Cristoforo Incorvaia

Mona Al-Ahmad

Ignacio Ansotegui

Stefania Arasi

Claus Bachert

Catherine Bos

Jean Bousquet

Andrzéj Bozek

Davide Caimmi

Moises Calderón

Ludger ; Klimek

Incorvaia Cristoforo

Al-Ahmad Mona

Ansotegui Ignacio

Arasi Stefania

Bos Catherine

Bousquet Jean

Bozek Andrzéj

Caimmi Davide

Calderón

Casale Thomas

Custovic Adnan

De Blay Frédéric

Demoly Pascal

Devillier Philippe

Didier Alain

Alessandro Fiocchi

Fox Adam

Gevaert Philippe

Gomez Maximiliano

Heffler Enrico
email: heffler.enrico@gmail.com

Ilina Natalia

Irani Carla

Jutel Marek

Karagiannis Efstrathios

Klimek Ludger

Kuna Piotr

O'hehir Robin

Oxana

Matricardi Paolo Maria

Morais De Almeida Mario

Mosges Ralph

Novak Natalija

Okamoto Yoshitaka

Panzner Petr

Papadopoulos Nikolaos

Park

Passalacqua Giovanni

Pawankar Ruby

Pfaar Oliver

Schmid- 13 Grendelmeier Peter

Scurati Silvia

Tortajada-Girbés Miguel

Vidal Carmen

Virchow Christian

Wahn Ulrich

Zieglmayer Petra

Giorgio Walter

Personalized medicine for allergy treatment: Allergen immunotherapy still a unique and unmatched model

Keywords: allergy treament, immunotherapy clinical, rhinitis

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We here respond point-by-point to your comments: Editor's comments:

1.Please communicate with our graphics editor to change the graphics to our journal's style. RESPONSE: We will contact Anna Głobińska just after having completed this submission 2. Convert the below part to a text box.

The clinician should then evaluate:

 The route of administration: presently SCIT and SLIT for aeroallergens, considering evidences and patient's needs/preference  The AIT product to be chosen as recommended by WAO83 and EAACI84 among the products supported by evidences.

 SCIT: different schedules upon the products (i.e. depot or aqueous)  SLIT: preference for drops or tablet, both considering evidences and patient's needs/preference  Dosages and schedules: continuous or pre-seasonal; long or short term.

 Most of the above can be influenced by costs and/or reimbursements. RESPONSE: We followed your suggestion and converted the text into a text-box 3. Make an algorithm graph for molecular diagnosis. RESPONSE: We thank the Editor for this suggestion, but we do believe that this is out of scope of our article and moreover almost impossible to produce a new graph and get the approval of it from all the Authors in F o r P e e r R e v i e w the due time (72 hours as requested). Therefore, we did not added such graph into this new version of the manuscript, hoping that you will anyway considering the article suitable for publication.

COMMENTS FOR THE AUTHOR(S)

In this revision authors made significant changes and improvements in the manuscript and they included some missing and most recent data to increase the quality of the paper. Im satisfied with the answers and replies to my questions. RESPONSE: We thank the Reviewer for his/her appreciation.

Best regards, Enrico Heffler (on behalf of all my co-Authors) The introduction of personalized medicine (PM) has been a milestone in the history of 101 medical therapy, because it has revolutionized the previous approach of treating the 102 disease with that of treating the patient. It is known today that diseases can occur in 103 different genetic variants, making specific treatments of proven efficacy necessary for a 104 given endotype. Allergic diseases are particularly suitable for PM, because they meet the 105 therapeutic success requirements, including a known molecular mechanism of the 106 disease, a diagnostic tool for such disease, and a treatment blocking the mechanism. The 107 stakes of PM in allergic patients are molecular diagnostics, to detect specific IgE to single 108 allergen molecules and to distinguish the causative molecules from those merely cross-109 reactive, pursuit of patient's treatable traits addressing genetic, phenotypic and 110 psychosocial features, and omics, such as proteomics, epi-genomics, metabolomics and 111 breathomics, to forecast patient's responsiveness to therapies, to detect biomarker and 112 mediators, and verify the disease control. This new approach has already improved the 113 precision of allergy diagnosis and is likely to significantly increase, through the higher 114 performance achieved with the personalized treatment, the effectiveness of allergen 115 immunotherapy by enhancing its already known and unique characteristics of treatment 116 that acts on the causes. In its millennial history, medicine has had the primary aim of treating diseases. The 126 development in the 1800s of pharmaceutical therapy with synthetic drugs was a 127 breakthrough 1 , but the concept that a given drug was suitable for treating a given disease, 128 regardless of the characteristics of the individual patient, have survived for a long time.

129

The conceptual framework of genotypes, endotypes and phenotypes 2 has provided crucial 130 tools to understand that the characteristics of the individual also affect the response to 131 drugs and paved the way to the new era of personalized (otherwise defined precision) 132 medicine. The new approach was first applied on oncology, concerning both diagnosis and 133 treatment 3 , but soon, with a significant boost from President Obama's announcement on 134 the great potential of personalized medicine 4 , many other fields of medicine gained its 135 benefits. As far as allergic and immune-mediated diseases are concerned, the introduction 136 of biologics to treat severe asthma provided a significant advance in personalized 137 treatment, through their ability to work on asthma according on molecular phenotypes 138 defined as high type 2 immunity asthma and low type 2 immunity asthma 5 . However, 139 allergen immunotherapy (AIT), a treatment for respiratory allergy first proposed in 1911 140 which in its long road has gone from empiricism to full scientific evidence 6 can actually be 141 proposed as "a medical model aiming to deliver customized healthcare, with medical 142 decisions, practices, and/or products tailored to the individual patient" 7 . The primary 143 therapeutic goal of AIT include reducing the frequency and intensity of symptoms, the use 144 of rescue medications, and improving the allergy-related quality of life. There is very strong 145 evidence to show that AIT achieves these goals in patients with allergic rhinitis 8 .

146 Furthermore, in mite-induced asthma AIT is likely to enable a successful step-down in the 147 inhaled corticosteroid dose in patients graded as step 3 or 4 of GINA scale 9 . Here we will 148 analyze the factors underlying the characteristics of AIT as an optimal model for 149 personalized medicine.

150

The birth of AIT and its embryonic properties as a personalized treatment.

151

As hinted above, AIT was introduced more than one hundred years ago, when the 152 knowledge on allergic disease was very limited 18 . This allergy diagnostics 203 approach is aimed at mapping the allergen sensitization of a patient at a molecular level, 204 using purified natural or recombinant allergenic molecules instead of allergen extracts.

205 Each allergen molecule is identified, classified according to the family to which it belongs, 206 and scientifically named according to the species to which it belongs. For example, the 207 major allergen from cockroach is a lipocalin named Bla g 4 (after Blatella germanica 19 .

208 Since its introduction, PAMD@ has been gradually used as a laboratory tool, and 209 presently more than 130 allergenic molecules commercially are accessible for in vitro 210 sIgE) testing 20 . Two platform to perform PAMD@ are available, which include a singleplex 228 using more than 700 different rules to mimic the experts' opinions on a complex ISAC 229 result 26 . In a study comparing sera from patients assayed with ImmunoCAP ISAC and 230 ALEX a good correlation between the results from the two methods was found 27 .

231

By focusing on applications for personalized medicine, numerous studies have assessed 232 the benefits of attaining with PAMD@ a more appropriate prescription for AIT. Table 1 233 summarizes the observations from these studies [28][29][30][31][32][33][34][35][36][37] . A vast literature is available, as 234 recently reviewed [38][39] , also for the advantages of PAMD@ in the diagnosis of food allergy 235 compared to the conventional diagnosis, but this will not be treated here since the purpose 236 of our analysis is to examine the progress achieved by PAMD@ in the appropriate 237 prescription of AIT. In fact, despite the numerous controlled AIT trials for food allergy 40 , 238 this therapy has not yet been approved.

239

As far as the identification of the really causative allergen in polysensitized patients is 240 concerned, the first in vitro test was the RAST inhibition 41 , which then evolved into CAP 241 inhibition assay. The latter has been shown to be comparable to PAMD@ in the ability to 242 identify the causative allergen 35,42 and to have a lower cost 43 [START_REF] Herland | Bjermer LHow representative are clinical 813 study patients with asthma or COPD for a larger "real life" population of patients with 814 obstructive lung disease?[END_REF] . Through this initiative we will be able define the real validity of the existing AIT RWEs.

411 This action has been also stated and promoted by the recent Real-Life Research 412 Manifesto, where it is pointed out the need to consider RWE in the complex scenario of 413 scientific clinical research [START_REF] Schünemann | guidelines: 18. How ROBINS-I and other tools to 818 assess risk of bias in nonrandomized studies should be used to rate the certainty of a 819 body of evidence[END_REF] . This document supported the use of tools, such as the ones 414 mentioned previously, to analyze RWE properly. Finally, the Manifesto also underlined the 415 major differences existing between retrospective and prospective RWE: where prospective 416 approach, whose typical example is a Disease or intervention's Registry, is the most 417 credible, as also stated by Sherman et al [START_REF] Roche | 822 Collaborators. The importance of real-life research in respiratory medicine: manifesto of 823 the Respiratory Effectiveness Group: Endorsed by the International Primary Care 824 Respiratory Group and the World Allergy Organization[END_REF] . In fact, it should be underlined that several AIT 418 RWE reports were retrospectively collected, combining different methodologies, thus 419 decreasing the validity of the final outcomes/conclusions. However, recent, well conducted 420 real-life studies, derived from a well-designed and set up database and including very 421 large patient populations, were of relevant importance, because they contributed to 422 increase the knowledge on the effectiveness of AIT in patients seen in daily clinical 423 practice, possibly not meeting the strict criteria to be included in a trial [START_REF] Sherman | Real-World Evidence -What Is It and What Can It Tell Us?[END_REF][START_REF] Wahn | Real-world benefit of allergen 830 immunotherapy for birch pollen-associated allergic rhinitis and asthma[END_REF] .

