Brief Report

Long-lasting humoral immune response induced in HIV-1-infected patients by a synthetic peptide (AT20) derived from the HIV-1 matrix protein p17 functional epitope Emanuele Foca`*1, Maria Luisa Iaria*2, Francesca Caccuri2, Simona Fiorentini2, Davide Motta1, Cinzia Giagulli2, Francesco Castelli1, Arnaldo Caruso2 1

Department of Clinical and Experimental Sciences, Section of Infectious Diseases, University of Brescia Medical School, Italy, 2Department of Molecular and Translational Medicine, Section of Microbiology, University of Brescia Medical School, Italy Objective: A therapeutic vaccination based on a synthetic peptide (AT20) representative of the HIV-1 matrix protein p17 (p17) functional region, coupled to keyhole limpet hemocyanin (KLH) AT20-KLH was capable of inducing the production of high-avidity antibodies (Abs) toward a previous untargeted p17 hotspot of functional activity in highly active antiretroviral therapy (HAART)-treated HIV-1-infected patients. Since avidity of Abs after immunization and the retention of antigens are important in sustaining the longlasting production of specific humoral responses, we asked whether AT20-KLH vaccination would result in development of a long-lived immune response. Methods: The long-term duration of Ab response to AT20-KLH has been evaluated in 10 patients previously enrolled for the AT20-KLH vaccination trial at day 898 post-immunization. Ab titer and their avidity was assessed using specifically designed ELISA assays, whereas their neutralizing capacity was estimated in vitro using a ‘wound sealing assay’. Results: Data obtained show that high titers of specific anti-AT20 Abs were maintained at more than 2 years after the last immunization. Furthermore, these Abs were capable to neutralize exogenous p17, as assessed by ability of sera derived from AT20-KLH-immunized patients to block the ability of p17 to promote cell migration in vitro. Conclusion: This finding attests for a successful AT20-KLH vaccine molecule formulation and for an effective HAART-dependent Ab persistence. Keywords: HIV-1, Matrix protein p17, AT20, Therapeutic vaccine, Long-lasting humoral response

Introduction HIV-1 matrix protein p17 (p17) exhibits different immunomodulatory and chemokine-like properties, which may be relevant in the context of viral pathogenesis.1 All p17 activities are mediated by its binding to specific cellular receptors,2,3 through a functional epitope located at the p17 NH2-terminal region.4 Antibodies (Abs) directed toward the p17 functional

Correspondence to: Arnaldo Caruso, University of Brescia Medical School, Department of Molecular and Translational Medicine, Section of Microbiology, Piazzale Spedali Civili, 1, 25123 Brescia, Italy. Email: [email protected] *These authors contributed equally to the work. ß W. S. Maney & Son Ltd 2015 DOI 10.1179/1528433614Z.0000000018

region were neutralizing all p17 biological activities by displacing the binding of viral protein to its cellular receptors.5,6 The occurrence of Ab response to the p17 functional region during the natural course of HIV-1 infection is rare and eventually, at a very low titer.1 Thus, despite the presence of high-titer anti-p17 Abs, sera from HIV-1-infected patients were not usually found to neutralize the biological activity of viral protein.1 On the basis of these evidences and successful preclinical data,7 a 20 amino acids-long synthetic peptide (AT20) representative of the p17 functional region, coupled to the carrier protein keyhole limpet hemocyanin (KLH) was selected as the active agent to perform HIV Clinical Trials

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Foca` et al.

Duration of antibody response to AT20-KLH vaccination

a therapeutic phase I clinical trial in highly active antiretroviral therapy (HAART)-treated HIV-1-infected patients. All tested AT20-KLH doses were safe and well tolerated. Moreover, all vaccinated patients developed high titers of high-avidity p17 neutralizing Abs.8 These findings provided the first evidence that the AT20 peptide-based approach was successful in its aim of redirecting HIV-1infected patients’ humoral responses toward a previous untargeted hotspot of functional activity. Avidity of Abs after immunization as well as antigen retention in lymph node germinal centers are known to correlate with the long-lasting production of specific humoral responses.9,10 Indeed, p17 is not only easily detected in blood of patients11 but it has been also demonstrated that it accumulates and persists in abundant amounts in germinal centers of patients’ lymph nodes before and after containment of HIV-1 by therapy.12 Consisting with such findings, we designed a study to prove whether immunization with AT20-KLH would result in development of a long-lived immune response sustained by a potent humoral immune response and by a continuous natural exposure to p17.

