(19)
(11)EP 2 985 342 A1

(12)EUROPEAN PATENT APPLICATION

(43)Date of publication:
17.02.2016 Bulletin 2016/07

(21)Application number: 14181113.3

(22)Date of filing:  15.08.2014
(51)Int. Cl.: 
C12N 5/0783  (2010.01)
G01N 33/50  (2006.01)
A61K 39/00  (2006.01)
G01N 33/569  (2006.01)
(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
Designated Extension States:
BA ME

(71)Applicants:
  • Miltenyi Biotec GmbH
    51429 Bergisch Gladbach (DE)
  • Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie e. V. -Hans-Knöll-Institut -
    07745 Jena (DE)

(72)Inventors:
  • Scheffold, Alexander
    10711 Berlin (DE)
  • Bacher, Petra
    10249 Berlin (DE)
  • Kniemeyer, Olaf
    07749 Jena (DE)
  • Teutschbein, Janka
    07743 Jena (DE)
  • Brakhage, Axel
    99425 Weimar (DE)

  


(54)Immunogenic antigens from Aspergillus fumigatus


(57) The present invention provides the in-vitro use of at least one in-vivo-target antigen of Aspergillus fumigatus for selective activation, detection and/or analysis of Aspergillus fumigatus specific CD4+ T cells in a sample comprising cells, wherein said at least one in-vivo-target antigen is selected from the group consisting of antigens Scw4, Pst1, Shm2, GliT and TpiA, the method therefor and pharmaceutical composition thereof.




Description

Field of the Invention



[0001] The present invention relates generally to in-vivo-target antigens from Aspergillus fumigatus, in particular to the use of these antigens for detecting, isolating and/or analyzing Aspergillus fumigatus-specific CD4+ T cells; and the use of these antigens as vaccines against Aspergillus fumigatus infection or allergy.

Background of the invention



[0002] Aspergillus fumigatus (A. fumigatus) is a ubiquitous spore-producing mold that can cause a diverse spectrum of human diseases, ranging from allergic hypersensitivity and non-invasive colonization to life-threatening invasive infections. Invasive aspergillosis (IA) is the most devastating disease caused by this fungus in immunocompromised patients. Despite new anti-fungal drugs, morbidity and mortality continue to be unacceptable high and invasive aspergillosis has become a major cause of infection-related mortality in hematopoietic stem cell recipients.

[0003] Although we routinely inhale several hundreds or thousands of A. fumigatus conidia per day, immune-competent individuals are efficiently protected by innate and adaptive immune mechanisms. Lung-resident alveolar macrophages and neutrophils ingest and kill A. fumigatus conidia and germlings and recruit other immune cells by secretion of pro-inflammatory cytokines. There is increasing evidence that CD4+ T cells orchestrate the anti-fungal immune response. In mouse models, monocytes and dendritic cells have been shown to prime A. fumigatus-specific CD4+ T cell responses that migrate to the airways. Adoptive transfer of A. fumigatus-specific IFN-γ producing T cells protected mice from invasive fungal disease and correlated with survival of IA patients. In accordance with the idea that humans are constantly confronted with fungal antigens it was recently shown that a small population of A. fumigatus-specific T cells is indeed consistently present in healthy donors (Bacher et al J Immunol 2013, Bacher et al Mucosal Immunol 2013). In IA patients, the frequencies of A. fumigatus-reactive T cells are strongly increased (unpublished observation) indicating the involvement of specific CD4+ T cells in antifungal immune defense.

[0004] Therefore, approaches supporting fungus-specific CD4+ T cells in immuno-compromised persons, e.g. by vaccination or adoptive T cell transfer seem to be promising for pre-emptive or therapeutic intervention against invasive fungal infections. However, in order to develop efficient immunotherapies or immunodiagnostic tools, a crucial first step is to define the antigen specificity of the human CD4+ T cells in vivo. Due to the complexity of the A. fumigatus proteome it is currently not known against which fungal antigens human T cells predominantly react and which T cell specificities are protective. The A. fumigatus genome contains several thousand open reading frames, encoding potential antigenic proteins. Bacher et al (J Immunol 2013 & Mucosal Immunol 2013) disclosed a highly specific and sensitive assay to enumerate and characterize antigen-specific CD4+ T cells directly ex vivo based on CD154+ pre-enrichment (Antigen-Reactive T cell Enrichment, ARTE). There is a need in the art for the identification of immunogenic antigens of Aspergillus fumigatus allowing the detection of Aspergillus fumigatus-specific T cells and/or which are useful for immunotherapy or immunodiagnostics.

Summary of the invention



[0005] Using the ARTE technology, we were able to identify very rare Aspergillus fumigatus specific T cells in all healthy donors, in accordance with the idea that humans are continuously exposed to fungal spores. Interestingly we found that using the complete Aspergillus lysate as an antigen, which covers most fungal proteins a large part of the reactive T cells had a naive phenotype. This data suggested that Aspergillus per se may not induce strong T cell responses in healthy individuals leaving most T cells untouched in a naive state. Surprisingly a completely different picture emerged, when we analysed a set of recombinant proteins from A. fumigatus, which were either known as immunogenic proteins or newly identified as strongly expressed by A. fumigatus and/or as target of specific antibodies in sera from patients with invasive Aspergillosis.

[0006] Surprisingly, our analysis identified from the large proteome of A. fumigatus several new in vivo target proteins, including Scw4, Pst1, Shm2, GliT and TpiA that have not been described as human T cell targets before. All these proteins belong to the "immunogenic" or "exhausting" protein group. These proteins can be used as in-vivo-target antigens to measure the magnitude and/or the quality of the T cell response against A. fumigatus in healthy persons or persons with A. fumigatus related diseases, i.e. invasive Aspergillosis (IA), allergies or allergic bronchopulmonary aspergillosis (ABPA) or Aspergillus colonisation of the lung (e.g. in cystic fibrosis patients) via the selective activation of Aspergillus fumigatus-specific CD4+ T cells in a blood or tissue sample or via MHC multimers loaded with peptides derived from the in vivo target proteins. In addition, they can be used in a method for detecting, isolating and/or analyzing Aspergillus fumigatus-specific CD4+ T cells. The frequency, phenotype or functional characteristics of the specific T cells may be used as a read-out to identify disease associated changes, which might have diagnostic or therapeutic relevance. Pharmaceutical compositions comprising Aspergillus-specific CD4+ T cells obtainable by the method of the present invention or comprising an in-vivo-target antigen of Aspergillus fumigatus may be used in adoptive cell therapy. Pharmaceutical compositions comprising one or more of the identified proteins or fragments thereof may also be used for vaccination against Aspergillus infections or allergies.

Brief description of the drawings



[0007] 

FIG 1. Memory CD4+ T cells from healthy human donors show specific reactivity against lysates from different A. fumigatus growth phases.
Following stimulation of PBMCs with the indicated crude lysates of different morphotypes, CD154+ expression on CD4+ T cells was analyzed directly ex vivo. (A) Cells were gated on lymphocytes and aggregates (scatter area versus height), dead cells and non-T cell lineages (CD14+, CD20+, dump) were excluded. Representative dot plot examples from one donor with frequencies of CD154+ cells among CD4+ lymphocytes and (B) summary of several donors with horizontal lines indicating mean values. (C-E) CD154+ cells were magnetically pre-enriched and stained for cytokine expression and phenotypic markers. (C) Number of CD154+ cells obtained from 1×10E7 stimulated PBMCs. (D) Percentages of cytokine-expressing cells among CD154+ T cells and (E) percentages of CD45RO+ memory cells among CD154+ T cells are shown. Significance was determined using paired Student's t-test. RC = resting conidia; SC = swollen conidia; GC = germinating conidia.

FIG 2. Resting conidia contain less T cell antigens than other A. fumigatus morphotypes. Following stimulation with the different crude growth phase lysates, CD154+ cells were magnetically isolated and expanded for 2 weeks. Upon re-stimulation with and without antigens as indicated, reactive CD4+ T cells were determined by CD154 and TNF-α expression. (A) Representative dot plot examples from one donor with percentage of reactive cells gated on CD4+ lymphocytes and (B) statistical analysis of several donors with horizontal lines indicating mean values. (C) The fungal lysate reactive T cell lines were re-stimulated with the specific lysate used for initial stimulation and analyzed for intracellular cytokine expression. Percentages of cytokine producing cells among CD154+ cells are depicted. Significance was determined using paired Student's t-test. RC = resting conidia; SC = swollen conidia; GC = germinating conidia.

