Publications by authors named "Weiguang Zeng"

65 Publications

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

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

The Toll-Like Receptor 2 agonist PEG-PamCys as an immunochemoprophylactic and immunochemotherapeutic against the liver and transmission stages of malaria parasites.

Int J Parasitol Drugs Drug Resist 2018 12 23;8(3):451-458. Epub 2018 Oct 23.

Malaria Unit, Department of Pathology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki, 852-8523, Japan. Electronic address:

Both vaccine and therapeutic approaches to malaria are based on conventional paradigms; whole organism or single antigen epitope-based vaccines administered with or without an adjuvant, and chemotherapeutics (anti-malaria drugs) that are toxic to the parasite. Two major problems that limit the effectiveness of these approaches are i) high levels of antigenic variation within parasite populations rendering vaccination efficacy against all variants difficult, and ii) the capacity of the parasite to quickly evolve resistance to drugs. We describe a new approach to both protection from and treatment of malaria parasites that involves the direct stimulation of the host innate immune response through the administration of a Toll-Like Receptor-2 (TLR2) agonist. The activity of PEG-PamCys against the hepatocytic stages, erythrocytic stages and gametocytes of the rodent malaria parasite Plasmodium yoelii was investigated in laboratory mice. We show that administration of PEG-PamCys, a soluble form of the TLR2 agonist S-[2,3-bis(palmitoyloxy)propyl] cysteine (PamCys), significantly and dramatically reduces the numbers of malaria parasites that grow in the livers of mice following subsequent challenge with sporozoites. We also show that treatment can also clear parasites from the liver when administered subsequent to the establishment of infection. Finally, PEG-PamCys can reduce the numbers of mosquitoes that are infected, and the intensity of their infection, following blood feeding on gametocytaemic mice. These results suggest that this compound could represent a novel liver stage anti-malarial that can be used both for the clearance of parasites following exposure and for the prevention of the establishment of infection.
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http://dx.doi.org/10.1016/j.ijpddr.2018.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6216107PMC
December 2018

A lipidated bi-epitope vaccine comprising of MHC-I and MHC-II binder peptides elicits protective CD4 T cell and CD8 T cell immunity against Mycobacterium tuberculosis.

J Transl Med 2018 10 11;16(1):279. Epub 2018 Oct 11.

CSIR-Institute of Microbial Technology, Chandigarh, India.

Background: The clinical trials conducted at Chingleput India suggest that BCG fails to protect against tuberculosis (TB) in TB-endemic population. Recent studies advocate that non-tuberculous mycobacteria and latent Mycobacterium tuberculosis (Mtb) infection interferes in the antigen processing and presentation of BCG in inducing protective immunity against Mtb. Thereby, indicating that any vaccine that require extensive antigen processing may not be efficacious in TB-endemic zones. Recently, we have demonstrated that the vaccine candidate L91, which is composed of lipidated promiscuous MHC-II binder epitope, derived from latency associated Acr1 antigen of Mtb is immunogenic in the murine and Guinea pig models of TB and conferred better protection than BCG against Mtb.

Methods: In this study, we have used a multi-stage based bi-epitope vaccine, namely L4.8, comprising of MHC-I and MHC-II binding peptides of active (TB10.4) and latent (Acr1) stages of Mtb antigens, respectively. These peptides were conjugated to the TLR-2 agonist Pam2Cys.

Results: L4.8 significantly elicited both CD8 T cells and CD4 T cells immunity, as evidenced by increase in the enduring polyfunctional CD8 T cells and CD4 T cells. L4.8 efficiently declined Mtb-burden and protected animals better than BCG and L91, even at the late stage of Mtb infection.

Conclusions: The BCG-L4.8 prime boost strategy imparts a better protection against TB than the BCG alone. This study emphatically denotes that L4.8 can be a promising future vaccine candidate for controlling active and latent TB.
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http://dx.doi.org/10.1186/s12967-018-1653-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180631PMC
October 2018

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

A lipidated peptide of Mycobacterium tuberculosis resuscitates the protective efficacy of BCG vaccine by evoking memory T cell immunity.

