Publications by authors named "Hyon-Xhi Tan"

23 Publications

  • Page 1 of 1

Coformulation with Tattoo Ink for Immunological Assessment of Vaccine Immunogenicity in the Draining Lymph Node.

J Immunol 2021 Jul 9. Epub 2021 Jul 9.

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

Characterization of germinal center B and T cell responses yields critical insights into vaccine immunogenicity. Nonhuman primates are a key preclinical animal model for human vaccine development, allowing both lymph node (LN) and circulating immune responses to be longitudinally sampled for correlates of vaccine efficacy. However, patterns of vaccine Ag drainage via the lymphatics after i.m. immunization can be stochastic, driving uneven deposition between lymphoid sites and between individual LN within larger clusters. To improve the accurate isolation of Ag-exposed LN during biopsies and necropsies, we developed and validated a method for coformulating candidate vaccines with tattoo ink in both mice and pigtail macaques. This method allowed for direct visual identification of vaccine-draining LN and evaluation of relevant Ag-specific B and T cell responses by flow cytometry. This approach is a significant advancement in improving the assessment of vaccine-induced immunity in highly relevant nonhuman primate models.
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http://dx.doi.org/10.4049/jimmunol.2001299DOI Listing
July 2021

Decay of Fc-dependent antibody functions after mild to moderate COVID-19.

Cell Rep Med 2021 Jun 9;2(6):100296. Epub 2021 May 9.

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

The capacity of antibodies to engage with immune cells via the Fc region is important in preventing and controlling many infectious diseases. The evolution of such antibodies during convalescence from coronavirus disease 2019 (COVID-19) is largely unknown. We develop assays to measure Fc-dependent antibody functions against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-expressing cells in serial samples from subjects primarily with mild-moderate COVID-19 up to 149 days post-infection. We find that S-specific antibodies capable of engaging Fcγ receptors decay over time, with S-specific antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADP) activity within plasma declining accordingly. Although there is significant decay in ADCC and ADP activity, they remain readily detectable in almost all subjects at the last time point studied (94%) in contrast with neutralization activity (70%). Although it remains unclear the degree to which Fc effector functions contribute to protection against SARS-CoV-2 re-infection, our results indicate that antibodies with Fc effector functions persist longer than neutralizing antibodies.
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http://dx.doi.org/10.1016/j.xcrm.2021.100296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106889PMC
June 2021

CD8 T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity.

Immunity 2021 05 15;54(5):1066-1082.e5. Epub 2021 Apr 15.

Department of Infectious Diseases, Austin Hospital, Heidelberg, VIC 3084, Australia; Department of Medicine and Radiology, The University of Melbourne, Parkville, VIC 3000, Australia; Data Analytics Research and Evaluation (DARE) Centre, Austin Health and The University of Melbourne, Heidelberg, VIC 3084, Australia.

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8 T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8 T cells directed toward subdominant epitopes (B7/N, A2/S, and A24/S) CD8 T cells specific for the immunodominant B7/N epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8 T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαβ repertoires and promiscuous αβ-TCR pairing within B7/NCD8 T cells. Our study demonstrates high naive precursor frequency and TCRαβ diversity within immunodominant B7/N-specific CD8 T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.
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http://dx.doi.org/10.1016/j.immuni.2021.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049468PMC
May 2021

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

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

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

The hallmarks of COVID-19 are higher pathogenicity and mortality in the elderly compared to children. Examining baseline SARS-CoV-2 cross-reactive immunological responses, induced by circulating human coronaviruses (hCoVs), is needed to understand such divergent clinical outcomes. Here we show analysis of coronavirus antibody responses of pre-pandemic healthy children (n = 89), adults (n = 98), elderly (n = 57), and COVID-19 patients (n = 50) by systems serology. Moderate levels of cross-reactive, but non-neutralizing, SARS-CoV-2 antibodies are detected in pre-pandemic healthy individuals. SARS-CoV-2 antigen-specific Fcγ receptor binding accurately distinguishes COVID-19 patients from healthy individuals, suggesting that SARS-CoV-2 infection induces qualitative changes to antibody Fc, enhancing Fcγ receptor engagement. Higher cross-reactive SARS-CoV-2 IgA and IgG are observed in healthy elderly, while healthy children display elevated SARS-CoV-2 IgM, suggesting that children have fewer hCoV exposures, resulting in less-experienced but more polyreactive humoral immunity. Age-dependent analysis of COVID-19 patients, confirms elevated class-switched antibodies in elderly, while children have stronger Fc responses which we demonstrate are functionally different. These insights will inform COVID-19 vaccination strategies, improved serological diagnostics and therapeutics.
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http://dx.doi.org/10.1038/s41467-021-22236-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016934PMC
April 2021

