Anthony L. Desbien

Targeting TLRs Expands the Antibody Repertoire in Response to a Malaria Vaccine

The use of TLR agonists in vaccination broadens the range of polymorphic variants against which the antibodies can be effective. Help! Sometimes one very talented athlete can carry a team to a championship. Yet, not even the best athletes can reach their full potential without the support of their teammates. Similarly, successful vaccines require strong and specific pathogen-derived antigens, but frequently, one of these essential players is not multitalented enough to elicit a protective immune response. To do so, these stars require help—which comes in the form of adjuvants. Whereas specific antigens induce a slower but pathogen-restricted immune response, adjuvants activate the faster but more general innate immune system. The addition of adjuvants to vaccine formulations is known to improve the quality, strength, and duration of the immune response by somewhat nebulous mechanisms. Now, Wiley et al. use massively parallel sequencing to quantify the immune response to a malarial antigen and find that a little help from an adjuvant results in added antibody diversity. The authors showed that adding a Toll-like receptor 4 agonist, which turns on a pattern-recognition receptor that activates innate immune cells, to a commonly used oil-in-water adjuvant in an antimalaria vaccine formulation greatly increased the diversity of antibodies made in response to the vaccine. These antibodies were better able to neutralize and could respond to more variants of the antigen. Therefore, in the context of an infection, these adjuvanted vaccines should be able to successfully fight more strains of a pathogen. What’s more, the sequencing method used by Wiley et al. should be broadly applicable to the characterization of immune responses to other vaccines and to infections, thus leading to improvements in the detection, diagnosis, and treatment of various diseases. Furthermore, this strategy can be used to scout out adjuvants that make the best teammates for pathogen-specific antigens in vaccine formulations. Vaccination with an isolated antigen is frequently not sufficient to elicit a protective immune response. The addition of adjuvants to the antigen can increase the magnitude and breadth of the response generated, but quantification of this increase as a function of adjuvant has been intractable. We have directly determined the variation of the immunoglobulin G variable-chain repertoire of an entire organism as a function of vaccination. Using the well-established Plasmodium vivax antigen, PvRII, and massively parallel sequencing, we showed that the use of a Toll-like receptor (TLR) agonist in the vaccine formulation increased the diversity of the variable region sequences in comparison to the use of an oil-in-water emulsion adjuvant alone. Moreover, increased variable domain diversity in response to the use of TLR agonist–based adjuvants correlated with improved antigen neutralization. The use of TLR agonists also broadened the range of polymorphic variants against which these antibodies could be effective. In addition, a peptide microarray demonstrated that inclusion of adjuvants changed the profile of linear epitopes from PvRII that were recognized by serum from immunized animals. The results of these studies have broad implications for vaccine design—they may enable tailored adjuvants that elicit the broad spectrum of antibodies required to neutralize drifted and polymorphic pathogen strains as well as provide a method for rapid determination of correlates of adjuvant-induced humoral immunity.

MyD88 and TRIF synergistic interaction is required for TH1‐cell polarization with a synthetic TLR4 agonist adjuvant

Glucopyranosyl lipid adjuvant‐stable emulsion (GLA‐SE) is a synthetic adjuvant TLR4 agonist that promotes potent poly‐functional TH1 responses. Different TLR4 agonists may preferentially signal via MyD88 or TIR‐domain‐containing adapter inducing IFN‐beta (TRIF) to exert adjuvant effects; however, the contribution of MyD88 and TRIF signaling to the induction of polyclonal TH1 responses by TLR4 agonist adjuvants has not been studied in vivo. To determine whether GLA‐SE preferentially signals through MyD88 or TRIF, we evaluated the immune response against a candidate tuberculosis (TB) vaccine Ag following immunization of mice lacking either signaling adapter compared with that of wild‐type mice. We find that both MyD88 and TRIF are necessary for GLA‐SE to induce a poly‐functional TH1 immune response characterized by CD4+ T cells producing IFN‐γ, TNF, and IL‐2, as well as IgG2c class switching, when paired with the TB vaccine Ag ID93. Accordingly, the protective efficacy of ID93/GLA‐SE immunization against aerosolized Mycobacterium tuberculosis was lost when either signaling molecule was ablated. We demonstrate that MyD88 and TRIF must be expressed in the same cell for the in vivo TH1‐skewing adjuvant activity, indicating that these two signaling pathways cooperate on an intracellular level. Thus engagement of both the MyD88 and TRIF signaling pathways are essential for the effective adjuvant activity of this TLR4 agonist.