424

425 Final message: AIT as personalized treatment 426 All the above is in line with the current view that collecting Big Data can lead to precision 427 medicine 79,80 . We previously pointed out AIT as a prototype of precision medicine 432 and responds perfectly to the three major needs to be met in personalized medicine, which 433 are the identification of the molecular mechanism of the disease, the availability of 434 diagnostic tools to recognize this mechanism and the availability of a treatment capable of 435 blocking the mechanism 82 . In Figure 1 we summarized all the possible aspects of the 436 updated journey to reach the best prescription of AIT, through a detailed and modern 437 diagnosis (including PAMD@) and definition of patient's eligibility to AIT. As to diagnosis, it 438 must be kept in mind that the medical history collected by a skilled doctor is the basis for 439 deciding the subsequent investigations in accordance with the individual characteristics.

440 The current and future tools to reach a AIT personalized prescription are summarized in 441 Figure 2 . Their variable characteristics suggest that personalized care is valuable in this 442 setting and may be also be preventive (by focusing on quality of life), predictive (by Tools still to be validated for AIT 226 information systems. For instance, the expert system named Allergenius ® is based on the 227 ImmunoCAP ISAC implemented with an advanced knowledge specification language 228 using more than 700 different rules to mimic the experts' opinions on a complex ISAC 229 result 26 . In a study comparing sera from patients assayed with ImmunoCAP ISAC and 230 ALEX a good correlation between the results from the two methods was found 27 .

443

231

By focusing on applications for personalized medicine, numerous studies have assessed 232 the benefits of attaining with PAMD@ a more appropriate prescription for AIT. Table 1 233 summarizes the observations from these studies [28][29][30][31][32][33][34][35][36][37] . A vast literature is available, as 234 recently reviewed [38][39] , also for the advantages of PAMD@ in the diagnosis of food allergy 235 compared to the conventional diagnosis, but this will not be treated here since the purpose 236 of our analysis is to examine the progress achieved by PAMD@ in the appropriate 237 prescription of AIT. In fact, despite the numerous controlled AIT trials for food allergy 40 , 238 this therapy has not yet been approved.

239

As far as the identification of the really causative allergen in polysensitized patients is 240 concerned, the first in vitro test was the RAST inhibition 41 , which then evolved into CAP 241 inhibition assay. The latter has been shown to be comparable to PAMD@ in the ability to 242 identify the causative allergen 35,42 and to have a lower cost 43 in patients with multiple 243 sensitization to Hymenoptera venom, in which a limited number of allergenic molecules 244 are responsible, while such outcomes was not reported for multi-sensitization to 245 respiratory allergens, involving a much higher number of molecules.

246 The basophil activation test (BAT), which measures basophil degranulation by flow 247 cytometry, has the unique characteristic to detect in vitro the reactivity of basophils to 248 allergens. Since these cells play a crucial role in allergic reactions, their in vitro detection 249 has a similar meaning to the in vivo provocation test with specific allergens, without 250 however the limit of false positive results from non-specific reactions, which represented a 432 and responds perfectly to the three major needs to be met in personalized medicine, which 433 are the identification of the molecular mechanism of the disease, the availability of 434 diagnostic tools to recognize this mechanism and the availability of a treatment capable of 435 blocking the mechanism 82 . In Figure 1 we summarized all the possible aspects of the Tools still to be validated for AIT

251
• Treatable traits (genetic, phenotypic, psychosocial features)

• Omics (proteomics, epigenomics, metabolomics, breathomics)

Tools of future applications

  much for your provisional acceptance of our manuscript.

117118

  Key words: allergen immunotherapy, personalized medicine, molecular diagnosis, 119 treatable traits, omics. 120 121 Short title: Allergen immunotherapy: a model of personalized medicine.

  7 and 428 now we would propose AIT as one of the best example of Personalized and Participatory 429 Medicine, aimed to satisfy the needs and preferences of patients and to strengthen the patient/doctor in jointly choosing the best therapeutic option. AIT is the only 431 medical treatment that has the capacity to change the natural history of allergic diseases81

459

  allowing treatment to be adjusted as a function of the individual's response) and 444 participative (empowering the patient). In particular, SLIT can be considered a precision 445 medicine treatment (especially with high quality NPP) that enable the physician to identify 446 the optimal treatment for each patient. This tailor-made approach to diagnosis, decision-447 making, product choice and treatment schedules may enhance effectiveness, minimize 448 adverse events, improve patient's quality of live and reduce the socio-economic impact. A 449 number of SLIT drops updosing regimens and maintenance treatment were shown to be 450 safe and effective. This means that SLIT liquid allergen extract formulations are extremely 451 flexible in terms of frequency of dosing and number of allergen doses delivered. Such 452 parameters can be fine-tuned to suit the patient's day-to-day treatment response and 453 intercurrent events. Depending on the sensitization profile and the clinical signs, the 454 specialist can develop a specific, tailor-made SLIT liquid-based approach that, due to its 455 ductility in clinical practice, may be an added value. In addition, SLIT liquid formulation 456 makes it possible to adapt the dose and regimen during the updosing and/or the 457 maintenance phase to target optimal effectiveness. Personalized medicine in AIT has the potential to improve various outcomes which have 460 so far produced limited results. This is true for preventive ability, which until now has been 461 shown only for the development of asthma in patients with allergic rhinitis85 , the 462 identification of biomarkers predictive of treatment efficacy (still not known)57 and cost-463 effectiveness compared to drug therapy, clearly demonstrated on the basis of the 464 persistence over time of the control of symptoms after AIT withdrawal86 , but further 465 improvable by greater diagnostic precision. Further issues could concern the expansion of 466 e-health systems 7 and the implementation of telemedicine87 .467 This journey is leading to the most appropriate choice of the AIT treatment thanks to the 468 multifaceted variety of AIT products empowering the professionality of the clinician in 469 choosing, through a shared decision that considers the patient's needs, a proper AIT 470 product for each patient according to eligibility and acceptability. 471 It is so conceivable to conclude that AIT is still a unique and unmatched model of 472 Personalized Medicine for allergy treatment, able to encompass the prediction of a 473 successful treatment and the potential prevention or progression of an allergic disease, 474 and possibly to face the challenge of providing such advances to at affordable costs. As 475 well, the characteristics of Personalized Medicine for allergy are likely to enable to face 476 challenges such as increasing prevalence, growing complexity and heterogeneity, impact 477 on patients, problem of access to care, therapeutic wandering, and successful 478 management of uncontrolled patients 88 . 479 480 Disclosure of Interests 481 M. Al-Ahamad has nothing to disclose.

Molecular diagnostics and other in vitro tests 193

  158 allergenic source directly from the doctor, was hugely lower than the allergen extracts 159 used today, but the positive results obtained paved the way to an evolution finally resulting 160 in high quality products. Another definition that underlines how the AIT was yet, but 161 improperly, proposed as personalized for the patient is "Named Patient Product" (NPP), 162 i.e. specifically prepared for a given patient according to the results of allergy testing.163 Nevertheless, NPPs were not a guarantee of clinical efficacy, because alongside products 164 with evidence of efficacy proven by placebo-controlled trials, others without such evidence 165 were equally admitted to the prescription. Moreover, extracts containing several allergens, 166 namely all the allergens giving positive results to allergy testing, were frequently used, 167 especially in the U.S.A, but they were found to be ineffective in a pivotal double-blind, 168 placebo-controlled trial in 199710 . Nowadays, registered products, whose effectiveness The diagnosis of allergy has been long based on skin prick test (SPT) with allergen 194 extracts and in vitro test measuring specific IgE antibodies to the suspected allergens, but 195 with both techniques the positive results indicated sensitization but not necessarily clinical

	192 1) 196 allergy. A great advance in diagnostic precision was achieved by molecular diagnosis,
	197	which allows to detect specific IgEs (sIgEs) to each single molecule, distinguishing those
	198	really causative (genuine) from those simply cross-reactive. Such diagnostic approach was
	199	variously named, including "Component resolved diagnosis" 15 , molecular-based allergy
	200	diagnostics 16 , and molecular allergy diagnostics 17 . The updated WAO-ARIA-GA 2 LEN
	201	consensus document on molecular-based allergy proposed the unifying definition of
	F o r r o F 202 "precision allergy molecular diagnostic applications" (PAMD@) 169
		P e e P e e
		r r
		R R
		e v i e w e v i e w
	185	than for European SLIT maintenance dosage. The authors argued that, based on the
	186	efficacy demonstrations from controlled trials, SLIT efficacy is likely to depend on
	187	additional factors apart from the extract dose 14 . Today, the pillars of AIT as a personalized
	188	therapy are represented by molecular diagnostics for achieving the highest possible
	189	precision, by the use of treatable traits, and by "omics".
	190
	191