Adaptive immunity to human coronaviruses is widespread but low in magnitude.

Clin Transl Immunology 2021 17;10(3):e1264. Epub 2021 Mar 17.

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

Objectives: Endemic human coronaviruses (hCoVs) circulate worldwide but cause minimal mortality. Although seroconversion to hCoV is near ubiquitous during childhood, little is known about hCoV-specific T-cell memory in adults.

Methods: We quantified CD4 T-cell and antibody responses to hCoV spike antigens in 42 SARS-CoV-2-uninfected individuals. Antigen-specific memory T cells and circulating T follicular helper (cTFH) cells were identified using an activation-induced marker assay and characterised for memory phenotype and chemokine receptor expression.

Results: T-cell responses were widespread within conventional memory and cTFH compartments but did not correlate with IgG titres. SARS-CoV-2 cross-reactive T cells were observed in 48% of participants and correlated with HKU1 memory. hCoV-specific T cells exhibited a CCR6 central memory phenotype in the blood, but were enriched for frequency and CXCR3 expression in human lung-draining lymph nodes.

Conclusion: Overall, hCoV-specific humoral and cellular memory are independently maintained, with a shared phenotype existing among coronavirus-specific CD4 T cells. This understanding of endemic coronavirus immunity provides insight into the homeostatic maintenance of immune responses that are likely to be critical components of protection against SARS-CoV-2.
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http://dx.doi.org/10.1002/cti2.1264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968850PMC
March 2021

Hemagglutinin Functionalized Liposomal Vaccines Enhance Germinal Center and Follicular Helper T Cell Immunity.

Adv Healthc Mater 2021 05 9;10(10):e2002142. Epub 2021 Mar 9.

Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, Parkville, VIC, 3052, Australia.

Despite remarkable successes of immunization in protecting public health, safe and effective vaccines against a number of life-threatening pathogens such as HIV, ebola, influenza, and SARS-CoV-2 remain urgently needed. Subunit vaccines can avoid potential toxicity associated with traditional whole virion-inactivated and live-attenuated vaccines; however, the immunogenicity of subunit vaccines is often poor. A facile method is here reported to produce lipid nanoparticle subunit vaccines that exhibit high immunogenicity and elicit protection against influenza virus. Influenza hemagglutinin (HA) immunogens are functionalized on the surface of liposomes via stable metal chelation chemistry, using a scalable advanced microfluidic mixing technology (NanoAssemblr). Immunization of mice with HA-liposomes elicits increased serum antibody titers and superior protection against highly pathogenic virus challenge compared with free HA protein. HA-liposomal vaccines display enhanced antigen deposition into germinal centers within the draining lymph nodes, driving increased HA-specific B cell, and follicular helper T cell responses. This work provides mechanistic insights into highly protective HA-liposome vaccines and informs the rational design and rapid production of next generation nanoparticle subunit vaccines.
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http://dx.doi.org/10.1002/adhm.202002142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206650PMC
May 2021

Immunogenicity of prime-boost protein subunit vaccine strategies against SARS-CoV-2 in mice and macaques.

Nat Commun 2021 03 3;12(1):1403. Epub 2021 Mar 3.