FIG 3. Ex vivo enumeration of CD4+ T cells reactive against single A. fumigatus proteins. 1×10E7 PBMCs were stimulated with A. fumigatus crude mycelia lysate, C. albicans lysate and MP65 as control antigens, or single A. fumigatus proteins as indicated. CD154+ cells were enriched and stained intracellularly for cytokine expression. (A) Representative dot plot examples from one donor. For an optimal detection of rare CD154+ events, aggregates, dead cells and non-target cells (CD8+, CD14+, CD20+) were excluded by using a dump channel.
The numbers of CD154+ cells obtained after enrichment are indicated. (B) Specificity of single protein-reactive CD154+ T cells. PBMCs were stimulated with the indicated proteins. CD154+ cells were isolated, subsequently expanded for 3 weeks and tested for specificity via antigen re-stimulation. Percentage of reactive cells among CD4+ lymphocytes are shown for several donors, as determined by CD154 and TNF-α expression. (C) Enumeration of reactive CD4+ T cells in several donors. The total number of CD154+ cells obtained after enrichment was normalized to the total number of CD4+ cells applied to the column. Background enriched from the non-stimulated control was subtracted. pp = peptide pool; r = recombinant protein

FIG 4. Combined characterization of frequency, phenotype and function enables classification of antigenic proteins.
Enriched CD154+ cells were ex vivo analyzed for frequency, expression of CD45RO and pro-inflammatory cytokine production and classified into "immunogenic", "non-target" and "exhausting" proteins, as indicated. Frequency was determined as in Fig. 4, percentages of CD45RO+ memory cells among CD154+ cells and percentages of cytokine-expressing cells among CD154+ T cells are shown. pp = peptide pool; r = recombinant

FIG 5. Stimulation of A. fumigatus specific Treg with pools of immunogenic, non-target and exhausted proteins. 2×10E7 PBMCs were stimulated with A. fumigatus crude mycelia lysate, membrane lysate or the indicated pools of single proteins. CD154+ and CD137+ cells were magnetically enriched and stained for Foxp3 expression. (A) Representative dot plot examples from one donor. Cells are gated on CD4+CD154- lymphocytes and Foxp3 expression on CD137+ cells is depicted. The numbers of CD137+Foxp3+ Treg cells obtained after enrichment are indicated. (B) Enumeration of reactive CD154+ Tcon and CD137+ Treg in several donors (n = 6). The total number of CD154+ and CD137+ cells obtained after enrichment was normalized to the total number of CD4+ cells applied to the column. Background enriched from the non-stimulated control was subtracted. (C) Percentages of CD45RO+ memory cells among CD154+ cells (Tmem). (D) Ratio of CD137+ Treg to CD 154+CD45RO+ Tmem.

FIG 6. Assessment of polyfunctional cytokine induction by immunogenic, non-target or exhausting proteins.
PBMCs were stimulated with A. fumigatus crude mycelia lysate or with pools of the proteins classified as immunogenic, non-target and exhausting. Antigen-specific T cells were isolated by ARTE and analyzed for polyfunctional cytokine expression of TNF-alpha, IL-2 and IFN-gamma. Statistical analysis from four donors with indicated mean values are shown.


Detailed description of the invention



[0008] The presence of A. fumigatus-specific CD4+ T cells in human blood has been described in several studies, using in vitro stimulation assays with whole conidia and hyphae, crude lysates, single proteins or epitopes. However, it has not been defined yet, which developmental stage (resting, swollen, germinating conidia or mycelia) and which subcellular protein fraction prime A. fumigatus-specific T cells in healthy human donors. Here, we demonstrate that the activated developmental stages of the fungus (swollen, germinating conidia and hyphae) contain the largest reservoir of potential T cell epitopes. Furthermore, T cell antigens in the metabolic active A. fumigatus morphotypes were largely overlapping, which is in line with recent results on the proteomic signature of A. fumigatus during early development. These studies show that the majority of mycelial proteins are also present in all early, metabolically active morphotypes and only the abundance varies.

[0009] The method ARTE (Antigen-Reactive T cell Enrichment; Bacher et al J Immunol 2013) was used for the direct quantification and multi-parameter characterization of rare human CD4+ T cells specific for various antigens of the important human pathogenic fungus Aspergillus fumigatus. We show that ARTE can be used for the direct quantification and multi-parameter characterization of rare human CD4+ T cells specific for various antigens of the important human-pathogenic fungus A. fumigatus. The sensitivity and flexibility of the method enabled the analysis of T cells specific for various developmental stages, subcellular compartments as well as a large set of selected single recombinant proteins. Importantly, the multi-parameter characterization of T cells reactive against single A. fumigatus proteins, i.e. the combination of frequencies, naive/memory distribution and effector cytokine production of specific T cells allowed the classification of proteins/antigens of A.fumigatus into two groups: the in-vivo-target antigens and the non-target antigens. One group (the non-target antigens) revealed a picture similar to the whole protein lysate, characterized by high frequencies of naive T cells (about 50%) and low frequencies of effector cytokine producers (<20% IFN-gamma, <5% IL-17) in most donors, suggesting that these proteins are no in vivo targets of T cells in healthy donors. The other group (in-vivo-target antigens) revealed a surprisingly high frequency of memory T cells (preferentially more than 60%) and only few naive T cells (preferentially less than 40%), suggesting that proteins of this group are in vivo targets of the T cell response in healthy donors. This group of in vivo targets can further be split up into two subgroups: "Immunogenic" proteins are characterized by high overall T cell frequencies (preferentially more than 1 in 10E3, more preferentially more than 1 in 10E4, most preferentially more than 1 in 10E5), mainly memory type cells (60-100%) and high IFN-γ (15-80%) and/or IL-17 (5-30%) production. "Exhausting" proteins were classified due to their low to intermediate overall frequencies (preferentially less than 1 in 10E4, more preferentially less than 1 in 10E5, most preferentially less than 1 in 10E6) and lack of effector cytokine production (<15% IFN-gamma, <5% IL-17), although the majority of cells had a clear memory phenotype (60-100%). These properties may indicate deletion and/or anergy of specific T cells due to overstimulation in vivo again supporting the idea that these proteins are in vivo recognized during in vivo contact with A. fumigatus. Thus the "immunogenic" and "exhausting" antigens are defined as "in-vivo-target antigens". Importantly these two subgroups with obvious immune reactivity in vivo contrast with the third group, which we termed "non-target" proteins, since they induce high overall T cell frequencies, but strikingly a large proportion of the cells is still in the naive state and also lacks effector cytokine production. This indicates that no immune reactivity is induced in vivo despite the fact that these proteins can stimulate T cells when present during in vitro stimulation. This suggests that non-target proteins are not relevant for T cell responses against A. fumigatus infections in healthy donors. The fact that the T cells reacting against the total A. fumigatus lysate also contain many naive T cells shows that indeed a large part of the A. fumigatus proteome actually belongs to the non-target protein group, i.e. is immunologically neutral. Thus identification of true in vivo target proteins, as disclosed in the present application, is an important step to identify proteins which possess relevance during in vivo A. fumigatus infections and thus possess potential value as diagnostic or therapeutic tools. However it has to be kept in mind that single proteins might be classified into different groups in different donors, indicating that donor-specific features may influence the reactivity against single proteins, e.g. MHC restriction elements.

[0010] From the identified immunogenic proteins, Crf1, Sod3 and Aspf22 have previously been described to elicit CD4+ T cell responses in humans. However, our analysis also identified new immunogenic proteins, including Scw4, Pst1, Shm2, GliT and TpiA that have not been described as human T cell targets before. The gliotoxin oxidase GliT has recently been identified via an immunoproteome screening approach and has been suggested to represent a novel antigen for serologic diagnosis of aspergillosis (Shi et al, BMC Microbiol 2012). Interestingly also two other "immunogenic" proteins, the enolase Aspf22 and Shm2 were detected in immunoblots using sera from patients with allergic bronchopulmonary aspergillosis. In addition, Shm2 belongs to the most abundant proteins identified in the mycelial proteome (Vödisch et al, Proteomics 2009).

[0011] However, it is important to note that the same characteristics also apply to other proteins that were tested in our study, e.g. CpcB, Aspf2 and Aspf3 which all belong to the "exhausting" group. Furthermore CatB, classified by our analysis as a "non-target" protein has previously been described to induce strong T cell proliferation in vitro but vaccination with CatB did not protect mice from invasive aspergillosis. Thus it is obvious, that other factors than protein localization, abundance or antibody reactivity are critical parameters to determine the true in vivo T cell stimulatory capacity and highlights the potential of our approach to systematically predict immunogenic and potential protective target proteins.