6

. Still, the different role of AIT compared

153

with the drug therapy available at the time was apparent, because drugs were aimed at 154 treating the symptoms of allergic rhinitis, while AIT was aimed at treating the specific 155 allergy of single patients, thus introducing a concept now acknowledged as personalized 156 medicine. The first studies addressed "hay fever" induced by grass pollen. It is obvious 157 that the quality of the products in that time, which consisted of extracts obtained from the has been plainly demonstrated by controlled trials on large patient populations, are 170 considered ideal for AIT 11 . However, good quality NPPs with demonstration of efficacy by 171 placebo-controlled trials, are still available to treat respiratory allergy with expectancy of 172 clinical success 12 . 173 Modern AIT as a prototype of personalized medicine. 174 The traditional diagnosis by skin tests relay on extracts, whose composition in minor and 175 major allergens can play a crucial role in defining sensitizations, as in the AIT products for 176 inducing clinical efficacy. Another critical factor is the extract potency, concerning both skin 177 testing and AIT. As to the latter, Larenas-Linneman et al. found a substantial variability in 178 allergen extract potency as measured and reported worldwide 13,14 . In particular, in Europe 179 the potency of extracts for sublingual immunotherapy (SLIT) is based on comparison with 180 in-house references, making it difficult to translate the dose to US extracts 13 . Moreover, 181 comparing SLIT maintenance solutions of grass pollen and Dermatophagoides 182 pteronyssinus from 4 leading European manufacturers to standardized concentrate 183 extracts of 3 US manufacturers measure in bioequivalent allergen units (BAU), the relative 184 potency was around 10 times higher for US standardized extracts (to be diluted for SLIT)

  294 recent years51 . For example, using treatable traits in a patient with COPD but symptoms 295 compatible also with asthma and positivity of tests for respiratory allergy due to 296 asymptomatic sensitization would prevent the incorrect prescription of AIT. The role of 297 treatable traits in the management of chronic respiratory diseases has been outlined by 298 some important studies. The first investigated the role of extra-pulmonary comorbidities as 299 treatable traits in patients with difficult-to-control asthma. The proposed comorbidities were 300 allergic and nonallergic rhinitis, chronic rhinosinusitis, vocal cord dysfunction, dysfunctional 301 breathing, gastroesophageal reflux, obesity, obstructive sleep apnoea, and anxiety/ 302 depression, of which the prevalence, impact and outcome of treatment were assessed, 303 also appraising the associations of single comorbidities and the potential of comorbidity 304 clusters on asthma control. The authors concluded that extra-pulmonary comorbidities are 328 possible synergistic effect of biologics and AIT. Precision medicine of allergic diseases is 329 nowadays highlighted by the consistent data of efficacy of biologics targeting cytokines ((IL 330 5) or their receptors (IL 5 rec. or IL4/IL13 rec.). Nonetheless, we should consider that the 331 eligibility of allergic patients to biologics or AIT is only partially overlapping, being the first 332 limited to severe asthma or CRSwNP whereas AIT has indication for allergic rhinitis and 333 mild/moderate asthma while severe asthma is a contraindication. Based on the 334 characteristics of the two treatments, a synergistic effect in allergic asthma is conceivable, 335 with biological achieving control of severe asthma but not changing its natural history while 336 AIT, once ceased the contraindication, can induce the long-term immunological changes.337Studies to investigate whether such synergy is achievable are warranted. The great advance provided by the introduction of personalized medicine in diagnosis of 365 allergy is likely to enable solving the long-standing problem of AIT related to the great 366 qualitative variability of AIT products and the consequent heterogeneity of the clinical 367 results. Actually, an updated way to correctly evaluate the effectiveness of AIT products 368 must avoid the mistakes of the past, when the results of a valid tool such as meta-analysis, 369 despite the clear differences between different products, tended in the conclusions to 370 extend the positive outcome to AIT in general65 . The latest generation of registered 371 products for SLIT, which were based on trials including large number of patients with 372 respiratory allergy to grass pollen and dust mites, meet the modern quality needs[66][67][68][69][70] . Still, 396 dust mites the development of a synthetic cultured medium free from proteins of animal 397 origin is needed 71 . As mentioned above, the use of omics is of marked importance in the 398 comprehensive characterization of allergen extracts, including transcriptome for de novo 399 sequencing, proteome for MS analysis, and allergome for IgE reactivity 72 . 400 In this context, the evaluation of the Real-World Evidences (RWE), should be properly 401 reappraised, since patients in clinical practice often do not meet the same criteria of the 402 subjects enrolled in the double-blind placebo-controlled trials (DBPCRT) leading to 403 registration by Regulatory Authorities 73 . In fact, distinction is made between the final 404 outcome of DBPCT efficacy and the one in RWE. Nonetheless, a correct evaluation of 405 these RWE has to be methodologically performed. To this purpose an ongoing European 406 Academy of Allergy and Clinical Immunology (EAACI) initiative is designed to ascertain the 407 validity of the existing RWE concerning AIT, applying the recent tools proposed by

	260 ImmunoSorbent Test (ELISA) or ImmunoCAP 45 and that in patients with severe reactions 362
	261 to stings, some of them with negative response to sIgE test and intradermal tests, BAT 363 Impact of personalized medicine on AIT
	F o r was positive in 81% of patients compared with to a rate 57% with intradermal testing, 14% with IgE test, 19% of patients being negative to either test except BAT 46 . A study analyzed the correlation between the BAT changes during venom immunotherapy and the treatment outcome. BAT was unchanged after one year, but in subsequent years a fourfold reduction was detected in all patients who developed tolerance to stings, while no change in BAT occurred in patients with a persistently positive sting challenge 47 . Such important outcome was found also in patients treated with subcutaneous immunotherapy (SCIT) for grass pollen allergy. In fact, basophil sensitivity showed a 447-fold decrease in the first year of treatment, remaining 100-fold lower than baseline in the 3 year-treatment period and 10-fold lower in the follow-up year. Notably, a decline in basophil sensitivity after three weeks of SCIT predicted long-F o r 305 F 262 263 264 265 266 267 268 269 270 271 272 o r F o r 364 F o r
	P e e term improvement in symptom and medication scores during the three years of treatment 48 . Further studies are needed to clarify the role of BAT as a biomarker or a predictive marker of AIT efficacy. P e e P e e P e e P e e in patients with multiple r r r r r 243 sensitization to Hymenoptera venom, in which a limited number of allergenic molecules 273 274 275 276 R 277 2 In vivo tests R R R R 244 are responsible, while such outcomes was not reported for multi-sensitization to 245 respiratory allergens, involving a much higher number of molecules. 246 The basophil activation test (BAT), which measures basophil degranulation by flow 247 cytometry, has the unique characteristic to detect in vitro the reactivity of basophils to 248 allergens. Since these cells play a crucial role in allergic reactions, their in vitro detection 249 has a similar meaning to the in vivo provocation test with specific allergens, without e v i e w 278 Theoretically, inducing allergic symptoms by a challenge with the suspected allergen in the 279 target organs can be a valuable in vivo test. However, as stated in the International 280 Consensus Statement on allergic rhinitis, the contrasting findings from different studies 281 and the absence thus far of a standardized technique limit the diagnostic utility of nasal 282 challenge, a pivotal role being currently acknowledged only in diagnosis of occupational e v i e w e v i e w e v i e w e v i e w
	250 283 rhinitis and local allergic rhinitis 49 . If future studies will achieve standardization of the however the limit of false positive results from non-specific reactions, which represented a
	251 284	limit of in vivo tests. BAT was found to be very useful in the personalized diagnosis of method, the nasal challenge could be used, alike BAT, as a test toward precision medicine.
	252 285	patients with allergic conditions including respiratory, food and Hymenoptera venom
	253 286 3.Treatable traits allergy who show multiple allergen reactivity, distinguishing the allergens as causative or
	254 287 The term "Treatable traits" was suggested by Agusti and coworkers to indicate a precision not based on the response of the basophils 44 . As mentioned above, our review is focused
	255 288 medicine methodology of diagnosis and treatment of chronic disorders of the airways on the effects of personalized diagnosis on the outcomes of AIT. As for CAP inhibition, the
	256 first studies concerned Hymenoptera venom allergy, demonstrating that BAT 289 based on finding genetic, phenotypic and psychosocial features which are associated with
	257 accomplished with the recombinant allergens from Vespula spp Ves v 1, Ves v 2, Ves v 3
	258 and Ves v 5 achieved a clear improvement in the specificity of diagnosis in patients with
	259 anaphylactic reactions to wasp stings over IgE detection by Enzyme-Linked

290 therapies able to improve respiratory health

50

. The field of application considered as most 291 appropriate for the use of treatable traits is represented by chronic respiratory diseases 292 and in particular by asthma and chronic obstructive pulmonary disease (COPD), which 293 share various clinical aspects but whose drug therapy has significantly differentiated in important treatable traits be evaluated in all asthmatic patients and especially in those with 306 difficult asthma. Such comorbidities may have a significant influence on asthma control, 307 thus their presence deserves treatment regardless of original asthma status and asthma 308 control 52 .The number of treatable traits involved in acute exacerbation differed according

309

to the respiratory disease, with a major role for C reacting protein in COPD, eosinophils 310 count and fractioned exhaled nitric oxide (FeNO) measurement in asthma, and bacterial 311 infections for bronchiectasis

53

.