Cooperative intracellular interactions between MyD88 and TRIF are required for CD4 T cell TH1 polarization with a synthetic TLR4 agonist adjuvant

Glucopyranosyl lipid adjuvant‐stable emulsion (GLA‐SE) is a synthetic adjuvant TLR4 agonist that promotes potent poly‐functional TH1 responses. Different TLR4 agonists may preferentially signal via MyD88 or TIR‐domain‐containing adapter inducing IFN‐beta (TRIF) to exert adjuvant effects; however, the contribution of MyD88 and TRIF signaling to the induction of polyclonal TH1 responses by TLR4 agonist adjuvants has not been studied in vivo. To determine whether GLA‐SE preferentially signals through MyD88 or TRIF, we evaluated the immune response against a candidate tuberculosis (TB) vaccine Ag following immunization of mice lacking either signaling adapter compared with that of wild‐type mice. We find that both MyD88 and TRIF are necessary for GLA‐SE to induce a poly‐functional TH1 immune response characterized by CD4+ T cells producing IFN‐γ, TNF, and IL‐2, as well as IgG2c class switching, when paired with the TB vaccine Ag ID93. Accordingly, the protective efficacy of ID93/GLA‐SE immunization against aerosolized Mycobacterium tuberculosis was lost when either signaling molecule was ablated. We demonstrate that MyD88 and TRIF must be expressed in the same cell for the in vivo TH1‐skewing adjuvant activity, indicating that these two signaling pathways cooperate on an intracellular level. Thus engagement of both the MyD88 and TRIF signaling pathways are essential for the effective adjuvant activity of this TLR4 agonist.

Squalene emulsion potentiates the adjuvant activity of the TLR4 agonist, GLA, via inflammatory caspases, IL-18, and IFN-γ.

The synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) is a potent Th1‐response‐inducing adjuvant when formulated in a squalene oil‐in‐water emulsion (SE). While the innate signals triggered by TLR4 engagement are well studied, the contribution of SE remains unclear. To better understand the effect of SE on the adjuvant properties of GLA‐SE, we compared the innate and adaptive immune responses elicited by immunization with different formulations: GLA without oil, SE alone or the combination, GLA‐SE, in mice. Within the innate response to adjuvants, only GLA‐SE displayed features of inflammasome activation, evidenced by early IL‐18 secretion and IFN‐γ production in memory CD8+ T cells and neutrophils. Such early IFN‐γ production was ablated in caspase‐1/11−/− mice and in IL‐18R1−/− mice. Furthermore, caspase‐1/11 and IL‐18 were also required for full Th1 CD4+ T‐cell induction via GLA‐SE. Thus, we demonstrate that IL‐18 and caspase‐1/11 are components of the response to immunization with the TLR4 agonist/squalene oil‐in‐water based adjuvant, GLA‐SE, providing implications for other adjuvants that combine oils with TLR agonists.

The TLR4 Agonist Vaccine Adjuvant, GLA-SE, Requires Canonical and Atypical Mechanisms of Action for TH1 Induction

The Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant formulated in a stable emulsion (GLA-SE) promotes strong TH1 and balanced IgG1/IgG2 responses to protein vaccine antigens. This enhanced immunity is sufficient to provide protection against many diseases including tuberculosis and leishmaniasis. To better characterize the adjuvant action it is important to understand how the different cytokines and transcription factors contribute to the initiation of immunity. In the present study using T-bet-/- and IL-12-/- mice and a blocking anti-IFNαR1 monoclonal antibody, we define mechanisms of adjuvant activity of GLA-SE. In accordance with previous studies of TLR4 agonist based adjuvants, we found that TH1 induction via GLA-SE was completely dependent upon T-bet, a key transcription factor for IFNγ production and TH1 differentiation. Consistent with this, deficiency of IL-12, a cytokine canonical to TH1 induction, ablated TH1 induction via GLA-SE. Finally we demonstrate that the innate immune response to GLA-SE, including rapid IFNγ production by memory CD8+ T cells and NK cells, was contingent on type I interferon, a cytokine group whose association with TH1 induction is contextual, and that they contributed to the adjuvant activity of GLA-SE.

Elimination of the cold-chain dependence of a nanoemulsion adjuvanted vaccine against tuberculosis by lyophilization

Next-generation rationally-designed vaccine adjuvants represent a significant breakthrough to enable development of vaccines against challenging diseases including tuberculosis, HIV, and malaria. New vaccine candidates often require maintenance of a cold-chain process to ensure long-term stability and separate vials to enable bedside mixing of antigen and adjuvant. This presents a significant financial and technological barrier to worldwide implementation of such vaccines. Herein we describe the development and characterization of a tuberculosis vaccine comprised of both antigen and adjuvant components that are stable in a single vial at sustained elevated temperatures. Further this vaccine retains the ability to elicit both antibody and TH1 responses against the vaccine antigen and protect against experimental challenge with Mycobacterium tuberculosis. These results represent a significant breakthrough in the development of vaccine candidates that can be implemented throughout the world without being hampered by the necessity of a continuous cold chain or separate adjuvant and antigen vials.

