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mmunogenicity and safety of a novel yeast Hansenula polymorpha-derivedecombinant Hepatitis B candidate vaccine in healthy adolescents and adultsged 10–45 years�
iguel W. Tregnaghia,∗, Ricardo Voelkerb, Eduardo Santos-Limac, Betzana Zambranod
Centro de Desarrollo de Proyectos Avanzados, Roma 1465, 5000 Córdoba, ArgentinaSistema de Emergencia Médico Móvil – Dirección de Medicina Preventiva, Montevideo, UruguaySanofi Pasteur, FranceSanofi Pasteur, Uruguay
r t i c l e i n f o
rticle history:eceived 24 June 2009eceived in revised form 1 February 2010ccepted 12 February 2010vailable online 26 February 2010
eywords:epatitis B vaccination
a b s t r a c t
The aim was to determine whether the immunogenicity of an investigational hepatitis B vaccine (spHB)is at least as high as that of a licensed control vaccine, Engerix B®, and to evaluate its safety beforeinclusion in new pediatric combination vaccines. Two randomized, controlled, blind-observer, Phase 3trials were performed: one in Argentina (344 participants aged 10–15 years, 10 �g HBsAg/dose) and onein Uruguay (344 participants aged 16–45 years, 20 �g HBsAg/dose). Both vaccines were given in a 0, 1, 6month schedule to all participants with a baseline anti-Hep B antibody titer <0.6 mIU/mL. Antibody titerswere measured pre-dose 1, 1 month after dose 2, pre-dose 3, and 1 month after dose 3. Statistical non-
epatitis B virusansenula polymorphaecombinant hepatits B surface antigenaccinationluminum hydroxide adjuvant
mmunogenicity
inferiority analyses were performed on seroprotection rates (SP) post-dose 3 (% with anti-Hep B titers≥10 mIU/mL; delta non-inferiority limit of −10%). In both studies, SP for the spHB vaccine was 100%and the spHB vaccine was non-inferior in terms of SP to the licensed control vaccine. GMTs post-dose3 were approximately 1.8- and 4.1-fold higher for spHB in the 10–15 year and 16–45 year age groups,respectively. Reactogenicity was low for each vaccine, after each dose. This highly immunogenic hepatitis
elect
B candidate vaccine was svaccines.
. Introduction
Despite prophylactic Hepatitis B (Hep B) vaccination being inlace for more than two decades, Hep B infection remains a majorublic health problem. According to World Health OrganizationWHO) data, approximately 350 million people live with chronicnfection, and approximately 75% of these live in areas of hyper-ndemicity where hepatitis B surface antigen (HBsAg) rates oferopositivity may reach 35%. Worldwide, more than 2 billion peo-le have been infected with the Hep B virus, and approximatelymillion deaths are attributable each year to Hep B-associated
irrhosis and hepatocellular carcinoma [1].
In 1992 the WHO recommended all countries to integrate Hep B
accination into their national vaccination calendar, and endorsedhe universal vaccination of infants against Hep B [2]. By Decem-er 2007, 171 countries had included Hep B vaccination in their
� Data presented orally at the XIII Latin American Congress of Infectology in Pedi-trics (SLIPE), Ecuador – 12–15 August 2009.∗ Corresponding author. Tel.: +54 351 425 18 00; fax: +54 351 425 63 73.
national program [3]. In highly endemic regions, Hep B infectionmost commonly occurs in the perinatal period (>20% of all infec-tions) or in early childhood (>60% of all infections), increasing therisk of chronic disease and its sequelae, with infants who becomeinfected having a 70–90% chance of becoming chronic Hep B carri-ers [4].
South America is considered as an area of intermediate HepB endemicity, although there is a wide variation in the incidenceof Hep B infection, generally with low prevalence rates occurringin southern parts of South America (e.g. about 0.6% in Chile) andhigh prevalence rates occurring in northern parts, with about 70%of the population in the Amazon Basin showing serological evi-dence of past or current Hep B infection [5]. In Latin America thehighest seroprevalence is found in the Dominican Republic (21.4%),followed by Brazil (7.9%), Venezuela (3.2%), Argentina (2.1%), Mex-ico (1.4%) and Chile (0.6%); in all of these countries, an increasein seroprevalence is found among those aged ≥16 years, sug-
gesting that sexual transmission is the major route of infection[6,7].