312

An important observation obtained through the review of the literature by Pavord and 313 Agusti concerned the identification of eosinophilic airway inflammation, as assessed by the 314 blood eosinophil count, as a treatable trait of particular importance in patients with airway 315 disease (including asthma and COPD, being significantly associated with long-term 316 outcomes 54 . The recent European Unbiased Biomarkers for the Prediction of Respiratory 317 Disease Outcomes (U-BIOPRED) project was aimed at identifying and quantifying, based 318 on the hypothesis that treatable traits are more common in severe asthma and may 319 significantly differ according to asthma phenotypes, the treatable traits recognized in the 320 severe and mild/moderate adult asthma cohorts and across previously identified 321 phenotypes 55 . 322 All studies available thus far support a chief role of treatable traits in evaluating patients 323 with respiratory diseases, however the preferable method of application and the cost-324 effectiveness of such a multidimensional intervention is still uncertain, making it necessary 325 to conduct randomized controlled trials involving large populations of patients 56 . 326 Concerning AIT, treatable traits of clinical interest may consist of the treatment outcome, 327 beyond the disease to be treated, on comorbidities 57 . Another trait may concern the 338 339 4.Omics 340 Following the definition of genomics in the 1960s as the complete nucleotide sequence of 341 an organism 58 , a large number of other "omics" were recently proposed, such as 342 proteomics (the complete proteins of a cell in any organism), epi-genomics (the 343 modification of nucleotides in an organism), metabolomics (the changes in gene activity in 344 response to metabolites 59 , breathomics (the multidimensional molecular analysis of 345 enxhaled breath 60 , but the list is very large and still expanding. In the field of allergy 346 diseases, asthma is the most investigated disease. In 2011, the detection of protein 347 changes by proteomics in different asthma stages was supported to improve the 348 understanding of the molecular mechanisms of the disease and to find novel mediators 349 and biomarkers 61 . Another omic of potential interest for personalized medicine is the 350 exposome, definable as a systematic approach to obtain large data sets on to 351 environmental exposures of an individual during the whole life 62 . 352 However, the available data on the state of omics technology in the management of 353 asthma and allergic diseases were recently analyzed by Donovan et al. After pointing out 354 that omics-based investigation are used increasingly to distinguish subtypes of 355 allergic diseases and asthma subtypes, forecast patient responsiveness to specific 356 therapies, detect biomarkers and mediators, and verify the disease control, the authors 357 concluded that omics testing in this field for are not yet a standard of care, and that key 358 factors need to be recognized before such technologies can be used successfully in 359 common clinical practice 63 . On the other hand, according to Fitzgerald et al., the promise 360 of new technology must leave room to an humanomics perspective, i.e. the 361 acknowledgement of patient's behavioral aspects 64 . 373 as mentioned above, the previous generation products include allergen extracts of efficacy 374 demonstrated by controlled trials, but also products missing such evidence. This implies 375 new properly designed clinical trials to demonstrate efficacy. Nonetheless, in order to 376 facilitate AIT product development it is advisable that the same product composition, 377 possibly using different formulation (i.e. drops/tablets) should have a mutual program and 378 recognition and possibly a full development program. Also, the relevance of testing all 379 allergens and the final cost of the studies for a limited effect size due to the huge placebo 380 effect in AIT trial is worthy of attention. These facets could provide a flexibility of treatment 381 otherwise difficult to reach in clinical practice, when batch to batch reproducibility is 382 ensured in terms of quality product and standardization. 383 Today, to achieve the highest quality any manufacturer of allergen products must follow 384 stringent scientific rules. The first is to ensure the presence in the allergen extracts of all 385 relevant allergens. As discussed above, PAMD@ provides the detections of all allergen 386 molecules, distinguishing those simply cross-reactive from genuine causative ones. 387 Similarly, the development of an AIT product requires during the manufacturing process 388 the identification of all component through mass-spectrometry based analysis, followed by 389 allergen extraction, purification and formulation, as well as yield cost, robustness and 390 scaling up. The subsequent step is the product characterization, which results in 391 development and implementation of analytical methods for identity, purity, consistency and 392 stability, which makes clinical development possible in its various phases. These phases 393 (I, II, III) are aimed at establishing safety, dosing and efficacy. For optimal quality level, 394 well-controlled source materials are also essential, which consist, for example for grass 395 pollen, in the harvesting and processing of pollens from cultured grasses, while for house 408 Respiratory Effectiveness Group (REG)/EAACI from one side (Relevant) and Grading of 409 Recommendations, Assessment, Development and Evaluation (GRADE) on the other side 410

Table 1 Studies that have assessed the benefits of attaining with PAMD a more 566 appropriate prescription for AIT

  The clinician should then evaluate:  The route of administration: presently SCIT and SLIT for aeroallergens, considering evidences and patient's needs/preference  The AIT product to be chosen as recommended by WAO 83 and EAACI 84 among the products supported by evidences. Dosages and schedules: continuous or pre-seasonal; long or short term. Most of the above can be influenced by costs and/or reimbursements. Virchow declares he has received fees from Allergopharma, ALK, LETI, Stallergenes 549 Greer. 550 U. Wahn declares he has received honoraria for consultancy as well as lecture fees from551Allergopharma, Stallergenes Greer, ALK, Roxall, Sanofi-Aventis, Novartis, Berlin-Chemie

