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Vaccine 32 (2014) 1072– 1078

Contents lists available at ScienceDirect

Vaccine

j our nal homep ag e: www.elsev ier .com/ locate /vacc ine

ynthetic HIV-1 matrix protein p17-based AT20-KLH therapeuticmmunization in HIV-1-infected patients receiving antiretroviralreatment: A phase I safety and immunogenicity study�

aria Luisa Iariaa,1, Simona Fiorentinia,1, Emanuele Focàb, Sonia Zicari a, Cinzia Giagulli a,rancesca Caccuria, Daniela Francisci c, Giovanni Di Perrid, Francesco Castelli b,ranco Baldelli c, Arnaldo Carusoa,∗

Department of Molecular and Translational Medicine, University of Brescia, Brescia, ItalyDepartment of Clinical and Experimental Sciences, University of Brescia, Brescia, ItalySection of Infectious Diseases, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, 06132 Perugia, ItalyDepartment of Medical Sciences, University of Turin, Turin, Italy

r t i c l e i n f o

rticle history:eceived 9 July 2013eceived in revised form6 December 2013ccepted 19 December 2013vailable online 20 January 2014

eywords:herapeutic vaccineIV-1 matrix protein p17eptide-based-immunotherapy

a b s t r a c t

Background: Therapeutic vaccination is a promising novel approach to treat HIV-1 infected people byboosting or redirecting immune system to neutralize critical HIV-1 antigens whose biological effects arerelevant in the context of viral pathogenesis. With the aim to induce neutralizing antibodies to the matrixprotein p17 we have developed a peptide-based immunogen (AT20-KLH) and evaluated its safety andimmunogenicity.Methodology: Twenty four asymptomatic HAART-treated HIV-1+ patients were enrolled in a phase I clin-ical study and were randomized to three groups: 2 groups were treated with five IM injection (Arm A:25 �g/inoculation; Arm B: 100 �g/inoculation) at day (D) D0, D28, D56, D84 and D112; the control group(Arm C) were not injected. Safety was assessed by monitoring local and systemic adverse events (AEs),recorded till D168. Evaluation of immunogenicity was by titering antibodies at D0, D35, D56, D63, D84,D91, D112, D140 and D168 using ELISA.Results: In all, 105 local and systemic AEs were reported across the three groups. Most were mild andresolved without sequelae. Also the few unsolicited events, deemed unrelated to the study vaccines,caused no problems. No significant changes in the routine laboratory parameters, CD4 T-cell count orHIV-1 viremia were found. At the time of enrollment 23 out of 24 patients had no anti-AT20 antibodies,whereas 11 exhibited anti-p17 antibodies. Irrespective of the presence of preimmunization antibodies,

all subjects developed high titers of anti-AT20 antibodies (GM 9775) in response to both AT20-KLH doses.These antibodies were also capable of recognizing AT20 within the p17 framework.Conclusions: The AT20 peptide-based approach has allowed to redirect HAART-treated patients’ humoralresponses toward a previously untargeted hotspot of functional activity. Overall, the tested AT20-KLHdoses were safe and well tolerated, supporting further exploration of AT20-KLH as an HIV-1 therapeuticvaccine candidate.

. Introduction

Highly active antiretroviral therapy (HAART), suppressing theIV-1 replication and ameliorating the immunologic response, has

� Clinical trial identifier MED-AT20-001.∗ Corresponding author at: Department of Molecular and Translational Medicine,

ection of Microbiology, Piazzale Spedali Civili, 1, 25123 Brescia, Italy.el.: +39 030 394491; fax: +39 030395258.

E-mail address: caruso@med.unibs.it (A. Caruso).1 These authors contributed equally to the work.

264-410X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.vaccine.2013.12.051

© 2013 Elsevier Ltd. All rights reserved.

led to reduced morbidity and mortality [1–3]. However, a discord-ant response on HAART with virologic suppression but impairedimmunologic reconstitution may be observed; in these individualsclinical disease progression is greater than in those with a com-plete response [4] and is an independent risk factor for mortality[5]. Moreover, multiclass drug resistance is a clinically importantissue in patients who cannot benefit from two fully active drugs [6].Therefore, lifelong adherence, side and long-term effects of HAART,

immune reconstitution and multiclass drug-resistant issues, alongwith cost, point for the need of novel therapeutic options.