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

SARS-CoV-2 vaccines are advancing into human clinical trials, with emphasis on eliciting high titres of neutralising antibodies against the viral spike (S). However, the merits of broadly targeting S versus focusing antibody onto the smaller receptor binding domain (RBD) are unclear. Here we assess prototypic S and RBD subunit vaccines in homologous or heterologous prime-boost regimens in mice and non-human primates. We find S is highly immunogenic in mice, while the comparatively poor immunogenicity of RBD is associated with limiting germinal centre and T follicular helper cell activity. Boosting S-primed mice with either S or RBD significantly augments neutralising titres, with RBD-focussing driving moderate improvement in serum neutralisation. In contrast, both S and RBD vaccines are comparably immunogenic in macaques, eliciting serological neutralising activity that generally exceed levels in convalescent humans. These studies confirm recombinant S proteins as promising vaccine candidates and highlight multiple pathways to achieving potent serological neutralisation.
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http://dx.doi.org/10.1038/s41467-021-21665-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930087PMC
March 2021

Evolution of immune responses to SARS-CoV-2 in mild-moderate COVID-19.

Nat Commun 2021 02 19;12(1):1162. Epub 2021 Feb 19.

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

The durability of infection-induced SARS-CoV-2 immunity has major implications for reinfection and vaccine development. Here, we show a comprehensive profile of antibody, B cell and T cell dynamics over time in a cohort of patients who have recovered from mild-moderate COVID-19. Binding and neutralising antibody responses, together with individual serum clonotypes, decay over the first 4 months post-infection. A similar decline in Spike-specific CD4 and circulating T follicular helper frequencies occurs. By contrast, S-specific IgG memory B cells consistently accumulate over time, eventually comprising a substantial fraction of circulating the memory B cell pool. Modelling of the concomitant immune kinetics predicts maintenance of serological neutralising activity above a titre of 1:40 in 50% of convalescent participants to 74 days, although there is probably additive protection from B cell and T cell immunity. This study indicates that SARS-CoV-2 immunity after infection might be transiently protective at a population level. Therefore, SARS-CoV-2 vaccines might require greater immunogenicity and durability than natural infection to drive long-term protection.
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http://dx.doi.org/10.1038/s41467-021-21444-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896046PMC
February 2021

Screening and development of monoclonal antibodies for identification of ferret T follicular helper cells.

Sci Rep 2021 Jan 21;11(1):1864. Epub 2021 Jan 21.

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

The ferret is a key animal model for investigating the pathogenicity and transmissibility of important human viruses, and for the pre-clinical assessment of vaccines. However, relatively little is known about the ferret immune system, due in part to a paucity of ferret-reactive reagents. In particular, T follicular helper (Tfh) cells are critical in the generation of effective humoral responses in humans, mice and other animal models but to date it has not been possible to identify Tfh in ferrets. Here, we describe the screening and development of ferret-reactive BCL6, CXCR5 and PD-1 monoclonal antibodies. We found two commercial anti-BCL6 antibodies (clone K112-91 and clone IG191E/A8) had cross-reactivity with lymph node cells from influenza-infected ferrets. We next developed two murine monoclonal antibodies against ferret CXCR5 (clone feX5-C05) and PD-1 (clone fePD-CL1) using a single B cell PCR-based method. We were able to clearly identify Tfh cells in lymph nodes from influenza infected ferrets using these antibodies. The development of ferret Tfh marker antibodies and the identification of ferret Tfh cells will assist the evaluation of vaccine-induced Tfh responses in the ferret model and the design of novel vaccines against the infection of influenza and other viruses, including SARS-CoV2.
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http://dx.doi.org/10.1038/s41598-021-81389-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820401PMC
January 2021

Aggregation by peptide conjugation rescues poor immunogenicity of the HA stem.

PLoS One 2020 2;15(11):e0241649. Epub 2020 Nov 2.

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

Influenza virus infection is a global public health threat. Current seasonal influenza vaccines are efficacious only when vaccine strains are matched with circulating strains. There is a critical need for developing "universal" vaccines that protect against all influenza viruses. HA stem is a promising target for developing broad-spectrum influenza vaccines due to its relatively conserved feature. However, HA stem is weakly immunogenic when administered alone in a soluble form. Several approaches have been employed to improve the immunogenicity of HA stem, including conjugation of HA stem with a highly immunogenic carrier protein or displaying HA stem on a nanoparticle scaffold. Converting a weakly immunologic protein into a multimer through aggregation can significantly enhance its immunogenicity, with some multimeric protein aggregates previously shown to be more immunogenic than their soluble counterparts in animal models. Here, we show that a chemically coupling a peptide derived from the head domain of PR8 HA (P35) with the poorly immunogenic HA stem protein results in aggregation of the HA stem which significantly increases stem-specific B cell responses following vaccination. Importantly, vaccination with this conjugate in the absence of adjuvant still induced robust B cell responses against stem in vivo. Improving HA stem immunogenicity by aggregation provides an alternative avenue to conjugation with exotic carrier proteins or nanoparticle formulation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241649PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605677PMC
December 2020

Suboptimal SARS-CoV-2-specific CD8 T cell response associated with the prominent HLA-A*02:01 phenotype.