[0012] In addition to the phenotypic characteristics, ARTE also allows to determine the functionally important production of effector cytokines, such as IFN-γ or IL-17. Although IL-17 is frequently claimed as an important cytokine for anti-fungal immune responses, the importance for protection against fungal infections versus immunopathology is currently under debate. In this study we observed a predominant IFN-γ production and only low IL-17, which is in line with previous reports suggesting that A. fumigatus elicits predominantly Th1 responses in vivo. Th2 cytokines (IL-4, IL-5, IL-13) were only marginal produced against the defined pools of single proteins (data not shown) and have previously been shown to be typically below 5% of all reactive CD154+ cells against the total soluble lysate (Bacher et al J. Immunol 2013). However, when single proteins or pools thereof were analyzed, polyfunctional cytokine production (co-expression of TNF-α, IL-2 and IFN-γ) and strong IL-17 cytokine production was only observed against proteins classified as "immunogenic". Also against the immunogenic proteins, IFN-γ was the dominating lineage defining cytokine confirming also on the level of single proteins that the in vivo response against A. fumigatus is rather biased towards a Th1 pattern. Moreover, some proteins (e.g. Scw4, Pst1, GliT, Aspf22) elicited in addition to IFN-γ the co-production of relatively high amounts of IL-17. The knowledge about the specific cytokine induction potential of certain proteins may help to improve vaccine design in the future. However, the functional importance of the various T cell cytokines has to be determined beforehand. Interestingly, by pooling proteins according to our classification, we found that not only conventional memory T cells, but also regulatory T cells were strongly activated by the immunogenic proteins, indicating that Tcon and Treg recognize the same antigens. In fact the number of Treg exactly paralleled the number of memory Tcon resulting in a stable Treg/Tmem ratio for all antigens. This indicates that Treg are not selectively generated against a subgroup of proteins but their expansion seems to be coupled to the expansion of conventional T cells, which may be mediated via growth factor supply, such as IL-2. Thus, the T cell response against all A. fumigatus proteins is controlled by Treg and therefore the depletion of Treg might be a promising strategy for releasing full T cell responses, e.g. for immunotherapeutic approaches. Alternatively Treg can be used directly ex vivo or following in vitro expansion to treat patients with overshooting or chronic inflammatory or allergic responses against A. fumigatus.

[0013] Finally, despite the fact that our analysis could define a set of immunogenic A. fumigatus proteins, the overall T cell response was directed against a multitude of different proteins. In addition, the T cell frequencies against single antigens were very low and there was significant donor-to-donor variation. This indicates that the A. fumigatus-specific T cell response is largely heterogeneous and also determined by host-specific or environmental factors, such as MHC restriction or variability in timing and dosage of antigen exposure. In line with this, a recent study identified 7 and 30 different T cell epitopes within the Crf1 and CatB protein, respectively, which are presented by different HLA-class II molecules. Thus, the existence of a single or even a few immuno-dominant antigens is rather unlikely emphasizing the importance to identify of single proteins or even peptides which are true in vivo targets in individual donors, as demonstrated here.

[0014] Therefore in a first aspect the present invention provides the in-vitro use of at least one in-vivo-target antigen of Aspergillus fumigatus for selective activation, detection and/or analysis of Aspergillus fumigatus-specific CD4+ cells in a sample comprising cells, wherein said at least one in-vivo-target-antigen is selected from the group consisting of antigens Scw4, Pst1, Shm2, GliT and TpiA.

[0015] In another aspect the present invention provides a method for detecting, isolating and/or analyzing Aspergillus fumigatus-specific CD4+ T cells, the method comprising:
  1. a) Adding to a sample comprising T cells at least one in-vivo-target antigen of Aspergillus fumigatus, wherein said at least one in-vivo-target antigen is selected from the group consisting of antigens Scw4, Pst1, Shm2, GliT and TpiA, thereby selectively activating Aspergillus fumigatus-specific CD4+ T cells
  2. b) Detection, isolation and/or analysis of said Aspergillus fumigatus-specific CD4+ T cells.


[0016] Detection and analysis of said Aspergillus fumigatus-specific CD4+ T cells may be performed by standard assays known in the field to analyse activated T cells, e.g. by detection of activation markers by flow cytometry or fluorescence microscopy, by quantitating secreted cytokines via ELISA, quantitation of proliferation (3H thymidine incorporation, CFSE dilution or comparable assays for cellular proliferation), intracellular cytokine staining, cytokine secretion assay, cytokine ELISPOT.

[0017] Isolation of said Aspergillus fumigatus-specific CD4+ T cells may be performed e.g. by flow-cytometry methods such as FACS® or by magnetic cell separation methods such as MACS®.

[0018] The method may comprise the additional step of enrichment of reactive T cells from a sample comprising T cells before adding the at least one in-vivo-target antigen. Enrichment of said reactive T cells may be performed by
  1. i) fluorescently, or magnetically labeling of one or more activation markers of said reactive T cells, wherein said activation markers are selected from the group consisting of CD154, CD137, cytokines (e.g. IL-2, TNF-alpha, IFN-gamma, IL-17, IL-4, IL-5, IL-13, IL-10, IL-22, IL-9), CD134, CD69, TGF-beta latency associated peptide (LAP), CD121, GARP
  2. ii) enriching the labeled cells of step i) via flow cytometry or magnetic cell separation methods.


[0019] The method may comprise the additional step of expansion of reactive T cells.

[0020] Exemplary, enrichment of reactive CD154+ or CD137+ T cells from a sample comprising cells may be performed by using magnetic cell separation technologies such as MACS® (Magnetic-activated cell sorting) or flow cytometric technologies such as FACS® (Fluorescence activated cell sorting) using an antigen-binding fragment such as an antibody against the marker CD154 or any other specific marker accessible on the cell surface.

[0021] Exemplary, expansion of said enriched reactive T cells can be performed with methods well known in the art such as culturing them alone or together with irradiated or mitomycin C treated autologous feeder cells and cytokines, such as IL-2. Alternatively specific T cells may be expanded by simply adding the antigens to a mixture of cells comprising the specific T cells. The mixture may also comprise antigen-presenting cells. Cytokines such as IL-2 may be added to enhance T cell proliferation.

[0022] In another aspect the present invention provides the use of peptides derived from at least one in-vivo-target antigen of A.fumigatus, for loading recombinant MHC class I or class II (HLA) proteins with said peptides to generate functional MHC/peptide complexes or multimeric composites thereof, such as tetramers, pentamers or other higher multimeric structures suitable to bind and/or activate peptide specific T cells, wherein said at least one in-vivo-target antigen is selected from the group consisting of antigens Scw4, Pst1, Shm2, GliT and TpiA

[0023] Such multimers would also be suitable to label and enrich specific T cells, if the multimers are also labelled, e.g. fluorescently, magnetically or by defined isotopes, similar as described above.

[0024] In an aspect the present invention provides a pharmaceutical composition comprising at least one in-vivo-target antigen of Aspergillus fumigatus for use as vaccine against aspergillosis, wherein said at least one in-vivo-target antigen is selected from the group consisting of antigens Scw4, Pst1, Shm2, GliT and TpiA.

[0025] In an aspect the present invention provides a pharmaceutical composition comprising Aspergillus fumigatus-specific CD4+ T cells for use in immunotherapy of aspergillosis, wherein said Aspergillus fumigatus-specific CD4+ T cells are obtained by the present method.

[0026] In an aspect the present invention provides a kit for detecting, isolating and/or analyzing Aspergillus fumigatus-specific CD4+ T cells comprising
  1. a) at least one in-vivo-target antigen, wherein said at least one in-vivo-target antigen is selected from the group consisting of antigens Scw4, Pst1, Shm2, GliT and TpiA
  2. b) an antigen-binding fragment specific for one or more activation markers of reactive T cells, wherein said activation markers are selected from the group consisting of CD154, CD137, cytokines (e.g. IL-2, TNF-alpha, IFN-gamma, IL-17, IL-4, IL-5, IL-13, IL-10, IL-22), CD134, CD69, TGF-beta latency associated peptide (LAP), CD121, GARP and wherein said antigen-binding fragment is coupled to a tag.


[0027] The tag may be a magnetic particle or fluorophore.

Definitions



[0028] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0029] The term "in-vivo-target antigen" as used herein has a further specific meaning as described herein. Since the method ARTE allows multi-parameter characterization of very rare single A. fumigatus protein-specific T cells, the combination of frequencies, naive/memory distribution and effector cytokine production allowed classification of the fungal antigens/proteins into three subgroups: "Immunogenic" antigens/proteins are characterized by high overall T cell frequencies, mainly memory type cells and high IFN-γ and/or IL-17 production. In contrast, "exhausting" antigens/proteins were classified due to their low to intermediate overall frequencies, and lack of effector cytokine production, although the majority of cells had a clear memory phenotype. These properties are indicative for an initial in vivo activation leading to acquisition of the memory phenotype but subsequent deletion and/or anergy of specific T cells, potentially due to inappropriate or missing costimulatory signals or alternatively by over-activation due to chronic presence of the antigen. These two subgroups with obvious immune reactivity in vivo contrast with the third group, which we termed "non-target" antigens/proteins, since they induce high overall T cell frequencies, but strikingly a large proportion of the cells is still in the naive state and also lacks effector cytokine production. This indicates that no immune reactivity is induced in vivo.

