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Please cite this article in press as: Iaria ML, et al. Synthetic HIV-1 matrix protein p17-based AT20-KLH therapeutic immuniza-

tion in HIV-1-infected patients receiving antiretroviral treatment: A phase I safety and immunogenicity study. Vaccine (2014),

http://dx.doi.org/10.1016/j.vaccine.2013.12.051

ARTICLE IN PRESSGModel

JVAC 14964 17

Vaccine xxx (2014) xxxxxx

Contents lists available at ScienceDirect

Vaccine

j ournal homepage: www.elsevier .com/ locate /vaccine

Synthetic HIV-1 matrix protein p17-based AT20-KLH therapeuticimmunization in HIV-1-infected patients receiving antiretroviraltreatment: A phase I safety and immunogenicity study

Maria Luisa Iaria a,1, Simona Fiorentinia,1, Emanuele Foc b, Sonia Zicari a, Cinzia Giagulli a,Q1Francesca Caccuri a, Daniela Francisci c, Giovanni Di Perri d, Francesco Castelli b,Franco Baldelli c, Arnaldo Caruso a,

a Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italyb Department of Clinical and ExperimentalSciences,University of Brescia, Brescia, Italyc Section of Infectious Diseases,Department of Experimental Medicine and Biochemical Sciences, University of Perugia, 06132 Perugia, Italyd Department of Medical Sciences, University of Turin, Turin, Italy

a r t i c l e i n f o

Article history:

Received 9 July 2013

Received in revised form

16 December 2013

Accepted 19 December 2013

Available online xxx

Keywords:

Therapeutic vaccine

HIV-1 matrix protein p17

Peptide-based-immunotherapy

a b s t r a c t

Background: Therapeutic vaccination is a promising novel approach to treat HIV-1 infected people by

boosting or redirecting immune system to neutralize critical HIV-1 antigens whose biological effects are

relevant in the context ofviral pathogenesis. With the aim to induce neutralizing antibodies to the matrix

protein p17 we have developed a peptide-based immunogen (AT20-KLH) and evaluated its safety and

immunogenicity.

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:

25g/inoculation; Arm B: 100g/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 ofimmunogenicity 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 and

resolved 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 or

HIV-1 viremia were found. At the time ofenrollment 23 out of24 patients had no anti-AT20 antibodies,

whereas 11 exhibited anti-p17 antibodies. Irrespective ofthe presence ofpreimmunization antibodies,

all subjects developed high titers ofanti-AT20 antibodies (GM 9775) in response to both AT20-KLH doses.

These antibodies were also capable ofrecognizing AT20 within the p17 framework.

Conclusions: The AT20 peptide-based approach has allowed to redirect HAART-treated patients humoral

responses toward a previously untargeted hotspot of functional activity. Overall, the tested AT20-KLH

doses were safe and well tolerated, supporting further exploration ofAT20-KLH as an HIV-1 therapeutic

vaccine candidate.

2013 Elsevier Ltd. All rights reserved.

1. Introduction

Highly active antiretroviral therapy (HAART), suppressing the

HIV-1 replication and ameliorating the immunologic response, has

Clinical trial identifier MED-AT20-001. Corresponding author at: Departmentof Molecular and Translational Medicine,Q2

Section of Microbiology, Piazzale Spedali Civili, 1, 25123 Brescia, Italy.

Tel.: +39 030 394491; fax: +39 030395258.

E-mail address: [email protected] (A. Caruso).1 These authors contributed equally to thework.

led to reduced morbidity and mortality [13]. However, a discord-ant response on HAART with virologic suppression but impaired

immunologic reconstitution may be observed; in these individuals

clinical 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 important

issue in patients who cannot benefitfrom two fully activedrugs [6].

Therefore, lifelong adherence, side and long-termeffects of HAART,

immune reconstitution and multiclass drug-resistant issues, along

with 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

0264-410X/$ seefront matter 2013 Elsevier Ltd. All rights reserved.


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effective, either more potent than those induced by the natural

infection.

HIV-1 protein triggers chronic immunological deregulation [7]

and, among them, several investigators have hypothesized an

important role of the matrix protein p17 that exhibits different

immunomodulatory properties which may be relevant in the con-

text of viral pathogenesis [811]. In addition, p17 is the target of

neutralizing Abs which correlate with slower progression to AIDS

[1215]. Thefinding that p17is exported from infected cells [16], is

detected in serum of HIV-1-infected patients [11] and accumulates

in lymph nodes of even successfully HAART-treated patients [17]

makes the mechanisms observed in vitro also possible in vivo.

