Magnitude of Therapeutic STING Activation Determines CD8+ T Cell-Mediated Anti-tumor Immunity.

Intratumoral (IT) STING activation results in tumor regression in preclinical models, yet factors dictating the balance between innate and adaptive anti-tumor immunity are unclear. Here, clinical candidate STING agonist ADU-S100 (S100) is used in an IT dosing regimen optimized for adaptive immunity to uncover requirements for a T cell-driven response compatible with checkpoint inhibitors (CPIs). In contrast to high-dose tumor ablative regimens that result in systemic S100 distribution, low-dose immunogenic regimens induce local activation of tumor-specific CD8+ effector T cells that are responsible for durable anti-tumor immunity and can be enhanced with CPIs. Both hematopoietic cell STING expression and signaling through IFNAR are required for tumor-specific T cell activation, and in the context of optimized T cell responses, TNFα is dispensable for tumor control. In a poorly immunogenic model, S100 combined with CPIs generates a survival benefit and durable protection. These results provide fundamental mechanistic insights into STING-induced anti-tumor immunity.

Recombinant Listeria promotes tumor rejection by CD8+ T cell-dependent remodeling of the tumor microenvironment.

Agents that remodel the tumor microenvironment (TME), prime functional tumor-specific T cells, and block inhibitory signaling pathways are essential components of effective immunotherapy. We are evaluating live-attenuated, double-deleted Listeria monocytogenes expressing tumor antigens (LADD-Ag) in the clinic. Here we show in numerous mouse models that while treatment with nonrecombinant LADD induced some changes in the TME, no antitumor efficacy was observed, even when combined with immune checkpoint blockade. In contrast, LADD-Ag promoted tumor rejection by priming tumor-specific KLRG1+PD1loCD62L- CD8+ T cells. These IFNγ-producing effector CD8+ T cells infiltrated the tumor and converted the tumor from an immunosuppressive to an inflamed microenvironment that was characterized by a decrease in regulatory T cells (Treg) levels, a proinflammatory cytokine milieu, and the shift of M2 macrophages to an inducible nitric oxide synthase (iNOS)+CD206- M1 phenotype. Remarkably, these LADD-Ag-induced tumor-specific T cells persisted for more than 2 months after primary tumor challenge and rapidly controlled secondary tumor challenge. Our results indicate that the striking antitumor efficacy observed in mice with LADD-based immunotherapy stems from TME remodeling which is a direct consequence of eliciting potent, systemic tumor-specific CD8+ T cells.

TNFα and Radioresistant Stromal Cells Are Essential for Therapeutic Efficacy of Cyclic Dinucleotide STING Agonists in Nonimmunogenic Tumors.

The cGAS-STING cytosolic DNA sensing pathway may play an integral role in the initiation of antitumor immune responses. Studies evaluating the immunogenicity of various cyclic dinucleotide (CDN) STING agonists administered by intratumoral (i.t.) injection showed potent induction of inflammation, tumor necrosis, and, in some cases, durable tumor-specific adaptive immunity. However, the specific immune mechanisms underlying these responses remain incompletely defined. The majority of these studies have focused on the effect of CDNs on immune cells but have not conclusively interrogated the role of stromal cells in the acute rejection of the CDN-injected tumor. Here, we revealed a mechanism of STING agonist-mediated tumor response that relied on both stromal and immune cells to achieve tumor regression and clearance. Using knockout and bone marrow chimeric mice, we showed that although bone marrow-derived TNFα was necessary for CDN-induced necrosis, STING signaling in radioresistant stromal cells was also essential for CDN-mediated tumor rejection. These results provide evidence for crosstalk between stromal and hematopoietic cells during CDN-mediated tumor collapse after i.t. administration. These mechanistic insights may prove critical in the clinical development of STING agonists. Cancer Immunol Res; 6(4); 422-33. ©2018 AACR.

Analysis of the Innate Response to Adjuvants: Characterization of the Draining Lymph Node by Fluorescence-Activated Cell Sorting.