[0030] The amino acid sequences of Scw4, Pst1, Shm2, GliT and TpiA are given in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively (in the one-letter code of amino acids). The gene/protein names ShmB or SHMT may also be used for Shm2. The gene/protein name Tpi1 may also be used for TpiA. The term "in-vivo-target antigen" Scw4, Pst1, Shm2, GliT and TpiA as used herein refers to all constellations of the respective antigen which retains the intended function of being an in-vivo-target antigen of Aspergillus fumigatus as defined herein. In other words, the divergences to the SEQ ID Nos:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively, should not affect their immunogenic potential as in-vivo-target antigen, respectively, of Aspergillus fumigatus as disclosed herein. Therefore, the in-vivo-target antigens Scw4, Pst1, Shm2, GliT and TpiA, respectively, can be the full length protein of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively. It can also be a variant thereof which have some amino acids deleted, added or replaced while still retaining the function of being an in-vivo-antigen target of Aspergillus fumigatus. Therefore, included in this definition are variants of the amino acid sequences in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively, such as amino acid sequences essentially similar to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively, having a sequence identity of at least 70%, or at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% at the amino acid sequence level. In the context of the present invention, "sequence identity" may be determined using pairwise alignments using alignments programs for amino acid sequences well known to the art.

[0031] The in-vivo-target antigen of Scw4, Pst1, Shm2, GliT and TpiA, respectively, can also be a fragment of the protein Scw4, Pst1, Shm2, GliT and TpiA, respectively, e.g. a minimal peptide of 6-15 amino acids in length. Preferentially, the in-vivo-target antigen Scw4, Pst1, Shm2, GliT and TpiA, respectively, is a peptide pool. The peptide pool may comprise several peptides covering the complete or part of the sequences of Scw4, Pst1, Shm2, GliT and TpiA, respectively, peptides with may or may have not overlapping amino acid sequences. More preferentially, the in-vivo-peptide antigen is a peptide pool comprising 15meric overlapping peptides (e.g. 11 amino acids overlap) and spanning the whole protein sequence.

[0032] In general all amino acid variations are included under this definition, which do not lead to intentional changes in the recognition or activation of the specific T cells which are recognizing the corresponding peptide or protein with the original peptides sequence as defined in the present application.

[0033] The term specific T cells as used herein refers to a T cell expressing a certain T cell antigen receptor with the capacity to recognize only a specific peptide bound to a certain MHC molecule but not to the same MHC molecule complexed with other irrelevant peptides. "Specific" does not exclude that several possible peptides may exist which can bind to the MHC molecule and which are then recognized by the same TCR. This can be peptides with high homology, i.e. similar amino acid sequences, but even complete unrelated peptides may have this capacity. Specificity does rather mean that a TCR does only react to small selection of peptide/MHC complexes out of large reservoire of possible combinations, and in this way the particular T cell is specific for the antigen, the peptide is derived of but it may not exclude the possibility that cross-reactivity against other peptide derived from similar or unrelated antigen occur. That a certain TCR recognized a certain MHC/peptide combination can be tested with various technologies. Direct binding of the TCR can be demonstrated by using multimeric complexes (tetramers, pentamers) of the specific MHC/peptide (antigen) complex labelled with a fluorochrome. Such multimeric MHC molecules are well known in the art. Briefly they consist of the extracellular protein domain of MHC class I or class II (in humans HLA class I or class II, Human Leukocyte Antigen) consisting of two protein chains, which may be linked via an peptide linker. Into the antigen binding "groove" of the MHC molecule antigenic peptides (e.g. 9-15mers), e.g. derived from the proteins identified in present invention, can be loaded. In this way a functional MHC/peptide monomer is generated which can bind to the specific TCR recognizing the combination of MHC + peptide. Typically this binding affinity is low. Therefore multimeric structures are generated via various technologies known in the art to fuse several MHC/peptide complexes (e.g. 4, 5, 6 or more) to generate a high avidity protein which can bind simultaneously to multiple TCRs on the T cell surface and generate stable interaction. Binding of such multimeric complexes may also induce specific T cell activation.

[0034] Alternatively recognition can be demonstrated by measuring the response of the T cells to activation by the specific peptide/MHC molecule, e.g. by adding the peptide to a mixture of autologous antigen presenting cells and the T cells for several hours or days and measuring cytokine expression, expression of surface markers such as CD154 or CD137 or proliferation of the T cells.

[0035] The term "antigen-reactive "T cell is used herein mostly overlapping with the term "antigen-specific" since T cells which can specifically recognize a specific peptide/MHC combination as described above become activated by this stimulus via their TCR, i.e. they express certain cytokines, activation markers or start to proliferate. This sign of activation can then be used to identify the cells as described above. "Antigen-reactive" refers to T cells which are activated via there TCR but not to T cells which are activated independently of the TCR, e.g. via cytokines produced by other activated cells in the culture. This type of "bystander" activation is not a cell intrinsic property but depends on the presence of other cells, whereas specific activation strictly depends on the presence of the specific peptide/MHC complex (see definition of a specific peptide above) and potentially co-stimulatory molecules, which do not induce activation on their own.

[0036] The groups of antigen-specific and antigen-reactive T cells do large overlap, however anergic or inactivated T cells may exist which still have the specific TCR but lost their potential to become activated, i.e. they are excluded from the group of antigen-reactive T cells.

[0037] Antigen-specificity and reactivity can be determined in all types of T cells. In particular conventional (Tcon) and regulatory T cells (Treg) can be distinguished. Treg are defined here as Foxp3+CD4+ T cells which typically do also express CD25 and lack expression of CD127. They represent a separate T cell lineage required to maintain tolerance against auto-antigens and harmless foreign antigens. Tcon cells as defined here comprise all T cells which are not Treg. In both populations Tcon and Treg naive (no previous contact to antigen) and antigen-experienced or memory T cells (Tmem) can be found, which substantially differ in many phenotypical and functional aspects. Each T cell have a specific antigen receptor (T cell receptor, TCR) for one particular antigen. Therefore T cells with specificity for a particular antigen are typically rare, i.e. about 1 in 1 million for naive T cells and typically <1% for memory T cells, except in certain acute infectious diseases. Therefore it requires analysis methods able to identify these rare cells to study the immune status of a certain patient against a defined antigen or pathogen.

[0038] The term "antigen-binding fragment" as used herein refers to any moiety that binds preferentially to the desired target molecule of the cell, i.e. the antigen. The term moiety comprises, e.g., an antibody or antibody fragment. The term "antibody" as used herein refers to polyclonal or monoclonal antibodies which can be generated by methods well known to the person skilled in the art. The antibody may be of any species, e.g. murine, rat, sheep, human. For therapeutic purposes, if non-human antigen binding fragments are to be used, these can be humanized by any method known in the art. The antibodies may also be modified antibodies (e.g. oligomers, reduced, oxidized and labelled antibodies). The term "antibody" comprises both intact molecules and antibody fragments, such as Fab, Fab', F(ab')2, Fv and single-chain antibodies. Additionally, the term "antigen-binding fragment" includes any moiety other than antibodies or antibody fragments that binds preferentially to the desired target molecule of the cell. Suitable moieties include, without limitation, oligonucleotides known as aptamers that bind to desired target molecules

[0039] The term "tag" as used herein refers to the coupling of the antigen-binding fragment, e.g. an antibody or fragment thereof, to other molecules, e.g. particles, fluorophores, haptens like biotin, or larger surfaces such as culture dishes and microtiter plates. In some cases the coupling results in direct immobilization of the antigen-binding fragment, e.g. if the antigen-binding fragment is coupled to a larger surface of a culture dish. In other cases this coupling results in indirect immobilization, e.g. an antigen-binding fragment coupled directly or indirectly (via e.g. biotin) to a magnetic bead is immobilized if said bead is retained in a magnetic field. In further cases the coupling of the antigen-binding fragment to other molecules results not in a direct or indirect immobilization but allows for enrichment, separation, isolation, and detection of cells according to the present invention, e.g. if the antigen-binding fragment is coupled to a fluorophore which then allows discrimination of stronger labeled cells, weaker labeled cells, and non-labeled cells, e.g. via flow cytometry methods, like FACSorting, or fluorescence microscopy.

[0040] The term "particle" as used herein refers to a solid phase such as colloidal particles, microspheres, nanoparticles, or beads. Methods for generation of such particles are well known in the field of the art. The particles may be magnetic particles. The particles may be in a solution or suspension or they may be in a lyophilized state prior to use in the present invention. The lyophilized particle is then reconstituted in convenient buffer before contacting the sample to be processed regarding the present invention.

[0041] The term "magnetic" in "magnetic particle" as used herein refers to all subtypes of magnetic particles which can be prepared with methods well known to the skilled person in the art, especially ferromagnetic particles, superparamagnetic particles and paramagnetic particles. "Ferromagnetic" materials are strongly susceptible to magnetic fields and are capable of retaining magnetic properties when the field is removed. "Paramagnetic" materials have only a weak magnetic susceptibility and when the field is removed quickly lose their weak magnetism. "Superparamagnetic" materials are highly magnetically susceptible, i.e. they become strongly magnetic when placed in a magnetic field, but, like paramagnetic materials, rapidly lose their magnetism.

[0042] The term "activation marker" as used herein refers to proteins specifically expressed on the cell surface of T cells following their activation via their specific antigen-receptor, usually within 1-48 hours after activation. Activated T cells may comprise CD4 T cells, CD8 T cells, naive T cells, memory T cells, gamma-delta T cells or regulatory T-cells. Such activation markers are, for example, CD154, CD137, secreted or membrane-anchored cytokines, e.g. IL-4, IL-5, IL-13, IFN-gamma, IL-10, IL-2, IL-22, TNF-alpha or "latent TGF-beta" (LAP), GARP (LRRC32), CD121a/b.