	 SCIT: different schedules upon the products (i.e. depot or aqueous)  SLIT: preference for drops or tablet, both considering evidences and patient's needs/preference o r P e e F o r P e e F o r P e e Co. KG, Lilly Deutschland.. P. Zieglmayer has nothing to disclose. C. Bos, E. Karagiannis and S. Scurati are employee of Stallergenes Greer 563 F 557 558 559 560 561 562 o r P e e bee and wasp venom extracts. Sastre, 2012 (29) Pollens 141 pts Agreement in AIT indication before and after ISAC(®) results in 62 (46%) patients; (kappa = 0.1057 ± 0.0413). F o r P e e Saltabayeva, 2017 (33) Pollens 95 pts (real-life study) A lower number of AIT treatments (n = 119) was needed according to MAD compared to 275 AIT treatments based on conventional diagnosis, with large reduction of the total costs for a 3-F o r P e e of the prescribed AIT in 52.87% of cases. Peveri, 2019 (37). Various aeroallergens 272 pts. In 50% of cases a change in the AIT prescription for respiratory allergy was found, resulting in a saving of financial F o r P e e F o r P e e F o r P e e F o r P e e F o r P e e F o r P e e F o r P e e F o r P e e 812 73)Herland K, Akselsen JP, Skjønsberg OH, Bjermer LHow representative are clinical 813 study patients with asthma or COPD for a larger "real life" population of patients with 814 obstructive lung disease? Respir Med. 2005;99(1):11-9. 815 74)Schünemann HJ, Cuello C, Akl EA, Mustafa RA, Meerpohl JJ, Thayer K, Morgan 816 RL, Gartlehner G, Kunz R, Katikireddi SV, Sterne J, Higgins P e e e e P JP, Guyatt F o r F r o  F 565 Author, year (ref) Allergens tested Population Results Mitterman, 2010 (28) Bee and wasp venom. 43 patients (pts) Recombinant information for allergists Vidal, 2014 (32) Pollens, dust mites, moulds, animal epithelia 62 Spanish allergists SPTs are insufficient to establish the clinical relevance of each for both venoms. A good concordance between the results of MAD and CAP-inhibition was found 602 12)Devillier P, Dreyfus JF, Demoly P, Calderón MA. A meta-analysis of sublingual allergen 603 immunotherapy and pharmacotherapy in pollen-induced seasonal allergic 604 rhinoconjunctivitis. BMC Med. 2014;12:71 605 13)Larenas-Linnemann D, Cox LS; Immunotherapy and allergy diagnostics Committee of 636 23)van Hage M, Schmid-Grendelmeier P, Skevaki C, Plebani M, Canonica W, Kleine-637 Tebbe J, Nystrand M, Jafari-Mamaghani M, Jakob T. Performance evaluation 638 of ImmunoCAP® ISAC 112: a multi-site study. Clin Chem Lab Med. 2017 Mar 670 of Allergen Molecule Based Diagnosis for Prescription of Immunotherapy in an Area 671 with Multiple Pollen Exposure. Int Arch Allergy Immunol. 2017;173(2):93-98. 672 34) Del-Río Camacho G, Montes Arjona AM, Fernández-Cantalejo Padial J, Rodríguez 704 44)Hemmings O, Kwok M, McKendry R, Santos AF. Basophil activation test: old and new 705 applications in allergy. Curr Allergy Asthma Rep. 2018 Nov 15;18(12):77. 706 45)Balzer L, Pennino D, Blank S, Seismann H, Darsow U, Schnedler M, McIntyre M, Ollert 736 51)Woodruff PG, Agusti A, Roche N, Singh D, Martinez FJ. Current concepts 737 in targeting chronic obstructive pulmonary disease pharmacotherapy: making 738 progress towards personalised management. Lancet. 2015;385(9979):1789-1798. 739 52)Tay TR, Hew M. Comorbid, "treatable traits" in difficult asthma: current evidence and 770 59)Abdel-Aziz MI, Neerincx AH, Vijverberg SJ, Kraneveld AD, Maitland-van der Zee AH. 771 Omics for the future in asthma. Semin Immunopathol. 2020;42(1):111-126. 772 60)Devillier P, Salvator H, Naline E, Couderc LJ, Gtassin-Delyle S. Metabolomics in the 773 diagnosis and pharmacotherapeutic management of respiratory diseases. Curr Pharm 803 71)Batard T, Hrabina A, Bi XZ, Chabre H, Lemoine P, Couret MN, Faccenda D, Villet 804 B, Harzic P, André F, Goh SY, André C, Chew FT, Moingeon P. Production and 805 proteomic characterization of pharmaceutical-grade Dermatophagoides pteronyssinus 806 and Dermatophagoides farinae extracts for allergy vaccines. Int Arch Allergy 871 87)Krishna MT, Knibb RC, Huissoon AP. Is there a role for telemedicine in adult allergy 872 services?.Clin Exp Allergy. 2016;46(5):668-77. 873 874 548 C 552 M. Worm declares she as received of honoraria or consultation fees by ALK-Abelló 553 Arzneimittel GmbH, Mylan Germany GmbH, Leo Pharma GmbH, Sanofi-Aventis 554 Deutschland GmbH, Regeneron Pharmaceuticals, DBV Technologies S.A, Stallergenes 555 Greer, HAL Allergie, Allergopharma, Bencard Allergie, Aimmune Therapeutics UK Limited, 556 Actelion Pharmaceuticals Deutschland G, Novartis AG, Biotest AG, AbbVie Deutschland & nonglycosylated allergen molecules (Api m 1, rApi m 2, rVes v 5) allowed to with positive test both allergic patients. Burgos, 2018 (36) specialist's composition 811 5;12(10):e0185830 venom in the 14 patients particularly in pollen-Martinez-Canavate Pollens 281 children MAD results changed the 810 the allergome of house dust mite Dermatophagoides species. PLoS One. 2017 Oct define the sensitizing allergen. Serum specific IgE adds value to SPT but MAD is strongly recommended, only when the value in 606 the American Academy of Allergy, Asthma and Immunology European allergen extract 807 Immunol. 2006;140(4):295-305) 875 88) Gómez RM, Ansotegui I, Canonica GW. Will precision medicine be available for all kU/l of Ves v 5 were d 5, and vice versa. 809 E, Mascarell L, Moingeon P. A combined transcriptome and proteome analysis extends about twice those of Pol 607 units and potency: review of available information. Ann Allergy Asthma Immunol. 808 72)Bordas-Le Floch V, Le Mignon M, Bussières L, Jain K, Martelet A, Baron-Bodo V, Nony 876 patients in the near future? Curr Opin Allergy Clin Immunol. 2019;19(1):75-80.
		Moreno, 2014 (30)	Pollens	r r r r r r r r r r r r r r r r	1263 pts	Based on history and year AIT treatment resources.
	567	Del-Rio Camacho, 2018	Pollens	R R R R R 70 children R R R R R R R R R R R	SPT, 73% of patients MAD modified AIT
	568	(34)		e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w	would prescribed AIT with a mix have been of grass and olive pollens, while only in 56.8% of patients the prescription in 54.3% of cases, and increasing the indication of single-allergen therapy from 18% to 51% reversing
						double positivity was the decision to prescribe
	827	confirmed by molecular AIT in 9.3% of cases LQ, Califf RM . Real-World Evidence -What Is It and What Can It Tell Us? N Engl J
	828	Savi, 2016 (35) Med. 2016;375(23):2293-2297.. Hymenoptera venoms	40 pts.	allergy diagnosis (MAD) By MAD data VIT would
	with Ole e 1 and Phl p have been prescribed to 829 77)Wahn U, Bachert C, Heinrich J, Richter H, Zielen S. Real-world benefit of allergen
	830	molecules. 7 pts for Polistes spp, to immunotherapy for birch pollen-associated allergic rhinitis and asthma. Allergy. 2018;
	Popescu, 2014 (31) 74(3):594-604. 832 78)Zielen S, Devillier P, Heinrich J, Richter H, Wahn U. Sublingual immunotherapy Grass pollen Not defined (review of published data) MAD biomarkers (including tolerogenic 6 for Vespula spp, and to 831 41 for both venoms. With the data from CAP dendtitic, regulatory T inhibition, it would have 833 provides long-term relief in allergic rhinitis and reduces the risk of asthma: a cells, IgE, IgA and IgG antibodies, and been a prescription to 15 834 retrospective, real-world database analysis. Allergy. 2018;165-177.
	835					apoptosis biomarkers patients for Polistes, to
						provide relevant 28 for Vespula, and to 11

817

G; GRADE Working Group GRADE guidelines: 18. How ROBINS-I and other tools to 818 assess risk of bias in nonrandomized studies should be used to rate the certainty of a 819 body of evidence. J Clin Epidemiol. 2019 Jul;111:105-114. 820 75)Roche N, Anzueto A, Bosnic Anticevich S, Kaplan A, Miravitlles M, Ryan D, Soriano JB, 821 Usmani O, Papadopoulos NG, Canonica GW; Respiratory Effectiveness Group 822 Collaborators. The importance of real-life research in respiratory medicine: manifesto of 823 the Respiratory Effectiveness Group: Endorsed by the International Primary Care 824 Respiratory Group and the World Allergy Organization. Eur Respir J. 2019 19;54(3) 825 76)Sherman RE, Anderson SA, Dal Pan GJ, Gray GW, Gross T, Hunter NL, LaVange 826 L, Marinac-Dabic D, Marks PW, Robb MA, Shuren J, Temple R, Woodcock J, Yue

for allergy treatment: allergen immunotherapy 2 still a unique and unmatched model.

  Division of Allergy/Immunology, University of South Florida, Tampa, FL United-States 36 14. Centre for Respiratory Medicine and Allergy, Institute of Inflammation and Repair, 37 University of Manchester and University Hospital of South Manchester, Manchester, Department of Dermatology and Allergy, University Hospital Bonn, Bonn, Germany 70 36. Department of Otorhinolaryngology, Chiba University Hospital, Chiba, Japan. 71 37. Department of Immunology and Allergology, Faculty of Medicine in Pilsen, Charles

	• Treatable traits (genetic, phenotypic, psychosocial features ) • Omics (proteomics, epigenomics, metabolomics, breathomics ) Tools of future applications w v R e r F o P e r e i e F o r P e e r R e v i e w University in Prague, Pilsen, Czech Republic 124 Introduction 69 35. 72 125 In its millennial history, medicine has had the primary aim of treating diseases. The 126 development in the 1800s of pharmaceutical therapy with synthetic drugs was a 127 breakthrough 1 , but the concept that a given drug was suitable for treating a given disease, 128 regardless of the characteristics of the individual patient, have survived for a long time. 129 The conceptual framework of genotypes, endotypes and phenotypes 2 has provided crucial 130 tools to understand that the characteristics of the individual also affect the response to 131 drugs and paved the way to the new era of personalized (otherwise defined precision) 132 medicine. The new approach was first applied on oncology, concerning both diagnosis and 133 treatment 3 , but soon, with a significant boost from President Obama's announcement on 35 13. F o r P e e r R e v i e w F o r P e e r R e v i e w 134 the great potential of personalized medicine 4 , many other fields of medicine gained its F 135 benefits. As far as allergic and immune-mediated diseases are concerned, the introduction o 136 of biologics to treat severe asthma provided a significant advance in personalized r 137 treatment, through their ability to work on asthma according on molecular phenotypes P e e r R e v i e 138 defined as high type 2 immunity asthma and low type 2 immunity asthma 146 Furthermore, in mite-induced asthma AIT is likely to enable a successful step-down in the w 147 inhaled corticosteroid dose in patients graded as step 3 or 4 of GINA scale 9 . Here we will
	148	analyze the factors underlying the characteristics of AIT as an optimal model for
	149	personalized medicine.
	150	The birth of

1 Personalized Medicine

5

. However,

139

allergen immunotherapy (AIT), a treatment for respiratory allergy first proposed in 1911 140 which in its long road has gone from empiricism to full scientific evidence 6 can actually be 141 proposed as "a medical model aiming to deliver customized healthcare, with medical 142 decisions, practices, and/or products tailored to the individual patient" 7 . The primary 143 therapeutic goal of AIT include reducing the frequency and intensity of symptoms, the use 144 of rescue medications, and improving the allergy-related quality of life. There is very strong 145 evidence to show that AIT achieves these goals in patients with allergic rhinitis 8 .

AIT and its embryonic properties as a personalized treatment.

