Microbiology & Immunology - Research Publications
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ItemInduction of vaginal-resident HIV-specific CD8 T cells with mucosal prime-boost immunization(NATURE PUBLISHING GROUP, 2018-05)Tissue-resident memory (TRM) CD8 T cells survey a range of non-lymphoid mucosal tissues where they rapidly mediate clearance of viral infections at the entry portals. Vaccines that establish CD8 TRM cells in the cervicovaginal mucosa hold promise for effective immunity against sexually transmitted HIV. We demonstrate that HIV-specific CD8 TRM cells can be established in the murine vaginal mucosa using a combined intranasal and intravaginal mucosal immunization with recombinant influenza-HIV vectors. Using in situ tetramer immunofluorescence microscopy, we found that this mucosally administered prime-boost immunization also resulted in the durable seeding of CD8 T cells in the frontline vaginal epithelial compartment as opposed to the vaginal submucosa. Upon cognate antigen recognition within the vaginal mucosa, these HIV-specific CD8 TRM cells rapidly initiated a tissue-wide state of immunity. The activation of HIV-specific CD8 TRM cells resulted in the upregulation of endothelial vessel addressin expression and substantial recruitment of both adaptive and innate immune cells in the vaginal mucosa. These findings suggest that the epithelial localization of HIV-specific CD8 TRM cell populations and their capacity to rapidly activate both arms of the immune system could significantly augment frontline defenses against vaginal HIV infection.
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ItemHIV-dependent depletion of influenza-specific memory B cells impacts B cell responsiveness to seasonal influenza immunisation(NATURE PORTFOLIO, 2016-05-25)Infection with HIV drives significant alterations in B cell phenotype and function that can markedly influence antibody responses to immunisation. Anti-retroviral therapy (ART) can partially reverse many aspects of B cell dysregulation, however complete normalisation of vaccine responsiveness is not always observed. Here we examine the effects of underlying HIV infection upon humoral immunity to seasonal influenza vaccines. Serological and memory B cell responses were assessed in 26 HIV+ subjects receiving ART and 30 healthy controls immunised with the 2015 Southern Hemisphere trivalent inactivated influenza vaccine (IIV3). Frequencies and phenotypes of influenza hemagglutinin (HA)-specific B cells were assessed by flow cytometry using recombinant HA probes. Serum antibody was measured using hemagglutination inhibition assays. Serological responses to IIV3 were comparable between HIV+ and HIV- subjects. Likewise, the activation and expansion of memory B cell populations specific for vaccine-component influenza strains was observed in both cohorts, however peak frequencies were diminished in HIV+ subjects compared to uninfected controls. Lower circulating frequencies of memory B cells recognising vaccine-component and historical influenza strains were observed in HIV+ subjects at baseline, that were generally restored to levels comparable with HIV- controls post-vaccination. HIV infection is therefore associated with depletion of selected HA-specific memory B cell pools.
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ItemTwist in the Tail: Escape from HIV Neutralising Antibodies at a Single Site Confers Broad Susceptibility to Others(ELSEVIER SCIENCE BV, 2016-10)
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ItemCross-lineage protection by human antibodies binding the influenza B hemagglutinin(NATURE PUBLISHING GROUP, 2019-01-18)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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ItemSubdominance and poor intrinsic immunogenicity limit humoral immunity targeting influenza HA stem(AMER SOC CLINICAL INVESTIGATION INC, 2019-02-01)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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ItemInducible Bronchus-Associated Lymphoid Tissues (iBALT) Serve as Sites of B Cell Selection and Maturation Following Influenza Infection in Mice(FRONTIERS MEDIA SA, 2019-03-29)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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ItemImproving immunological insights into the ferret model of human viral infectious disease(WILEY, 2019-11)Ferrets are a well-established model for studying both the pathogenesis and transmission of human respiratory viruses and evaluation of antiviral vaccines. Advanced immunological studies would add substantial value to the ferret models of disease but are hindered by the low number of ferret-reactive reagents available for flow cytometry and immunohistochemistry. Nevertheless, progress has been made to understand immune responses in the ferret model with a limited set of ferret-specific reagents and assays. This review examines current immunological insights gained from the ferret model across relevant human respiratory diseases, with a focus on influenza viruses. We highlight key knowledge gaps that need to be bridged to advance the utility of ferrets for immunological studies.
