Elaine Tritto

Molecular and cellular signatures of human vaccine adjuvants.

Oil-in-water emulsions are potent human adjuvants used for effective pandemic influenza vaccines; however, their mechanism of action is still unknown. By combining microarray and immunofluorescence analysis, we monitored the effects of the adjuvants MF59 oil-in-water emulsion, CpG, and alum in the mouse muscle. MF59 induced a time-dependent change in the expression of 891 genes, whereas CpG and alum regulated 387 and 312 genes, respectively. All adjuvants modulated a common set of 168 genes and promoted antigen-presenting cell recruitment. MF59 was the stronger inducer of cytokines, cytokine receptors, adhesion molecules involved in leukocyte migration, and antigen-presentation genes. In addition, MF59 triggered a more rapid influx of CD11b+ blood cells compared with other adjuvants. The early biomarkers selected by microarray, JunB and Ptx3, were used to identify skeletal muscle as a direct target of MF59. We propose that oil-in-water emulsions are the most efficient human vaccine adjuvants, because they induce an early and strong immunocompetent environment at the injection site by targeting muscle cells.

Mechanism of action of licensed vaccine adjuvants

Despite the fact that alum and oil-in-water emulsions have been used for decades as human vaccine adjuvants in a large number of individuals, their mechanism of action is not completely understood. It has been reported that these particulate adjuvants act by increasing antigen availability and uptake by immune cells. However, recent work on alum and on the squalene-based emulsion MF59, has demonstrated that besides antigen delivery functions, these classes of adjuvants can also activate innate immunity pathways in vivo, generating an immunocompetent environment at injection site. Interestingly, it has been demonstrated that alum adjuvanticity depends on the activation of a protein complex called NLPR3/inflammasome, which is required for the correct processing of a number of pro-inflammatory cytokines, including IL1beta. More work needs to be performed to investigate if the inflammasome is also required for the activity of MF59 and of other particulate vaccine adjuvants.

Alum adjuvanticity: Unraveling a century old mystery

The development of vaccine adjuvants for human use has been one of the slowest processes in the history of medicine. For almost one century, aluminium hydroxide (alum) has been the only vaccine adjuvant approved worldwide. Only in the past decade have two oil‐in‐water emulsions and one TLR agonist been approved by the European authorities as new vaccine adjuvants. Despite the fact that alum has been injected into billions of people, its mechanism of action is not fully understood. Recently, several reports have greatly increased our knowledge of the molecular and cellular events triggered by alum; however, the contribution of each of these processes to alum adjuvanticity is still unclear. A study published in this issue of the European Journal of Immunology, together with two recent publications, have demonstrated that the NOD‐like receptor, pyrin domain containing 3 (Nlrp3)‐inflammasome is the molecular target of alum immunostimulatory activity in vitro. Surprisingly, these three studies reported conflicting results on the requirement of the Nlrp3 inflammasome complex for alum adjuvant effects in vivo. This commentary attempts to resolve some of these discrepancies.

Rational design of small molecules as vaccine adjuvants

Small-molecule immune potentiators can be engineered to be potent adjuvants with localized innate immune activation and short in vivo residence times. Better Adjuvants Through Chemistry Vaccine development has come a long way since Jenner first noticed that cowpox protected against smallpox. And yet, many vaccines do not work well alone; adjuvants are included with the vaccine to boost the immune response. Despite the critical role of adjuvants in vaccine efficacy, new adjuvant development has been empirical. Now, Wu et al. report the rational optimization of small-molecule immune potentiators (SMIPs) as adjuvants. These SMIPs were engineered to have limited bioavailability and remain localized, inducing temporally and spatially restricted inflammation. This systematic approach to optimizing adjuvant properties may allow for improved immune responses to vaccines with fewer side effects. Adjuvants increase vaccine potency largely by activating innate immunity and promoting inflammation. Limiting the side effects of this inflammation is a major hurdle for adjuvant use in vaccines for humans. It has been difficult to improve on adjuvant safety because of a poor understanding of adjuvant mechanism and the empirical nature of adjuvant discovery and development historically. We describe new principles for the rational optimization of small-molecule immune potentiators (SMIPs) targeting Toll-like receptor 7 as adjuvants with a predicted increase in their therapeutic indices. Unlike traditional drugs, SMIP-based adjuvants need to have limited bioavailability and remain localized for optimal efficacy. These features also lead to temporally and spatially restricted inflammation that should decrease side effects. Through medicinal and formulation chemistry and extensive immunopharmacology, we show that in vivo potency can be increased with little to no systemic exposure, localized innate immune activation and short in vivo residence times of SMIP-based adjuvants. This work provides a systematic and generalizable approach to engineering small molecules for use as vaccine adjuvants.