A clear index for a response to adjuvants is a change in the cellular composition of lymph nodes draining the site of adjuvant injection (Didierlaurent et al., J Immunol 183:6186-6197, 2009; Caproni et al., J Immunol 188:3088-98, 2012; Desbien et al., Eur J Immunol 1-11, 2014). During the steady state, lymph nodes (LNs) are composed of a fixed ratio of innate and adaptive cells awaiting activation signals from tissue draining lymph. Upon exposure to innate stimulants, lymph nodes undergo dramatic changes. The most apparent change to the lymph node is an increase in size. Antigen-independent activation of naïve T cells and B cells, as a consequence of type I interferon signaling, results in upregulation of CD69 (Sun and Zhang, J. Exp. Med 188:2335-2342, 1998), causing increased retention of cells in the lymph node and transient lymphopenia in the blood (Shiow et al., Nature 440:540-544, 2006). In addition tissue-resident dendritic cells, macrophages, as well as circulating inflammatory monocytes will migrate into draining LNs and display maturation markers associated with activation. Such features can provide powerful discrimination of adjuvant potencies.

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.

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.

Antigen presentation by B cells guides programing of memory CD4+ T-cell responses to a TLR4-agonist containing vaccine in mice.

The contribution of B cells to immunity against many infectious diseases is unquestionably important and well characterized. Here, we sought to determine the role of B cells in the induction of T-helper 1 (TH 1) CD4+ T cells upon vaccination with a tuberculosis (TB) antigen combined with a TLR4 agonist. We used B-cell deficient mice (µMT-/- ), tetramer-positive CD4+ T cells, markers of memory "precursor" effector cells (MPECs), and T-cell adoptive transfers and demonstrated that the early antigen-specific cytokine-producing TH 1 responses are unaffected in the absence of B cells, however MPEC induction is strongly impaired resulting in a deficiency of the memory TH 1 response in µMT-/- mice. We further show that antigen-presentation by B cells is necessary for their role in MPEC generation using B-cell adoptive transfers from wt or MHC class II knock-out mice into µMT-/- mice. Our study challenges the view that B-cell deficiency exclusively alters the TH 1 response at memory time-points. Collectively, our results provide new insights on the multifaceted roles of B cells that will have a high impact on vaccine development against several pathogens including those requiring TH 1 cell-mediated immunity.

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.

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.

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.

TLR4 ligand formulation causes distinct effects on antigen-specific cell-mediated and humoral immune responses.

The formulation of TLR ligands and other immunomodulators has a critical effect on their vaccine adjuvant activity. In this work, the synthetic TLR4 ligand GLA was formulated with three distinct vaccine delivery system platforms (aqueous suspension, liposome, or oil-in-water emulsion). The effect of the different formulations on the adaptive immune response to protein subunit vaccines was evaluated in the context of a recombinant malaria antigen, Plasmodium berghei circumsporozoite protein (PbCSP). Antibody responses in vaccinated mice were similar for the different formulations of GLA. However, cell-mediated responses differed significantly depending on the adjuvant system; in particular, the emulsion formulation of the TLR4 ligand induced significantly enhanced cellular IFN-γ and TNF-α responses compared to the other formulations. The effects of differences in adjuvant formulation composition and physical characteristics on biological activity are discussed. These results illustrate the importance of formulation of immunostimulatory adjuvants (e.g. TLR ligands) on the resulting immune responses to adjuvanted vaccines and may play a critical role for combating diseases where T cell immunity is advantageous.

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.

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 T(H)1 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 T(H)1 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 T(H)1 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 T(H)1-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.

Targeting TLRs expands the antibody repertoire in response to a malaria vaccine.

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.

The Epstein-Barr virus Bcl-2 homolog, BHRF1, blocks apoptosis by binding to a limited amount of Bim.