Proc Natl Acad Sci U S A 2020 09 10;117(39):24384-24391. Epub 2020 Sep 10.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3000, Australia;

An improved understanding of human T cell-mediated immunity in COVID-19 is important for optimizing therapeutic and vaccine strategies. Experience with influenza shows that infection primes CD8 T cell memory to peptides presented by common HLA types like HLA-A2, which enhances recovery and diminishes clinical severity upon reinfection. Stimulating peripheral blood mononuclear cells from COVID-19 convalescent patients with overlapping peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the clonal expansion of SARS-CoV-2-specific CD8 and CD4 T cells in vitro, with CD4 T cells being robust. We identified two HLA-A*02:01-restricted SARS-CoV-2-specfic CD8 T cell epitopes, A2/S and A2/Orf1ab Using peptide-HLA tetramer enrichment, direct ex vivo assessment of A2/S CD8 and A2/Orf1ab CD8 populations indicated that A2/S CD8 T cells were detected at comparable frequencies (∼1.3 × 10) in acute and convalescent HLA-A*02:01 patients. These frequencies were higher than those found in uninfected HLA-A*02:01 donors (∼2.5 × 10), but low when compared to frequencies for influenza-specific (A2/M1) and Epstein-Barr virus (EBV)-specific (A2/BMLF) (∼1.38 × 10) populations. Phenotyping A2/S CD8 T cells from COVID-19 convalescents ex vivo showed that A2/S CD8 T cells were predominantly negative for CD38, HLA-DR, PD-1, and CD71 activation markers, although the majority of total CD8 T cells expressed granzymes and/or perforin. Furthermore, the bias toward naïve, stem cell memory and central memory A2/S CD8 T cells rather than effector memory populations suggests that SARS-CoV-2 infection may be compromising CD8 T cell activation. Priming with appropriate vaccines may thus be beneficial for optimizing CD8 T cell immunity in COVID-19.
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http://dx.doi.org/10.1073/pnas.2015486117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533701PMC
September 2020

Humoral and circulating follicular helper T cell responses in recovered patients with COVID-19.

Nat Med 2020 09 13;26(9):1428-1434. Epub 2020 Jul 13.

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

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has dramatically expedited global vaccine development efforts, most targeting the viral 'spike' glycoprotein (S). S localizes on the virion surface and mediates recognition of cellular receptor angiotensin-converting enzyme 2 (ACE2). Eliciting neutralizing antibodies that block S-ACE2 interaction, or indirectly prevent membrane fusion, constitute an attractive modality for vaccine-elicited protection. However, although prototypic S-based vaccines show promise in animal models, the immunogenic properties of S in humans are poorly resolved. In this study, we characterized humoral and circulating follicular helper T cell (cTFH) immunity against spike in recovered patients with coronavirus disease 2019 (COVID-19). We found that S-specific antibodies, memory B cells and cTFH are consistently elicited after SARS-CoV-2 infection, demarking robust humoral immunity and positively associated with plasma neutralizing activity. Comparatively low frequencies of B cells or cTFH specific for the receptor binding domain of S were elicited. Notably, the phenotype of S-specific cTFH differentiated subjects with potent neutralizing responses, providing a potential biomarker of potency for S-based vaccines entering the clinic. Overall, although patients who recovered from COVID-19 displayed multiple hallmarks of effective immune recognition of S, the wide spectrum of neutralizing activity observed suggests that vaccines might require strategies to selectively target the most potent neutralizing epitopes.
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http://dx.doi.org/10.1038/s41591-020-0995-0DOI Listing
September 2020

High CD26 and Low CD94 Expression Identifies an IL-23 Responsive Vδ2 T Cell Subset with a MAIT Cell-like Transcriptional Profile.