The aim of therapeutic vaccination in HIV-1-infected individ-uals is to induce or better direct immune responses that are

cine 32 (2014) 1072– 1078 1073

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ffective, either more potent than those induced by the naturalnfection.

HIV-1 protein triggers chronic immunological deregulation [7]nd, among them, several investigators have hypothesized anmportant role of the matrix protein p17 that exhibits differentmmunomodulatory properties which may be relevant in the con-ext of viral pathogenesis [8–11]. In addition, p17 is the target ofeutralizing Abs which correlate with slower progression to AIDS12–15]. The finding that p17 is exported from infected cells [16], isetected in serum of HIV-1-infected patients [11] and accumulates

n lymph nodes of even successfully HAART-treated patients [17]akes the mechanisms observed in vitro also possible in vivo.P17 activities are all mediated by its binding to specific cellular

eceptors [18–20] through a functional epitope, shaped as a par-ially unfolded �-helix located at the p17 NH2-terminal region [8].

ice immunized with a 20 amino acids (aa)-long synthetic pep-ide (AT20) representative of the p17 functional region, coupled tohe carrier protein Keyhole Limpet Hemocyanin (KLH), developed17-neutralizing Abs capable of blocking p17/p17 receptor(s) inter-ction and, consequently, all biological activities of the viral protein8,21]. Further, being that AT20 resides within a highly conservedonformational epitope, immunization induces Abs capable of neu-ralizing the p17 proteins derived from divergent strains displayingritical mutations within AT20 [22]. On the basis of these preclin-cal data, AT20-KLH was selected as the active agent to perform aherapeutic phase I clinical trial in HIV-1-infected patients.

. Materials and methods

.1. Study product

A contractor for the good manufacturing practice (GMP), iden-ified in Italy (Areta International S.r.l., Gerenzano), produced andeleased the AT20-KLH vaccine according to current regulations.he released product was obtained conjugating the GMP-gradeT20 peptide (OPC, Germany) with the GMP-grade KLH (Byosin, CA,SA) as carrier protein. The final product containing the drug sub-

tance and the excipients was packaged as ready-to-use vials (25 �gnd 100 �g) and stored at 5 ± 3 ◦C. The immunogenic product wasmulsified extemporaneously with the adjuvant (GMP-grade Mon-anide ISA-51, Seppic, France) in a 1:1 ratio and administeredntramuscularly deeply in the usual regions.

.2. Study design

The therapeutic phase I study (MED-AT20-001, EudraCT n.008-001465-29) was a multi-centre, randomized, dose escalationlinical trial whose primary endpoint was safety assessment andhe secondary endpoint was immunogenicity.

Study was conducted in HIV-1-infected, clinically asymptomaticndividuals, in HAART therapy for at least 1 year prior to vacci-ation, with undetectable (<50 copies/ml) viral load for at least

months prior to immunization, with CD4 nadir ≥200 cell/mm3

ince infection, and with CD4+ T-cell count ≥350 cells/mm3 fort least 3 months prior to inclusion at entry. Pregnancy or preg-ancy risk were exclusion criteria. All patients provided informedonsent. The study was conducted in accordance with the ICHuidelines on Good Clinical Practice and the Helsinki/Edinburgheclaration. Patients enrolled (n = 24) were randomized to one of

he two treatment arms (n = 18) and to the control arm (n = 6, noreatment) (Fig. 1). The patients were allocated to a treatment or

ontrol arm in a 3:1 ratio into 2 subsequent cohorts of 12 patientsach, (in each cohort 9 patients were allocated to treatment and 3o control), corresponding to 2 vaccine doses (25 �g/inoculation;00 �g/inoculation), by a centralized randomization starting from

Fig. 1. Participant flow diagram. Enrolled patients were randomized to two AT20-KLH peptide dosage groups to receive the vaccine (Arm A, 25 �g dose and Arm B,100 �g dose) or no treatment (Arm C, control).

the lower vaccine dose. Allocation of the patients in the nextcohort did not occur before all participants in the previous cohorthave received at least 3 vaccinations and the safety results havebeen reviewed by an Independent Drug Safety Monitoring Board(IDSMB). Five intramuscular administrations were provided topatients randomized in the treated groups: days D0, D28, D56, D84and D112 (±3 days).