[0043] CD154 is a member of the TNF gene family and, inter alia, is expressed by various cells, particularly by T lymphocytes. CD154 is rapidly (within minutes) downregulated by the stimulated T lymphocytes upon contact with its receptor CD40. CD154 can be used to detect T lymphocytes independently of their functional potential. The use of CD 154 in the detection and separation of T lymphocytes leads to reliable detection and isolation of T lymphocytes, possible independently of their function, i.e. all antigen-specific T lymphocytes in a sample can be determined and separated.

[0044] A method is disclosed in WO2004/027428 for the detection and/or isolation of antigen-specific T lymphocytes in a suspension following activation with an antigen, in which method the suspension is contacted with a CD40/CD154 system inhibitor, intra- and/or extracellular determination of CD154 is effected, and the cells having CD154 are detected and/or isolated. Addition of a CD40/CD154 system inhibitor impairs or inhibits the interaction and signalling between CD40 and CD154. CD40/CD154 system inhibitors can be any of molecules or even physical exposures capable of blocking or inhibiting the interaction between CD40 and CD154. Accordingly, the inhibiting agent can be an antibody, e.g. one directed against CD40, a molecule, a caesium or lithium ion having an effect on the interaction between CD40 and CD154. Of course, said agent can also be a substance inhibiting the secretion or endocytosis in the cell, such as brefeldin A (Bref-A). Bref-A inhibits the Golgi apparatus and the secretion of a variety of cytokines. These substances ensure that CD40, CD154, the interaction between the two of them, or the CD40/CD154 system are modified in such a way that CD154 either is no longer down-regulated and/or degraded on the cell surface, or, provided it is still within the cell, no longer transported therein. Such interruption of the transport within the cell prevents degradation of CD154. Consequently, CD154 is stabilized inside or outside the cell as an external receptor, thereby allowing detection and subsequent isolation using detection methods well-known to those skilled in the art.

[0045] EP2306191B1 disclosed that the use of CD154 as a negative selection marker greatly increases the window of time for the use of the marker CD 137 for the positive selection of regulatory T cells (Treg) and that 2 to 24 hours after activation, regulatory T-cells can be isolated with great purity and yield. Therefore it is possible with the present invention to analyse rare antigen-specific Treg cells, i.e. Aspergillus fumigatus-specific CD4+ T cells, using the combination of activation markers CD137 and CD154.

[0046] For isolation (selection or enrichment) in principle any sorting technology can be used. This includes for example affinity chromatography or any other antibody-dependent separation technique known in the art, which allows processing. Any ligand-dependent separation technique known in the art may be used in conjunction with both positive and negative separation techniques that rely on the physical properties of the cells.

[0047] An especially potent parallel sorting technology is magnetic cell sorting. The term "magnetic cell sorting" or "magnetic cell sorting process" is used herein to refer to procedures for cell separation (cell sorting) including, but are not limited to, magnetic separation using antibodies linked to colloidal magnetic particles or micron-sized magnetic particles (e.g. 1-10 µm). Methods to separate cells magnetically are commercially available e.g. from Invitrogen, Stem cell Technologies, in Cellpro, Seattle or Advanced Magnetics, Boston. For example, autologous monoclonal antibodies can be directly coupled to magnetic polystyrene particles like Dynal M 450 or similar magnetic particles and used e.g. for cell separation. Alternatively, antibodies can be biotinylated or conjugated with digoxigenin and used in conjunction with avidin or anti-digoxigenin coated affinity columns. In a preferred embodiment however, monoclonal antibodies are used in conjunction with colloidal superparamagnetic microparticles having an organic coating by e.g. polysaccharides (Miltenyi et al., 1990, Cytometry 11:231-8). These particles can be used having a size of 10 to 200 nm, preferably between 40 and 100 nm, and can be either directly conjugated to autologous antibodies or used in combination with anti-immunoglobulin, avidin or antihapten-specific microbeads. Polysaccharide-coated superparamagnetic particles are commercially available from Miltenyi Biotec GmbH, Germany.

[0048] The cells can be analyzed (characterized) after selective activation with one or more of the in-vivo-target antigens of Aspergillus fumigatus as disclosed herein according to all methods known to the person skilled in the art. Preferred for the characterization of cells are in particular cell sorting (e.g. further magnetic cell sorting (MACS)), fluorescence activated cell sorting (FACS), ELISA, PCR and/or all fluorescence microscopes known in the art.

[0049] The sample comprising T cells can be any sample comprising T cells. E.g. the sample is directly derived from blood, peripheral mononuclear blood cells (PBMC), body tissue or cells from tissue fluids of animals, preferentially mammals such as humans, mouse, rat, sheep or dogs. The sample comprising T cells may also be a sample in which T cells are enriched from e.g. a blood sample. The T cells may be the only subtype of cells in the sample, or in addition to the T cells may be present antigen presenting cells, or further cells of the blood. The sample comprising T cells may encompass expanded T cells or non-expanded T cells.

[0050] The separated Aspergillus fumigatus-specific CD4+ T cells can be used before and/or after cloning and/or growing and/or concentrated in cell mixtures as pharmaceutical composition in the immunotherapy of aspergillosis. It is additionally possible for the coding gene sequences of the TCR (T-cell receptor) to be isolated from the separated Aspergillus fumigatus-specific CD4+ T cells and be used for further therapeutic purposes such as, for example, for cellular therapies. It is additionally possible to employ the Aspergillus fumigatus-specific CD4+ T cells in the form mentioned in further investigations and/or analyses. The pharmaceutical composition can be used for the treatment and/or prevention of diseases in mammals, possibly including administration of a pharmaceutically effective amount of the pharmaceutical composition to the mammal.

Embodiments



[0051] In one embodiment of the invention a human sample, e.g. blood or tissue from a patient suffering from IA or at risk for an A. fumigatus infection is obtained. Patients at risk for fungal infections are immunocompromised patients, e.g. following chemotherapy, hematopoetic stem cell transplantation, HIV patients, or patients with intrinsic genetic defects leading to immune deficiency. One or more in-vivo-target antigens of Aspergillus fumigatus, wherein said at least one in-vivo-target antigen is selected from the group consisting of antigens Scw4, Pst1, Shm2, GliT and TpiA is added to the sample. After incubation of 6-18 hours, the Aspergillus fumigatus-specific CD4+ T cells are detected via expression of activation markers such as CD154, CD137, cytokines or proliferation and quantitated and/or characterized, e.g. by flow-cytometry or purified via FACS sorting. Alternatively, the Aspergillus fumigatus-specific CD4+ T cells are isolated by labeling the cells of the sample with an antibody against the marker molecules mentioned above coupled to a magnetic bead and separated magnetically by applying a magnetic field.

[0052] Alternatively, the Aspergillus fumigatus-specific CD4+ T cells are directly labelled with an MHC multimer loaded with one or several peptides derived from the in vivo target antigens identified in the present invention and the labelled cells are similarly characterized or isolated as described above by well known methods in the art. The information about the frequency, phenotype and functional properties of the A. fumigatus specific cells identified as described above may be used to diagnose invasive fungal infection or to identify sensitization of the patient's immune system against the fungus.

[0053] In a similar way other A. fumigatus related diseases may be diagnosed or analysed via characterization of the A. fumigatus specific T cells from the patients. Such diseases include cystic fibrosis, allergies, asthma, chronic obstructive pulmonary disease (COPD). In these patients overshooting or chronic immune activation against the fungus contributes to the disease phenotype but is often difficult to be specifically determined. Analysing the specific T cell response against the in vivo target proteins can be used to identify the type of immune response and help to select the appropriate therapeutic concept. Increased frequencies identify invasive aspergillosis. Alterations in the Treg / effector T cell ratio may identify not properly regulated immune reactions, e.g. allergies or chronic activation, certain cytokine patterns identify patient subgroups: allergic or asthmatic patients have increased Th2 cytokines alone or together with Th17 cytokines, patients with strong neutrophil contribution to the inflammation in the lung have increased Th17 cytokines, or patients with a defect in the immune response (no/low effector cytokine production), e.g. T cell exhaustion as a result of in vivo overstimulation.

[0054] In one embodiment of the invention T cells specific for one or more in-vivo-target antigens of Aspergillus fumigatus, wherein said at least one in-vivo-target antigen is selected from the group consisting of antigens Scw4, Pst1, Shm2, GliT and TpiA, are isolated as described above, e.g. using CD154 as a marker for activated effector T cells. The cell can be isolated from the patient or from the donor in case of bone marrow transplantation or be isolated from a third party donor with related MHC molecules. The isolated T cells can then be directly infused into a patient with an A. fumigatus infection, e.g. invasive fungal infection. Alternatively the T cells can be expanded in vitro and infused into the patient with the aim to enhance the immune defense against the fungal infection and cure the patient.

