Jenny M. Reimer

Expansion of the mast cell chymase locus over the past 200 million years of mammalian evolution

The acidic granules of natural killer (NK) cells, T cells, mast cells, and neutrophils store large amounts of serine proteases. Functionally, these proteases are involved, e.g., in the induction of apoptosis, the recruitment of inflammatory cells, and the remodeling of extra-cellular matrix. Among the granule proteases are the phylogenetically related mast cell chymases, neutrophil cathepsin G, and T-cell granzymes (Gzm B to H and Gzm N), which share the characteristic absence of a Cys191–Cys220 bridge. The genes of these proteases are clustered in one locus, the mast cell chymase locus, in all previously investigated mammals. In this paper, we present a detailed analysis of the chymase locus in cattle (Bos taurus) and opossum (Monodelphis domestica). The gained information delineates the evolution of the chymase locus over more than 200 million years. Surprisingly, the cattle chymase locus contains two α-chymase and two cathepsin G genes where all other studied chymase loci have single genes. Moreover, the cattle locus holds at least four genes for duodenases, which are not found in other chymase loci. Interestingly, duodenases seem to have digestive rather than immune functions. In opossum, on the other hand, only two chymase locus-related genes have been identified. These two genes are not arranged in one locus, but appear to have been separated by a marsupial-specific chromosomal rearrangement. Phylogenetic analyses place one of the opossum genes firmly with mast cell α-chymases, which indicates that the α-chymase had already evolved as a separate, clearly identifiable gene before the separation of marsupials and placental mammals. In contrast, the second gene in opossum is positioned phylogenetically between granzymes, cathepsin G, and the duodenases. These genes, therefore, probably evolved as separate subfamilies after the separation of placental mammals from marsupials. In platypus, only one chymase locus-like sequence could be identified. This previously published “granzyme” does not cluster clearly with any of the chymase locus gene families, but shares the absence of the Cys191–Cys220 bridge with the other chymase locus proteases. These findings indicate that all chymase locus genes are derived from a single ancestor that was present more than 200 million years ago.

Immune enhancing properties of the novel Matrix-M™ adjuvant leads to potentiated immune responses to an influenza vaccine in mice.

The novel saponin based adjuvant Matrix-M™ was recently used in a Phase I study of seasonal influenza in elderly. The present study is a pre-clinical evaluation of the efficacy and mode-of-action of Matrix-M™ formulated influenza vaccine in mice. A manuscript on safety profile and immunogenicity in elderly humans is under preparation. We have previously shown that subcutaneous injections of Matrix-M™, without coformulated antigen, results in a dose-dependent recruitment of leukocytes to draining lymph nodes (dLNs). Herein we compared the mode of action of Matrix-M™ with Alum, FCA and AS03 alone or formulated with influenza split virion antigen injected intramuscularly. The elicited responses in dLNs and spleen were investigated 48h later. Matrix-M™ was particularly efficient in activation of central innate immune cells such as neutrophils, DCs and macrophages compared to the other adjuvants analyzed. Moreover, the adjuvant influence on the recall immune response to influenza antigen was studied by in vitro re-stimulation of splenocytes from mice immunized with influenza antigen adjuvanted with Matrix-M™, Alum or AS03. Splenocytes from mice immunized with influenza antigen and Matrix-M™ produced both Th1 and Th2 cytokines upon re-stimulation. This response was significantly stronger than that induced by the other adjuvants studied. Interestingly, increased levels of the neutrophil chemoattractant KC were produced by antigen stimulated splenocytes from mice immunized with Matrix-M™ adjuvanted vaccine, which is in agreement with the increase of neutrophils into dLNs and spleen after Matrix-M™ injection. Furthermore, influenza antigen adjuvanted with Matrix-M™ induced significantly higher antigen-specific IgG1 and IgG2a responses compared to antigen alone. In conclusion, adjuvant Matrix-M™ activates the innate immune system without antigen present. This activation may explain the enhanced immunity to influenza seen with Matrix-M™ adjuvant. Despite this potent immune activation mediated by Matrix-M™, GLP-toxicity studies and clinical data suggest that Matrix-M™ adjuvant has a mild to moderate safety profile.

Matrix-M™ adjuvanted envelope protein vaccine protects against lethal lineage 1 and 2 West Nile virus infection in mice

West Nile virus (WNV) is a mosquito-transmitted flavivirus and an emerging pathogen in many parts of the world. In the elderly and immunosuppressed, infection can progress rapidly to debilitating and sometimes fatal neuroinvasive disease. Currently, no WNV vaccine is approved for use in humans. As there have been several recent outbreaks in the United States and Europe, there is an increasing need for a human WNV vaccine. In this study, we formulated the ectodomain of a recombinant WNV envelope (E) protein with the particulate saponin-based adjuvant Matrix-M™ and studied the antigen-specific immune responses in mice. Animals immunized with Matrix-M™ formulated E protein developed higher serum IgG1 and IgG2a and neutralizing antibody titers at antigen doses ranging from 0.5 to 10 μg compared to those immunized with 3 or 10 μg of E alone, E adjuvanted with 1% Alum, or with the inactivated virion veterinary vaccine, Duvaxyn(®) WNV. This phenotype was accompanied by strong cellular recall responses as splenocytes from mice immunized with Matrix-M™ formulated vaccine produced high levels of Th1 and Th2 cytokines. Addition of Matrix-M™ prolonged the duration of the immune response, as elevated humoral and cellular responses were maintained for more than 200 days. Importantly, mice vaccinated with Matrix-M™ formulated E protein were protected from lethal challenge with both lineage 1 and 2 WNV strains. In summary, Matrix-M™ adjuvanted E protein elicited potent and durable immune responses that prevented lethal WNV infection, and thus is a promising vaccine candidate for humans.

