rPhl p 1 Timothy

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Code: g205
Latin name: Phleum pratense
Source material: rPhl p 1 is a CCD-free recombinant protein

Timothy grass allergen components

Available ImmunoCAP®:

Summary

Allergen components from timothy grass, Phleum pratense, available for allergen-specific IgE antibody testing, are produced either with recombinant technique or as purified native proteins (1). The next generation of immunotherapy may be based on recombinant allergen components, possibly modified to reduce the risk of anaphylaxis. If the sensitisation profile to an allergen such as timothy is known, only those components to which the patient is actually sensitised should be relevant for therapy. This would eliminate the risk that the therapeutic reagent would induce IgE antibodies to additional components. The allergen components of timothy in IgE antibody tests may also be used for monitoring immunotherapy that is done with the naturalextract.

Studies have evaluated different combinations of recombinant allergens for diagnostic use in grass pollen allergy. The fact that only a limited number of recombinant timothy grass pollen allergens account for the detection of a high percentage of patients with grass pollen-specific IgE suggests the usefulness of recombinant allergens not only for in vitro diagnosis but also for patient-tailored immunotherapy (2).

For example, a study used sera from 193 European, American, and Asian subjects to evaluate the percentage of IgE directed to rPhl p 1, rPhl p 2, rPhl p 5, and rBet v 2. The study also used natural pollen extracts from Anthoxanthum odoratum, Avena sativa, Cynodon dactylon, Lolium perenne, Phragmites australis, Poa pratensis, Secale cereale, Triticum sativum, Zea mays, IgE antibodies directed to these 4 recombinant pollen allergens was detected in 59% of these patients (3).

A similar study, examined the in vitro IgE antibody-binding capacity to the 3 recombinant timothy allergens, rPhl p 1, rPhl p 2, rPhl p 5, and birch profilin in sera from 183 patients allergic to grass pollen from different populations in Europe, Japan, and Canada. More than ninety-four percent of the patients could be diagnosed with a combination of recombinant Phl p 1, Phl p 2, Phl p 5, and Birch profilin. Sera that did not react with the recombinant allergens contained low levels of timothy grass pollen-specific IgE antibodies. The study pointed out that although considerable variability in the IgE antibody recognition frequency of the recombinant allergens was observed in certain populations, a good correlation was found between natural timothy-serum specific IgE antibodies and the combination of recombinant allergens in all 183 tested sera. The authors suggested that the addition of other recombinant allergens (e.g., recombinant Phl p 4) would only slightly improve the in vitro test sensitivity (4).

rPhl p 1, rPhl p 2, rPhl p 5 and Birch pollen recombinant allergens (rBet v 1, rBet v 2) were used for the measurement of allergen-specific IgE and IgG subclass antibody responses in fifty-five pollen-allergic patients, allowing allergy diagnosis in 52 of 54 of the grass pollen and in 35 of 36 of the Birch pollen-allergic patients (5).

A larger study, evaluating sensitisation to timothy grass pollen using sera from 749 patients and a timothy extract compared to 8 recombinant timothy allergens, found that 95% had detectable IgE antibodies to the timothy extract. The prevalence of IgE antibody reactivity increased from 86.8% to 93.3% as the number of combined recombinant allergens rose from 2 to 8. The prevalences for each allergen were: rPhl p 1 = 83%, rPhl p 2 = 55%, nPhl p 4 = 70%, rPhl p 5 = 50%, rPhl p 6 = 44%, rPhl p 7 = 7%, rPhl p11 = 43% and rPhl p 12 = 15%. Monosensitisation to rPhl p 1 occurred in 6% patients and was negligible for the remaining molecules (6).

A study evaluating the same group of 8 allergens, using sera of 77 patients allergic to grass pollen, found a similar frequency of sensitisation to these allergens. This study also demonstrated a good correlation, as expected, between the calcium-binding proteins of rPhl p 7 and Bet v 4, and between the profilin of rPhl p 12 and rBet v 2. Nevertheless, as with other studies, highly variable individual sensitisation patterns were seen (7).

