Recombinant allergens can be produced as molecules that exactly mimic the properties of the natural allergens, or modified variants with advantageous properties such as reduced allergenic activity or increased immunogenicity, or alternatively as hybrid molecules resembling the epitopes of several different allergens to include the relevant epitopes of complex allergen sources . Recombinant allergens can be used instead of timothy grass pollen extracts, that exhibit a considerable heterogeneity regarding the presence of individual allergens and hence yield a certain variability of the results of the in vivo test. A single recombinant allergen or a combination of a few major recombinant allergens can substitute the crude extract for diagnostic purposes in vitro and in vivo.
rPhl p 1 is considered by many authors the most important timothy grass pollen “Species Specific” allergen [18–22], whereas other authors maintain that the rPhl p 5 is the most significant [23–27].
In a recent study, Casquete - Román et al. detected in a pediatric population a total of 99.4% of the patients classified as sensitized to grass pollen who yielded positive values (> 0.1 kUA/l) for the recombinant “Species Specific” allergens (rPhl p 1 + rPhl p 5), while 46% of them proved positive for the Cross-Reactive (rPhl p 7 + rPhl p 12) allergens . Rossi et al. found the following frequency of sensitization in 77 patients (mean age 21.6 years): rPhl p1 = 93.5%; rPhl p 5 = 72.7%; rPhl p 7 = 7.8%; rPhl p 12 = 35.1% . In a subsequent very recent study (2010), Rossi detected in 33 patients (age range 9–62 years) rPhl p 1 in 100% of patients, rPhl p 5 in 76%, rPhl p 7 in 3% and rPhl p 12 in 45% . Mari found the following data in sera of 749 grass-sensitized patients, selected on a population of 4,606 unselected subjects, with an age range of 2 to 70 years: rPhl p 1 = 83%, rPhl p 5 = 50%, rPhl p 7 = 7%, rPhl p 12 = 15% and isolated reactivity to rPhl p 1 in 6%, whereas it was negligible for the remaining molecules .
Among the most important recombinant allergens we measured rPhl p 1 and rPhl p 5, because they are considered the major “Species Specific Allergens” in the international literature [29–31] and rPhl p 12 and rPhl p 7, the principal Cross-Reactive components. Our study has shown the predominance in children with grass pollen allergy of both of rPhl p 1 and rPhl p 5, found in 99% and 67% of them, respectively.
The pan-allergen rPhl p 12 was detected in a notable percentage of children (32%), while Valenta et al.  reported that 20% of patients allergic to grass displayed IgE reactivity to profilins.
The discrepancy between these frequencies of sensitization is probably related to a geographical variation in allergen exposure.
Sensitization only to “Species Specific” allergenic molecules of Pp was observed very frequently, in more than half of children (65%), while sensitization to “Species Specific” and Cross-Reactive allergenic molecules together was found in a smaller percentage (35%). This may reflect the impact of large amounts of grass pollen in this geographic area, as observed also by Rossi et al. .
rPhl p 1 was the only allergenic molecule of Pp detected alone in a relevant percentage of patients (26%), while rPhl p 5 was rarely found as the only sensitizing allergen (1%). No patients were uniquely sensitized against rPhl p 7 and rPhl p 12. Consequently, this study has shown that rPhl p 1 is the predominant grass pollen allergenic molecule, sensitizing almost all patients both in association with other rPhl and acting as a single allergen.
There was a significant correlation between serum IgE against timothy grass pollen extract and Pp IgE levels.
Laffer et al. examined in sera of 183 patients allergic to grass pollen from different populations (Europe, Japan, and Canada) the in vitro IgE antibody-binding capacity to three recombinant timothy allergens, rPhl p 1, rPhl p 2, rPhl p 5, and birch profilin. More than 94% of patients could be diagnosed with a combination of recombinant rPhl p 1, rPhl p 2, rPhl p 5, and birch profilin, while the sera that did not react with the recombinant allergens contained low levels of timothy grass pollen-specific IgE antibodies. A good correlation was observed between natural timothy-serum specific IgE antibodies and the combination of recombinant allergens in all 183 tested sera .
The analysis of the IgE positivity for Pp allergenic molecules according to timothy grass pollen-specific IgE levels showed that rPhl p 1 IgE were detectable at any level of grass pollen IgE (in 97% - 100% of patients), both in children with a low grade of sensitization and in high sensitized grass pollen patients, while monosensitization to rPhl p 1 was very important in patients with lower grass pollen IgE levels, disappearing with the increase in timothy grass pollen-specific IgE antibodies (with statistical significance). However, rPhl p 5 IgE positivity was raised with the increase in grass pollen IgE level, with statistically significant differences, and it became the same as rPhl p 1 in patients with higher grass pollen IgE level (≥ 100 kUA/l). Moreover, rPhl p 12 IgE positivity increased with IgE against timothy grass pollen extract, once again with significant differences.
Studying the pattern of sensitization to Pp allergens according to the patients’ age, we have observed some interesting age-related differences. Sensitization to rPhl 1 seems to appear early, even before the age of 5 years, because it was found in 100% of our patients at this age. rPhl p 5 sensitization, although detected in a smaller percentage of children at the age < 5 years, was already present at this age and its positivity increased with the rise of the age (from 59% to 69% of children), although without statistical significance.