MF59 and Pam3CSK4 Boost Adaptive Responses to Influenza Subunit Vaccine through an IFN Type I-Independent Mechanism of Action

The innate immune pathways induced by adjuvants required to increase adaptive responses to influenza subunit vaccines are not well characterized. We profiled different TLR-independent (MF59 and alum) and TLR-dependent (CpG, resiquimod, and Pam3CSK4) adjuvants for the ability to increase the immunogenicity to a trivalent influenza seasonal subunit vaccine and to tetanus toxoid (TT) in mouse. Although all adjuvants boosted the Ab responses to TT, only MF59 and Pam3CSK4 were able to enhance hemagglutinin Ab responses. To identify innate immune correlates of adjuvanticity to influenza subunit vaccine, we investigated the gene signatures induced by each adjuvant in vitro in splenocytes and in vivo in muscle and lymph nodes using DNA microarrays. We found that flu adjuvanticity correlates with the upregulation of proinflammatory genes and other genes involved in leukocyte transendothelial migration at the vaccine injection site. Confocal and FACS analysis confirmed that MF59 and Pam3CSK4 were the strongest inducers of blood cell recruitment in the muscle compared with the other adjuvants tested. Even though it has been proposed that IFN type I is required for adjuvanticity to influenza vaccines, we found that MF59 and Pam3CSK4 were not good inducers of IFN-related innate immunity pathways. By contrast, resiquimod failed to enhance the adaptive response to flu despite a strong activation of the IFN pathway in muscle and lymph nodes. By blocking IFN type I receptor through a mAb, we confirmed that the adjuvanticity of MF59 and Pam3CSK4 to a trivalent influenza vaccine and to TT is IFN independent.

The Acquired Immune Response to the Mucosal Adjuvant LTK63 Imprints the Mouse Lung with a Protective Signature

LTK63, a nontoxic mutant of Escherichia coli heat labile enterotoxin (LT), is a potent and safe mucosal adjuvant that has also been shown to confer generic protection to several respiratory pathogens. To understand the mechanisms of action underlying the LTK63 protective effect, we analyzed the molecular and cellular events triggered by its administration in vivo. We show here that LTK63 intrapulmonary administration induced in the mouse lung a specific gene expression signature characterized by the up-regulation of cell cycle genes, several host defense genes, chemokines, chemokine receptors, and immune cell-associated genes. Such a transcriptional profile reflected the activation of alveolar macrophages and the recruitment to the lung of T and B cells and innate immune cells such as granulocytes, NK, and dendritic cells. All of these events were T cell dependent and specific for LTK63 because they were absent in SCID and nude mice. Additionally, we showed that LTK63 induces a potent adaptive immune response against itself directed to the lung. We propose that acquired response to LTK63 is the driving force for the local recruitment of both adaptive and innate immune cells. Our data suggest that LTK63 acts as an airway infection mimic that establishes a generic protective environment limiting respiratory infection by innate immune mechanisms and by improving adaptive responses to invading pathogens.

Intranasal Administration of CpG Induces a Rapid and Transient Cytokine Response Followed by Dendritic and Natural Killer Cell Activation and Recruitment in the Mouse Lung

CpG-containing oligodeoxynucleotides are potent mucosal adjuvants and effective as stand-alone treatment of respiratory infections in mice. Although CpG is also used as a type 1 helper immunomodulator in the treatment of asthma and allergic disease, immune modulation following intranasal application has not been fully characterized yet. Using a B-type CpG, we monitored RNA expression profiles, cytokine production and cellular activation in lung tissue and bronchoalveolar lavages ex vivo and cytokine production of purified cell populations in vitro. CpG triggered the upregulation of many transcripts, including interferon response genes and proinflammatory cytokine genes, between 3 h and 4 days. Overlapping subsets of these cytokine proteins were induced in vitro in purified CD11c+ cells, B cells and alveolar macrophages from the lung, thus identifying these cells as direct targets of CpG. While lung B cells strongly respond to CpG in vitro, less activation is found ex vivo, suggesting efficient CpG sequestering or rapid B cell migration after activation. In contrast, a type II alveolar epithelial cell line did not respond to CpG in vitro. We noted selective recruitment of plasmacytoid dendritic cells (DCs) into the lung tissue, and of conventional DCs and natural killer (NK) cells into the lung tissue and bronchoalveolar space. Furthermore, CpG induced activation of intrapulmonary DCs, NK and T cells. We hypothesize that CpG-linked adjuvanticity and clearance of respiratory pathogens are mediated by two major mechanisms: transient induction of the interferon pathway limiting microbial survival and selective recruitment of DCs and NK cells, which allows for better adaptive responses.