This study was conducted in parallel in four clinical cen-tres in Italy (Santa Maria Misericordia Hospital, Perugia; SpedaliCivili, Brescia; Amedeo di Savoia Hospital, Turin; IRCCS FondazioneOspedale Maggiore/Policlinico/Mangiagalli/Regina Elena, Milan)under the sponsorship of Medestea Research & Production SpA,Turin.

2.3. Laboratory procedures

2.3.1. Safety and laboratory assessmentsEvaluation of safety included adverse events (AEs) monitor-

ing, physical examination and clinical and laboratory assessment(hematology, serum chemistry and urinalysis). AEs have been cat-egorized according to the ICH guidelines as grade 1 (Mild), grade 2(Moderate), grade 3 (Severe), and grade 4 (Serious). As additionalparameters to assess immunological safety of the vaccine, CD4+ T-cell count and plasmatic HIV-1 RNA load were also evaluated bystandard procedures on visit (V) 1 (screening) and on D0, D28, D56,D84, D112, D140 and D168 (±3 days).

2.3.2. Blood sample processingBlood sampling for specific antibody (Ab) detection was per-

formed on V1 and on D0, D7, D28, D35, D56, D63, D84, D91, D112,D119, D140 and D168 (±3 days). Sera obtained were aliquoted andfrozen at −80 ◦C until use.

2.3.3. Measurement of serum antibodiesAnti-AT20 and anti-p17 Ab titers were evaluated by ELISA as

described [21,22]. Ab titers were determined as the reciprocal ofthe sample dilution where the absorbance value of the tested sam-ple arises at least 2-fold (cut-off value) from the absorbance value

detected using a pool of HIV-1-negative sera.

Abs avidity index (AI) was evaluated as described [23] usingammonium thiocyanate (NH4SCN) as dissociating agent. AI wasthe NH4SCN molar concentration capable to halve the Ab/antigen

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inding as compared to NH4SCN-untreated wells (M ≤ 1, low avid-ty; 1 < M < 2 medium avidity; M ≥ 2 high avidity).

.4. Statistical analysis

Even if this was not a remit of MED-AT20-001 study, enroll-ent of 24 subjects provides a sufficient number of observed AEs

o describe the safety endpoint by number, percentage and con-dence interval (95%CI). Difference in Ab levels was evaluated byann–Whitney test. To determine whether Ab AI increases during

reatments, we used Wilcoxon test.

. Results

.1. Demographics

The 24 enrolled individuals were subdivided into three arms:rm A (n = 9) patients treated with the AT20-KLH lower dose

25 �g/inoculation); Arm B (n = 9), patients receiving the AT20-LH higher dose (100 �g/inoculation); and Arm C (n = 6), controlntreated patients. The mean age ± standard deviation (SD) oftudy participants was 43.56 ± 9.42 in Arm A, 40.00 ± 9.38 in Arm Bnd 39.83 ± 10.59 in Arm C and 95.8% were Caucasian (Table 1S). Athe time of enrolment, CD4 count (mean ± SD) was 760 ± 138.5 inrm A, 802 ± 147.5 in Arm B and 698 ± 188.2 in Arm C and CD4/CD8atio (mean ± SD) was 1.28 ± 0.6 in Arm A, 1.33 ± 1.1 in Arm B and.99 ± 0.4 in Arm C. No difference in baseline demographics wasresent. All 24 patients have completed the study. The according-o-protocol analysis for the presence of anti-p17 and anti-AT20 Absncluded a screening phase (D-15), a treatment phase of 112 days,onsisting in five vaccination (D0, D28, D56, D84, D112), five visitsfter one week to each vaccination (V4, V5, V7, V9, V11), and aost-vaccination follow-up period (V12) ending 56 days after lastaccination (V13) (Table 1).