Clearly IgE antibody reactivity profiles will vary from country to country and will depend on the prevalence of pollen allergens. In an evaluation of the IgE antibody reactivity profile to individual recombinant and native allergens in sera from 1,177 subjects sensitised to timothy and/or birch pollen and living in Finnish and Russian Karelia, the IgE antibody reactivity to pollen extracts and 8 Timothy allergens (rPhl p 1, 2, 5, 6, 7, 11, 12 and nPhl p 4) and 3 Birch pollen allergens (rBet v 1, 2 and 4) revealed that the levels of IgE antibodies to timothy and Birch pollen were higher in Finnish (median 5.2 kUA/L) than in Russian Karelia (median 1.8 kUA/L). There was a significantly higher prevalence of IgE reactivity to 3 timothy pollen allergens in Finnish (n=57) than in Russian Karelia (n=12): rPhl p 2, 28 vs. 0%; rPhl p 5, 60 vs. 0%; rPhl p 6, 47 vs. 0%. The prevalence of IgE antibody reactivity to the birch pollen allergens was similar in the 2 populations. IgE antibody reactivity to rPhl p 2, 5, 6 and 11 was associated with hayfever symptoms (8).

Because of patients being sensitised to minor timothy allergens, occasional subjects may demonstrate allergen-specific IgE antibodies to timothy extract but not to individual recombinants (9).

Assessing patients’ sera for allergen-specific IgE and IgG4 antibody reactivity to individual recombinant P. pratense allergens after immunotherapy has been reported to be useful in defining optimal allergen extract doses. For example, a study that found no significant rPhl p 12-specific IgG4 antibody increase after immunotherapy, suggesting that Phl p 12 was underrepresented in the extract used. The simple detection of specific serum IgG4 antibodies a few weeks after the start of immunotherapy was a valuable tool for estimating the presence of relevant allergens in a given immunotherapeutic allergen extract (10). Grass pollen immunotherapy elicits an array of antibody specificities and these reflect the allergen content and the potency of allergen extracts, which may contribute to defining optimal allergen extract doses (11).


Allergens from Phleum pratense listed by IUIS*

Phl p 1 Phl p 2 Phl p 4
Phl p 5 Phl p 6 Phl p 7
Phl p 11 Phl p 12 Phl p 13

*International Union of Immunological Societies (www.allergen.org) Jan. 2008.


g205 rPhl p 1

Recombinant non-glycosylated protein produced in an E. coli strain carrying a cloned cDNA encoding Phleum pratense allergen Phl p 1

Common name: Group 1 grass allergen
Biological function: b-Expansin
Mw: 27 kDa

Allergen description

Phl p 1 (1-4,7-9,12-26)  is a group 1 grass pollen allergen, a family of allergens present in all grass species (12). Group 1-grass pollen allergens are glycosylated proteins that show 60-70 % sequence identity to expansins, a family of proteins involved in cell wall loosening and extension in plants 1. IgE antibodies in almost 40% of allergic individuals, representing around 400 million allergic patients (15,19), recognize group 1 allergens. More than 95% of grass pollen-allergic patients display IgE-reactivity to group 1 grass pollen allergens of different grass species (17). A major IgE-reactive segment of Phl p 1 also exhibits a significant sequence identity of 43% with the family of immunoglobulin domain-like group 2/3 grass pollen allergens (12).

Recombinant Phl p 1 has been shown to resemble native Phl p 1, closely binding to IgE in up to 87% of patients with grass pollen allergy, indicating that rPhl p 1 shares many of the IgE epitopes with natural group 1 grass pollen allergens (17,20). rPhl p 1 produced in Escherichia coli (E. coli) is not glycosylated in difference to the native molecule. Group1 allergens have been cloned from at least 10 grass species (1).

rPhl p 1 has also been shown to inhibit IgE antibody binding to most of group 1 isoallergens from 7 to 8 grass species in studies (17,20), showing extensive cross-reactivity between species. Thus, a single recombinant group 1 allergen contains many of the IgE epitopes of group 1 isoallergens from a number of different grass species (20)  and may represent a useful tool for specific diagnosis and therapy of grass pollen allergy (1).