[0055] The antigens can also be used to isolate and expand A. fumigatus specific Foxp3+ regulatory T cells (Treg), e.g. by sorting specifically CD137 expressing T cells or CD154- CD137+ T cells after a short 6-12 hours stimulation with the antigen in vitro. These Treg can similarly be expanded in vitro or be directly used for infusion into patients, e.g. with chronic inflammatory reactions or allergies against A. fumigatus.

[0056] In one embodiment of the invention one or several in-vivo-target antigens of Aspergillus fumigatus, which are selected from the group consisting of antigens Scw4, Pst1, Shm2, GliT and TpiA are used to vaccinate people at risk for getting A. fumigatus infections or allergies or chronic inflammatory reactions.

[0057] The components necessary to perform the methods disclosed herein may be provided as a kit. Each kit contains the components necessary to perform selective activation of Aspergillus fumigatus specific CD4+ T cells in a sample comprising T cells and detection, isolation and/or analysis of said Aspergillus fumigatus-specific CD4+ T cells. A kit for detecting, isolating and/or analyzing Aspergillus fumigatus-specific CD4+ T cells comprises

a) at least one in-vivo-target-antigen, wherein said at least one in-vivo-target antigen is selected from the group consisting of antigens Scw4, Pst1, Shm2, GliT and TpiA

b) Antigen-binding fragment specific for one or more activation markers of reactive T cells, wherein said activation markers are selected from the group consisting of CD154, CD137, cytokines (e.g. IL-2, IL-4, IL-5, IL-9, IL-13, IL-17, IL-22, IFN-gamma, TNF-alpha), CD134, CD69, TGF-beta latency associated peptide (LAP), and wherein said antigen-binding fragment is coupled to a tag.



[0058] Preferentially, the tag is a magnetic particle such as a magnetic bead or a fluorophore.

[0059] For use in magnetic cell sorting the antigen binding fragments are coupled to magnetic particles as described herein. The magnetic particles, e.g. MicroBeads (Miltenyi Biotec GmbH), of the kit may be in a solution or suspension or they may be in a lyophilized state prior to use in a method of the present invention. The lyophilized particle is then reconstituted in convenient buffer before contacting with the sample containing neuronal cells to be processed regarding the present invention.

Examples


Example 1: Materials and Methods


Blood donors



[0060] Buffy coats from healthy donors were obtained from the Institute for Transfusion Medicine, University Hospital Dortmund after informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (GE Healthcare Life Sciences, Freiburg, Germany) density gradient centrifugation.

Preparation of A. fumigatus lysates



[0061] For the generation of all A. fumigatus (ATCC46645) protein extracts, except for the lysate of resting conidia (RC), 2x10E8 conidia were inoculated in 200 ml YPD medium and cultured at 37 °C and 200 rpm. Swollen conidia (SC) were harvested after 6 h, germinated conidia (GC) after 8 h, and mycelium after 20 h of cultivation. Cells were recovered by centrifugation (RC, SC, GC) or filtration (mycelium) and washed with water before storage at -80 °C. Total RC, SC, or GC lysates were generated by disruption of frozen cells in saline [0.9 % (w/v) NaCl] using a Micro-Dismembrator (Sartorius). For total mycelial lysate, frozen mycelium was ground in liquid nitrogen by using a mortar and pestle and resuspendend in PBS supplemented with 2 mM MgCl2. Total soluble protein fractions of the lysates (crude lysates) were obtained after removal of insoluble material (cell wall pellet) by centrifugation for 15 min at 10,000 × g. Fractionated mycelial protein extracts were obtained by sequential centrifugation of total mycelial lysate. The cell wall protein fraction was processed by resuspension of the cell wall pellet (15 min of centrifugation at 10000 xg) in PBS/2 mM MgCl2. By centrifugation of the crude mycelial lysate at 100,000 × g for 60 min, the cell membrane-enriched protein fraction (pellet) was separated from the cytosolic protein fraction. Cell membrane extract was generated by resuspension of the membrane pellet in PBS/2 mM MgCl2.

Generation of recombinant A. fumigatus proteins



[0062] The recombinant proteins used were generated by standard technologies known in the field, i.e. cloning of the coding sequences into a suitable expression vector adding a suitable tag for subsequent protein purification (e.g. 6 his tag or MBP-HIS-tagged). The proteins were expressed in E. coli, namely Crf2, GliT, Scw4, Aspf3, Shm2, CpcB, Aspf22, Pst1 and TpiA or in Pichia pastoris FG-GAP repeat protein, [devoid of the sequence encoding the 25 amino acids N-terminal secretion signal peptide (Δ25FG-GAP)] and of AspF2. All peptide sequences relevant to the present invention are listed below as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5. All proteins were purified by affinity chromatography using an Äkta explorer purification system (GE Healthcare). If necessary, an ion exchange column (SOURCE 15Q, GE Healthcare) was used for further purification. Generally, all buffer exchanges were conducted using HiPrep 26/10 desalting columns (GE Healthcare). All HIS-tagged proteins were applied to a Ni Sepharose 6 Fast Flow (GE Healthcare) column, and eluted with 250 mM imidazole. MPP-tagged proteins were loaded onto an Amylose Resin HF (New England Biolabs) column and eluted with 10 mM maltose. The MBP-HIS-tag of MBP-tagged proteins and the HIS-tag of AspF3 were cleaved using TEV-protease and removed by its binding to Ni Sepharose. After buffer exchange to 20 mM Tris-HCl pH 8 (CpcB, Pst1, TpiA) or 20 mM Tris-HCl pH 8.5, 6 M urea (Scw4), corresponding proteins were further purified by ion exchange chromatography using an NaCl gradient for elution. Scw4 was further purified by reversed phase chromatopraphy (Source 15RPC, GE Healthcare) after exchanging the buffer to 0.05% (v/v) trifluoroacetic acid, 10 % (v/v) acetonitril. Purified Scw4 was lyophilized and resolved in PBS. For all other purified proteins, the buffer was exchanged to PBS (FG-GAP, Pst1, AspF22, AspF2, GliT) or 0.9 % NaCl (Shm2, CpcB, AspF3, TpiA)

Stimulation of antigen-reactive T cells



[0063] PBMCs were resuspended at a concentration of 1×10E7/ml in RPMI-1640 (Miltenyi Biotec, Bergisch Gladbach, Germany), supplemented with 5 % (v/v) human AB-serum (BioWhittaker/Lonza, Walkersville, MD, USA), and 2 mM L-glutamine (PAA Laboratories, Pasching, Austria). Cells were stimulated for 7 hours with the following antigens: A. fumigatus lysates (each 40 µg/ml), C. albicans-lysate (20 µg/ml; Greer Laboratories, Lenoir, NC, USA), CMV-lysate (10 µg/ml, Siemens Healthcare Diagnostics, Marburg, Germany), recombinant A. fumigatus proteins (Crf2, Pst1, Aspf2, Aspf3, Shm2, FG-GAP, GliT, Aspf22, CpcB, TpiA, Scw4; each 20 µg/ml) or peptide pools (C. albicans MP65, Gel1, Crf1, Aspf3, CatB, Sod3, Shm2 ; each 0.6 nmol/peptide/ml; all from Miltenyi Biotec), or pools of proteins according to the classification into immunogenic (Scw4, CRF1, CRF2, Pst1, Shm2, each 20 µg/ml), non-target (Gel1, CatB; each 20 µg/ml) and exhausted (Aspf2, CpcB, Aspf3, FG-GAP; each 20 µg/ml). 1µg/ml CD40 and 1µg/ml CD28 functional grade pure antibody (both Miltenyi Biotec) was added. In some experiments, CD45RA+ cells were depleted from PBMCs prior stimulation using CD45RA microbeads and LD columns (Miltenyi Biotec).

Enrichment and characterization of antigen-reactive T cells



[0064] Enrichment of reactive CD154+ T cells or combined enrichment of CD154+/CD137+ T cells was performed using the CD154 MicroBead Kit alone, or in combination with the CD137 MicroBead Kit (both Miltenyi Biotec). In brief, cells were indirectly magnetically labeled with CD154-Biotin and CD137-PE followed by anti-Biotin Microbeads and anti-PE Microbeads and enriched by two sequential MS MACS columns (Miltenyi Biotec). For analysis of cytokine expression, 1 µg/ml Brefeldin A (Sigma Aldrich) was added for the last 2 hours of stimulation. Surface staining was performed on the first column, followed by fixation, permeabilization (Inside stain Kit; Miltenyi Biotec) and intracellular cytokine staining on the second column, as described (Bacher et al J Immunol 2013), or staining of Foxp3 using the Foxp3 Staining Buffer Set (Miltenyi Biotec).

In vitro expansion and re-stimulation of antigen-reactive T cell lines



[0065] Magnetically enriched CD154+ T cells were expanded with 1:100 mitomycin C (Sigma Aldrich) treated autologous feeder cells in X-Vivo™15 (BioWhittaker/Lonza), supplemented with 5 % (v/v) AB-serum (BioWhittaker/Lonza), 200 U/ml IL-2 (Proleukin®; Novartis, Nürnberg, Germany) and 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B (Antibiotic Antimycotic Solution, Sigma Aldrich) at a density of 2.5x10E6 cells/cm2. During expansion for 2-3 weeks, medium was replenished and cells were split as needed.