.2. Safety

Safety was assessed by monitoring local and systemic AEs andere reported according to the MedDRA Dictionary. No significant

lteration of clinical and laboratory assessments were observed.he number of patients with at least one local and/or non-local AEelated or not to vaccination is reported in Table 2. A total num-er of 105 AEs were observed. Notably, most of the registeredEs were mild [Arm A: 70/75, 94% (95%CI: 87.4–98.6%); Arm B:3/25, 92% (95%CI: 81.7–102%); Arm C: 2/5, 40% (95%CI: 0–83%)]nd recovered (71/75, 95%, 95%CI: 89–99% in Arm A and 23/25, 92%,5%CI: 81.7–102% in Arm B) in all groups of participants (Table 2S).oderate AEs were rare, being only 6.7% in Arm A (5/75, 95%CI:

–11.2%), 8% in Arm B (2/25, 95%CI: 0–18.5%) and 40% in Arm C2/5, 95%CI: 0–83%). No serious AEs occurred in any patients receiv-ng the AT20-KLH vaccine, whereas a severe AE (Phosphokinasencrease) was observed in one Arm C patient. The most frequentEs category was “General disorders and administration site condi-

ions” (43/105, 41%, 95%CI: 31–49%) with more widespread eventsf pyrexia (13/43, 30%, 95%CI: 16.5–43.5%), swelling (10/43, 23%,5%CI: 10.5–35.5%) and pain (9/43, 21%, 95%CI: 9.1–32.9%). Most ofhese AEs were showed by patients in Arm A (39/43, 90%, 95%CI:1–98%). Moreover, in Arm A the majority of AEs (53/75, 71%, 95%CI:0–79%) was related with the treatment while, in Arm B, just 20%f them (5/25, 95%CI: 4.4–35.6%) were treatment-related AEs.

As a further safety parameter, assessment of CD4+ T-cell countnd viral load was performed. Data collected during the treatment

hase and at D168 are consistent with a remarkable immunologicnd virologic safety of the vaccination. No variation of CD4+ T-cellount was observed throughout the whole study duration (Fig. 2A).

comparative analysis between the individual level of CD4 T-cells, Tab

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M.L. Iaria et al. / Vaccine 32 (2014) 1072– 1078 1075

Table 2Incidence of AEs.

Arm A (n = 9) Arm B (n = 9) Arm C (n = 6)

Any system 9(100.0%) 6(66.7%) 3(50%)

Blood and lymphatic system disorders 1(11.1%) 2(22.2%) 0(0.0%)Lymphadenopathy 1(11.1%) 2(22.2%) 0(0.0%)

Ear and labirinth disorders 2(22.2%) 1(11.1%) 0(0.0%)Vertigo 2(22.2%) 1(11.1%) 0(0.0%)

Gastrointestinal disorders 2(22.2%) 4(44.4%) 0(0.0%)Diarrhea 2(22.2%) 2(22.2%) 0(0.0%)Costipation 0(0.0%) 1(11.1%) 0(0.0%)Gastric ulcer 0(0.0%) 1(11.1%) 0(0.0%)Tongue disorders 1(11.1%) 0(0.0%) 0(0.0%)Toothache 0(0.0%) 1(11.1%) 0(0.0%)

General disorders and administration site conditions 8(88.9%) 2(22.2%) 1(16.7%)Pain 5(55.6%) 1(11.1%) 0(0.0%)Pyrexia 4(44.4%) 1(11.1%) 1(16.7%)Swelling 4(44.4%) 0(0.0%) 0(0.0%)Malaise 2(22.2%) 0(0.0%) 0(0.0%)Asthenia 0(0.0%) 1(11.1%) 0(0.0%)Influenza-like illness 1(11.1%) 0(0.0%) 0(0.0%)Injection site pain 1(11.1%) 0(0.0%) 0(0.0%)Nodule 1(11.1%) 0(0.0%) 0(0.0%)Tenderness 1(11.1%) 0(0.0%) 0(0.0%)

Infections and infestations 6(66.7%) 4(44.4%) 1(16.7%)Influenza 3(33.3%) 1(11.1%) 1(16.7%)Nasopharyngitis 1(11.1%) 2(22.2%) 0(0.0%)Pharingitis 1(11.1%) 0(0.0%) 1(16.7%)Fungal infection 0(0.0%) 1(11.1%) 0(0.0%)Gastroenteritis 1(11.1%) 0(0.0%) 0(0.0%)Helicobacter infection 0(0.0%) 1(11.1%) 0(0.0%)Oral Herpes 1(11.1%) 0(0.0%) 0(0.0%)

Investigations 0(0.0%) 1(11.1%) 1(16.7%)Blood creatinine phosphokinase increase 0(0.0%) 0(0.0%) 1(16.7%)HIV test positive 0(0.0%) 1(11.1%) 0(0.0%)