Cell Rep 2020 06;31(11):107773

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3000, Australia. Electronic address:

Vδ2 T cells play a critical role in immunity to micro-organisms and cancer but exhibit substantial heterogeneity in humans. Here, we demonstrate that CD26 and CD94 define transcriptionally, phenotypically, and functionally distinct Vδ2 T cell subsets. Despite distinct antigen specificities, CD26CD94 Vδ2 cells exhibit substantial similarities to CD26 mucosal-associated invariant T (MAIT) cells, although CD26 Vδ2 cells exhibit cytotoxic, effector-like profiles. At birth, the Vδ2Vγ9 population is dominated by CD26CD94 cells; during adolescence and adulthood, Vδ2 cells acquire CD94/NKG2A expression and the relative frequency of the CD26CD94 subset declines. Critically, exposure of the CD26CD94 subset to phosphoantigen in the context of interleukin-23 (IL-23) and CD26 engagement drives the acquisition of a cytotoxic program and concurrent loss of the MAIT cell-like phenotype. The ability to modulate the cytotoxic potential of CD26CD94 Vδ2 cells, combined with their adenosine-binding capacity, may make them ideal targets for immunotherapeutic expansion and adoptive transfer.
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http://dx.doi.org/10.1016/j.celrep.2020.107773DOI Listing
June 2020

Sequencing B cell receptors from ferrets (Mustela putorius furo).

PLoS One 2020 29;15(5):e0233794. Epub 2020 May 29.

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

The domestic ferret (Mustela putorius furo) provides a critical animal model to study human respiratory diseases. However immunological insights are restricted due to a lack of ferret-specific reagents and limited genetic information about ferret B and T cell receptors. Here, variable, diversity and joining genes within the ferret kappa, lambda and heavy chain immunoglobulin loci were annotated using available genomic information. A multiplex PCR approach was derived that facilitated the recovery of paired heavy and light chain immunoglobulin sequences from single sorted ferret B cells, allowing validation of predicted germline gene sequences and the identification of putative novel germlines. Eukaryotic expression vectors were developed that enabled the generation of recombinant ferret monoclonal antibodies. This work advances the ferret as an informative immunological model for viral diseases by allowing the in-depth interrogation of antibody-based immunity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0233794PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259655PMC
September 2020

Self-assembling influenza nanoparticle vaccines drive extended germinal center activity and memory B cell maturation.

JCI Insight 2020 05 21;5(10). Epub 2020 May 21.

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

Protein-based, self-assembling nanoparticles elicit superior immunity compared with soluble protein vaccines, but the immune mechanisms underpinning this effect remain poorly defined. Here, we investigated the immunogenicity of a prototypic ferritin-based nanoparticle displaying influenza hemagglutinin (HA) in mice and macaques. Vaccination of mice with HA-ferritin nanoparticles elicited higher serum antibody titers and greater protection against experimental influenza challenge compared with soluble HA protein. Germinal centers in the draining lymph nodes were expanded and persistent following HA-ferritin vaccination, with greater deposition of antigen that colocalized with follicular dendritic cells. Our findings suggest that a highly ordered and repetitive antigen array may directly drive germinal centers through a B cell-intrinsic mechanism that does not rely on ferritin-specific T follicular helper cells. In contrast to mice, enhanced immunogenicity of HA-ferritin was not observed in pigtail macaques, where antibody titers and lymph node immunity were comparable to soluble vaccination. An improved understanding of factors that drive nanoparticle vaccine immunogenicity in small and large animal models will facilitate the clinical development of nanoparticle vaccines for broad and durable protection against diverse pathogens.
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http://dx.doi.org/10.1172/jci.insight.136653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259527PMC
May 2020

Inducible Bronchus-Associated Lymphoid Tissues (iBALT) Serve as Sites of B Cell Selection and Maturation Following Influenza Infection in Mice.

Front Immunol 2019 29;10:611. Epub 2019 Mar 29.