Current knowledge suggests that the balance between life and death within a cell can be controlled by the stable engagement of Bcl-2-related proapoptotic proteins such as Bak, Bax, and Bim by survival proteins such as Bcl-2. BHRF1 is a prosurvival molecule from Epstein-Barr virus that has a high degree of homology to Bcl-2. To understand how BHRF1 blocks apoptosis, BHRF1 and mutants of BHRF1 were expressed in primary cells and an IL-2-dependent T cell line. BHRF1 bound the Executioner Bak and, when cells were cultured without cytokines, BHRF1 associated with Bim. A point mutation that lost the ability to bind Bak retained its ability to bind Bim and to protect cells. This result demonstrated that it was the capacity of BHRF1 to bind Bim, not Bak, that provided protection. Interestingly, the amount of Bim bound by BHRF1 was minimal when compared with the amount of Bim induced by apoptosis. Thus, BHRF1 does not act by simply absorbing the excess Bim produced while cells prepare for death. Rather, BHRF1 may act either by binding preferentially the most lethal form of Bim or by acting catalytically on Bim to block apoptosis.

Impaired viral entry cannot explain reduced CD4+ T cell susceptibility to HIV type 1 in certain highly exposed individuals.

Rare individuals report repeated unprotected HIV-1 sexual exposures, yet remain seronegative for years. We investigated the possibility that reduced in vitro CD4(+) T cell susceptibility to HIV-1 infection protects such highly exposed seronegative (ES) individuals. Susceptibility to three R5-tropic HIV-1 isolates, regardless of inoculating dose, was remarkably similar between 81 ES and 33 low-risk controls. In 94% (99/105) of donors, we observed a 1.36 log-unit range in HIV-1(JR-CSF) production, with similar results for HIV-1(1192). The median frequency of intracellular Gag(+) T cells after single-round infection was similar in ES (5.2%) and controls (7.2%), p = 0.456. However, in repeated testing, CD4(+) T cells from two controls (6.1%) and four ES (4.9%) exhibited a 10- to 2500-fold reduction in HIV-1 production and required 5- to 12-fold greater HIV-1(1192) and HIV-1(JR-CSF) inocula to establish infection (TCID(50)). Reduced viral entry cannot explain the low producer phenotype; no differences in CCR5 receptor density or beta-chemokine production were observed. In conclusion, we have identified a remarkably narrow range of HIV-1 susceptibility in seronegative donors regardless of risk activity, which can be applied as a benchmark to assess vaccine-induced antiviral effector activities. However, CD4(+) T cells from a subset of individuals demonstrated reduced HIV-1 susceptibility unexplained by impaired entry, lending support to the possibility that cellular restriction of HIV-1 may account for continued seronegativity in some of those having repeated sexual exposure. Identifying the host-virus interactions responsible for diminished in vitro susceptibility may contribute to the development of novel therapeutic strategies.

Most highly exposed seronegative men lack HIV-1-specific, IFN-gamma-secreting T cells.

Naturally acquired cellular immunity in individuals who have been exposed to HIV-1 but have remained uninfected may hold clues for the design of an effective HIV vaccine. To determine the presence and nature of such an HIV-1-specific immune response, we evaluated the quantity and fine specificity of HIV-1-reactive IFN-gamma-secreting T cells in a group of highly exposed seronegative men having sex with men. All 46 ES reported frequent unprotected anal sex with known HIV-1-infected partners at enrollment, and high risk activities continued in at least one-half of the volunteers for up to >6 years of observation. Despite the high frequency of unprotected anal intercourse and potential HIV-1 exposure, the vast majority of individuals demonstrated no or very low numbers of HIV-1-specific, IFN-gamma-secreting T cells. Even when HIV-1 epitopes were presented by peptide-pulsed autologous dendritic cells in 15 of the highest risk volunteers, HIV-1-specific T cells remained infrequent, and the proportion of responders was not significantly different from that in a lower risk seronegative control cohort. Only PBMC from two individuals who have remained uninfected to date exhibited distinctly positive responses. However, these responses rarely persisted over time, single epitope specificities were identified in only one volunteer, and HIV-1-specific memory T cell clones did not expand in vitro. HIV-1-specific, IFN-gamma-secreting T cells are thus unlikely to substantially contribute to resistance against infection in most exposed seronegative men having sex with men.