J Transl Med 2017 10 6;15(1):201. Epub 2017 Oct 6.

CSIR-Institute of Microbial Technology, Chandigarh, India.

Background: The current BCG vaccine induces only short-term protection against Mycobacterium tuberculosis (Mtb), suggesting its failure to generate long-lasting memory T cells. Previously, we have demonstrated that a self-adjuvanting peptide of Mtb (L91), successfully generated enduring memory Th1 cells. Consequently, we investigated if L91 was able to recuperate BCG potency in perpetuating the generation of memory T cells and protection against Mtb infected mice.

Methods: In the present study, we evaluated the potency of a self adjuvanting Mtb peptide vaccine L91 in invigorating BCG immune response against Mtb in mice. Female BALB/c mice were immunized with BCG. Later, they were boosted twice with L91 or an antigenically irrelevant lipidated influenza virus hemagglutinin peptide (LH). Further, PBMCs obtained from BCG vaccinated healthy subjects were cultured in vitro with L91. T cell responses were determined by surface markers and intracellular cytokine staining. Secretion of cytokines was estimated in the culture supernatants (SNs) by ELISA.

Results: Compared to the BCG-vaccinated controls, L91 booster significantly enhanced the percentage of memory Th1 cells and Th17 cells and reduced the mycobacterial burden in BCG primed and L91-boosted (BCG-L91) group, even after 229 days of BCG vaccination. Further, substantial augmentation in the central (CD44CD62LCD127) and effector memory (CD44CD62LCD127) CD4 T cells was detected. Furthermore, greater frequency of polyfunctional Th1 cells (IFN-γTNF-α) and Th17 cells (IFN-γIL-17A) was observed. Importantly, BCG-L91 successfully prevented CD4 T cells from exhaustion by decreasing the expression of PD-1 and Tim-3. Additionally, augmentation in the frequency of Th1 cells, Th17 cells and memory CD4 T cells was observed in the PBMCs of the BCG-vaccinated healthy individuals following in vitro stimulation with L91.

Conclusions: Our study demonstrated that L91 robustly reinvigorate BCG potency to invoke enduring protection against Mtb. This novel vaccination stratagem involving BCG-priming followed by L91-boosting can be a future prophylactic measure to control TB.
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http://dx.doi.org/10.1186/s12967-017-1301-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389088PMC
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

Mycobacterium ulcerans low infectious dose and mechanical transmission support insect bites and puncturing injuries in the spread of Buruli ulcer.

PLoS Negl Trop Dis 2017 04 14;11(4):e0005553. Epub 2017 Apr 14.

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

Addressing the transmission enigma of the neglected disease Buruli ulcer (BU) is a World Health Organization priority. In Australia, we have observed an association between mosquitoes harboring the causative agent, Mycobacterium ulcerans, and BU. Here we tested a contaminated skin model of BU transmission by dipping the tails from healthy mice in cultures of the causative agent, Mycobacterium ulcerans. Tails were exposed to mosquito (Aedes notoscriptus and Aedes aegypti) blood feeding or punctured with sterile needles. Two of 12 of mice with M. ulcerans contaminated tails exposed to feeding A. notoscriptus mosquitoes developed BU. There were no mice exposed to A. aegypti that developed BU. Eighty-eight percent of mice (21/24) subjected to contaminated tail needle puncture developed BU. Mouse tails coated only in bacteria did not develop disease. A median incubation time of 12 weeks, consistent with data from human infections, was noted. We then specifically tested the M. ulcerans infectious dose-50 (ID50) in this contaminated skin surface infection model with needle puncture and observed an ID50 of 2.6 colony-forming units. We have uncovered a biologically plausible mechanical transmission mode of BU via natural or anthropogenic skin punctures.
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http://dx.doi.org/10.1371/journal.pntd.0005553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406025PMC
April 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

A novel therapeutic strategy of lipidated promiscuous peptide against Mycobacterium tuberculosis by eliciting Th1 and Th17 immunity of host.