  158 allergenic source directly from the doctor, was hugely lower than the allergen extracts 159 used today, but the positive results obtained paved the way to an evolution finally resulting 160 in high quality products. Another definition that underlines how the AIT was yet, but 161 improperly, proposed as personalized for the patient is "Named Patient Product" (NPP), 162 i.e. specifically prepared for a given patient according to the results of allergy testing.163 Nevertheless, NPPs were not a guarantee of clinical efficacy, because alongside products 164 with evidence of efficacy proven by placebo-controlled trials, others without such evidence 165 were equally admitted to the prescription. Moreover, extracts containing several allergens, 166 namely all the allergens giving positive results to allergy testing, were frequently used, 167 especially in the U.S.A, but they were found to be ineffective in a pivotal double-blind, 168 placebo-controlled trial in 199710 . Nowadays, registered products, whose effectiveness 169 has been plainly demonstrated by controlled trials on large patient populations, are 170 considered ideal for AIT11 . However, good quality NPPs with demonstration of efficacy by

	192 1)
	171 172	F o r P e e F o r placebo-controlled trials, are still available to treat respiratory allergy with expectancy of clinical success 12 . P e e
		r r
		R R
		e v i e w e v i e w
	181	comparing SLIT maintenance solutions of grass pollen and Dermatophagoides
	182	pteronyssinus from 4 leading European manufacturers to standardized concentrate
	183	extracts of 3 US manufacturers measure in bioequivalent allergen units (BAU), the relative
	184	potency was around 10 times higher for US standardized extracts (to be diluted for SLIT)
	151 185	As hinted above, AIT was introduced more than one hundred years ago, when the than for European SLIT maintenance dosage. The authors argued that, based on the
	152 186	knowledge on allergic disease was very limited 6 . Still, the different role of AIT compared efficacy demonstrations from controlled trials, SLIT efficacy is likely to depend on
	153 187	with the drug therapy available at the time was apparent, because drugs were aimed at additional factors apart from the extract dose 14 . Today, the pillars of AIT as a personalized
	154 188	treating the symptoms of allergic rhinitis, while AIT was aimed at treating the specific therapy are represented by molecular diagnostics for achieving the highest possible
	189	precision, by the use of treatable traits, and by "omics".
	190
	191

155 allergy of single patients, thus introducing a concept now acknowledged as personalized 156 medicine. The first studies addressed "hay fever" induced by grass pollen. It is obvious 157 that the quality of the products in that time, which consisted of extracts obtained from the 173 Modern AIT as a prototype of personalized medicine. 174 The traditional diagnosis by skin tests relay on extracts, whose composition in minor and 175 major allergens can play a crucial role in defining sensitizations, as in the AIT products for 176 inducing clinical efficacy. Another critical factor is the extract potency, concerning both skin 177 testing and AIT. As to the latter, Larenas-Linneman et al. found a substantial variability in 178 allergen extract potency as measured and reported worldwide 13,14 . In particular, in Europe 179 the potency of extracts for sublingual immunotherapy (SLIT) is based on comparison with 180 in-house references, making it difficult to translate the dose to US extracts 13 . Moreover,

Molecular diagnostics and other in vitro tests 193

  The diagnosis of allergy has been long based on skin prick test (SPT) with allergen 194 extracts and in vitro test measuring specific IgE antibodies to the suspected allergens, but 195 with both techniques the positive results indicated sensitization but not necessarily clinical

	196	allergy. A great advance in diagnostic precision was achieved by molecular diagnosis,
		F o r
		P e e
		r
		R
		e v i e w

  limit of in vivo tests. BAT was found to be very useful in the personalized diagnosis of ImmunoSorbent Test (ELISA) or ImmunoCAP[START_REF][END_REF] and that in patients with severe reactions 261 to stings, some of them with negative response to sIgE test and intradermal tests, BAT262was positive in 81% of patients compared with to a rate 57% with intradermal testing, 294 recent years 51 . For example, using treatable traits in a patient with COPD but symptoms 295 compatible also with asthma and positivity of tests for respiratory allergy due to 296 asymptomatic sensitization would prevent the incorrect prescription of AIT. The role of 297 treatable traits in the management of chronic respiratory diseases has been outlined by 298 some important studies. The first investigated the role of extra-pulmonary comorbidities as 299 treatable traits in patients with difficult-to-control asthma. The proposed comorbidities were 300 allergic and nonallergic rhinitis, chronic rhinosinusitis, vocal cord dysfunction, dysfunctional 301 breathing, gastroesophageal reflux, obesity, obstructive sleep apnoea, and anxiety/ 302 depression, of which the prevalence, impact and outcome of treatment were assessed, 303 also appraising the associations of single comorbidities and the potential of comorbidity 304 clusters on asthma control. The authors concluded that extra-pulmonary comorbidities are 305 important treatable traits be evaluated in all asthmatic patients and especially in those with 306 difficult asthma. Such comorbidities may have a significant influence on asthma control, 307 thus their presence deserves treatment regardless of original asthma status and asthma 308 control 52 .The number of treatable traits involved in acute exacerbation differed according 328 possible synergistic effect of biologics and AIT. Precision medicine of allergic diseases is 329 nowadays highlighted by the consistent data of efficacy of biologics targeting cytokines ((IL 330 5) or their receptors (IL 5 rec. or IL4/IL13 rec.). Nonetheless, we should consider that the 331 eligibility of allergic patients to biologics or AIT is only partially overlapping, being the first 332 limited to severe asthma or CRSwNP whereas AIT has indication for allergic rhinitis and 333 mild/moderate asthma while severe asthma is a contraindication. Based on the 334 characteristics of the two treatments, a synergistic effect in allergic asthma is conceivable, 335 with biological achieving control of severe asthma but not changing its natural history while 336 AIT, once ceased the contraindication, can induce the long-term immunological changes. 396 dust mites the development of a synthetic cultured medium free from proteins of animal 397 origin is needed 71 . As mentioned above, the use of omics is of marked importance in the 398 comprehensive characterization of allergen extracts, including transcriptome for de novo 430 cooperation patient/doctor in jointly choosing the best therapeutic option. AIT is the only 431 medical treatment that has the capacity to change the natural history of allergic diseases 81

	252 253 254 255 256 first studies concerned Hymenoptera venom allergy, demonstrating that BAT patients with allergic conditions including respiratory, food and Hymenoptera venom allergy who show multiple allergen reactivity, distinguishing the allergens as causative or not based on the response of the basophils 44 . As mentioned above, our review is focused on the effects of personalized diagnosis on the outcomes of AIT. As for CAP inhibition, the 257 accomplished with the recombinant allergens from Vespula spp Ves v 1, Ves v 2, Ves v 3 F o r P e e r R e v i e w 337 Studies to investigate whether such synergy is achievable are warranted. 260 F o r P e e r R e v i e w 309 to the respiratory disease, with a major role for C reacting protein in COPD, eosinophils 310 count and fractioned exhaled nitric oxide (FeNO) measurement in asthma, and bacterial 311 infections for bronchiectasis 53 . 312 An important observation obtained through the review of the literature by Pavord and 313 Agusti concerned the identification of eosinophilic airway inflammation, as assessed by the 314 blood eosinophil count, as a treatable trait of particular importance in patients with airway 315 disease (including asthma and COPD, being significantly associated with long-term 316 outcomes 54 . The recent European Unbiased Biomarkers for the Prediction of Respiratory 317 Disease Outcomes (U-BIOPRED) project was aimed at identifying and quantifying, based 318 on the hypothesis that treatable traits are more common in severe asthma and may 319 significantly differ according to asthma phenotypes, the treatable traits recognized in the 320 severe and mild/moderate adult asthma cohorts and across previously identified 321 phenotypes 55 . 322 All studies available thus far support a chief role of treatable traits in evaluating patients 323 with respiratory diseases, however the preferable method of application and the cost-324 effectiveness of such a multidimensional intervention is still uncertain, making it necessary 325 to conduct randomized controlled trials involving large populations of patients 56 . F o r P e e r R e v i e w 338 339 4.Omics 340 Following the definition of genomics in the 1960s as the complete nucleotide sequence of 341 an organism 58 , a large number of other "omics" were recently proposed, such as 342 proteomics (the complete proteins of a cell in any organism), epi-genomics (the 343 modification of nucleotides in an organism), metabolomics (the changes in gene activity in 344 response to metabolites 59 , breathomics (the multidimensional molecular analysis of 345 enxhaled breath 60 , but the list is very large and still expanding. In the field of allergy 346 diseases, asthma is the most investigated disease. In 2011, the detection of protein 347 changes by proteomics in different asthma stages was supported to improve the 348 understanding of the molecular mechanisms of the disease and to find novel mediators 349 and biomarkers 61 . Another omic of potential interest for personalized medicine is the 350 exposome, definable as a systematic approach to obtain large data sets on to 351 environmental exposures of an individual during the whole life 62 . 352 However, the available data on the state of omics technology in the management of 353 asthma and allergic diseases were recently analyzed by Donovan et al. After pointing out 354 that omics-based investigation are used increasingly to distinguish subtypes of 355 allergic diseases and asthma subtypes, forecast patient responsiveness to specific 356 therapies, detect biomarkers and mediators, and verify the disease control, the authors 357 concluded that omics testing in this field for are not yet a standard of care, and that key 358 factors need to be recognized before such technologies can be used successfully in 359 o r P e e r R F o r P e e r R F o r P e e r R common clinical practice F e v i e w e v i e w e v i e w
	258 and Ves v 5 achieved a clear improvement in the specificity of diagnosis in patients with 326 Concerning AIT, treatable traits of clinical interest may consist of the treatment outcome,
	259 anaphylactic reactions to wasp stings over IgE detection by Enzyme-Linked 327 beyond the disease to be treated, on comorbidities 57 . Another trait may concern the

63

. On the other hand, according to Fitzgerald et al., the promise 360 of new technology must leave room to an humanomics perspective, i.e. the 361 acknowledgement of patient's behavioral aspects

64

.