[0066] 5×10E5 expanded T cells were re-stimulated with autologous CD3-depleted (CD3 MicroBeads; Miltenyi Biotec) PBMC in a ratio of 1:1 in 96-well flat bottom plates with different antigens in presence of 1µg/ml CD28 functional grade pure Ab for 2h plus additional 4h with 1µg/ml Brefeldin A (Sigma Aldrich). After fixation and permeabilization cells were stained intracellularly for CD 154 and cytokines.

Flow cytometry



[0067] Different combinations of the following monoclonal antibodies were used according to manufacturer's recommendations: CD4-VioBlue, CD4-FITC, CD4-APC-Vio770 (VIT4), CD3-APC (BW264/56), CD14-VioGreen, CD14-PerCP (TÜK4), CD20-VioGreen, CD20-PerCP (LT20), CD8-VioGreen (BW135/80), CD45RO-FITC, CD45RO-PerCP (UCHL-1), CCR7-PE (REA108), CD45RA-APC, CD45RA-FITC (T6D11), anti-Biotin-PE, anti-Biotin-VioBlue (Bio3-18E7), CD154-PE, CD154-APC, CD154-VioBlue (5C8), TNF-α-FITC, TNF-α-PE-Vio770 (cA2), IFN-γ-FITC, IFN-γ-APC, IFN-γ-PE (45-15), IL-2-APC (N7.48A), IL-17-FITC, IL-17-PE (CZ8-23G1), IL-10-PE (B-T10), IL-4-PE (7A3-3), CD137-PE (4B4-1) (all Miltenyi Biotec), CD45RO-PE.Cy7 (UCHL-1, BD Bioscience, San Jose, CA, USA), IFNγ-PerCP-Cy5.5 (4S.B3; BioLegend, San Diego, CA, USA), IL-22-PE (142928; R&D Systems Europe, Ltd., Abingdon, UK), Foxp3-PerCP-Cy5.5 (PCH101; eBioscience, San Diego, CA, USA). Data were acquired on a MACSQuant® analyzer and MACSQuantify™ software was used for analysis (both Miltenyi Biotec).

Statistical analysis



[0068] Statistical tests were performed with GraphPad PRISM® software 5.0 (GraphPad Software, La Jolla, CA, USA) using two-tailed paired Student's t-test. P values of < 0.05 were considered statistically significant.

Example 2: Human CD4+ T cell response against lysates of different A. fumigatus morphotypes



[0069] CD4+ T cells specifically reacting against A. fumigatus can be identified using CD154 expression as a specific read-out for antigen activated CD4+ T cells after short in vitro stimulation with fungal lysate (Bacher et al, J Immunol 2013; Frentsch et al, Nat Med 2005) To analyze against which A. fumigatus morphotype the human T cell response is directed, peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated for 7 hours with crude lysates from resting, swollen and germinating conidia or mycelia, containing the total soluble fraction of the mechanically disrupted fungal cells. Reactive CD154-expressing CD4+ T cells were identified by flow-cytometry. Although all lysates induced a small population of CD154 expressing CD4+ T cells, the frequency of reactive cells stimulated with resting conidia lysate was significantly lower, compared to stimulation with lysates from other morphotypes (Fig. 1A, B). To enable the direct ex vivo phenotypic and functional characterization of the specific T cells, we next magnetically pre-enriched the rare antigen-reactive CD154+ T cells from larger cell numbers (1×10E7 PBMC). Again, a significantly higher number of target cells could be detected after stimulation with lysates of the more progressed development stages versus the resting conidia lysate (Fig. 1C). However, analysis of cytokine production and phenotype revealed no major differences of T cells stimulated with the different A. fumigatus morphotype lysates: as shown in Fig. 1D, against all lysates a high frequency of TNF-α and IL-2 producers could be detected, whereas the production of the lineage defining cytokines IFN-γ and IL-17 was only low, although IFN-γ production was clearly predominant over IL-17, as already described before (Bacher et al J Immunol 2013, Schönbrunn et al J Immunol 2012). Similarly, irrespective of the A. fumigatus lysate used for stimulation a comparable amount of reactive memory T cells was detected (Fig. 1E).

[0070] In summary, these results demonstrate that reactive memory CD4+ T cells against different morphotypes of A. fumigatus are present in healthy human donors and suggest that the strongest T cell response is directed against the actively growing fungus.

Examples 3: Swollen and germinating conidia as well as mycelia contain overlapping T cell antigens



[0071] We next addressed the question, whether the reactive CD4+ T cells recognize different or the same antigens expressed by the various A. fumigatus morphotypes. To this end, specific T cell lines were generated by expanding the magnetically enriched CD154+ T cells after stimulation with the different morphotype lysates. Upon re-stimulation the T cell lines initially stimulated with protein extracts of swollen conidia, germinating conidia and mycelia were equally reactive to either lysate, as shown by re-expression of CD154 and production of cytokines (Fig. 2A, B). However, the re-stimulation with resting conidia lysate was in each case significantly lower, suggesting that a considerable proportion of T cell antigens, which are present in the metabolically active morphotypes (swollen and germinating conidia, mycelia), are missing in the resting conidia lysate. Furthermore, resting conidia-reactive T cell lines reacted equally well to re-stimulation with each lysate, suggesting that resting conidia do not contain a significant fraction of T cell target proteins solely present in the dormant stage. As expected, none of the expanded cell lines reacted upon re-stimulation with CMV-lysate as a control antigen, providing evidence for the specificity of the expanded fungus-reactive T cell lines. As for the ex vivo response, we observed no differences in the cytokine producing capacities of the different T cell lines upon re-stimulation (Fig. 2C).

Example 4: ARTE allows direct characterization of human CD4+ T cells reacting against single A. fumigatus proteins



[0072] So far, only few single proteins of A. fumigatus have been analyzed and directly compared in their capacity to elicit CD4+ T cell responses in humans. In particular the direct qualitative and quantitative characterization of the responding T cells is missing, which avoids an experimental bias due to prolonged in vitro culture. However, the quality of the T cell response generated in vivo may provide important insight into the immunogenic properties of specific antigenic proteins.

[0073] Therefore we performed multi-parameter analysis of the T cells specific for a panel of 15 selected A. fumigatus proteins with different biological functions and cellular localization. Either recombinant proteins or synthesized 15mer peptide pools covering the complete protein sequence were used for stimulation. The analyzed proteins included cell wall, GPI-anchored, secreted, as well as cytosolic proteins and were chosen based on their high abundance within the conidial, mycelia or secreted proteome and/or their previous description as being immunogenic, based on T cell or serum reactivity. A concentration of 20 µg/ml of recombinant proteins for the stimulation of 1×10E7 PBMCs was determined based on titration of the single proteins on expanded total mycelia-reactive T cell lines (data not shown). The C. albicans protein MP65 had previously been described as a major antigen target of human T cell responses and served as a positive control.

[0074] Against the majority of the analyzed single A. fumigatus proteins no reactive CD4+ T cells above background could be detected by standard flow-cytometry without pre-enrichment (data not shown). To enable the direct ex vivo detection of reactive CD4+ T cells against the single A. fumigatus proteins, we performed ARTE from 1×10E7 stimulated PBMCs. Although the frequencies upon single protein stimulation were significantly lower as upon stimulation with A. fumigatus or C. albicans crude lysates, specific T cells against single proteins could be clearly detected compared to the non-stimulated control (Fig. 3A).

[0075] The specificity of the ex vivo detected single protein-reactive CD154+T cells was confirmed by expansion and re-stimulation of specific T cell lines (Fig. 3B). To this end PBMC were stimulated with the proteins indicated in the figure 3B for 7 hours. ppMP65 is a control peptide pool of the protein MP65 of C. albicans. CD154+ cells were isolated, subsequently expanded for 3 weeks and tested for specificity via antigen re-stimulation (MP65, whole Aspergillus lysate, CMV lysate or the A. fumigatus single protein used for initial isolation of the cells). Percentage of reactive cells among CD4+ lymphocytes are shown for several donors, as determined by CD154 and TNF-α expression.

[0076] Interestingly, the T cell responses against the different proteins were quite variable with frequencies ranging from 1.2×10E-6 to 3.1×10E-4 (Fig. 3C) and showed strong intra- and inter-donor variability (Fig. 3A, C). As expected, this indicates an overall diverse repertoire of A. fumigatus-reactive CD4+ T cells, probably due to different exposure and/or HLA-restriction. The subcellular location of the proteins did not result in a clear-cut phenotypic/functional characteristic of the resulting T cell response, although a trend towards a strong reactivity against membrane-associated proteins was observed.