Sci Rep 2016 Apr 7;6:23917. Epub 2016 Apr 7.

CSIR-Institute of Microbial Technology, Chandigarh, India.

Regardless of the fact that potent drug-regimen is currently available, tuberculosis continues to kill 1.5 million people annually. Tuberculosis patients are not only inflicted by the trauma of disease but they also suffer from the harmful side-effects, immune suppression and drug resistance instigated by prolonged therapy. It is an exigency to introduce radical changes in the existing drug-regime and discover safer and better therapeutic measures. Hence, we designed a novel therapeutic strategy by reinforcing the efficacy of drugs to kill Mtb by concurrently boosting host immunity by L91. L91 is chimera of promiscuous epitope of Acr1 antigen of Mtb and TLR-2 agonist Pam2Cys. The adjunct therapy using drugs and L91 (D-L91) significantly declined the bacterial load in Mtb infected animals. The mechanism involved was through enhancement of IFN-γ(+)TNF-α(+) polyfunctional Th1 cells and IL-17A(+)IFN-γ(+) Th17 cells, enduring memory CD4 T cells and downregulation of PD-1. The down-regulation of PD-1 prevents CD4 T cells from undergoing exhaustion and improves their function against Mtb. Importantly, the immune response observed in animals could be replicated using T cells of tuberculosis patients on drug therapy. In future, D-L91 therapy can invigorate drugs potency to treat tuberculosis patients and reduce the dose and duration of drug-regime.
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http://dx.doi.org/10.1038/srep23917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4823727PMC
April 2016

A lipidated form of the extracellular domain of influenza M2 protein as a self-adjuvanting vaccine candidate.

Vaccine 2015 Jul 3;33(30):3526-32. Epub 2015 Jun 3.

Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Melbourne 3010, Victoria, Australia.

The highly conserved extracellular domain of Matrix protein 2 (M2e) of influenza A virus has been previously investigated as a potential target for an universal influenza vaccine. In this study we prepared four lipopeptide influenza vaccine candidates in which the TLR2 agonist S-[2,3-bis(palmitoyloxy)propyl] cysteine, (Pam2Cys) was attached to either the N- or C-terminus of the M2e consensus sequence SLLTEVETPIRNEWGCRCNDSSDP and its analogue sequence with the two cysteine residues replaced with serine residues. The results of animal study show that each of these lipopeptides induced strong M2e-specific antibody responses in the absence of extraneous T helper cell epitope(s) which are normally incorporated in the previous studies or addition of extraneous adjuvant and that these antibodies are protective against lethal challenge with influenza virus. Comparison of different routes of inoculation demonstrated that intranasal administration of M2e lipopeptide induced higher titers of IgA and IgG2b antibodies in the bronchoalveolar lavage than did subcutaneous vaccination and was better at mitigating the severity of viral challenge. Finally, we show that anti-M2e antibody specificities absent from the antibody repertoire elicited by a commercially available influenza vaccine and by virus infection can be introduced by immunization with M2e-lipopeptide and boosted by viral challenge. Immunization with this lipidated form of the M2e epitope therefore offers a means of using a widely conserved epitope to generate protective antibodies which are not otherwise induced.
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http://dx.doi.org/10.1016/j.vaccine.2015.05.053DOI Listing
July 2015

Bioactivity in an Aggrecan 32-mer Fragment Is Mediated via Toll-like Receptor 2.

Arthritis Rheumatol 2015 May;67(5):1240-9

University of Melbourne, Murdoch Childrens Research Institute, and Royal Children's Hospital, Parkville, Victoria, Australia.

Objective: To determine whether an aggrecan 32-mer fragment derived from dual ADAMTS and matrix metalloproteinase (MMP) cleavage in the aggrecan interglobular domain was bioactive and, if so, to elucidate its mechanism of action.