IL-18 and Subcapsular Lymph Node Macrophages are Essential for Enhanced B Cell Responses with TLR4 Agonist Adjuvants.

Designing modern vaccine adjuvants depends on understanding the cellular and molecular events that connect innate and adaptive immune responses. The synthetic TLR4 agonist glycopyranosyl lipid adjuvant (GLA) formulated in a squalene-in-water emulsion (GLA-SE) augments both cellular and humoral immune responses to vaccine Ags. This adjuvant is currently included in several vaccines undergoing clinical evaluation including those for tuberculosis, leishmaniasis, and influenza. Delineation of the mechanisms of adjuvant activity will enable more informative evaluation of clinical trials. Early after injection, GLA-SE induces substantially more Ag-specific B cells, higher serum Ab titers, and greater numbers of T follicular helper (TFH) and Th1 cells than alum, the SE alone, or GLA without SE. GLA-SE augments Ag-specific B cell differentiation into germinal center and memory precursor B cells as well as preplasmablasts that rapidly secrete Abs. CD169+ SIGNR1+ subcapsular medullary macrophages are the primary cells to take up GLA-SE after immunization and are critical for the innate immune responses, including rapid IL-18 production, induced by GLA-SE. Depletion of subcapsular macrophages (SCMф) or abrogation of IL-18 signaling dramatically impairs the Ag-specific B cell and Ab responses augmented by GLA-SE. Depletion of SCMф also drastically reduces the Th1 but not the TFH response. Thus the GLA-SE adjuvant operates through interaction with IL-18–producing SCMф for the rapid induction of B cell expansion and differentiation, Ab secretion, and Th1 responses, whereas augmentation of TFH numbers by GLA-SE is independent of SCMф.

A structure-function approach to optimizing TLR4 ligands for human vaccines.

Adjuvants are combined with vaccine antigens to enhance and modify immune responses, and have historically been primarily crude, undefined entities. Introducing toll‐like receptor (TLR) ligands has led to a new generation of adjuvants, with TLR4 ligands being the most extensively used in human vaccines. The TLR4 crystal structures demonstrate extensive contact with their ligands and provide clues as to how they discriminate a broad array of molecules and activate or attenuate innate, as well as adaptive, responses resulting from these interactions. Leveraging this discerning ability, we made subtle chemical alterations to the structure of a synthetic monophosphoryl lipid‐A molecule to produce SLA, a designer TLR4 ligand that had a number of desirable adjuvant effects. The SLA molecule stimulated human TLR4 and induced Th1 biasing cytokines and chemokines. On human cells, the activity of SLA plateaued at lower concentrations than the lipid A comparator, and induced cytokine profiles distinct from other known TLR4 agonists, indicating the potential for superior adjuvant performance. SLA was formulated in an oil‐in‐water emulsion, producing an adjuvant that elicited potent Th1‐biased adaptive responses. This was verified using a recombinant Leishmania vaccine antigen, first in mice, then in a clinical study in which the antigen‐specific Th1‐biased responses observed in mice were recapitulated in humans. These results demonstrated that using structure‐based approaches one can predictably design and produce modern adjuvant formulations for safe and effective human vaccines.

Development of a high density hemagglutinin protein microarray to determine the breadth of influenza antibody responses

We have developed an influenza hemagglutinin protein microarray to assess humoral recognition of diverse influenza strains induced by vaccination and infection. Each array consists of controls and 127 hemagglutinin antigens from 60 viruses, spotted in replicates to generate a single array of 1296 spots. Six arrays are configured on a single slide, which in the following analysis was probed simultaneously with 2 isotype-specific fluorescent secondary antibodies yielding over 15,000 data points per slide. Here we report the use of this system to evaluate mouse, ferret, and human sera. The array allows simultaneous examination of the magnitude of antibody responses, the isotype of such antibodies, and the breadth of influenza strain recognition. We are advancing this technology as a platform for rapid, simple, high-throughput assessment of homologous and heterologous antibody responses to influenza disease and vaccination.

Comment on “The Common R71H-G230A-R293Q Human TMEM173 Is a Null Allele”

STING is a signaling protein encoded by the human TMEM173 gene that is central to the host response to infection. Upon binding cyclic dinucleotides (CDNs), STING activates transcriptional programs that orchestrate innate and adaptive immune responses ([1][1], [2][2]). For this reason, STING is a