P17 activities are all mediated by its binding to specific cellular

receptors [1820] through a functional epitope, shaped as a par-

tially unfolded-helix located at the p17 NH2-terminal region [8].

Mice immunized with a 20 amino acids (aa)-long synthetic pep-

tide (AT20) representative of the p17 functional region, coupled to

the carrier protein Keyhole Limpet Hemocyanin (KLH), developed

p17-neutralizingAbs capableof blockingp17/p17receptor(s) inter-

action and,consequently, all biological activities of the viral protein

[8,21]. Further, being that AT20 resides within a highly conserved

conformational epitope immunization it induces Abs capable of

neutralizing the p17 proteins derived from divergent strains dis-

playing critical mutations within AT20 [22]. On the basis of thesepreclinical data, AT20-KLH was selected as the active agent to per-

form a therapeutic phase I clinical trial in HIV-1-infected patients.

2. Materials and methods

2.1. Study product

A contractor for the good manufacturing practice (GMP) was

identified in Italy (Areta International S.r.l., Gerenzano), pro-

duced and released the AT20-KLH vaccine according to current

regulations. The released product was obtained conjugating the

GMP-grade AT20 peptide (OPC, Germany) with the GMP-grade

KLH (Byosin, CA, USA) as carrier protein. The final product con-taining the drug substance and the excipients was packaged as

ready-to-use vials (25g and 100g) and stored at 53 C. The

immunogenic product was emulsified extemporaneously with the

adjuvant (GMP-grade Montanide ISA-51, Seppic, France) in a 1:1

ratio and administered intramuscularly deeply in the usualregions.

2.2. Study design

The therapeutic phase I study (MED-AT20-001, EudraCT n.

2008-001465-29) was a multi-centre, randomized, dose escalation

clinical trial whose primary endpoint was safety assessment and

the secondary endpoint was immunogenicity.

Studywas conducted in HIV-1-infected, clinically asymptomatic

individuals, in HAART therapy for at least 1 year prior to vacci-nation, with undetectable (


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JVAC 14964 17

M.L. Iaria et al. / Vaccinexxx (2014) xxxxxx 3

binding as compared to NH4SCN-untreated wells (M1, low avid-

ity; 1< M< 2 medium avidity; M2 high avidity).

2.4. Statistical analysis

Even if this was not a remit of MED-AT20-001 study, enroll-

ment of 24 subjects provides a sufficient number of observed AEs

to describe the safety endpoint by number, percentage and con-

fidence interval (95%CI). Difference in Ab levels was evaluated by

MannWhitney test. To determine whether Ab AI increases during

treatments, we used Wilcoxon test.

3. Results

3.1. Demographics

The 24 enrolled individuals were subdivided into three arms:

Arm A (n =9) patients treated with the AT20-KLH lower dose

(25g/inoculation); Arm B (n =9), patients receiving the AT20-

KLH higher dose (100g/inoculation); and Arm C (n = 6), control

untreated patients. The mean age standard deviation (SD) of

study participants was 43.569.42in Arm A,40.009.38in Arm B

and 39.8310.59 in ArmC and95.8% were Caucasian(Table 1S). At

the time of enrolment, CD4 count (meanSD) was 760138.5 in

Arm A,802147.5 inArmB and 698188.2 in ArmC and CD4/CD8

ratio (meanSD) was 1.280.6 in Arm A, 1.331.1 in ArmB and

0.990.4 in Arm C. No difference in baseline demographics was

present. All 24 patients have completed the study. The according-

to-protocol analysis for the presence of anti-p17 and anti-AT20Abs

included a screening phase (D-15), a treatment phase of 112 days,

consisting in five vaccination (D0, D28, D56, D84, D112), five visits

after one week to each vaccination (V4, V5, V7, V9, V11), and a

post-vaccination follow-up period (V12) ending 56 days after last

vaccination (V13) (Table 1).

3.2. Safety

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

alteration of clinical and laboratory assessments were observed.

The number of patients with at least one local and/or non-local AE

related or not to vaccination is reported in Table 2. A total num-

ber of 105 AEs were observed. Notably, most of the registered

AEs were mild [Arm A: 70/75, 94% (95%CI: 87.498.6%); Arm B:

23/25, 92% (95%CI: 81.7102%); Arm C: 2/5, 40% (95%CI: 083%)]

and recovered(71/75,95%,95%CI: 8999% in ArmA and 23/25,92%,

95%CI: 81.7102% in Arm B) in all groups of participants (Table 2S).