Example 5: Integration of phenotypic and functional markers of specific T cells allows classification of antigenic proteins



[0077] Since our method allows multi-parameter characterization of very rare single A. fumigatus protein-specific T cells we integrated cytokine production, as well as phenotypic T cell markers, into our further analyses (Fig. 4). The combination of frequencies, naive/memory distribution and effector cytokine production allowed classification of the fungal proteins into three groups: "Immunogenic" proteins are characterized by high overall T cell frequencies, mainly memory type cells and high IFN-γ and/or IL-17 production. In contrast, "exhausting" proteins were classified due to their low to intermediate overall frequencies, and lack of effector cytokine production, although the majority of cells had a clear memory phenotype. These properties are indicative for deletion and/or anergy of specific T cells. These two groups with obvious immune reactivity in vivo contrast with the third group, which we termed "non-target" proteins, since they induce high overall T cell frequencies, but strikingly a large proportion of the cells is still in the naive state and also lacks effector cytokine production. This indicates that no immune reactivity is induced in vivo. Interestingly, the reactivity against the mycelia crude lysate as shown in figure 4 is also characterized by a high frequency of naive T cells and rather low effector cell frequencies, when compared to the immunogenic protein group, suggesting that a large fraction of the fungal proteins actually belong to the exhausting or non-target group.

Example 6: A. fumigatus-specific Tcon and Treg recognize the same antigens



[0078] We recently demonstrated that A. fumigatus surprisingly generates a strong Treg response in vivo, which even exceeds conventional memory T cells (Tmem) (Bacher, P. et al, Mucosal Immunol. 2013). We therefore analyzed whether the same or different antigens are recognized by A. fumigatus-specific Tmem and Treg. To this end, the single proteins were pooled according to our previous classification into a immunogenic group, containing proteins with the highest reactivity (Scw4, Crf1, Crf2, Pst1 and Shm2) as well as a non-target (Gel1, CatB) and exhausting (Aspf2, Aspf3, CpcB, Fg-Gap) group and used for stimulation in comparison to the mycelia crude lysate or the mycelia membrane fraction. CD137 which is expressed by Treg after 6 hours of stimulation was used together with CD154 enrichment to differentiate between Treg (CD137+CD154-) and Tcon (CD137-CD154+) (Bacher, P. et al, Mucosal Immunol. 2013; Schoenbrunn, A., et al, J Immunol 2012). As shown in figure 5A and B the Treg response mirrored the response of the Tcon, in that a high reactivity of specific CD137+ Treg was found in response to the A. fumigatus crude lysate, as well as the membrane fraction and the immunogenic protein pool. Again, the majority of reactive CD154+ Tcon against the immunogenic and exhausting protein pools displayed a memory phenotype, whereas a larger proportion against the non-target pool was still in the naive state (Fig. 5C). This results in an equally high Treg to Tmem ratio for all fractions (Fig. 5D), indicating that A. fumigatus-specific Treg and Tmem are directed against the same target antigens and that the A. fumigatus-specific T cell response is balanced by specific Treg cells.

References:



[0079] 

Bacher, P., C. Schink, J. Teutschbein, O. Kniemeyer, M. Assenmacher, A. A. Brakhage, and A. Scheffold. 2013. Antigen-reactive T cell enrichment for direct, high-resolution analysis of the human naive and memory Th cell repertoire. J Immunol 190: 3967-3976.

Bacher, P., O. Kniemeyer, A. Schonbrunn, B. Sawitzki, M. Assenmacher, E. Rietschel, A. Steinbach, O. A. Cornely, A. A. Brakhage, A. Thiel, and A. Scheffold. 2013. Antigen-specific expansion of human regulatory T cells as a major tolerance mechanism against mucosal fungi. Mucosal Immunol. 2013

Frentsch, M., O. Arbach, D. Kirchhoff, B. Moewes, M. Worm, M. Rothe, A. Scheffold, and A. Thiel. 2005. Direct access to CD4+ T cells specific for defined antigens according to CD154 expression. Nat Med 11: 1118-1124.

Schoenbrunn, A., M. Frentsch, S. Kohler, J. Keye, H. Dooms, B. Moewes, J. Dong, C. Loddenkemper, J. Sieper, P. Wu, C. Romagnani, N. Matzmohr, and A. Thiel. 2012. A converse 4-1BB and CD40 ligand expression pattern delineates activated regulatory T cells (Treg) and conventional T cells enabling direct isolation of alloantigen-reactive natural Foxp3+ Treg. J Immunol 189: 5985-5994.

Shi, L. N., F. Q. Li, M. Huang, J. F. Lu, X. X. Kong, S. Q. Wang, and H. F. Shao. 2012. Immunoproteomics based identification of thioredoxin reductase GliT and novel Aspergillus fumigatus antigens for serologic diagnosis of invasive aspergillosis. BMC Microbiol 12: 11. Vödisch, M., D. Albrecht, F. Lessing, A. D. Schmidt, R. Winkler, R. Guthke, A. A. Brakhage, and O. Kniemeyer. 2009. Two-dimensional proteome reference maps for the human pathogenic filamentous fungus Aspergillus fumigatus. Proteomics 9: 1407-1415.


Sequences



[0080] 

SEQ ID NO:1 Aspergillus fumigatus cell wall glucanase Scw4 (protein)

SEQ ID NO:2 Aspergillus fumigatus GPI-anchored cell wall protein Pst1 (protein)

SEQ ID NO:3 Aspergillus fumigatus serine hydroxymethyltransferase Shm2 (protein)

SEQ ID NO:4 Aspergillus fumigatus thioredoxin reductase GliT (protein)

SEQ ID NO:5 Aspergillus fumigatus triosephosphate isomerase TpiA (protein)














Claims

1. The in-vitro use of at least one in-vivo-target antigen of Aspergillus fumigatus for selective activation, detection and/or analysis of Aspergillus fumigatus specific CD4+ T cells in a sample comprising cells, wherein said at least one in-vivo-target antigen is selected from the group consisting of antigens Scw4, Pst1, Shm2, GliT and TpiA.
 
2. The use according to claim 1 wherein said at least one in-vivo-target antigen is a peptide for loading recombinant MHC class I or class II proteins to generate functional MHC/peptide complexes or multimeric composites thereof suitable to bind and/or activate peptide specific T cells.
 
3. A method for detecting, isolating and/or analyzing Aspergillus fumigatus-specific CD4+ T cells, the method comprising:

a) Adding to a sample comprising T cells at least one in-vivo-target antigen of Aspergillus fumigatus, wherein said at least one in-vivo-target antigen is selected from the group consisting of antigens Scw4, Pst1, Shm2, GliT and TpiA

b) Detection, isolation and/or analysis of said Aspergillus fumigatus-specific CD4+ T cells.


 
4. The method according to claim 3, wherein said Aspergillus fumigatus specific CD4+ T cells comprise regulatory T cells, memory T cells and naive T cells.
 
5. The method according to claim 3 or 4 comprising the additional step of enrichment of reactive T cells from a sample comprising T cells before adding the at least one in-vivo-target-antigen.
 
6. The method according to claim 5, wherein enrichment of said reactive T cells is performed by

i) fluorescently or magnetically labeling of one or more activation markers of said reactive T cells, wherein said activation markers are selected from the group consisting of CD154, CD137, IL-2, IL-4, IL-5, IL-9, IL-13, Il-17, IL-22, IFN-gamma, TNF-alpha, CD134, CD69, TGF-beta latency associated peptide (LAP)

ii) enriching the labeled cells of step i) via flow cytometry or magnetic cell separation methods.


 
7. The method according to any one of claim 3 to 6 comprising the additional step of expansion of reactive T cells.
 
8. The method according to any one of claims 3 to 7, wherein said at least one in-vivo-target antigen is a full length protein, a fragment thereof, a peptide, a peptide pool, a nucleic acid encoding the antigen or fragments thereof.
 
9. A pharmaceutical composition comprising an at least one in-vivo-target antigen of Aspergillus fumigatus for use as vaccine against aspergillosis, wherein the at least one in-vivo-target antigen is selected from the group consisting of antigens Scw4, Pst1, Shm2, GliT and TpiA.
 
10. A pharmaceutical composition comprising Aspergillus fumigatus-specific CD4+ T cells for use in immunotherapy of aspergillosis, wherein said Aspergillus fumigatus-specific CD4+ T cells are obtained by the method of any one of claims 3 to 8.
 
11. A kit for detecting, isolating and/or analyzing Aspergillus fumigatus-specific CD4+ T cells comprising

a) at least one in-vivo-target antigen, wherein said at least one in-vivo-target antigen is selected from the group consisting of antigens Scw4, Pst1, Shm2, GliT and TpiA

b) Antigen-binding fragment specific for one or more activation markers of reactive T cells, wherein said activation markers are selected from the group consisting of CD 154, CD137, IL-2, IL-4, IL-5, IL-9, IL-13, Il-17, IL-22, IFN-gamma, TNF-alpha, CD134, CD69, TGF-beta latency associated peptide (LAP), and wherein said antigen-binding fragment is coupled to a tag.


 
12. The kit of claim 11, wherein said tag is a magnetic particle or fluorophore.
 




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REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description




Non-patent literature cited in the description