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

Seasonally recurrent influenza virus infections are a significant cause of global morbidity and mortality. In murine models, primary influenza infection in the respiratory tract elicits potent humoral responses concentrated in the draining mediastinal lymph node and the spleen. In addition to immunity within secondary lymphoid organs (SLO), pulmonary infection is also associated with formation of ectopic inducible bronchus-associated tissues (iBALT) in the lung. These structures display a lymphoid organization, but their function and protective benefits remain unclear. Here we examined the phenotype, transcriptional profile and antigen specificity of B cell populations forming iBALT in influenza infected mice. We show that the cellular composition of iBALT was comparable to SLO, containing populations of follicular dendritic cells (FDC), T-follicular helper (Tfh) cells, and germinal center (GC)-like B cells with classical dark- and light-zone polarization. Transcriptional profiles of GC B cells in iBALT and SLO were conserved regardless of anatomical localization. The architecture of iBALT was pleiomorphic and less structurally defined than SLO. Nevertheless, we show that GC-like structures within iBALT serve as a distinct niche that independently support the maturation and selection of B cells primarily targeted against the influenza virus nucleoprotein. Our findings suggest that iBALT, which are positioned at the frontline of the lung mucosa, drive long-lived, and unique GC reactions that contribute to the diversity of the humoral response targeting influenza.
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http://dx.doi.org/10.3389/fimmu.2019.00611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450362PMC
August 2020

Identification of murine antigen-specific T follicular helper cells using an activation-induced marker assay.

J Immunol Methods 2019 04 22;467:48-57. Epub 2019 Feb 22.

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

Protective antibody (Ab) responses induced by natural infection or vaccination play a central role in defense against invasive pathogens. Germinal centers (GCs) are the sites of Ab affinity maturation and T follicular helper (Tfh) cells are a critical factor for driving GC formation and B cell selection. Therefore characterization of antigen (Ag)-specific Tfh cells is increasingly essential to define the mechanistic basis of protective antibody responses. However, since Tfh are weak producers of cytokines it is difficult to detect Ag-specific Tfh cells using conventional intracellular cytokine staining (ICS). Here, we report an assay identifying mouse Ag-specific Tfh cells by assessing the upregulation of surface activation-induced markers (AIM). Murine lymph node (LN)-derived Tfh cells largely retained CXCR5 and PD-1 expression following 18-hour cell culture. After influenza infection or influenza hemagglutinin (HA) protein vaccination of mice, stimulation of lymph node cell suspensions with peptide pools or whole protein drove upregulation of CD25, OX40 (CD134), ICOS (CD278) and CD154 on Tfh cells. Upregulation of either CD154 or CD25/OX40 proved a sensitive method for delineating HA-specific Tfh cells. This assay provides the opportunity to quantify antigen-specific Tfh cells in mice without the need for transgenic models or MHC-II tetramer reagents restricted to specific epitopes.
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http://dx.doi.org/10.1016/j.jim.2019.02.008DOI Listing
April 2019

Cross-lineage protection by human antibodies binding the influenza B hemagglutinin.

Nat Commun 2019 01 18;10(1):324. Epub 2019 Jan 18.

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

Influenza B viruses (IBV) drive a significant proportion of influenza-related hospitalisations yet are understudied compared to influenza A. Current vaccines target the head of the viral hemagglutinin (HA) which undergoes rapid mutation, significantly reducing vaccine effectiveness. Improved vaccines to control IBV are needed. Here we developed novel IBV HA probes to interrogate humoral responses to IBV in humans. A significant proportion of IBV HA-specific B cells recognise both B/Victoria/2/87-like and B/Yamagata/16/88-like lineages in a distinct pattern of cross-reactivity. Monoclonal antibodies (mAbs) were reconstituted from IBV HA-specific B cells, including mAbs providing broad protection in murine models of lethal IBV infection. Protection was mediated by neutralising antibodies targeting the receptor binding domain, or via Fc-mediated functions of non-neutralising antibodies binding alternative epitopes including the IBV HA stem. This work defines antigenic cross-recognition between IBV lineages and provides guidance for the rational design of improved IBV vaccines for broad and durable protection.
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http://dx.doi.org/10.1038/s41467-018-08165-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338745PMC
January 2019

Influenza Virus Infection Enhances Antibody-Mediated NK Cell Functions via Type I Interferon-Dependent Pathways.