Methods: Mouse primary chondrocytes, synovial fibroblasts, or peritoneal macrophages, human primary chondrocytes, and cells or cell lines from myeloid differentiation factor 88 (MyD88)-deficient and Toll-like receptor 2 (TLR-2)-deficient mice were stimulated with synthetic mouse 32-mer peptide, human 32-mer peptide, a 32-mer scrambled peptide, or native, glycosylated 32-mer peptide. Cells stimulated with 32-mer peptide were analyzed for changes in messenger RNA (mRNA) expression by quantitative polymerase chain reaction. Conditioned medium was analyzed for levels of interleukin-6 protein by an AlphaLISA or for levels of MMP-3 and MMP-13 protein by Western blotting. NF-κB activation was measured in a luciferase reporter assay.

Results: Treatment of mouse cells or cartilage explants with 32-mer peptide or scrambled peptide revealed that the 32-mer peptide, but not the scrambled peptide, had antianabolic, procatabolic, and proinflammatory bioactivity in vitro. Chondrocytes, synovial fibroblasts, and macrophages from MyD88-deficient mice failed to respond to 32-mer peptide stimulation. A macrophage cell line derived from TLR-2-deficient mice also failed to respond to 32-mer peptide stimulation. Stimulation of human chondrocytes with human 32-mer peptide increased the expression of catabolic markers at the mRNA and protein levels. Mouse and human 32-mer peptide stimulated NF-κB activation in a TLR-2-dependent reporter assay, and the response of chondrocytes from both species to native, glycosylated 32-mer peptide was similar to the response to synthetic peptides.

Conclusion: The aggrecan 32-mer fragment is a novel endogenous ligand of TLR-2 with the potential to accelerate cartilage destruction in vivo.
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http://dx.doi.org/10.1002/art.39063DOI Listing
May 2015

Different arms of the adaptive immune system induced by a combination vaccine work in concert to provide enhanced clearance of influenza.

PLoS One 2014 18;9(12):e115356. Epub 2014 Dec 18.

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

Current split influenza virus vaccines that induce strain-specific neutralising antibodies provide some degree of protection against influenza infection but there is a clear need to improve their effectiveness. The constant antigenic drift of influenza viruses means that vaccines are often not an exact match to the circulating strain and so levels of relevant antibodies may not be sufficiently high to afford protection. In the situation where the emergent influenza virus is completely novel, as is the case with pandemic strains, existing vaccines may provide no benefit. In this study we tested the concept of a combination vaccine consisting of sub-optimal doses of split influenza virus vaccine mixed with a cross-protective T-cell inducing lipopeptide containing the TLR2 ligand Pam2Cys. Mice immunised with combination vaccines showed superior levels of lung viral clearance after challenge compared to either split virus or lipopeptide alone, mediated through activation of enhanced humoral and/or additional cellular responses. The mechanism of action of these vaccines was dependent on the route of administration, with intranasal administration being superior to subcutaneous and intramuscular routes, potentially through the induction of memory CD8+ T cells in the lungs. This immunisation strategy not only provides a mechanism for minimising the dose of split virus antigen but also, through the induction of cross-protective CD8+ T cells, proves a breadth of immunity to provide potential benefit upon encounter with serologically diverse influenza isolates.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115356PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4270762PMC
December 2015

The influenza virus-specific CTL immunodominance hierarchy in mice is determined by the relative frequency of high-avidity T cells.

J Immunol 2014 May 2;192(9):4061-8. Epub 2014 Apr 2.

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

Virus-specific CTL responses typically fall into reproducible hierarchies with particular epitopes eliciting either immunodominant or subdominant responses after viral challenge. The recently acquired capacity to directly enumerate naive CTL precursors (CTLps) in both mice and humans has implicated CTLp frequency as a key predictor of immune response magnitude after Ag challenge. However, recent studies have indicated that naive CTLp frequencies do not necessarily predict the size of the Ag-driven response, indicating an important role for differential CTLp recruitment and/or expansion. This study characterizes the early emergence of various influenza epitope-specific CTL responses at multiple sites in C57BL/6 mice, and probes the role of Ag dose and TCR avidity in dictating immune response hierarchies. Despite large naive CTLp numbers, subdominance was found to arise largely as a consequence of the abrupt and premature cessation of CTL proliferation, at least for one epitope specificity. Investigation into the possible drivers of the poor proliferation observed for subdominant specificities showed that the immunodominance hierarchy endured irrespective of epitope abundance, and correlated with the prevalence of high-avidity T cells in both the naive and immune compartments. Our study strongly indicates that the quality, and not simply the quantity, of antiviral CTLs dictate response magnitude.
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http://dx.doi.org/10.4049/jimmunol.1301403DOI Listing
May 2014

Lipidated promiscuous peptide augments the expression of MHC-II molecules on dendritic cells and activates T cells.