Musculoskeletal and connective tissue disorders 1(11.1%) 2(22.2%) 0(0.0%)Arthralgia 1(11.1%) 1(11.1%) 0(0.0%)Back pain 0(0.0%) 1(11.1%) 0(0.0%)

Nervous system disorders 1(11.1%) 2(22.2%) 1(16.7%)Headache 1(11.1%) 2(22.2%) 0(0.0%)Sciatica 0(0.0%) 0(0.0%) 1(16.7%)

Psychiatric disorders 0(0.0%) 1(11.1%) 0(0.0%)Insomnia 0(0.0%) 1(11.1%) 0(0.0%)

Respiratory, thoracic and mediastinal disorders 2(22.2%) 0(0.0%) 0(0.0%)Cough 2(22.2%) 0(0.0%) 0(0.0%)Oropharingeal pain 1(11.1%) 0(0.0%) 0(0.0%)

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easured at D-15 and D168, confirmed that they did not signifi-antly differ in 100% of subjects enrolled (Fig. 2B). Preservation ofirculating CD4+ T cells was mirrored by the absence of significantlasma viremia rebounds.

Based on data obtained, after a careful evaluation of the AEs,DSMB qualified the AT20-KLH vaccine candidate as safe and wellolerated both locally and systemically. A report has been depositedo the Italian Regulatory Agencies.

.3. Immunogenicity

Serum samples were analyzed at the Brescia University coreaboratory. In agreement with previous observation, showing thaturing the natural course of HIV-1 infection the occurrence of Ab

esponse to AT20 is rare and – eventually – at a very low titer [7,24],ost patients (23/24, 95.9%, 95%CI: 94.8–95%) had not detectable

nti-AT20 Abs at the time of enrolment. The only patient posi-ive for AT20 Abs (Ab titer: 100) was randomly enrolled in Arm A.

0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%)

Eleven out of 24 patients (45.8%, 95%CI: 25.4–64.6%) (5 randomizedin Arm A, 3 in Arm B and 3 in Arm C) exhibited p17 Abs with titersranging from 100 to 12,800 (Ab GMT = 852.4). Subjects immunizedwith AT20-KLH developed high titers of anti-AT20 Abs comparedto untreated patients (p < 0.001). As a consequence of the risinglevel of anti-AT20 Abs, difference in sera reactivity were no moreobserved when patient’s samples were tested in ELISA plates coatedwith the AT20 peptide or with the entire recombinant native p17protein (Fig. 3A). These results demonstrate that the Abs generatedto the synthetic AT20 peptide are capable to recognize the epitopewithin the viral protein framework. Moreover, sera derived frompatients immunized with AT20-KLH were capable to displace thebinding between p17 and p17 receptor(s), whereas sera derivedfrom patients belonging to the control Arm did not (Figure 1S).

The clinical trial was designed as a dose escalation trial. There-fore, we evaluated if one of the two doses tested have elicited abetter immune response. As shown in Fig. 4A, four weeks afterthe first dose of vaccine (D28), the rate of seroconversion to AT20

1076 M.L. Iaria et al. / Vaccine 32

Fig. 2. Profile of CD4+ T cell count along clinical trial. (A) Diagrams represent mean(±SD) absolute CD4+ T cell count of patients enrolled in Arm A, Arm B and Arm C.Data were collected at the indicated visits. Arrows indicate time for each vaccination.(sb

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B) Comparison between CD4+ T cell number, as measured in each patient, at thecreening visit (V1) and during the follow up period (V13). P value was calculatedy Mann–Whitney test.

as 55% in Arm A (5/9) and 33% (3/9) in Arm B. Seroconvertion in00% of vaccinated subjects occurred from the second dose onward.oreover, as soon as four weeks after the second dose of vaccine