Currently, most Hep B vaccines are based on recombinant Hep-atitis B surface antigen (HBsAg) produced from recombinant yeast(e.g. Saccharomices cerevisiae, Pichia pastoris) or recombinant mam-
alian cells (e.g. Chinese Hamster Ovary [CHO]). Such Hep Baccines are highly immunogenic, inducing protective anti-Hep Bntibody titres (≥10 mIU/mL) in approximately 95% of vaccinees3,8].
Previous data have shown that CHO-derived recombinant Hepvaccines, which contain the S-protein component of the Hep B
urface protein, together with the pre-S1 and pre-S2 domains, areore immunogenic than S. cerevisiae-derived recombinant Hep B
accines made using the adw2 Hep B virus subtype [9–11]. Moreecently, a new, easily scalable yeast expression system based onansenula polymorpha, with high expression of recombinant HBsAgnd downstream purification, has been performed and the resultingntigen particles have been shown to be very similar to CHO-erived particles, with limited differences in peptide composition12,13].
Although HBsAg vaccines with local strain specificity haveeen produced, the quality of the protection of H. polymorpha-erived HBsAg/adw2 vaccines to infections with heterologousirus strains remains poorly documented, even though sometudies have shown differences in antigen-antibody recognitionatterns using H. polymorpha-derived HBsAg in three differentntigenic forms (adw2, ayw3 and adr) [14]. Several such vac-ines have been developed, evaluated in clinical trials and licensed15–18].
Within the scope of pediatric combination product develop-ent, sanofi pasteur (formerly Aventis Pasteur) has taken on the
evelopment of a novel yeast H. polymorpha-derived recombinantep B vaccine candidate using 2-phenoxy-ethanol/formaldehydes preservative; this candidate was initially described by Dimin-ky et al. [13] and is now being developed by sanofi pasteur as aomponent for inclusion in future multivalent pediatric combina-ion vaccines. In this paper, we report the results of two clinicalrials in which this new candidate vaccine (spHB) is compared ton existing S. cerevisiae-derived recombinant Hep B, thimerosal-ontaining reference vaccine that was commercially available inrgentina (Engerix B® Pediatrico) and Uruguay (Engerix B®) at the
ime of the studies.
. Materials and methods
.1. Study design and participants
Two randomized, controlled, blind-observer clinical studiesere performed using the same study design. The first study waserformed in adolescent participants (aged 10–15 years) in 30 careentres affiliated with the Hospital Infantil de Córdoba in Argentina,nd the second study was performed in participants aged 16–45ears at the Sistema de Emergencia Médico in Uruguay. For eachtudy, the protocol and the informed consent form were approvedy the appropriate local institutional review boards and indepen-ent ethics committees, as well as at the national level by thedministración Nacional de Medicamentos, Alimentos y Tecnologíaédica (ANMAT) in Argentina and by the Unidad Asesora de Inves-
igación de la División Control de Calidad (DICOCA) in Uruguay.oth studies were conducted according to local regulations, Goodlinical Practice, and applicable International Conference on Har-onisation guidelines, and conformed to the ethical principles of
he Declaration of Helsinki. Written informed consent was obtainedrom each participant and at least one parent (or legally acceptableepresentative) (for minors) or from each participant (for adults)
efore enrolment into the study.
Healthy participants were eligible if they complied with alltudy inclusion/exclusion criteria and with the study timetable. Allarticipants were randomly assigned (1:1) to receive a total of threeoses of spHB or the comparator Engerix B® Pediatrico (Argentina)
e 28 (2010) 3595–3601
or Engerix B® (Uruguay), administered at Day 0 and at 1 month and6 months post-dose 1 (0, 1, 6 month schedule).
Participants were excluded from enrollment in the study if theywere a breast-feeding mother or intended to become pregnant dur-ing the course of the study; if they had been treated with cadavericpituitary-derived growth hormone; if they had an evolving mod-erate or severe illness, a known allergy to any vaccine component,a blood disorder contraindicating intramuscular (IM) injection, animmunological deficiency, a history of Hep B infection or previousHep B vaccination, or if they had received an immunosuppressantdrug or corticosteroid in the 8 weeks prior to study inclusion (withthe exception of participants on a dose-tapering schedule of oralsteroids of less than 7 days, as long as not more than one coursehad been received in the 2 weeks prior to inclusion), any vaccinein the 15 days prior to inclusion or any live attenuated vaccine inthe 2 weeks prior to inclusion, any human blood-derived productin the 6 months prior to inclusion or any had the administrationof any such product planned during the course of the study, or hadany abnormality that the Investigator considered might interferewith the assessment of the trial objectives.