Indian J Med Res 2013 Nov;138(5):744-8

CSIR-Institute of Microbial Technology, Chandigarh, India.

Background & Objectives: In spite of the fact that BCG is the most widely used vaccine, tuberculosis (TB) continues to be a major killer disease in TB-endemic regions. Recently, many emerging evidences from the published literature indicate the role of environmental mycobacteria in blocking the processing and presentation of BCG antigens and thereby impairing with suboptimal generation of protective T cells. To surmount this problem associated with BCG, we constructed a novel lipopeptide (L91) by conjugating a promiscuous peptide consisting of CD4 + T-helper epitope of sequence of 91-110 of 16 kDa antigen of Mycobacterium tuberculosis to Pam2Cys, an agonist of Toll-like receptor-2.

Methods: Mice were immunized subcutaneously with 20 nmol of L91, followed by a booster with 10 nmol, after an interval of 21 days of primary immunization. Animals were sacrificed after seven days of post-booster immunization. L91 induced immune response was characterized by the expression of MHC-II and CD74 on the surface of dendritic cells (DCs) by flowcytometry. Cytokines (IL-4, IL-10, IFN-γ) secretion and anti-peptide antibodies were measured by ELISA.

Results: Self-adjuvanting lipopeptide vaccine (L91) was directly bound to MHC-II molecules and without requiring extensive processing for its presentation to T cells. It stimulated and activated dendritic cells and augmented the expression of MHC-II molecules. Further, it activated effector CD4 T cells to mainly secrete interferon (IFN)-γ but not interleukin (IL)-4 and IL-10. L91 did not elicit anti-peptide antibodies.

Interpretation & Conclusions: The findings suggest that L91 evokes maturation and upregulation of MHC class II molecules and promotes better antigen presentation and, therefore, optimum activation of T cells. L91 mainly induces effector Th1 cells, as evidenced by predominant release of IFN-γ, consequently can mount favourable immune response against M. tuberculosis . As L91 does not provoke the generation of anti-peptide antibodies, there is no fear of the efficacy of the vaccine being neutralized by pre-existing anti-mycobacterial antibodies in TB-endemic population. In conclusion, L91 may be considered as a future potential candidate vaccine against TB.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928704PMC
November 2013

Activation of the NLRP3 inflammasome by IAV virulence protein PB1-F2 contributes to severe pathophysiology and disease.

PLoS Pathog 2013 30;9(5):e1003392. Epub 2013 May 30.