J Virol 2019 03 19;93(5). Epub 2019 Feb 19.

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

Natural killer (NK) cells are an important component in the control of influenza virus infection, acting to both clear virus-infected cells and release antiviral cytokines. Engagement of CD16 on NK cells by antibody-coated influenza virus-infected cells results in antibody-dependent cellular cytotoxicity (ADCC). Increasing the potency of antibody-mediated NK cell activity could ultimately lead to improved control of influenza virus infection. To understand if NK cells can be functionally enhanced following exposure to influenza virus-infected cells, we cocultured human peripheral blood mononuclear cells (PBMCs) with influenza virus-infected human alveolar epithelial (A549) cells and evaluated the capacity of NK cells to mediate antibody-dependent functions. Preincubation of PBMCs with influenza virus-infected cells markedly enhanced the ability of NK cells to respond to immune complexes containing hemagglutinin (HA) and anti-HA antibodies or transformed allogeneic cells in the presence or absence of a therapeutic monoclonal antibody. Cytokine multiplex, RNA sequencing, supernatant transfer, Transwell, and cytokine-blocking/cytokine supplementation experiments showed that type I interferons released from PBMCs were primarily responsible for the influenza virus-induced enhancement of antibody-mediated NK cell functions. Importantly, the influenza virus-mediated increase in antibody-dependent NK cell functionality was mimicked by the type I interferon agonist poly(I·C). We conclude that the type I interferon secretion induced by influenza virus infection enhances the capacity of NK cells to mediate ADCC and that this pathway could be manipulated to alter the potency of anti-influenza virus therapies and vaccines. Protection from severe influenza may be assisted by antibodies that engage NK cells to kill infected cells through ADCC. Studies have primarily focused on antibodies that have ADCC activity, rather than the capacity of NK cells to become activated and mediate ADCC during an influenza virus infection. We found that type I interferon released in response to influenza virus infection primes NK cells to become highly reactive to anti-influenza virus ADCC antibodies. Enhancing the capacity of NK cells to mediate ADCC could assist in controlling influenza virus infections.
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http://dx.doi.org/10.1128/JVI.02090-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384076PMC
March 2019

Subdominance and poor intrinsic immunogenicity limit humoral immunity targeting influenza HA stem.

J Clin Invest 2019 02 22;129(2):850-862. Epub 2019 Jan 22.

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

Both natural influenza infection and current seasonal influenza vaccines primarily induce neutralizing antibody responses against highly diverse epitopes within the "head" of the viral hemagglutinin (HA) protein. There is increasing interest in redirecting immunity toward the more conserved HA stem or stalk as a means of broadening protective antibody responses. Here we examined HA stem-specific B cell and T follicular helper (Tfh) cell responses in the context of influenza infection and immunization in mouse and monkey models. We found that during infection, the stem domain was immunologically subdominant to the head in terms of serum antibody production and antigen-specific B and Tfh cell responses. Similarly, we found that HA stem immunogens were poorly immunogenic compared with the full-length HA with abolished sialic acid binding activity, with limiting Tfh cell elicitation a potential constraint to the induction or boosting of anti-stem immunity by vaccination. Finally, we confirm that currently licensed seasonal influenza vaccines can boost preexisting memory responses against the HA stem in humans. An increased understanding of the immune dynamics surrounding the HA stem is essential to inform the design of next-generation influenza vaccines for broad and durable protection.
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http://dx.doi.org/10.1172/JCI123366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6355240PMC
February 2019

Recombinant influenza virus expressing HIV-1 p24 capsid protein induces mucosal HIV-specific CD8 T-cell responses.

Vaccine 2016 Feb 28;34(9):1172-9. Epub 2016 Jan 28.

