vaccination with lipopeptide-protein Ag complexes produced proinflammatory cytokines, exhibited in vivo lytic activity, and protected mice from challenge with an infectious chimeric influenza virus containing a single OVA epitope as part of the influenza neuraminidase protein. Induction of a CD8(+) T cell response correlated with the ability of lipopeptide to facilitate Ag uptake by DCs followed by trafficking of Ag-bearing cells into draining lymph nodes. Oppositely charged but not similarly charged lipopeptides were more effective in DC uptake and trafficking. Very high protein-specific Ab titers were also achieved by vaccination with complexes composed of oppositely charged lipopeptide and protein, whereas vaccination with similarly charged constituents resulted in significant but lower Ab titers. Regardless of whether similarly or oppositely charged lipopeptides were used in the induction of Ab, vaccination generated dominant IgG1 isotype Abs rather than IgG2a.
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http://dx.doi.org/10.4049/jimmunol.1100486DOI Listing
August 2011

Synthesis of toll-like receptor-2 targeting lipopeptides as self-adjuvanting vaccines.

Methods Mol Biol 2008 ;494:247-61

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

Effective Th1- and Th2-type immune responses that result in protective immunity against pathogens can be induced by self-adjuvanting lipopeptides containing the lipid moiety dipalmitoyl-S-glyceryl cysteine (Pam2Cys). The potent immunogenicity of these lipopeptides is due to their ability to activate dendritic cells by targeting and signaling through Toll-like receptor-2 (TLR-2). In addition, the simplicity and flexibility in their design as well as their ease of chemical definition and characterisation makes them highly attractive vaccine candidates for humans and animals. We describe in this chapter the techniques involved in the synthesis of an immunocontraceptive lipopeptide vaccine as well as the experimental assays carried out to evaluate its efficiency.
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http://dx.doi.org/10.1007/978-1-59745-419-3_14DOI Listing
November 2008

A self-adjuvanting lipopeptide-based vaccine candidate for the treatment of hepatitis C virus infection.

Vaccine 2008 Sep 7;26(37):4866-75. Epub 2008 Apr 7.

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

Effective CD8(+) T cell responses have been induced using totally synthetic self-adjuvanting lipopeptides containing the dipalmitoyl-S-glyceryl cysteine lipid moiety, which is a ligand for Toll-like receptor 2 (TLR2) on dendritic cells (DC). In this study, we evaluated the use of lipopeptide vaccine candidates containing HLA-A2-restricted epitopes for DC-based immunotherapy of HCV infection. Lipopeptides were able to induce specific CD8(+) T cell responses in HLA-A2 transgenic mice and consistently activated human monocyte-derived DC from both healthy individuals and HCV infected patients. Lipopeptide-pulsed human DC were also found to secrete the pro-inflammatory cytokine IL-12p70 and were able to activate antigen-specific IFN-gamma production by autologous CD8(+) T cells obtained from a hepatitis C patient. These results show that DC from HCV patients can be matured and antigen loaded with TLR2-targeting lipopeptides for effective presentation of CD8(+) T cell epitopes; the use of autologous lipopeptide-pulsed DC or direct lipopeptide vaccination may be successful approaches for the priming or boosting of anti-HCV CD8(+) T cell responses to aid in the clearance of the virus in chronically infected individuals.
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http://dx.doi.org/10.1016/j.vaccine.2008.03.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526279PMC
September 2008