Phl p 1 displays sequence identities of greater than 85% and homologies of greater than 90% with Lol p 1 (rye grass) and Hol I 1 (velvet grass) (27). However, despite the high degree of homology, amino acid differences occur in immunodominant positions, which may be responsible for the differing immune response also found to group 1 allergens of different grass species (3,27).

g206 rPhl p 2

Recombinant non-glycosylated protein produced in an E. coli strain carrying a cloned cDNA encoding Phleum pratense allergen Phl p 2

Common name: Group 2 grass allergen
Biological function: Unknown
Mw: 13 kDa

Allergen description

Phl p 2 (1-3,7-9,18-19,22-24,28-34) is representative of the large family of cross-reacting plant allergens classified as grass allergens group 2/3.  These comprise 10-12 kDa non-glycosylated proteins of 95-98 amino acid residues which exhibit 85-90 % sequence identity between grass species. Group 2 and 3 allergens share a high degree of sequence homolgy with the C-terminal part of group 1 allergens but are sufficiently different to give a  more or less separate antibody recognition. Cross-reactivity between group 1 and group 2/3 allergens has not so far been shown for human IgE antibodies (1). Recombinant Phl p 2 has been demonstrated by immunological cross-reactivity studies to be immunologically equivalent to the natural protein (30).

g208 nPhl p 4

Natural protein purified from Phleum pratense

Common name: Group 4 grass allergen
Biological function: Berberine bridge enzyme
Mw: 55 kDa

Allergen description

Phl p 4 (7-8,35) is a major allergen which reacts with IgE antibodies of approximately 75% of grass pollen-allergic patients (35-39). Phl p 4 belongs to the Group 4 grass pollen allergens, which are present in many grass species, including timothy grass and Mugwort (35,40). Group 4 allergens are highly basic glycoproteins with Mw 50-67 kDa. They carry 10-15% carbohydrates and some of the IgE antibody responses obtained are probably to the carbohydrate determinants.

This group of allergens has been located in the wall of pollens, and in timothy grass and birch pollens also in the cytoplasm. In the foods peanut, apple, celery, and carrot, only cytoplasmic areas contained this allergen. As Group 4-related allergens occur in a range of pollens of unrelated plants and in plant foods, they contribute to cross-reactivity between some pollens and foods (41).

It is therefore not surprising that Phl p 4-specific IgE antibodies will cross-react with allergens present in pollen of trees, grasses, and weeds, as well as in plant-derived food (36). Cross-reactivity has been demonstrated between the pollen allergen Dac g 4 in orchard grass (Dactylis glomerata) (42) and similar allergens in pollen of Secale cereale (cultivated rye), Lolium perenne (rye grass), Festuca elatior (meadow fescue), Holcus lanatus (velvet grass), Bromus arvensis (field brome), Poa pratense (Kentucky blue grass), Hordeum sativum (barley), and Phleum pratense (timothy grass) (39,42). Nevertheless, the expression of Group 4 allergens in these plants varies considerably (35).

g215 rPhl p 5b

Recombinant non-glycosylated protein produced in an E. coli strain carrying a cloned cDNA encoding Phleum pratense allergen Phl p 5b

Common name: Group 5 grass allergen, Ag25
Biological function: Not confirmed but possibly a ribonuclease
Mw: 32 kDa
Isoforms:  Phl p 5a, a 38 kDa protein; Phl p 5b, a 32 kDa protein (53).

Allergen description

Phl p 5 (1-3,7-9,18-19,22-24,26,43-52) is a major allergen from Timothy grass pollen and is one of the most reactive of the group 5 allergens, inducing allergic rhinitis and bronchial asthma in grass pollen-allergic patients. Group 5 allergens seem to be restricted to the Pooideae subfamily of grasses. Between 65-90% of grass pollen-allergic patients in temperate climate areas are reported to be sensitized against group 5 grass pollen allergens (1,45). Rainfall contributes to an increase in respirable particles containing group 5 allergens, which bursts the pollen grains (54).