Allergic disorders display a clinical evolution during childhood and adolescence, related from an immunological viewpoint to the appearance of sensitizations to food allergens during the first years of life, followed by the onset of sensitization to inhalant allergens. The number of allergen sensitizations increase from childhood, when mono- or oligo-sensitizations are common, to adolescence, when polysensitization is more frequent [35, 36]. This concept may be at the basis of the different frequency of sensitization to rPhl p1 and rPhl p 5 in children with different ages. According to the majority of studies, rPhl p 1 causes IgE reactivity in more than 90% of allergic subjects and exists in all grass species , while the rate of detection of rPhl p 5 is 65%- 85% among populations of individuals allergic to grass pollen from temperate regions . The predominant role of rPhl p 1 that dominates the immune response to grass pollen extract may explain the detection of Phl p 1 IgE at any level of grass pollen IgE and at all ages, detectable also in very young children, in whom IgE against rPhl p 5 have poor relevance.
rPhl p 7 sensitization is insignificant in our population and is completely absent in patients aged < 5 years.
The frequency of sensitization to rPhl p 12 increased with the rise of the age (from 30% to 36% ones), although without statistical significance.
Scala et al., using allergen-based microarray for the detection of IgE-related sensitization to panels of allergens, gave a more precise and comprehensive evaluation for an IgE-based epidemiology. They observed that seven thousand two hundred and forty-six patients (44.16% of the total allergic cohort; 51% female) were IgE reactive on ISAC to at least one of the timothy grass pollen allergens as representative markers of other homologous grass allergens. Phl p 1 was the allergen most frequently recognized. The mean age was 30.1 ± 16.5 years, significantly higher if compared with mite allergen IgE-reactive subjects (p < 0.001), with the highest value of IgE recognition reached between 15 and 25 years of age. Phl p 2, 5, and 6 showed a similar behaviour of IgE recognition, whereas the pan-allergens Phl p 7 and Phl p 12 showed a different pattern of IgE recognition, because they did not generate any cluster with the molecules belonging to their parental biological source. Pan-allergens showed clustering trends with their homologous molecules suggesting different pathways of primary immunological sensitization .
Profilins (Phl p 12, Bet v 2) occur as highly Cross-Reactive allergens in a variety of plants unrelated from a botanical point of view and plant products. Patients producing specific IgE antibodies are either sensitized or at risk of developing allergic reactions to various plant pollens and plant-derived foods .
In our pediatric population, children with IgE against “Species Specific” and Cross-Reactive allergens were more frequently sensitized to aeroallergens Parietaria judaica and Olea and to foods peanuts and tomato compared to patients with IgE against “Species Specific” allergenic molecules only, with statistical significance. It is possible that this difference can be related to a sensitization for Cross-Reactive molecules. Indeed, among patients sensitized to “Species Specific” and Cross-Reactive molecules, only 50% were really allergic to Parietaria (IgE against Parietaria + IgE against Par j 2), while among patients only sensitized for “Species Specific” Pp allergens, 88% were really sensitized to Parietaria. It is also very remarkable that patients with IgE only against “Species Specific” allergenic molecules of Pp had not IgE vs Bet v 2, another profilin.
Considering possible correlations between clinical symptoms and the pattern of sensitization to allergenic molecules of Pp, we have observed that patients also positive for Cross-Reactive allergens were more often affected with anaphylaxis, urticaria and angioedema caused by peanuts, tomato and fruits. Further studies are needed to clarify the possible role of Cross-Reactive allergens in the development of anaphylactic symptoms due to plant-related foods.
The international literature largely reports that only a limited number of recombinant timothy grass pollen allergens account for a high percentage of IgE against grass pollen extract; it is suggested the possible utility of recombinant allergens not only for in vitro diagnosis, but probably also for specific immunotherapy . With the use of defined molecules instead of crude allergen extract–based mixtures, it would be possible to know more precisely the mechanisms underlying immunotherapy and to develop new forms and perhaps prophylactic immunotherapy strategies .
In agreement with this concept, a very recent study by Tripodi et al. defined the compatibility of the profiles of IgE sensitization to Pp with a mixture of recombinant allergenic molecules of Pp previously proposed for specific immunotherapy. Sera reacting against Pp were tested for rPhl p 1, rPhl p 2, rPhl p 4, nPhl p 4, rPhl p 5b, rPhl p 6, rPhl p 7, rPhl p 11, and Phl p 12 and the IgE individual sensitization profiles were matched against an experimental allergen-specific immunotherapy preparation containing Phl p 1, Phl p 2, Phl p 5, and Phl p 6. The authors concluded that molecularly designed immunotherapy preparations tailored to patients’ needs should consider the high heterogeneity of IgE sensitization profiles to Pp, suggesting that trials are needed to test whether different molecular sensitization profiles to grass pollen underlie different clinical responses to the same immunotherapy preparation .
Our data show the predominant role of rPhl p 1 in pediatric populations as the most relevant sensitizing allergen detectable at all ages and at all levels of timothy grass pollen-specific IgE antibodies, while the frequency of rPhl p 5 rises with the increase in patients’ age and with grass pollen IgE levels, supporting the possible use of both allergens to create an immunotherapy tailored to the single patient.