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

Influenza viruses are promising mucosal vaccine vectors for HIV but their use has been limited by difficulties in engineering the expression of large amounts of foreign protein. We developed recombinant influenza viruses incorporating the HIV-1 p24 gag capsid into the NS-segment of PR8 (H1N1) and X31 (H3N2) influenza viruses with the use of multiple 2A ribosomal skip sequences. Despite the insertion of a sizable HIV-1 gene into the influenza genome, recombinant viruses were readily rescued to high titers. Intracellular expression of p24 capsid was confirmed by in vitro infection assays. The recombinant influenza viruses were subsequently tested as mucosal vaccines in BALB/c mice. Recombinant viruses were attenuated and safe in immunized mice. Systemic and mucosal HIV-specific CD8 T-cell responses were elicited in mice that were immunized via intranasal route with a prime-boost regimen. Isolated HIV-specific CD8 T-cells displayed polyfunctional cytokine and degranulation profiles. Mice boosted via intravaginal route induced recall responses from the distal lung mucosa and developed heightened HIV-specific CD8 T-cell responses in the vaginal mucosa. These findings demonstrate the potential utility of recombinant influenza viruses as vaccines for mucosal immunity against HIV-1 infection.
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http://dx.doi.org/10.1016/j.vaccine.2016.01.030DOI Listing
February 2016

Contemporary HIV Vaccines: Tissue Resident T-Cells and Strategies to Prevent Mucosal Infection.

Curr Top Med Chem 2016 ;16(10):1107-17

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

HIV is primarily transmitted to women via the cervicovaginal mucosa, with the infection remaining localized for several days prior to systemic dissemination and irreversible damage to the immune system. The early phase during which HIV infection is localized and exhibits little or no viral diversity presents a vantage point for HIV vaccines that stimulate T-cell mediated clearance. CD(8+) resident memory T-cells (TRM) are positioned at mucosal entry sites and are established upon resolution of infection by mucosal pathogens. TRM cells are long-lived and locally patrol mucosal tissues. Upon antigenic reactivation, the sentinel-like functions of TRM cells mediate rapid clearance of subsequent infection by recruitment of additional immune cells from circulation and initiate a tissue-wide antiviral state, thus preventing the recurrence of disease. These properties are ideally suited for an HIV vaccine aimed at halting the infection cycle of HIV during the earliest phases. In this review, we summarize recent vaccine developments from parallel research areas incorporating the use of live mucosal vectors complemented with chemokine-regulating compounds, which can induce the seeding of the vaginal mucosa with TRM cells. We present the proposition that similar novel vaccine regimens can be translated into approaches for future HIV vaccines aimed at inducing heightened immunity in vaginal tissues against HIV.
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http://dx.doi.org/10.2174/1568026615666150901114920DOI Listing
November 2016

Standard trivalent influenza virus protein vaccination does not prime antibody-dependent cellular cytotoxicity in macaques.

J Virol 2013 Dec 9;87(24):13706-18. Epub 2013 Oct 9.

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

Yearly vaccination with the trivalent inactivated influenza vaccine (TIV) is recommended, since current vaccines induce little cross neutralization to divergent influenza strains. Whether the TIV can induce antibody-dependent cellular cytotoxicity (ADCC) responses that can cross-recognize divergent influenza virus strains is unknown. We immunized 6 influenza-naive pigtail macaques twice with the 2011-2012 season TIV and then challenged the macaques, along with 12 control macaques, serially with H1N1 and H3N2 viruses. We measured ADCC responses in plasma to a panel of H1 and H3 hemagglutinin (HA) proteins and influenza virus-specific CD8 T cell (CTL) responses using a sensitive major histocompatibility complex (MHC) tetramer reagent. The TIV was weakly immunogenic and, although binding antibodies were detected by enzyme-linked immunosorbent assay (ELISA), did not induce detectable influenza virus-specific ADCC or CTL responses. The H1N1 challenge elicited robust ADCC to both homologous and heterologous H1 HA proteins, but not influenza virus HA proteins from different subtypes (H2 to H7). There was no anamnestic influenza virus-specific ADCC or CTL response in vaccinated animals. The subsequent H3N2 challenge did not induce or boost ADCC either to H1 HA proteins or to divergent H3 proteins but did boost CTL responses. ADCC or CTL responses were not induced by TIV vaccination in influenza-naive macaques. There was a marked difference in the ability of infection compared to that of vaccination to induce cross-reactive ADCC and CTL responses. Improved vaccination strategies are needed to induce broad-based ADCC immunity to influenza.
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http://dx.doi.org/10.1128/JVI.01666-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3838265PMC
December 2013
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