2.2. Vaccines and vaccine administration
The investigational vaccine (batch number PFAGL003-04 for thestudy in Argentina, and batch numbers PFAGL003-06-A (first andsecond doses) and PFAGL003-06-B (third dose) for the study inUruguay), which includes using 2-phenoxy-ethanol/formaldehydeas preservative, was produced and supplied by sanofi pasteur(formerly Aventis Pasteur), Argentina. For the study in Argentina(participants aged 10–15 years), each 0.5 mL dose contained 10 �gof purified recombinant HBsAg adsorbed onto 0.3 mg of aluminumhydroxide; for the study in Uruguay (participants aged 16–45years), each 1 mL dose contained 20 �g purified recombinantHBsAg adsorbed onto 0.6 mg of aluminum hydroxide. For both stud-ies, the investigational vaccine was supplied as a liquid in a vial.The physicochemical properties of the investigational vaccine aredescribed by Diminsky et al. [13], and full details including thesequence are provided in the Universal Protein Resource (UniProt)as number P03141 [19].
The comparator vaccine (Engerix B® Pediatrico [Argentina] orEngerix B® [Uruguay]) was produced by GlaxoSmithKline. For thestudy in Argentina (participants aged 10–15 years), each 0.5 mLdose contained 10 �g of purified recombinant HBsAg adsorbed onto0.25 mg of aluminum hydroxide; for the study in Uruguay (partic-ipants aged 16–45 years), each 1 mL dose contained 20 �g purifiedrecombinant HBsAg adsorbed onto 0.5 mg of aluminum hydroxide.For both studies, the comparator vaccine was supplied as a liquidin a pre-filled syringe.
For each group, the vaccine was administered by IM injectioninto the deltoid muscle.
2.3. Serologic testing
Blood samples for determination of HBsAg antibodies were col-lected from each participant just before the first dose, at 1 monthpost-dose 2, and just before and at 1 month post-dose 3. Sero-logic analyses for HBsAg antibodies were done in a blinded mannerwith respect to group but not with respect to visit at the sanofipasteur central clinical immunology laboratory (Swiftwater, Penn-sylvania, USA) using a commercially available radioimmunoassay
kit (AUSAB®, Abbott Laboratories, North Chicago, Illinois, USA). Thekit contained polystyrene beads coated with HBsAg (human origin),which were incubated with test samples, reference standards, orquality controls. Specific antibodies present in the samples boundto the solid phase HBsAg on the beads. The beads were washed
nd radiolabeled [125I]-HBsAg was added. The radiolabeled anti-en bound to antibodies on the beads and created a radioactiventigen-antibody-antigen complex. Radioactivity levels were theneasured in counts per minute (CPM) with a gamma counter. The
PM was proportional to the level of HBsAg antibodies present inhe sample and was converted to mIU/mL using the formula devel-ped by Hollinger [20].
.4. Reactogenicity and safety
All participants were included in the evaluation of reacto-enicity and safety. After each vaccination, each participant wasbserved for 30 min to monitor any immediate reactions. Byonvention, any event occurring within the 30 min following a vac-ination was considered to be related to the vaccination and wasermed a ‘reaction’. For the next 7 days, each participant recordedhe onset, duration and intensity of pre-defined (solicited) injec-ion site reactions (erythema/redness, edema, induration, pain)nd solicited systemic reactions (pyrexia/fever, asthenia, headache,alaise, myalgia, nausea, vomiting, diarrhea), and also of any non-
olicited injection site or systemic event, daily on diary cards (byonvention, solicited events were considered to be related to the
accination, and hence termed ‘reactions’). Non-solicited injectionite and systemic events that occurred between 8 days after eachaccination and the next visit were collected by the participant,nd were assessed by the Investigator for their relationship to theaccination. From 1 month to 6 months post-dose 3 only serious
Fig. 1. Summary of parti
e 28 (2010) 3595–3601 3597
adverse events (SAEs) were collected by the participant; these SAEdata were collected by the Investigator by telephone at 6 monthsafter the third vaccination.