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

The ability for a host to recognize infection is critical for virus clearance and often begins with induction of inflammation. The PB1-F2 of pathogenic influenza A viruses (IAV) contributes to the pathophysiology of infection, although the mechanism for this is unclear. The NLRP3-inflammasome has been implicated in IAV pathogenesis, but whether IAV virulence proteins can be activators of the complex is unknown. We investigated whether PB1-F2-mediated activation of the NLRP3-inflammasome is a mechanism contributing to overt inflammatory responses to IAV infection. We show PB1-F2 induces secretion of pyrogenic cytokine IL-1β by activating the NLRP3-inflammasome, contributing to inflammation triggered by pathogenic IAV. Compared to infection with wild-type virus, mice infected with reverse engineered PB1-F2-deficient IAV resulted in decreased IL-1β secretion and cellular recruitment to the airways. Moreover, mice exposed to PB1-F2 peptide derived from pathogenic IAV had enhanced IL-1β secretion compared to mice exposed to peptide derived from seasonal IAV. Implicating the NLRP3-inflammasome complex specifically, we show PB1-F2 derived from pathogenic IAV induced IL-1β secretion was Caspase-1-dependent in human PBMCs and NLRP3-dependent in mice. Importantly, we demonstrate PB1-F2 is incorporated into the phagolysosomal compartment, and upon acidification, induces ASC speck formation. We also show that high molecular weight aggregated PB1-F2, rather than soluble PB1-F2, induces IL-1β secretion. Furthermore, NLRP3-deficient mice exposed to PB1-F2 peptide or infected with PB1-F2 expressing IAV were unable to efficiently induce the robust inflammatory response as observed in wild-type mice. In addition to viral pore forming toxins, ion channel proteins and RNA, we demonstrate inducers of NLRP3-inflammasome activation may include disordered viral proteins, as exemplified by PB1-F2, acting as host pathogen 'danger' signals. Elucidating immunostimulatory PB1-F2 mediation of NLRP3-inflammasome activation is a major step forward in our understanding of the aetiology of disease attributable to exuberant inflammatory responses to IAV infection.
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http://dx.doi.org/10.1371/journal.ppat.1003392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3667782PMC
December 2013

The design and proof of concept for a CD8(+) T cell-based vaccine inducing cross-subtype protection against influenza A virus.

Immunol Cell Biol 2013 Jan 13;91(1):96-104. Epub 2012 Nov 13.

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

In this study, we examined the reactivity of human peripheral blood mononuclear cells to a panel of influenza A virus (IAV) CD8(+) T-cell epitopes that are recognised by the major human leukocyte antigen (HLA) groups represented in the human population. We examined the level of recognition in a sample of the human population and the potential coverage that could be achieved if these were incorporated into a T-cell epitope-based vaccine. We then designed a candidate influenza vaccine that incorporated three of the examined HLA-A2-restricted influenza epitopes into Pam2Cys-based lipopeptides. These lipopeptides do not require the addition of an adjuvant and can be delivered directly to the respiratory mucosa enabling the generation of local memory cell populations that are crucial for clearance of influenza. Intranasal administration of a mixture of three lipopeptides to HLA-A2 transgenic HHD mice elicited multiple CD8(+) T-cell specificities in the spleen and lung that closely mimicked the response generated following natural infection with influenza. These CD8(+) T cells were associated with viral reduction following H3N1 influenza virus challenge for as long as 3 months after lipopeptide administration. In addition, lipopeptides containing IAV-targeting epitopes conferred substantial benefit against death following infection with a virulent H1N1 strain. Because CD8(+) T cell epitopes are often derived from highly conserved regions of influenza viruses, such vaccines need not be reformulated annually and unlike current antibody-inducing vaccines could provide cross-protective immunity against newly emerging pandemic viruses.
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http://dx.doi.org/10.1038/icb.2012.54DOI Listing
January 2013

Intranasal administration of the TLR2 agonist Pam2Cys provides rapid protection against influenza in mice.

Mol Pharm 2012 Sep 3;9(9):2710-8. Epub 2012 Aug 3.

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

The protective role played by the innate immune system during early stages of infection suggests that compounds which stimulate innate responses could be used as antimicrobial or antiviral agents. In this study, we demonstrate that the Toll-like receptor-2 agonist Pam2Cys, when administered intranasally, triggers a cascade of inflammatory and innate immune signals, acting as an immunostimulant by attracting neutrophils and macrophages and inducing secretion of IL-2, IL-6, IL-10, IFN-γ, MCP-1 and TNF-α. These changes provide increased resistance against influenza A virus challenge and also reduce the potential for transmission of infection. Pam2Cys treatment also reduced weight loss and lethality associated with virulent influenza virus infection in a Toll-like receptor-2-dependent manner. Treatment did not affect the animals' ability to generate an adaptive immune response, measured by the induction of functional influenza A virus-specific CD8(+) T cells following exposure to virus. Because this compound demonstrates efficacy against distinct strains of influenza, it could be a candidate for development as an agent against influenza and possibly other respiratory pathogens.
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http://dx.doi.org/10.1021/mp300257xDOI Listing
September 2012

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

Polyfunctional CD8(+) T cells are associated with the vaccination-induced control of a novel recombinant influenza virus expressing an HCV epitope.