Two isoforms exists, denoted “a” and “b,” where Phl p 5b, although beeing the smaller of the two isoforms, have been demonstrated to contain at least one more IgE antibody binding epitope than Phl p 5a (1).

rPhl p 5 has been shown to be very similar to natural Phl p 5, and to have a moderately high homology to other Group 5 allergens (1,49). rPhl p5 reacts with serum IgE antibodies in up to 90% of grass pollen-allergic patients (43,51).

rPhl p 5, has been shown to be cross-reactive with similar Group 5 allergens from several grass and grain species 45, including Lol p 5 from Rye grass pollen (Lolium perenne) and Poa p 9 from Meadow grass (Poa pratensis). Nevertheless, variable IgE immunoreactivity does occur to these allergens and more diversity has been shown for goup 5 allergens than for group 1 allergens (1,50).

g209 rPhl p 6

Recombinant non-glycosylated protein produced in an E. coli strain carrying a cloned cDNA encoding Phleum pratense allergen Phl p 6

Common name: Group 6 grass allergen
Biological function: Unknown
Mw: 15 kDa

Allergen description

Phl p 6 (1,7-8,18,55-58)  binds IgE antibodies from 60-75% of grass pollen-sensitised subjects (1,55). Phl p 6 is one of the group 6 grass allergens and has so far only been identified in timothy and Kentucky blue grass.

Group 6 allergens are acidic non-glycosylated proteins with a Mw of about 13 kDa. They have a fairly high degree of amino-acid sequency homolgy to the C-terminal part of group 5 allergens and IgE antibodies to Phl p 6 in most cases cross-react to group 5 allergens (1).

Studies, including structural and detailed localisation (55), have resulted in the development of recombinant rPhl p 6, which has been shown to have the same reactivity with serum IgE antibodies as the native molecule.

g210 rPhl p7

Recombinant non-glycosylated protein produced in an E. coli strain carrying a cloned cDNA encoding Phleum pratense allergen Phl p 7

Common names: 2-EF-hand, Ca2+-binding protein, CBP, Polcalcin
Biological function: Calcium-binding protein
Mw: 9 kDa

Allergen description

Phl p 7 (1,7-8,59-60) is a minor, non- glycosylated, allergen of Timothy grass pollen, recognising serum IgE antibodies in 10-15% of grass pollen-sensitized subjects. It is a 2-EF-hand, Ca2+-binding protein with a high sequence identity to homologous pollen proteins found in a number of other plants (1,60-61).

Ca2+-binding plant allergens can be grouped in different families according to the number of Ca2+-binding domains (EF hands). 2 EF-hand Ca2+-binding proteins include Phl p 7 (Timothy grass) and Aln g 4 (Alder), 3 EF-hand Ca2+-binding proteins include Bet v 3 (Birch), and 4 EF-hand Ca2+-binding proteins Jun o 4 (Prickly juniper). Through molecular modeling, structural similarities have been found among the allergens with 2, 3, and 4 EF-hands. In a study evaluating pollens from 16 unrelated plants, 22% of the patients with multiple pollen sensitization reacted to at least one of the Ca2+-binding allergens. A hierarchy of IgE antibody cross-reactivity was noted (rPhl p7 > rAln g 4 > rJun o 4 > rBet v 3). rPhl p 7 was identified as the EF-hand allergen containing the most IgE antibody-binding epitopes in the population studied (59).