D56), 22% of vaccinated persons (4/18; 3 belonging to Arm A andne belonging to Arm B) reached their anti-AT20 Ab titer peak12,800). At D84, four weeks after the third vaccination, a furthernhancement of Ab titers in 12 patients was observed (5 belong-ng to Arm A and 7 belonging to Arm B). At this time 55% (n = 5)f Arm A patients reached the highest titer (12,800) of anti-AT20gG, whereas only 22% (n = 2) of Arm B subjects reached their anti-T20 Ab peak (Ab titer 6400, n = 1; Ab titer 12,800, n = 1) (p = 0.09).t D112, in Arm A two patients (22.2%) had not reached yet theirT20 Ab peak and showed an Ab titer of 6400 (n = 1) and 800 (n = 1).t this timepoint, 5 patients in Arm B showed a further increment

n anti-AT20 Ab titer whereas three of them reached their Ab peakAb titer 12,800, n = 2; Ab titer 6400, n = 1). By the end of vaccina-ion, 100% of treated patients reached a plateau of anti-AT20 Abiter that was maintained till the follow-up observation time point.he highest titer observed following this immunization protocol12,800) was reached by 78% (n = 7) patients from Arm A and 55%n = 5) patients from Arm B (Fig. 4A). However, as shown in Fig. 4B,he anti-AT20 Abs GM titers among vaccinated persons belongingo Arm A and Arm B did not significantly differ at any time point. Tonvestigate the possibility that AT20-specific memory B-cells coulde recruited in response to AT20-KLH vaccination, anti-AT20 IgGvidity was also evaluated (Fig. 4C). To this purpose, anti-AT20 IgGI was calculated when Abs first appeared and the follow-up phase

D168). Abs with high avidity at the first appearance were devel-ped by 67% (n = 6) of patients in Arm A and 33% (n = 3) of patientsn Arm B. At D168 all these patients showed Abs with an avidityrade ≥3. The remaining patients’ population (n = 9) showed AT20

(2014) 1072– 1078

IgG at low (n = 2 in Arm A; n = 3 in Arm B) or medium avidity (n = 1in Arm A; n = 3 in Arm B) when Abs were first detected. Avidityincreased over time in 77% of these subjects (n = 7) reaching a highAI. Among them, 2 patients in Arm A and 4 patients in Arm B dis-played an AI > 3. Only two subjects (n = 1 in Arm A and n = 1 in ArmB) displayed anti-AT20 IgG at a medium AI.

In summary, these results attest for the capability of AT20-KLHvaccine to elicit a “de novo” anti-AT20 Ab synthesis, as well as totrigger anti-AT20 Ab production from a pre-existing pool of B lym-phocytes by retrieving an immunological memory. In both cases,AI reached at the follow-up phase was significantly higher (p = 0.01in Arm A, p = 0.008 in Arm B) than the AI observed when Absfirst appeared. Furthermore, when anti-AT20 Abs were producedthrough a de novo induction, 77% of vaccinated patients (n = 7) wereenabled to develop anti-AT20 Abs with high avidity by the end ofthe study.

4. Discussion

This is a first-in-man evaluation of a synthetically manufacturedvaccine consisting of a structurally conserved HIV-1 matrix proteinregion (AT20), functionally involved in p17/p17 receptor(s) interac-tion [8]. Being an exploratory study, patients received two differentAT20-KLH doses. Notably, none of them caused major AEs and themost common mild AE was a dose-independent pain at the siteof injection, consistent with the presence of adjuvant in the for-mulation. Preservation of CD4+ T-cell number and the absence ofplasma viremia rebounds attest for a remarkable immunologic andvirologic safety of the vaccine preparation. These results prove thatAT20-KLH is safe and well tolerated.

All patients, except one, had no detectable anti-AT20 Abs at thetime of enrolment. This is in line with previous studies showingthat Abs to p17 NH2-terminal region are less common than thoserecognizing other p17 epitopes, and that these Abs disappear signif-icantly before than AIDS-related reduction in Ab titer occurs [24].All enrolled subjects immunized with AT20-KLH developed hightiters of anti-AT20 Abs. Such an immunogenic activity is uncom-mon amongst HIV-1 vaccines, since results recorded up to datenever reached 100%, even in HIV-1-seronegative volunteers [25].This finding points to a strong immunogenicity of the AT20-KLHmolecule from one side, confirming the capability of HAART tofully restore patients’ humoral response on the other side. Phe-notypic and functional B cell abnormalities commonly observed inuntreated HIV-1-infected patients are indeed completely restoredby HAART [26].

Our study has shown that Abs generated to the synthetic AT20peptide recognize the epitope residing within the native p17 con-firming that AT20, as immunogenic peptide, is presented in vivoin a conformation that mimics the structure it acquires within theviral protein. These data are strengthen by the observation that Absdeveloped by AT20-KLH-vaccinated patients are able to neutralizep17/p17 receptor(s) interaction and p17 biological activity [27]. Thelatter results are in agreement with our preclinical data [21] show-ing that Abs elicited in mice by AT20-KLH injection possess potentp17 neutralizing activity.