Mild, moderate and severe pain was defined as ‘spontaneouspain and well tolerated’, ‘pain restricting movement’, and ‘pain pre-venting normal daily activity’, respectively. For erythema/rednessand edema, a diameter of 0.5 to <2 cm was graded as mild, a diam-eter of 2 to <5 cm was graded as moderate, and a diameter of≥5 cm was graded as severe. For induration and for all systemicevents except for pyrexia/fever, assessments of mild, moderate andsevere were defined as ‘easily tolerated’, ‘sufficiently discomfort-ing to interfere with normal activity’ and ‘prevent normal activity’,respectively. For fever, mild, moderate and severe were defined asaxillary temperature 37.1–38 ◦C (inclusive), 38.1–39 ◦C (inclusive),and ≥39.1 ◦C, respectively.
2.5. Statistical analyses
For the two studies, the statistical hypothesis for the pri-mary objective was that the anti-Hep B seroprotection rate(defined as the percentage of participants with an anti-Hep B anti-body titre ≥10 mIU/mL at 1 month post-dose 3) achieved in the
investigational vaccine group was non-inferior to that achievedin participants receiving the comparator vaccine. For the non-inferiority test, the 95% confidence interval [CI] of the differencebetween the investigational (test) minus comparator (reference)vaccines was calculated based on the Wilson score method with-
cipant disposition.
3598 M.W. Tregnaghi et al. / Vaccine 28 (2010) 3595–3601
Table 1Seroprotection rates (anti-Hep B ≥10 mIU/mL) 1 month post-dose 3 (full analysis set population).
Timepoint Age group (country) spHBa Engerix B®b Difference(spHB minus Engerix B®)
Pre-dose 1 10–15 years (Argentina) (N = 164) 0 (0;2.2) 0 (0;2.2) NP16–45 years (Uruguay) 0 0 NP
7 monthsc (post-dose 3) 10–15 years (Argentina) (N = 164) 100 (97.8;100) 99.4 (96.6;100) 0.6 (-1.7;3.4)16–45 years (Uruguay) (N = 151 [spHB]and N = 147 [Engerix B])
100 (97.6;100) 95.9 (91.3;98.5) 4.1 (0.77;8.62)
Data are seroprotection rate (% participants with anti-Hep B ≥10 mIU/mL) (95% CI). spHB = sanofi pasteur Hansenula polymorpha-derived recombinant Hep B candidate.N
t HBs
otd
H[mcttduf
P = not performed.a 10 �g purified recombinant HBsAg in Argentina and 20 �g purified recombinanb Engerix B® Pediatrico in Argentina and Engerix B® in Uruguay.c Data from per protocol analysis set.
ut continuity correction, and the hypothesis of non-inferiority waso be rejected if the lower bound of the two-sided 95% CI for theifference of the seroprotection rates was below −10%.
Descriptive statistics were calculated for continuous data (anti-Bs: log10 mean titre and standard deviation, geometric mean titre
GMT], 95% CI of the GMT [calculated using the normal approximateethod]) and for categorical data (seroprotection: number, per-
entage of participants, 95% CI of the percentage [calculated using
he exact binomial method]). The GMT was calculated assuminghat the log10 transformation of the titer data follows a normalistribution. First, the mean was calculated on the log10 titer datasing the usual calculation for normal distribution; anti-log trans-ormations were than applied to these results to provide GMTs. The
Fig. 2. Anti-Hep B geometric mean titres following spHB or Engerix B® Pediatrico
Ag in Uruguay.
geometric mean is defined as follows:
GM =(
n∏i=1
yi
)1/n
= 10
((1/n)∑n
i=1log10(yi)
)
where (y1, y2, . . ., yn) are the observed titers or individual ratios for
each subject.
A sample size of 344 participants in each study (to obtain 258evaluable participants assuming about 25% non-evaluability) wascalculated using the Farrington and Manning formula to obtain anoverall power of 90% for the non-inferiority testing [21].
/Engerix B® in Argentina (10–15 years) (a) and Uruguay (16–45 years) (b).
M.W. Tregnaghi et al. / Vaccine 28 (2010) 3595–3601 3599
Table 2Solicited injection site and systemic reactions within 7 days after each dose in Argentinean participants aged 10–15 years (safety analysis set population).
spHB = sanofi pasteur Hansenula polymorpha-derived recombinant Hep B candidate. Data are number and percentage of participants with at least one reaction.