Similarly, a high degree of cross-reactivity has been demonstrated among plants containing a Ca2+-binding protein, including members of the Brassica species, and Alnus glutinosa, Olea europea, Betula verrucosa (Bet v 4) and Cynodon dactylon (Cyn d 7) (60,62). Che a 3 from Chenopodium album pollen has also been reported to have a high similarity with calcium-binding protein allergens from pollens of olive, birch, alder, rapeseed, and timothy grass (63-64).

rPhl p 7 is therefore likely to cross-react with pollen proteins from most plants, in particular with other grass species, trees of the Fagales order such as birch tree, and olive trees and weeds (65).

g211 rPhl p 11

Recombinant non-glycosylated protein produced in an E. coli strain carrying a cloned cDNA encoding Phleum pratense allergen Phl p 11

Common name: Group 11 grass allergen
Biological function: Unknown
Mw: 20 kDa

Allergen description

Phl p 11 (7-8,66) is an allergen with structural similarity to the Soybean trypsin inhibitor family of proteins, however no enzymatic activity has been found. Note that the allergen now known as Phl p 12 was initially described as Phl p 11 (67-68).

Group 11 allergens are glycoproteins where the carbohydrate moities have been found to consist mainly of MUXF3 and MMXF3 structures. These glycan determinants are frequently found in a number of plants and are commonly called CCD (Cross-reactive Carbohydrate Determinants).

Up to 70% of grass pollen sensitized individuals in temperate climates have been reported to react with group 11 allergens. It has been suggested that a part  (up to 25%) of the IgE-binding to group 11 allergens might be directed to the carbohydrate epitopes (1,69).

Recombinant Phl p 11 lacks carbohydrate modification. One-third of 184 grass pollen-sensitised subjects showed allergen-specific IgE reactivity to recombinant Phl p 11 (66).

This class of grass pollen allergen was first described in Lolium perenne (Lol p 11) (69); significant levels of IgE antibodies binding to the purified native protein were found in 66% (n=270) of grass pollen-sensitised subjects. Phl p 11 shows 94% sequence identity to the homologous Lol p 11, and 33%-47% to described pollen proteins from a wider range of different plant species, including Oryza sativa, Zea mays, Betula pendula, Olea europea (Ole e 1), Syringa vulgaris (Syr v 1) and Ligustrum vulgare (Lig v 1).

g212 rPhl p 12

Recombinant non-glycosylated protein produced in an E. coli strain carrying a cloned cDNA encoding Phleum pratense allergen Phl p 12

Common name: Profilin
Biological function: Actin-binding protein
Mw: 14 kDa

Allergen description

Phl p 12 (7-8,26,28,67-68,70-71) is a pollen profilin. It has the characteristics of a minor allergen, binding IgE antibodies from approximately 15-30% of grass pollen-allergic subjects with varying proportions in different geographical regions (1,72).

Note that the allergen now known as Phl p 12 was initially described as Phl p 11 (67-68).

Profilins are 14 kDa acidic proteins involved in cytoskeleton dynamics by binding to actin (1).

Profilin are ubiquitous proteins present in all eukaryotic organisms. Phl p 12 has >75% sequence identity with profilins of a wide range of species, from pollen as well as various plant-derived foods and latex (28,70). The sequence identity between Phl p 12 and animal profilins ranges between approximately 30% and 45%. Immunological cross-reactivity among pollen profilins and profilins of plant-derived foods is well documented. Profilins with high sequence identity have been described in Phleum pratense, Olea europaea, Cynodon dactylon, Parietaria judaica, and Helianthus annuus pollen (73-74). Nonetheless, it has been reported that Phl p profilin is in part responsible for the T-cell mediated immunological response in patients allergic to timothy, but that the response is very specific, since Phl p profilin-specific T-cell lines did not show cross-reactivity with a highly homologous profilin from Parietaria judaica (68,75).

Other profilins include Bet v 2 from birch (Betula verrucosa), Hev b 8 from latex (Hevea braziliensis), and Pho d 2 from date palm pollen (76-77). Profilin allergens also play an important role in banana and pineapple allergy, and other exotic fruits (78). Similarly, 2 rice profilin cDNAs were reported to have an 83% to 89% similarity to profilin from maize, C. dactylon, H. brasiliensis and timothy grass (79).

Compiled by Dr Harris Steinman, harris@zingsolutions.com

References

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As in all diagnostic testing, the diagnosis is made by the physican based on both test results and the patient history.