Antiviral Res 2012 May 4;94(2):168-78. Epub 2012 Apr 4.

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

In hepatitis C virus (HCV) infection, CD8(+) T cell responses have been shown to be important in viral clearance. Examining the efficacy of CD8(+) T cell vaccines against HCV has been limited by the lack of an HCV infectious model in mice and the differences between MHC restriction in humans and mice. Using HLA-A2 transgenic HHD mice, we demonstrate that intranasally delivered Pam2Cys-based lipopeptides containing HLA-A2-restricted HCV epitopes can induce polyfunctional CD8(+) T cell responses in several organs including the liver. To examine the activity of these responses in an infectious context, we developed a recombinant influenza virus that expresses the NS5B(2594-2602) epitope from non-structural protein 5B of hepatitis C virus (PR8-HCV(NS5B)). We showed that mice inoculated with a lipopeptide containing the NS5B epitope had reduced viral loads following challenge with the PR8-HCV(NS5B) virus. This reduction was associated with the induction of NS5B(2594-2602)-specific IFN-γ and TNF-α co-producing CD8(+) T cells. The T cell receptor usage in the NS5B(2594-2602) response was found to exhibit a Vβ8.1/8.2 bias that was characterized by a narrow repertoire and a common CDR3β motif. This work has identified CD8(+) T cell functions induced by lipopeptides that are associated with viral control and demonstrate the potential of lipopeptide-based vaccines as candidates for treatment of HCV infection.
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http://dx.doi.org/10.1016/j.antiviral.2012.03.009DOI Listing
May 2012

Herpes simplex virus antigens directly activate NK cells via TLR2, thus facilitating their presentation to CD4 T lymphocytes.

J Immunol 2012 May 30;188(9):4158-70. Epub 2012 Mar 30.

Centre for Virus Research, Westmead Millennium Institute, Westmead, New South Wales 2145, Australia.

NK cells infiltrate human herpetic lesions, but their role has been underexplored. HSV can stimulate innate immune responses via surface TLR2, which is expressed on monocyte-derived dendritic cells (DCs) and NK cells. In this study, UV-inactivated HSV1/2 and immunodominant HSV2 glycoprotein D peptides conjugated to the TLR2 agonist dipalmitoyl-S-glyceryl cysteine stimulated CD4 T lymphocyte IFN-γ responses within PBMCs or in coculture with monocyte-derived DCs. NK cells contributed markedly to the PBMC responses. Furthermore, NK cells alone were activated directly by both Ags, also upregulating HLA-DR and HLA-DQ and then they activated autologous CD4 T lymphocytes. Using Transwells, Ag-stimulated NK cells and CD4 T lymphocytes were shown to interact through both cell-to-cell contact and cytokines, differing in relative importance in different donors. A distinct immunological synapse between Ag-stimulated NK cells and CD4 T lymphocytes was observed, indicating the significance of their cell-to-cell contact. A large proportion (57%) of NK cells was also in contact with CD4 T lymphocytes in the dermal infiltrate of human recurrent herpetic lesions. Thus, NK cells stimulated by TLR2-activating HSV Ags can present Ag alone or augment the role of DCs in vitro and perhaps in herpetic lesions or draining lymph nodes. In addition to DCs, NK cells should be considered as targets for adjuvants during HSV vaccine development.
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http://dx.doi.org/10.4049/jimmunol.1103450DOI Listing
May 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

Affinity thresholds for naive CD8+ CTL activation by peptides and engineered influenza A viruses.

J Immunol 2011 Dec 28;187(11):5733-44. Epub 2011 Oct 28.