Moderate AEs were rare, being only 6.7% in Arm A (5/75, 95%CI:

811.2%), 8% in Arm B (2/25, 95%CI: 018.5%) and 40% in Arm C

(2/5,95%CI: 083%). No serious AEsoccurred in anypatients receiv-

ing the AT20-KLH vaccine, whereas a severe AE (Phosphokinase

increase) was observed in one Arm C patient. The most frequentAEs categorywas General disordersand administration site condi-

tions (43/105, 41%, 95%CI: 3149%) with more widespread events

of pyrexia (13/43, 30%, 95%CI: 16.543.5%), swelling (10/43, 23%,

95%CI: 10.535.5%) and pain (9/43, 21%, 95%CI: 9.132.9%). Most of

these AEs were showed by patients in Arm A (39/43, 90%, 95%CI:

8198%).Moreover,inArmAthemajorityofAEs(53/75,71%,95%CI:

6079%) was related with the treatment while, in Arm B, just 20%

of them (5/25, 95%CI: 4.435.6%) were treatment-related AEs.

As a further safety parameter, assessment of CD4+ T-cell count

and viral load was performed. Data collected during the treatment

phase and at D168 are consistent with a remarkable immunologic

and virologic safety of the vaccination. No variation of CD4+ T-cell

countwas observed throughout the whole study duration (Fig.2A).

A comparative analysis between the individual level of CD4T-cells, T

able

1

T

rialschedule;13visits,5administrations,168daysduration/subject.

Visitnumber

V1

V2

V3

V4

V5

V6

V7

V8

V9

V10

V11

V12

V13

Trialtimelines(indicative)

D-15a

D0

D7

D28

D35

D56

D63

D84

D91

D112

D119

D140

D168

Timewindows(day)

[3days]

[3days]

[3days]

[3days]

[3days]

[3days]

[3days]

[3days]

[3days]

[3days]

[3days]

Vaccinationdose

X

X

X

X

X

Anti-AT20Abtiterandavidityindex(ELISA)

X

X

X

X

X

X

X

X

X

X

X

X

X

Anti-p17proteinAbtiter(ELISA)

X

X

X

X

X

X

X

X

X

X

X

X

X

Cellularimmunityanalysisb

X

X

X

X

X

X

X

X

X

Local&systemicevents/reactions

X

X

X

X

X

X

X

X

X

X

X

X

Trialphase

SCREENING

PRIMING

FOLLOW-UP

aWithin1monthto1weekpriortodesign.

bForpatientsenrolledinclinicalcentertoBrescia.

A

llpatientshaveundergonealltheproceduresde

scribedunlessexplicitlyexplained.

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Table 2

Incidence of AEs.

A r m 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%)

Skin and subcutaneous tissue disorders 3(33.3%) 0(0.0%) 0(0.0%)

Pruritus 3(33.3%) 0(0.0%) 0(0.0%)

Erithema 1(11.1%) 0(0.0%) 0(0.0%)

measured at D-15 and D168, confirmed that they did not signifi-

cantly differ in 100% of subjects enrolled (Fig. 2B). Preservation of

circulating CD4+ T cells was mirrored by the absence of significant

plasma viremia rebounds.Based on data obtained, after a careful evaluation of the AEs

IDSMB qualified the AT20-KLH vaccine candidate as safe and well

toleratedboth locally and systemically.A report has beendeposited

to the Italian Regulatory Agencies.

3.3. Immunogenicity

Serum samples were analyzed at the Brescia University core

laboratory. In agreement with previous observation, showing that

during the natural course of HIV-1 infection the occurrence of Ab

responseto AT20is rareand eventually ata verylowtiter [7,24],

most patients (23/24, 95.9%, 95%CI: 94.895%) had not detectable

anti-AT20 Abs at the time of enrolment. The only patient posi-

tive for AT20 Abs (Ab titer: 100) was randomly enrolled in Arm A.