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ItemLow pH Exposure During Immunoglobulin G Purification Methods Results in Aggregates That Avidly Bind Fcg Receptors: Implications for Measuring Fc Dependent Antibody Functions(FRONTIERS MEDIA SA, 2019-10-11)Evaluating the biophysical and functional nature of IgG is key to defining correlates of protection in infectious disease, and autoimmunity research cohorts, as well as vaccine efficacy trials. These studies often require small quantities of IgG to be purified from plasma for downstream analysis with high throughput immunoaffinity formats which elute IgG at low-pH, such as Protein G and Protein A. Herein we sought to compare Protein G purification of IgG with an immunoaffinity method which elutes at physiological pH (Melon Gel). Critical factors impacting Fc functionality with the potential to significantly influence FcγR binding, such as IgG subclass distribution, N-glycosylation, aggregation, and IgG conformational changes were investigated and compared. We observed that transient exposure of IgG to the low-pH elution buffer, used during the Protein G purification process, artificially enhanced recognition of Fcγ Receptors (FcγRs) as demonstrated by Surface Plasmon Resonance (SPR), FcγR dimer ELISA, and a functional cell-based assay. Furthermore, low-pH exposed IgG caused conformational changes resulting in increased aggregation and hydrophobicity; factors likely to contribute to the observed enhanced interaction with FcγRs. These results highlight that methods employed to purify IgG can significantly alter FcγR-binding behavior and biological activity and suggest that the IgG purification approach selected may be a previously overlooked factor contributing to the poor reproducibility across current assays employed to evaluate Fc-mediated antibody effector functions.
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ItemLink between Low-Fouling and Stealth: A Whole Blood Biomolecular Corona and Cellular Association Analysis on Nanoengineered Particles(American Chemical Society, 2019-05-28)Upon exposure to human blood, nanoengineered particles interact with a multitude of plasma components, resulting in the formation of a biomolecular corona. This corona modulates downstream biological responses, including recognition by and association with human immune cells. Considerable research effort has been directed toward the design of materials that can demonstrate a low affinity for various proteins (low-fouling materials) and materials that can exhibit low association with human immune cells (stealth materials). An implicit assumption common to bio–nano research is that nanoengineered particles that are low-fouling will also exhibit stealth. Herein, we investigated the link between the low-fouling properties of a particle and its propensity for stealth in whole human blood. High-fouling mesoporous silica (MS) particles and low-fouling zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) particles were synthesized, and their interaction with blood components was assessed before and after precoating with serum albumin, immunoglobulin G, or complement protein C1q. We performed an in-depth proteomics characterization of the biomolecular corona that both identifies specific proteins and measures their relative abundance. This was compared with observations from a whole blood association assay that identified with which cell type each particle system associates. PMPC-based particles displayed reduced association both with cells and with serum proteins compared with MS-based particles. Furthermore, the enrichment of specific proteins within the biomolecular corona was found to correlate with association with specific cell types. This study demonstrates how the low-fouling properties of a material are indicative of its stealth with respect to immune cell association.
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ItemCirculating TFH cells, serological memory, and tissue compartmentalization shape human influenza-specific B cell immunity(AMER ASSOC ADVANCEMENT SCIENCE, 2018-02-14)Immunization with the inactivated influenza vaccine (IIV) remains the most effective strategy to combat seasonal influenza infections. IIV activates B cells and T follicular helper (TFH) cells and thus engenders antibody-secreting cells and serum antibody titers. However, the cellular events preceding generation of protective immunity in humans are inadequately understood. We undertook an in-depth analysis of B cell and T cell immune responses to IIV in 35 healthy adults. Using recombinant hemagglutinin (rHA) probes to dissect the quantity, phenotype, and isotype of influenza-specific B cells against A/California09-H1N1, A/Switzerland-H3N2, and B/Phuket, we showed that vaccination induced a three-pronged B cell response comprising a transient CXCR5-CXCR3+ antibody-secreting B cell population, CD21hiCD27+ memory B cells, and CD21loCD27+ B cells. Activation of circulating TFH cells correlated with the development of both CD21lo and CD21hi memory B cells. However, preexisting antibodies could limit increases in serum antibody titers. IIV had no marked effect on CD8+, mucosal-associated invariant T, γδ T, and natural killer cell activation. In addition, vaccine-induced B cells were not maintained in peripheral blood at 1 year after vaccination. We provide a dissection of rHA-specific B cells across seven human tissue compartments, showing that influenza-specific memory (CD21hiCD27+) B cells primarily reside within secondary lymphoid tissues and the lungs. Our study suggests that a rational design of universal vaccines needs to consider circulating TFH cells, preexisting serological memory, and tissue compartmentalization for effective B cell immunity, as well as to improve targeting cellular T cell immunity.