Isolation of transcriptionally active umbilical cord blood-derived basophils expressing FcɛRI, HLA-DR and CD203c

Background:  Basophils are inflammatory cells associated with allergy and parasite infections. Investigation of their true biological function has long been hampered by the difficulty in obtaining sufficient amounts of pure basophils and by the lack of phenotypic markers. Moreover, it has been very difficult to clone and identify basophil‐specific granule proteins, partially because of an almost complete lack of mRNA in mature circulating basophils.

Matrix-M™ adjuvant: enhancing immune responses by ‘setting the stage’ for the antigen

Adjuvants belong to a diverse collection of substances or formulations that, in one way or another, increase or improve immune responses to antigen(s). Classical adjuvants target the physical presentation of antigens by adsorption or emulsification with a physical stabilization of the antigen and/or prolonged release of antigen from the site of the injection as a major adjuvant activity [1]. In recent years, the formulation of emulsion types of adjuvants has been improved, new types of adjuvants have been developed and the knowledge on adjuvant modes of action has increased.

High degree of conservation of the multigene tryptase locus over the past 150–200 million years of mammalian evolution

Activated mast cells release a number of potent inflammatory mediators including histamine, proteoglycans, cytokines, and serine proteases. The proteases constitute the majority of the mast cell granule proteins, and they belong to either the chymase or the tryptase family. In mammals, these enzymes are encoded by two different loci, the mast cell chymase and the multigene tryptase loci. In mice and humans, a relatively large number of tryptic enzymes are encoded from the latter locus. These enzymes can be grouped into two subfamilies, the group 1 tryptases, with primarily membrane-anchored enzymes, and the group 2 tryptases, consisting of the soluble mast cell tryptases. In order to study the appearance of these enzymes during vertebrate evolution, we have analyzed the dog, cattle, opossum, and platypus genomes and sought orthologues in the genomes of several bird, frog, and fish species as well. Our results show that the overall structure and the number of genes within this locus have been well conserved from marsupial to placental mammals. In addition, two relatively distantly related group 2 tryptase genes and several direct homologues of some of the group 1 genes are present in the genome of the platypus, a monotreme. However, no direct homologues of the individual genes of either group 1 or 2 enzymes were identified in bird, amphibian, or fish genomes. Our results indicate that the individual genes within the multigene tryptase locus, in their present form, are essentially mammal-specific.

Human Cord Blood Derived Immature Basophils Show Dual Characteristics, Expressing Both Basophil and Eosinophil Associated Proteins

Basophils are blood cells of low abundance associated with allergy, inflammation and parasite infections. To study the transcriptome of mature circulating basophils cells were purified from buffy coats by density gradient centrifugations and two-step magnetic cell sorting. However, after extensive analysis the cells were found to be transcriptionally inactive and almost completely lack functional mRNA. In order to obtain transcriptionally active immature basophils for analysis of their transcriptome, umbilical cord blood cells were therefore cultured in the presence of interleukin (IL)-3 for 9 days and basophils were enriched by removing non-basophils using magnetic cell sorting. The majority of purified cells demonstrated typical metachromatic staining with Alcian blue dye (95%) and expression of surface markers FcεRI and CD203c, indicating a pure population of cells with basophil-like phenotype. mRNA was extracted from these cells and used to construct a cDNA library with approximately 600 000 independent clones. This library served as tool to determine the mRNA frequencies for a number of hematopoietic marker proteins. It was shown that these cells express basophil/mast cell-specific transcripts, i.e. β-tryptase, serglycin and FcεRI α-chain, to a relatively low degree. In contrast, the library contained a high number of several eosinophil-associated transcripts such as: major basic protein (MBP), charcot leyden crystal (CLC), eosinophil cationic protein (ECP), eosinophil derived neurotoxin (EDN) and eosinophil peroxidase (EPO). Out of these transcripts, MBP and EPO were the most frequently observed, representing 8% and 3.2% of the total mRNA pool, respectively. Moreover, in a proteome analysis of cultured basophils we identified MBP and EPO as the two most prominent protein bands, suggesting a good correlation between protein and mRNA analyses of these cells. The mixed phenotype observed for these cells strengthens the conclusion that eosinophils and basophils are closely linked during human hematopoietic development. The dual phenotype also indicates that other cytokines than IL-3 or cell surface interactions are needed to obtain the full basophil specific phenotype in vivo.