Eleven out of 24 patients (45.8%, 95%CI: 25.464.6%) (5 random-

ized in Arm A, 3 in Arm B and 3 in Arm C) exhibiting p17 Abs

with titers ranging from 100 to 12,800 (Ab GMT = 852.4). Subjects

immunized with AT20-KLH developed high titers of anti-AT20 Abscompared to untreatedpatients (p < 0.001). As a consequence of the

rising level of anti-AT20 Abs, difference in sera reactivity were no

more observed when patients samples were tested in ELISA plates

coated withthe AT20peptide or withthe entire recombinantnative

p17 protein (Fig. 3A). These results demonstrate that the Abs gen-

erated to the synthetic AT20 peptide are capable to recognize the

epitope within the viral protein framework. Moreover,sera derived

from patients immunized with AT20-KLH were capable to displace

thebinding between p17and p17receptor(s)whereas sera derived

from 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 a

better immune response. As shown in Fig. 4A, four weeks after

the first dose of vaccine (D28), the rate of seroconversion to AT20

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JVAC 14964 17

M.L. Iaria et al. / Vaccinexxx (2014) xxxxxx 5

Fig. 2. Profileof CD4+ T cell count along clinical trial. (A) Diagrams representmean

(SD) absolute CD4+ T cell count of patients enrolled in Arm A, Arm B and Arm C.

Datawere collectedat theindicatedvisits. Arrows indicatetime foreach vaccination.

(B) Comparison between CD4+ T cell number, as measured in each patient, at the

screening visit (V1) and during the follow up period (V13). Pvalue was calculated

by MannWhitney test.

was 55% in Arm A (5/9) and 33% (3/9) in Arm B. Seroconvertion

in 100% of vaccinated subjects was occurred from the second dose

onward. Moreover, as soon as four weeks after the second dose ofvaccine(D56), 22%of vaccinated persons (4/18; 3 belongingto Arm

A andonebelonging toArm B)reachedtheiranti-AT20Ab titerpeak

(12,800). At D84, four weeks after the third vaccination, a further

enhancement of Ab titers in 12 patients was observed (5 belong-

ing to Arm A and 7 belonging to Arm B). At this time 55% (n = 5)

of Arm A patients reached the highest titer (12,800) of anti-AT20

IgG, whereas only 22% (n = 2) of Arm B subjects reached their anti-

AT20 Ab peak (Ab titer 6400, n = 1; Ab titer 12,800, n = 1 ) (p = 0.09).

At D112, in Arm A two patients (22.2%) had not reached yet their

AT20Ab peakand showedan Abtiter of6400 (n =1)and800(n =1).

At this timepoint, 5 patients in Arm B showed a further increment

in anti-AT20 Ab titer whereas three of them reached their Ab peak

(Ab titer 12,800, n = 2; Ab titer 6400, n = 1). By the end of vaccina-

tion, 100% of treated patients reached a plateau of anti-AT20 Abtiter that was maintained till the follow-up observation time point.

The highest titer observed following this immunization protocol

(12,800) was reached by 78% (n =7) patients from Arm A and 55%

(n = 5)patients fromArm B (Fig. 4A). However, as shown in Fig. 4B,

the anti-AT20 Abs GMT among vaccinated persons belonging to

Arm A and Arm B did not significantly differ at any time point. To

investigate the possibility that AT20-specific memory B-cells could

be recruited in response to AT20-KLH vaccination, anti-AT20 IgG

avidity was also evaluated (Fig. 4C). To this purpose, anti-AT20 IgG

AI was calculated when Abs first appeared andthe follow-upphase

(D168). Abs with high avidity at the first appearance were devel-

oped by 67% (n =6) of patients inArm A and 33%(n = 3) of patients

in Arm B. At D168 all these patients showed Abs with an avidity

grade 3. The remaining patients population (n = 9) showed AT20

IgG at low(n = 2 in Arm A; n =3 inArm B)or mediumavidity (n = 1

in Arm A; n =3 in Arm B) when Abs were first detected. Avidity

increased over time in 77% of these subjects (n = 7) reaching a high

AI. 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 Arm

B) displayed anti-AT20 IgG at a medium AI.

In summary, these results attest for the capability of AT20-

KLH vaccine to elicit a de novo anti-AT20 Ab synthesis, as well

as to trigger anti-AT20 Ab production from a pre-existing pool of

B lymphocytes by retrieving an immunological memory. In both

cases, AIs reached at the follow-up phase was significantly higher

(p =0.01inArmA,p =0.008inArmB)thantheAIobservedwhenAbs

first appeared. Furthermore, when anti-AT20 Abs were produced

through a de novo induction, 77% of vaccinated patients (n =7)were

enabled to develop anti-AT20 Abs with high avidity by the end of

the study.