Table 1 Studies that have assessed the benefits of attaining with PAMD a more 566 appropriate prescription for AIT

  Personalized medicine in AIT has the potential to improve various outcomes which have 460 so far produced limited results. This is true for preventive ability, which until now has been 461 shown only for the development of asthma in patients with allergic rhinitis85 , the 462 identification of biomarkers predictive of treatment efficacy (still not known)57 and cost-463 effectiveness compared to drug therapy, clearly demonstrated on the basis of the 464 persistence over time of the control of symptoms after AIT withdrawal86 , but further 465 improvable by greater diagnostic precision. Further issues could concern the expansion of 466 e-health systems 7 and the implementation of telemedicine87 .467 This journey is leading to the most appropriate choice of the AIT treatment thanks to the 468 multifaceted variety of AIT products empowering the professionality of the clinician in 469 choosing, through a shared decision that considers the patient's needs, a proper AIT 470 product for each patient according to eligibility and acceptability.471 It is so conceivable to conclude that AIT is still a unique and unmatched model of 472 Personalized Medicine for allergy treatment, able to encompass the prediction of a 473 successful treatment and the potential prevention or progression of an allergic disease, 474 and possibly to face the challenge of providing such advances to at affordable costs. As 475 well, the characteristics of Personalized Medicine for allergy are likely to enable to face 602 12)Devillier P, Dreyfus JF, Demoly P, Calderón MA. A meta-analysis of sublingual allergen 603 immunotherapy and pharmacotherapy in pollen-induced seasonal allergic 604 rhinoconjunctivitis. BMC Med. 2014;12:71 605 13)Larenas-Linnemann D, Cox LS; Immunotherapy and allergy diagnostics Committee of 606 the American Academy of Allergy, Asthma and Immunology European allergen extract 607 units and potency: review of available information. Ann Allergy Asthma Immunol. 636 23)van Hage M, Schmid-Grendelmeier P, Skevaki C, Plebani M, Canonica W, Kleine-637 Tebbe J, Nystrand M, Jafari-Mamaghani M, Jakob T. Performance evaluation 638 of ImmunoCAP® ISAC 112: a multi-site study. Clin Chem Lab Med. 2017 Mar 670 of Allergen Molecule Based Diagnosis for Prescription of Immunotherapy in an Area 671 with Multiple Pollen Exposure. Int Arch Allergy Immunol. 2017;173(2):93-98. 672 34) Del-Río Camacho G, Montes Arjona AM, Fernández-Cantalejo Padial J, Rodríguez 704 44)Hemmings O, Kwok M, McKendry R, Santos AF. Basophil activation test: old and new 705 applications in allergy. Curr Allergy Asthma Rep. 2018 Nov 15;18(12):77. 706 45)Balzer L, Pennino D, Blank S, Seismann H, Darsow U, Schnedler M, McIntyre M, Ollert Woodruff PG, Agusti A, Roche N, Singh D, Martinez FJ. Current concepts 737 in targeting chronic obstructive pulmonary disease pharmacotherapy: making 738 progress towards personalised management. Lancet. 2015;385(9979):1789-1798. 739 52)Tay TR, Hew M. Comorbid, "treatable traits" in difficult asthma: current evidence and Abdel-Aziz MI, Neerincx AH, Vijverberg SJ, Kraneveld AD, Maitland-van der Zee AH. 803 71)Batard T, Hrabina A, Bi XZ, Chabre H, Lemoine P, Couret MN, Faccenda D, Villet 804 B, Harzic P, André F, Goh SY, André C, Chew FT, Moingeon P. Production and 805 proteomic characterization of pharmaceutical-grade Dermatophagoides pteronyssinus 806 and Dermatophagoides farinae extracts for allergy vaccines. Int Arch Allergy Krishna MT, Knibb RC, Huissoon AP. Is there a role for telemedicine in adult allergy 872 services?.Clin Exp Allergy. 2016;46(5):668-77. Gómez RM, Ansotegui I, Canonica GW. Will precision medicine be available for all 876 patients in the near future? Curr Opin Allergy Clin Immunol. 2019;19(1):75-80.

	F o r P e e r R e v i e w r R e v i e w r R e v i e w r R e v i e w r R e v i e w Pollens 1263 pts Grass pollen Not defined (review of published data) r R e v i e w Pollens 70 children Hymenoptera venoms 40 pts. r R e v i e w r R e v i e w r R e v i e w r R e v i e w r R e v i e w r R e v i e w r R e v i e w r R e v i e w r R e v i e w r R e v i e w r R e v i e w 459 476 challenges such as increasing prevalence, growing complexity and heterogeneity, impact 458 Moreno, 2014 (30) Based on history and SPT, 73% of patients would have been prescribed AIT with a mix of grass and olive pollens, while only in 56.8% of patients the double positivity was confirmed by molecular allergy diagnosis (MAD) with Ole e 1 and Phl p molecules. Popescu, 2014 (31) MAD biomarkers (including tolerogenic year AIT treatment Del-Rio Camacho, 2018 (34) MAD modified AIT prescription in 54.3% of cases, and increasing the indication of single-allergen therapy from 18% to 51% reversing the decision to prescribe AIT in 9.3% of cases Savi, 2016 (35) By MAD data VIT would have been prescribed to 7 pts for Polistes spp, to 6 for Vespula spp, and to 41 for both venoms. With resources. 567 568 The clinician should then evaluate:  The route of administration: presently SCIT and SLIT for aeroallergens, considering evidences and patient's needs/preference  The AIT product to be chosen as recommended by WAO 83 and EAACI 84 among the products supported by evidences.  SCIT: different schedules upon the products (i.e. depot or aqueous)  SLIT: preference for drops or tablet, both considering evidences and patient's needs/preference  Dosages and schedules: continuous or pre-seasonal; long or short term.  Most of the above can be influenced by costs and/or reimbursements. F o r P e e F o r P e e F o r P e e 548 C Virchow declares he has received fees from Allergopharma, ALK, LETI, Stallergenes 549 Greer. 550 U. Wahn declares he has received honoraria for consultancy as well as lecture fees from 551 Allergopharma, Stallergenes Greer, ALK, Roxall, Sanofi-Aventis, Novartis, Berlin-Chemie 552 M. Worm declares she as received of honoraria or consultation fees by ALK-Abelló 553 Arzneimittel GmbH, Mylan Germany GmbH, Leo Pharma GmbH, Sanofi-Aventis 554 Deutschland GmbH, Regeneron Pharmaceuticals, DBV Technologies S.A, Stallergenes 555 Greer, HAL Allergie, Allergopharma, Bencard Allergie, Aimmune Therapeutics UK Limited, 556 Actelion Pharmaceuticals Deutschland G, Novartis AG, Biotest AG, AbbVie Deutschland & 557 Co. KG, Lilly Deutschland.. 558 P. Zieglmayer has nothing to disclose. 559 C. Bos, E. Karagiannis and S. Scurati are employee of Stallergenes Greer 560 561 562 563 F o r P e e 565 Author, year (ref) Allergens tested Population Results Mitterman, 2010 (28) Bee and wasp venom. 43 patients (pts) Recombinant nonglycosylated allergen molecules (Api m 1, rApi m 2, rVes v 5) allowed to define the sensitizing venom in the 14 patients with positive test both bee and wasp venom extracts. Sastre, 2012 (29) Pollens 141 pts Agreement in AIT indication before and 62 (46%) patients; (kappa = 0.1057 ± 0.0413). P e e conventional diagnosis, with large reduction of the total costs for a 3-P e e respiratory allergy was found, resulting in a saving of financial P e e P e e P e e P e e P e e P e e P e e P e e P e e e e P after ISAC(®) results in F o r information for allergists Vidal, 2014 (32) Pollens, dust mites, moulds, animal epithelia 62 Spanish allergists SPTs are insufficient to establish the clinical relevance of each allergen. Serum specific IgE adds value to SPT but MAD is strongly recommended, particularly in pollen-allergic patients. Saltabayeva, 2017 (33) Pollens 95 pts (real-life study) A lower number of AIT treatments (n = 119) was needed according to MAD compared to 275 AIT treatments based on F o r for both venoms. A good concordance between the results of MAD and CAP-inhibition was found only when the value in kU/l of Ves v 5 were about twice those of Pol d 5, and vice versa. Martinez-Canavate Burgos, 2018 (36) Pollens 281 children MAD results changed the specialist's composition of the prescribed AIT in 52.87% of cases. Peveri, 2019 (37). Various aeroallergens 272 pts. In 50% of cases a change in the AIT prescription for F o r F o r F o r F o r F o r F o r 736 51)F o r o r F o r F r o 871 87)873 874 770 59)F 875 88)
	477 on patients, problem of access to care, therapeutic wandering, and successful dendtitic, regulatory T the data from CAP
	478 management of uncontrolled patients 88 .	cells, IgE, IgA and IgG inhibition, it would have
	479 480 481	Disclosure of Interests M. Al-Ahamad has nothing to disclose.	antibodies, and been a prescription to 15 apoptosis biomarkers patients for Polistes, to provide relevant 28 for Vespula, and to 11

Acknowledgments 564 This is an initiative supported by Stallergenes Greer with an unrestricted grant Acknowledgments 564 This is an initiative supported by Stallergenes Greer with an unrestricted grant


Figure 1: Movable cages on pasture for organic rabbit farming (© INRAE, T. Gidenne) DIGITAL BREEDING AND ASSISTED MANAGEMENT IN ORGANIC RABBIT FARMING: THE FIRST RESULTS

Keywords: Smartphone application, Performance referencing, Organic rabbit farming

A smartphone application GAELA combining decision support (breeding management) and performance recording (single, direct and secure entry on a public server) for rabbit farming using individual monitoring of breeders was created. Performance of reproduction were compiled for 6 farms over 2 years of production (2018)(2019)(2020). The livestock size averaged 30 does and varied largely among the farms. With 3.9 matings, 2.6 parturitions per female/year were obtained (66.8% fertility rate). Total number of kits born by parturition averaged 7.8 and total number of kits born alive averaged 7.1. The present study validated the utility of GAELA application and confirmed the modest performances in organic rabbit farming. The new version of GAELA application is available since the end of 2020. It provides new functions facilitating daily management and is improved to avoid entering incorrect information. New functions such as "Pregnancy Palpation", "Adoption" and "Fattening Management" are also available in this version. The future version of GAELA will further provide the calculation and analysis of performances of breeders or of the flock, and it will build a national reference system for "non-conventional" rabbit farming.