3
3
eiilPcrp
aiia
oUamricad
a 10 �g purified recombinant HBsAg.
. Results
.1. Participant characteristics
In each country a total of 344 participants (172 participants inach vaccine group) were enrolled as planned (aged 10–15 yearsn Argentina and 16–45 years in Uruguay). The number of partic-pants in the Full Analysis Set (FAS; participants who received ateast one injection, analyzed by randomized group), and the Perrotocol Analysis Set (PPAS; participants who had no major proto-ol deviation) and the Safety Analysis Set (SAS; participants whoeceived at least one injection, analyzed by vaccine received) areresented in Fig. 1.
The PPAS, which included only participants who were seroneg-tive to HBsAg prior to the first dose, was used for the primarymmunogenicity analysis of non-inferiority and for the descriptivemmunogenicity data at 30 days post-dose 3. The SAS was used forll safety analyses.
In each study, the participants receiving each vaccine weref similar age (approximately 13 and 30 years in Argentina andruguay, respectively) and body mass index (approximately 19nd 25 kg/m2 in Argentina and Uruguay, respectively). In Argentina,ore females that males were included in each group (female:male
atio 1.2–1.3), whereas in Uruguay more males than females werencluded (female:male ratio 0.41–0.55); this difference was notonsidered to be clinically relevant and was not considered to haveny impact on the interpretation of the immunogenicity or safetyata.
3.2. Immunogenicity
The proportion of seroprotected participants (anti-Hep B≥10 mIU/mL) at each timepoint and the non-inferiority analysis 1month post-dose 3 for each study (PPAS) is presented in Table 1.
For each age group, the anti-Hep B seroprotection rate was simi-lar for the investigational vaccine and the comparator. For both agegroups, statistical analysis confirmed non-inferiority of the inves-tigational vaccine, with the lower bound of the two-sided 95% CIfor the difference being greater than the clinical limit set for non-inferiority (-10%) in each age group.
The GMT data at each timepoint are shown in Fig. 2a (Argentina,10–15 years of age) and Fig. 2b (Uruguay, 16–45 years of age). Inboth age groups, in terms of descriptive assessment of the GMT(95% CI) data, the response to the investigational spHB at 1 monthpost-dose 3 (PPAS) was 1.8-fold higher than the response to EngerixB® Pediatrico (Argentina; 10–15 years) (8799 [7413;10444] versus4854 [3844;6129] mIU/mL) and 4.1-fold higher than the responseto Engerix B® (Uruguay; 16–45 years) (5782 [4419;7565] versus1409 [943;2104] mIU/mL, respectively), with no overlap of 95% CIs.Similarly, at 1 month post-dose 1 and prior to dose 3, the GMTs forthe spHB groups were markedly higher than for the comparator,with no overlap of 95% CIs.
3.3. Safety and reactogenicity
The incidence of immediate reactions was low in both vaccinegroups for each age range, and was slightly higher for the spHB vac-
3600 M.W. Tregnaghi et al. / Vaccine 28 (2010) 3595–3601
Table 3Solicited injection site and systemic reactions within 7 days after each dose in Uruguayan participants aged 16–45 years (safety analysis set population).
pHB = sanofi pasteur Hansenula polymorpha-derived recombinant Hep B candidatea 20 �g purified recombinant HBsAg.
ine than for the comparator (1.7% versus 0.0% of participants aged0–15 years and 7.6% versus 3.5% of participants aged 16–45 yearsith at least one immediate reaction). Most immediate reactionsere considered to be mild or moderate in severity and included
pisodes of injection site pain, injection site paresthesia, vomiting,aso-vagal syncope, dental traumatism, and dizziness.
Solicited injection site and systemic reactions that occurredfter each vaccination are summarized in Table 2 (Argentinean par-icipants aged 10–15 years) and Table 3 (Uruguayan participantsged 16–45 years).
In both age groups, and for both the investigational and com-arator products, the incidence of solicited adverse reactions wasomparable and generally decreased with successive vaccinations.ew adverse reactions were rated as severe.