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

High-avidity interactions between TCRs and peptide + class I MHC (pMHCI) epitopes drive CTL activation and expansion. Intriguing questions remain concerning the constraints determining optimal TCR/pMHCI binding. The present analysis uses the TCR transgenic OT-I model to assess how varying profiles of TCR/pMHCI avidity influence naive CTL proliferation and the acquisition of effector function following exposure to the cognate H-2K(b)/OVA(257-264) (SIINFEKL) epitope and to mutants provided as peptide or in engineered influenza A viruses. Stimulating naive OT-I CD8(+) T cells in vitro with SIINFEKL induced full CTL proliferation and differentiation that was largely independent of any need for costimulation. By contrast, in vitro activation with the low-affinity EIINFEKL or SIIGFEKL ligands depended on the provision of IL-2 and other costimulatory signals. Importantly, although they did generate potent endogenous responses, infection of mice with influenza A viruses expressing these same OVA(257) variants failed to induce the activation of adoptively transferred naive OT-I CTLps, an effect that was only partially overcome by priming with a lipopeptide vaccine. Subsequent structural and biophysical analysis of H2-K(b)OVA(257), H2-K(b)E1, and H2-K(b)G4 established that these variations introduce small changes at the pMHCI interface and decrease epitope stability in ways that would likely impact cell surface presentation and recognition. Overall, it seems that there is an activation threshold for naive CTLps, that minimal alterations in peptide sequence can have profound effects, and that the antigenic requirements for the in vitro and in vivo induction of CTL proliferation and effector function differ substantially.
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http://dx.doi.org/10.4049/jimmunol.1003937DOI Listing
December 2011

Promiscuous peptide of 16 kDa antigen linked to Pam2Cys protects against Mycobacterium tuberculosis by evoking enduring memory T-cell response.

J Infect Dis 2011 Nov 20;204(9):1328-38. Epub 2011 Sep 20.

Immunology and Cell Biology Division, Institute of Microbial Technology, Chandigarh, India.

One of the main reasons considered for BCG failure in tuberculosis-endemic areas is impediment by environmental mycobacteria in its processing and generation of memory T-cell response. To overcome this problem, we developed a unique lipopeptide (L91) by linking the promiscuous peptide (sequence 91-110) of 16 kDa antigen of Mycobacterium tuberculosis to Pam2Cys. L91 does not require extensive antigen processing and generates enduring Th1 memory response. This is evidenced by the fact that L91 significantly improved the activation, proliferation, and generation of protective T cells. Furthermore, L91 surmounts the barrier of major histocompatibility complex polymorphism and induces better protection than BCG. This peptide has self-adjuvanting properties and activates dendritic cells. Importantly, L91 activates T cells isolated from purified protein derivative-positive healthy volunteers that responded weakly to free peptide (F91). In essence, L91 can be a potent future vaccine candidate against tuberculosis.
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http://dx.doi.org/10.1093/infdis/jir548DOI Listing
November 2011

Precursor frequency and competition dictate the HLA-A2-restricted CD8+ T cell responses to influenza A infection and vaccination in HLA-A2.1 transgenic mice.

J Immunol 2011 Aug 15;187(4):1895-902. Epub 2011 Jul 15.

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

The human HLA-A2-restricted CD8(+) T cell response to influenza A virus (IAV) is largely directed against the matrix protein-derived M1(58-66) epitope and represents an archetypal example of CD8(+) T cell immunodominance. In this study, we examined the CD8(+) T cell hierarchy to M1(58-66) and two subdominant IAV-specific epitopes: NS1(122-130) and PA(46-55) in HLA-A2(+) human subjects and HLA-A2.1 transgenic (HHD) mice. Using epitope-based lipopeptides, we show that the CD8(+) T cell hierarchy induced by IAV infection could also be induced by lipopeptide vaccination in a context outside of viral infection when the Ag load is equalized. In the HHD HLA-A2.1 mouse model, we show that the naive T cell precursor frequencies, and competition at the Ag presentation level, can predict the IAV-specific CD8(+) T cell hierarchy. Immunization of mice with subdominant epitopes alone was unable to overcome the dominance of the M1(58-66)-specific response in the face of IAV challenge; however, a multiepitope vaccination strategy was most effective at generating a broad and multispecific response to infection.
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http://dx.doi.org/10.4049/jimmunol.1100664DOI Listing
August 2011