4. Discussion

This is a first-in-man evaluation of a synthetically manufactured

vaccine consisting of a structurally conserved HIV-1 matrix protein

region (AT20),functionallyinvolved in p17/p17receptor(s) interac-

tion [8]. Being an exploratory study, patients received two differentAT20-KLH doses. Notably, none of them caused major AEs and the

most common mild AE was a dose-independent pain at the site

of injection, consistent with the presence of adjuvant in the for-

mulation. Preservation of CD4+ T-cell number and the absence of

plasma viremia rebounds attest for a remarkable immunologic and

virologicsafety of the vaccine preparation.These results prove that

AT20-KLH is safe and well tolerated.

All patients, except one, had no detectable anti-AT20 Abs at the

time of enrolment. This is in line with previous studies showing

that Abs to p17 NH2-terminal region are less common than those

recognizingother p17epitopes,and thatthese Abs disappearsignif-

icantly before than AIDS-related reduction in Ab titer occurs [24].

All enrolled subjects immunized with AT20-KLH developed high

titers of anti-AT20 Abs. Such an immunogenic activity is uncom-mon amongst HIV-1 vaccines, since results recorded up to date

never reached 100%, even in HIV-1-seronegative volunteers [25].

This finding points to a strong immunogenicity of the AT20-KLH

molecule from one side, confirming the capability of HAART to

fully restore patients humoral response on the other side. Phe-

notypic and functional B cell abnormalities commonly observed in

untreated HIV-1-infected patients are indeed completely restored

by HAART [26].

Ourstudy have shown that Abs generated to the syntheticAT20

peptide recognize the epitope residing within the native p17 con-

firming that AT20, as immunogenic peptide, is presented in vivo

in a conformation that mimics the structure it acquires within the

viral protein. These data arestrengthenby theobservationthat Abs

developed by AT20-KLH-vaccinated patients are able to neutralizep17/p17receptor(s) interaction andp17 biological activity[27]. The

latter results are in agreement with our preclinical data [21] show-

ing that Abs elicited in mice by AT20-KLH injection possess potent

p17 neutralizing activity.

The finding that Abs elicited by AT20-KLH recognizes both lin-

ear and conformational epitopes on the viral protein [22], makes

possible neutralization of p17 with major amino acid substitutions

within this epitope. This opens to the opportunity of evaluating

the efficacy of the peptide-based vaccine in patients infected with

HIV-1 strains displaying divergent p17 proteins. Interestingly, by

evaluating anti-AT20 Abs AI, we were able to discriminate de novo

patients from individuals with a pre-existing pool of B-cells that

can be triggered by AT20-KLH to recover a specific immunological

memory. It will be interesting to investigate if differentmodality of

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Fig. 3. Profile of the Ab response to AT20-KLH vaccination. (A) Level of anti-AT20 Abs were evaluated by ELISA, using platescoated with unconjugated AT20 peptide (filled

signs) or with the entire recombinant p17 protein (empty signs). Circles represents 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 were

grouped by dose ofAT20-KLH received (Arm A, empty circles; ArmB, filled circles).(B) Trend ofanti-AT20 Absincreasealong theclinicaltrial. (C)Anti-AT20Abs avidity grade

(AI) in each AT20-KLH treated patient assessed at the time of first anti-AT20 Ab appearance and at the follow-up phase (V13). Each patient is represented with a different

symbol. If AI1, anti-AT20 IgG wereconsidered at low avidity; if 1< AI< 2, at medium avidity and if AI2 at high avidity. Pvalue was calculated with Wilcoxon test.

Ab development (natural vs. induced) may reflect a different capa-

bility of Abs to recognize linear and conformational epitopes and

therefore, a differentresponseof patientsto thetherapeuticvaccine

in terms of efficacy.

All data indicate the achievement of both the primary (safety)

and the secondary (immunogenicity) endpoints of the study, high-

lighting the effectiveness of our approach of using an immunogen

incorporating a selected determinant in order to induce potent

and specific Abs against a crucial p17 functional epitope, rather

than the whole virus or viral subunits, which are known to elicit

adverse immunosuppressive, immunoenhancing and autoimmune

responses [2830].

In conclusion, using a peptide-based approach for therapeutic

vaccinationit has beenpossibleto redirect HAART-treatedpatients

humoral responses toward a previously untargeted hotspot of

functional activity. The AT20-KLH-based clinical trial provides an

incentive to expand present efforts in therapeutic immunizations,

and to boost international partnerships with the aim to speedy

achieve effective immune-based interventions for the long-lasting

repair 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 escalation

clinical trial fully sponsored by Medestea Research & Production

S.p.A., a privately owned biopharmaceutical company.

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