In all groups the most commonly reported solicited injectionite reaction was pain, the incidence of which decreased in eachge and vaccine group with successive vaccinations. Slightly moreain was reported for the investigational vaccine group than forhe comparator. This incidence of the other solicited injection siteeactions was low, and was similar in each age and vaccine group.
In all groups except following spHB in participants aged 16–45ears for whom pyrexia/fever was the most common solicited sys-emic reaction, the most commonly reported solicited systemiceaction was headache, the incidence being highest after the post-ose 1 and similar in each age and vaccine group.
Very few injection site or systemic reactions were reported morehan 7 days after each vaccination in either age or vaccine group,nd for each age group there was descriptively no difference inhese reactions between the investigational and comparator vac-ines.
0 0.0 1 0.6 0 0.0 1 0.6
are percentage of participants with at least one reaction.
In each age group the incidence of serious adverse events (SAEs)reported throughout the study (including the 6-month follow-up period) was low and was similar for each vaccine group. InArgentina 2.3% (investigational vaccine) and 1.7% (Engerix B® Pedi-atrico) of participants aged 10–15 years reported an SAE, and inUruguay 5.2% of participants aged 16–45 years in each vaccinegroup reported an SAE (the majority being due to an outbreak of QFever). No SAE was considered to be related to the investigationalor comparator vaccine.
4. Discussion
These two studies evaluated the immunogenicity and safety ofan investigational recombinant Hep B H. polymorpha-derived vac-cine (spHB) administered at Day 0 and at 1 month and 6 monthspost-dose 1 (0, 1, 6 month schedule) in comparison to Engerix B®
Pediatrico (participants aged 10–15 years in Argentina) or EngerixB® (participants aged 16–45 years in Uruguay). Participants wererandomized at Day 0 to receive either the investigational or com-parator vaccine.
The immunogenicity of each vaccine was strong with >95%of participants in each age and vaccine group being consideredseroprotected (using the correlate marker for seroprotection ofanti-HBs ≥10 mIU/mL) at 1 month post-dose 3. Following the
investigational vaccine the seroprotection rate was statisticallynon-inferior to that following the commercially available compara-tor. These results suggest that the majority of participants in bothvaccine and age groups would be expected to be protected againstHep B infection.
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In terms of GMT, the immune response to the comparator vac-ine in participants aged 10–15 years (4854 mIU/mL) and 16–45ears (1409 mIU/mL) was similar to that reported in previous stud-es using 10 �g Engerix B® in adolescents [22,23,24] and 20 �gngerix B® in adults [25,26,27] at 1 month after a standard 0, 1,month dosing schedule. In comparison, in our study, the GMT
esponse to the investigational vaccine was approximately 1.8- and.1-fold higher than the response to the comparator in participantsged 10–15 years and 16–45 years, respectively. Higher peak anti-ep B antibody titres have been shown to be associated with longerntibody persistence [28] and it has been shown that high titres fol-owing primary series vaccination correlate with the retention oferoprotection up to 15 years later [29].
The investigational vaccine was well tolerated in both ageroups, and the overall safety profile was comparable to thatbserved for the comparator vaccine and to that described in theiterature for yeast-derived Hep B vaccines [30]. There were fewmmediate adverse reactions in any group and most adverse events
ere of mild or moderate intensity. No serious adverse eventsccurred in either age or vaccine group.
The investigational candidate spHB vaccine, based on its highmmunogenicity and good safety, qualified for further clinicalevelopment as a component in future pediatric combination vac-ines. Combination vaccines that contain HBsAg have facilitated thentegration of Hep B vaccination into existing infant immunizationrograms and have contributed to improved compliance [8]. It islanned that such integration of the candidate Hep B vaccine willelp to increase vaccine coverage in the pediatric population.
cknowledgements
The authors wish to acknowledge Siham B’Chir for the statisti-al analysis and Andrew Lane and Emmanuel Vidor for assistancen the preparation of this manuscript. All are employees of sanofiasteur, France. In addition, the authors acknowledge the follow-
ng for assistance in the trial conduct: Dr J. Usher, Dr M. Grana, Dr. Calvari, Dr M. Tregnaghi (jr.), Dr P Vanadia, Dr A.M. Baudagna,
ioch. C. Lludgar, Dr R Ceni, Dr A Dini, Dr N Giordano, Dr J Saquieres,r P Baglivi, Dr E Prosper, Dr E Saldias.
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