The finding that Abs elicited by AT20-KLH recognizes both lin-ear and conformational epitopes on the viral protein [22], makespossible neutralization of p17 with major amino acid substitutionswithin this epitope. This opens to the opportunity of evaluatingthe efficacy of the peptide-based vaccine in patients infected withHIV-1 strains displaying divergent p17 proteins. Interestingly, by

evaluating anti-AT20 Abs AI, we were able to discriminate de novopatients from individuals with a pre-existing pool of B-cells thatcan be triggered by AT20-KLH to recover a specific immunologicalmemory. It will be interesting to investigate if different modality of

M.L. Iaria et al. / Vaccine 32 (2014) 1072– 1078 1077

Fig. 3. Profile of the Ab response to AT20-KLH vaccination. (A) Level of anti-AT20 Abs were evaluated by ELISA, using plates coated with unconjugated AT20 peptide (filledsigns) or with the entire recombinant p17 protein (empty signs). Circles represent data obtained from vaccine-treated patients, squares are for untreated control patients.Data are represented as Ab geometric mean (GM) titers (log scale). Arrows indicate time for each vaccination (0, 28, 56, 84, 112 days).

Fig. 4. Quality of AT20-KLH-induced Ab response. (A) Diagram represents titers of anti-AT20-Abs evaluated at the indicated day of observation. Vaccinated subjects weregrouped by dose of AT20-KLH received (Arm A, empty circles; Arm B, filled circles). (B) Trend of anti-AT20 Abs increase along the clinical trial. (C) Anti-AT20 Abs AI in eachA nd at

A and i

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aliatar

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T20-KLH treated patient assessed at the time of first anti-AT20 Ab appearance aI ≤ 1, anti-AT20 IgG were considered at low avidity; if 1 < AI < 2, at medium avidity

b development (natural vs. induced) may reflect a different capa-ility of Abs to recognize linear and conformational epitopes and,herefore, a different response of patients to the therapeutic vaccinen terms of efficacy.

All data indicate the achievement of both the primary (safety)nd the secondary (immunogenicity) endpoints of the study, high-ighting the effectiveness of our approach of using an immunogenncorporating a selected determinant in order to induce potentnd specific Abs against a crucial p17 functional epitope, ratherhan the whole virus or viral subunits, which are known to elicit

dverse immunosuppressive, immunoenhancing and autoimmuneesponses [28–30].

In conclusion, using a peptide-based approach for therapeuticaccination it has been possible to redirect HAART-treated patients’

the follow-up phase (V13). Each patient is represented with a different symbol. Iff AI ≥ 2 at high avidity. P value was calculated with Wilcoxon test.

humoral responses toward a previously untargeted hotspot offunctional activity. The AT20-KLH-based clinical trial provides anincentive to expand present efforts in therapeutic immunizations,and to boost international partnerships with the aim to speedyachieve effective immune-based interventions for the long-lastingrepair of HIV-1-related immune defects.

Acknowledgements

The therapeutic phase I study (MED-AT20-001, EudraCT n.2008-001465-29) was a multi-centre, randomized, dose escalationclinical trial fully sponsored by Medestea Research & ProductionS.p.A., a privately owned biopharmaceutical company.

1 cine 32

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078 M.L. Iaria et al. / Vac

The authors are grateful to all the participants for their commit-ent, time and participation in this study. We particularly thankntonio Soleti of Medestea Research & Production S.p.A., Turin,

taly, for his assistance in coordinating efforts to provide the GMPrade vaccine.

Conflict of interest statement: Emanuele Focà received speak-rs’ honoraria and grants for participating to Conferences fromristol-Myers Squibb, Merck-Sharp & Dohme, Gilead Sciences, Viivealthcare, Abbvie, Janssen. Francesco Castelli acts as principal

nvestigator in clinical trials sponsored by BMS, Janssen, Roche,SD and Novartis. He received research grant from Pfizer, Viiv

nd Abbott. Arnaldo Caruso and Simona Fiorentini are inventorsn patents owned by Medestea. All the other authors declare noonflicts of interest.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.vaccine.013.12.051.

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