Antibody blockade of IL-17 family cytokines in immunity to acute murine oral mucosal candidiasis

Antibodies targeting IL‐17A or its receptor, IL‐17RA, are approved to treat psoriasis and are being evaluated for other autoimmune conditions. Conversely, IL‐17 signaling is critical for immunity to opportunistic mucosal infections caused by the commensal fungus Candida albicans, as mice and humans lacking the IL‐17R experience chronic mucosal candidiasis. IL‐17A, IL‐17F, and IL‐17AF bind the IL‐17RA‐IL‐17RC heterodimeric complex and deliver qualitatively similar signals through the adaptor Act1. Here, we used a mouse model of acute oropharyngeal candidiasis to assess the impact of blocking IL‐17 family cytokines compared with specific IL‐17 cytokine gene knockout mice. Anti‐IL‐17A antibodies, which neutralize IL‐17A and IL‐17AF, caused elevated oral fungal loads, whereas anti‐IL‐17AF and anti‐IL‐17F antibodies did not. Notably, there was a cooperative effect of blocking IL‐17A, IL‐17AF, and IL‐17F together. Termination of anti‐IL‐17A treatment was associated with rapid C. albicans clearance. IL‐17F‐deficient mice were fully resistant to oropharyngeal candidiasis, consistent with antibody blockade. However, IL‐17A‐deficient mice had lower fungal burdens than anti‐IL‐17A‐treated mice. Act1‐deficient mice were much more susceptible to oropharyngeal candidiasis than anti‐IL‐17A antibody‐treated mice, yet anti‐IL‐17A and anti‐IL‐17RA treatment caused equivalent susceptibilities. Based on microarray analyses of the oral mucosa during infection, only a limited number of genes were associated with oropharyngeal candidiasis susceptibility. In sum, we conclude that IL‐17A is the main cytokine mediator of immunity in murine oropharyngeal candidiasis, but a cooperative relationship among IL‐17A, IL‐17AF, and IL‐17F exists in vivo. Susceptibility displays the following hierarchy: IL‐17RA‐ or Act1‐deficiency > anti‐IL‐17A + anti‐IL‐17F antibodies > anti‐IL‐17A or anti‐IL‐17RA antibodies > IL‐17A deficiency.

Controlled Mycobacterium tuberculosis infection in mice under treatment with anti-IL-17A or IL-17F antibodies, in contrast to TNFα neutralization

Antibodies targeting IL-17A or its receptor IL-17RA show unprecedented efficacy in the treatment of autoimmune diseases such as psoriasis. These therapies, by neutralizing critical mediators of immunity, may increase susceptibility to infections. Here, we compared the effect of antibodies neutralizing IL-17A, IL-17F or TNFα on murine host responses to Mycobacterium tuberculosis infection by evaluating lung transcriptomic, microbiological and histological analyses. Coinciding with a significant increase of mycobacterial burden and pathological changes following TNFα blockade, gene array analyses of infected lungs revealed major changes of inflammatory and immune gene expression signatures 4 weeks post-infection. Specifically, gene expression associated with host-pathogen interactions, macrophage recruitment, activation and polarization, host-antimycobacterial activities, immunomodulatory responses, as well as extracellular matrix metallopeptidases, were markedly modulated by TNFα blockade. IL-17A or IL-17F neutralization elicited only mild changes of few genes without impaired host resistance four weeks after M. tuberculosis infection. Further, the absence of both IL-17RA and IL-22 pathways in genetically deficient mice did not profoundly compromise host control of M. tuberculosis over a 6-months period, ruling out potential compensation between these two pathways, while TNFα-deficient mice succumbed rapidly. These data provide experimental confirmation of the low clinical risk of mycobacterial infection under anti-IL-17A therapy, in contrast to anti-TNFα treatment.

Histone Deacetylase Inhibitors Prolong Cardiac Repolarization through Transcriptional Mechanisms

Histone deacetylase (HDAC) inhibitors are an emerging class of anticancer agents that modify gene expression by altering the acetylation status of lysine residues of histone proteins, thereby inducing transcription, cell cycle arrest, differentiation, and cell death or apoptosis of cancer cells. In the clinical setting, treatment with HDAC inhibitors has been associated with delayed cardiac repolarization and in rare instances a lethal ventricular tachyarrhythmia known as torsades de pointes. The mechanism(s) of HDAC inhibitor-induced effects on cardiac repolarization is unknown. We demonstrate that administration of structurally diverse HDAC inhibitors to dogs causes delayed but persistent increases in the heart rate corrected QT interval (QTc), an in vivo measure of cardiac repolarization, at timepoints far removed from the Tmax for parent drug and metabolites. Transcriptional profiling of ventricular myocardium from dogs treated with various HDAC inhibitors demonstrated effects on genes involved in protein trafficking, scaffolding and insertion of various ion channels into the cell membrane as well as genes for specific ion channel subunits involved in cardiac repolarization. Extensive in vitro ion channel profiling of various structural classes of HDAC inhibitors (and their major metabolites) by binding and acute patch clamp assays failed to show any consistent correlations with direct ion channel blockade. Drug-induced rescue of an intracellular trafficking-deficient mutant potassium ion channel, hERG (G601S), and decreased maturation (glycosylation) of wild-type hERG expressed by CHO cells in vitro correlated with prolongation of QTc intervals observed in vivo The results suggest that HDAC inhibitor-induced prolongation of cardiac repolarization may be mediated in part by transcriptional changes of genes required for ion channel trafficking and localization to the sarcolemma. These data have broad implications for the development of these drug classes and suggest that the optimal time to assess potentially transcriptionally mediated physiologic effects will be delayed relative to an epigenetic drugs Tmax/Cmax.