INTRODUCTION

The development of organic production is growing significantly since 2009. However, organic rabbit farming (ORF) remains a niche market in France (about 50 farms), and the consumer demand exceeds the supply [START_REF] Roinsard | Lapin Bio : développer une production cunicole durable en agriculture biologique[END_REF]. ORF French specifications contain several rules and recommendations, such as whole year grazing, natural breeding, and slaughter after 100 days of age, housing in movable cages (Figure 1) or in paddocks, and use of breeds adapted to grazing and adapted to outdoor management. In indoor conventional rabbit farming, the breeding performance is referenced in a national database for more than 30 years. This allows identifying the technical progress or difficulties of rabbit farms, and to guide development and research efforts. In independent or organic rabbit farming there is no performance benchmarking. This lack of technical references is an obstacle to the development of the "alternative" rabbit sector, since such information allows farmers and supervisors to establish, organise and size an installation project.

The first effort was made to develop a computer tool to create a referencing system for ORF [START_REF] Gidenne | A referencing system to analyse performances of French organic rabbit farms[END_REF]. A referencing system to collect and analyse performance of French organic rabbit farms was created using an Excel application World Rabbit Science Association 12th World Rabbit Congress -November 3-5 2021 -Nantes, France, Communication F-06, 4 pp 2 (RTS2CuniBio). Nonetheless, the RTS2CuniBio tool is suitable for processing a limited number of farms (<5000 lines), and still needs a transfer of entry from traditional livestock paper notebook. We therefore aimed to expand this database to a larger number of farms, thanks to a new database management tool, associated with a smartphone application "GAELA". GAELA can also assist the farmer for the daily management of the rabbit.

MATERIALS AND METHODS

Animals and experimental design

The first version of GAELA was achieved in 2018 (Figure 2). It allows the direct entry of information concerning the reproducers (male or female), mating, parturition and weaning. It includes a calendar of actions and alerts (Figure 3), as well as a data synchronization with a secure database. With a fast, direct and unique entry of livestock data "at field". The internet connection is not necessary for entry of data. The synchronization by using internet allows transmission of data into a server, which constitutes the whole data set. The user can show in a page the reproduction performances of each female or male previously recorded (Figure 4). There were more than 10 farmers participated in the tests since 2018. We selected 6 farms that recorded regularly their data (from 2018 to 2020) for a preliminary analysis (Table 1). The analysis was restricted to reproductive performances.

RESULTS AND DISCUSSION

Descriptive analysis of the farms selected

The female livestock size among the farms was quite variable (Table 1), ranging from 9 to more than 70. It is noteworthy that many weaning results were lacking, especially in Farm D, only 31.5% of parturitions has records of weaning results. There was also a high variability in the management of the reproductive females, with a female to male ratio averaging 6.2 but ranging from 2.6 to 7.0. The reproductive rhythm (natural mating) was extensive, with 3.9 mating per female per year, leading to an average of 2.6 parturitions (or kindlings). The parturition interval averaged 3.3 months. This World Rabbit Science Association 12th World Rabbit Congress -November 3-5 2021 -Nantes, France, Communication F-06, 4 pp 3 information should be taken with caution because the entry of data may not be complete in some, especially in farm D and E there is a lot of lacking data of parturition.

Performances of the reproductive livestock (Table 2)

The fertility rate (averaged 66.8%) was higher than that previous study (60.3%, [START_REF] Gidenne | A referencing system to analyse performances of French organic rabbit farms[END_REF], but varied according to the farm. The litter size (7.1 born alive) varied widely between farms (ranging from 3.9 to 9.0), it was lower that previous study (8.0 born alive, [START_REF] Gidenne | A referencing system to analyse performances of French organic rabbit farms[END_REF] based on results of 6 farms during 3 years (2015-2017), and was much lower than conventional rabbit farming (≈10, [START_REF] Coutelet | Performances moyenne des élevages cunicoles en France pour l'année 2014[END_REF]. Weaning performances (Table 3)

The age at weaning averaged 57.2 days (ranged from 42.5 to 74.2 days among the 6 farms). The survival rate of suckling kits at weaning averaged 69.3%, but with a large range between farms (46.2-89.0%). Except the results of farm E and F, the data were comparable to those observed by previous studies [START_REF] Lebas | Lapins Bio sur prairie : des résultats chiffrés[END_REF][START_REF] Gidenne | A referencing system to analyse performances of French organic rabbit farms[END_REF]. Accordingly, the number of weaned rabbits per parturition averaged 5.1. It should be noted that the Farm F (1.8 weaned rabbits per parturition) recorded abnormally a lot parturitions (n = 25) resulted in 0 kit weaned, which deserves to check if there were any health problems or typing errors.

The result confirmed the existence of a large progress margin in the management of the maternity unit [START_REF] Gidenne | A referencing system to analyse performances of French organic rabbit farms[END_REF], by improving the survival rate before weaning (housing management, prophylaxy, etc.), while reducing the parturition interval, and without impairing the survival rate after weaning.

Improvement of GAELA application

The preliminary analysis permitted identification of technical progress potential. The second version of GAELA application (available since the end of 2020) added new alerts to avoid entering incorrect information, for example entering a parturition when the mating took place less than 29 days ago or more than 33 days (5% of the cases in present study). Other new functions are available, such as "Pregnancy Palpation" (notification alerts in the diary, between 10 and 15 days after mating), "Adoption" (allows also to record adoptions or withdrawals of kits at birth) and "Fattening Management" (possibility to withdraw one or more weaned rabbits, with recording of a cause of exit: sale, death or renewal).

The future version of GAELA will further provide the calculation and analysis automatic of performances of breeders or of the flock, and it will build a national reference system for "nonconventional" rabbit farming (such organic one). GAELA is not restricted to organic rabbit farmers, but to any rabbit farm wishing to monitor the individual career of each reproducer of its flock. GAELA can also be easily adapted for other farming systems (pigs, etc.).

CONCLUSION

GAELA application is simple and ergonomic, and is available on request to INRAE. It is intended to replace the traditional livestock notebook, and will allow analysis of individual farm performance. The present study validated the utility of GAELA application and confirmed the modest performances in ORF. The new version of GAELA application is available since the end of 2020, it has new functions facilitating daily management and is improved to avoid entering incorrect information.

Figure 2 :

 2 Figure 2: Homepage.Figure 4: Performances of a doe.

Figure 4 :

 4 Figure 2: Homepage.Figure 4: Performances of a doe.

Figure 3 :

 3 Figure 3: Reminders to enter the upcoming event.

Table 2 :

 2 Reproduction performances

	Farm	A	B	C	D	E	F	Average
	Matings/female/year	3.7	5.7	3.4	3.9	3.9	3.0	3.9
	Matings/male/year	21.8	38.9	23.0	27.1	24.9	7.8	23.9
	Fertility rate, %	63.0	59.6	79.4	65.5	56.0	77.2	66.8
	Parturitions/female/year	2.3	3.4	2.7	2.5	2.2	2.4	2.6
	Parturition interval, days	89.2	88.9	53.2	98.4	112.9	146.8	98.2
	Total born/parturition	7.7	9.5	7.9	8.5	7.8	5.3	7.8
	Born alive/parturition	7.3	9.0	6.6	8.3	7.3	3.9	7.1

Table 3 :

 3 Weaning performances

	Farm	A	B	C	D	E	F	Average
	Age at weaning, days	42.5	65.0	45.1	74.2	61.1	55.3	57.2
	Weaned number/parturition	6.1	6.4	5.9	6.0	4.0	1.8	5.1
	Weaned number/female/year	14.4	21.8	9.6	15.2	9.0	4.3	16.8
	Survival rate at weaning %	81.9	70.2	89.0	72.6	56.7	46.2	69.3

Table 1 :

 1 Descriptive data of the livestock

	Farm	A	B	C	D	E	F	Average
	Reproductive females (mean number/year/farm)	29.1	70.8	32.0	21.0	9.1	15.5	29.6
	Reproductive males (mean number/year/farm)	4.9	10.4	3.8	3.0	1.4	6.1	4.9
	Female to male ratio	5.9	6.8	8.5	7.0	6.4	2.6	6.2
	Matings/year	108	405	87	81	35	47	127
	Parturitions/year	68	241	69	53	20	38	81
	Weanings/year	64	204	45	17	16	26

ACKNOWLEDGEMENTS

The authors thank INRAE funding program for organic agriculture (AgriBio4, Cunipat project), INRAE-Phase division, 3L consortium, French Region Occitanie and the EU (FEDER) for financial support for the development of the GAELA smartphone application.