Methods This is a single-center prospective observational study where we enrolled all the patients followed up at the Clinic of Infectious and Tropical Diseases of Brescia who participated at the therapeutic phase I study MED-AT20-001, (EudraCT n.2008001465-29) a multi-center, randomized, dose escalation clinical trial. For a thorough explanation of the study protocol and inclusion criteria, please refer the main study article.8 Briefly, the patients were HIV-1-infected, clinically asymptomatic individuals, in HAART therapy for at least 1 year, HIV-1 viral load v50 copies/ml for at least 6 months and CD4zT-cell count i350 cells mm{3 for at least 3 months prior to inclusion at entry, and CD4 nadir i200 cell mm{3. They were randomized 3: 1 to receive AT20-KLH vaccine, obtained conjugating the GMP-grade AT20 peptide (OPC, Heidelberg, Germany) with the GMP-grade KLH (Byosin, CA, USA) as carrier protein. The patients in the treatment arm were then randomized 1: 1 to receive via intramuscular injection either 25 mg (Arm A) or 100 mg (Arm B) of vaccine. The patients subsequently attended our outpatient services with a schedule of 3–6 months, up to 24 months (day 898) after the study conclusion (day 168): at every visit, standard laboratory tests including CD4zT-cell count, HIV–RNA, basic hematology, and 158

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biochemistry were performed. After that, the patients underwent clinical examination, and adverse events (AEs) and concomitant medications were recorded. Finally, antiretroviral therapy was evaluated and modified according to patients’ needs. Blood samples were collected at day 898, and detection of specific Ab and Ab avidity index were performed by specifically developed ELISA assays as already described.8 Neutralization capability of Abs developed following AT20-KLH vaccination was assessed by impairing the p17 pro-migratory activity on human umbilical vein endothelial cells (HUVECs), as already described.3

Results and Discussion Ten out of the 24 total study patients were followed up in our outpatient clinic, 7 of whom received the AT20-KLH. In particular, six were in the Arm A (25 mg for each dose) and one was in the Arm B (100 mg for each dose) of vaccine; 3 belonged to the control arm. Seven out of 10 patients were males and median age was 43 (range 23–59) years. Median CD4zT-cell count at nadir was 347 cells ml{1 (range 243–500 cells ml{1). We recorded only two AEs occurring from day 168 to day 898 and unrelated to the vaccine administration: one patient was hospitalized for acute gastroenteritis and another patient had a mild elevation of serum aminotransferase of unknown origin. This data demonstrated a good long-term safety of the AT20-KLH vaccine. All patients remained virologically suppressed throughout the 2 years of observation, and no virological blips were recorded. Moreover, no significant differences in the trend of CD4zT-cell count, CD4% and CD4/CD8 ratio were observed over the 2-year long observation period (Table 1). As previously described,8 patients enrolled in the MED-AT20-001 trial did not show consistent levels of anti-AT20 Abs at day 0 (Ab titer v10 in 6/7 patients belonging to Arm A and Arm B of the study; Ab titer 5 100 in 1/7 patients) whereas 100% of immunized subjects developed high titers of AT20-specific Abs by the end of vaccination (day 168). Notably, at day 898, anti-AT20 Abs were consistently maintained in all patients immunized with AT20-KLH. In fact, comparison of Ab titers between day 168 and day 898 post-immunization showed only a modest fold reduction in anti-AT20 Ab levels [median fold decrease: 2.5 (range: 1–5)]. The same trend was observed also when Abs to the entire p17 protein were evaluated. At day 0, 6/7 immunized subjects had negligible or low levels of anti-p17 Abs (Ab titer v10 in 3/7 patients; Ab titer j 100 in 3/7

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Duration of antibody response to AT20-KLH vaccination

Table 1 Trends of CD4zT-cell count. CD4% and CD4/CD8 ratio upon AT20-KLH immunization. 3 months

6 months

12 months

24 months

Arm

CD4z

CD4%

CD4/CD8

CD4z

CD4%

CD4/CD8

CD4z

CD4%

CD4/CD8

CD4z

CD4%

CD4/CD8

A A A A A A B C C C

912 565 901 758 1159 599 595 650 930 652

48.3 40.3 34 36.5 50.2 48.5 33.4 33.4 39.8 36

2.6 0.9 0.7 1.1 1.5 1.3 1 0.7 1.3 0.9

964 614 1340 n.a. 1094 804 826 659 976 906

51.2 41.5 36.8 n.a. 51.8 50.2 33.6 36.2 39.4 36.4

3.4 0.9 0.8 n.a. 1.5 1.3 1.1 0.8 1.3 0.9

956 700 716 1015 1040 746 567 663 877 613

48.4 45.5 39.2 41.6 49.8 49.3 33.5 33.6 42.8 32

2.5 1.1 0.9 1.3 1.3 1.2 1 0.7 1.5 0.9

1216 444 1304 988 1087 741 651 642 787 1181

49.6 42.2 37.9 37.3 52.9 45.5 36.3 32.4 42.6 37.3

2.7 0.9 0.9 1.1 1.4 1.2 1.1 0.6 1.4 1

patients) and AT20-KLH vaccination consistently increased anti-p17 Ab levels in all of them. Again, titers of anti-p17 Abs induced by AT20-KLH vaccination were long lasting [anti-p17 titer median fold decrease at day 898 as compared to day 168: 1 (range: 1–3)]. Patients belonging to the control group displayed no detectable anti-AT20 and anti-p17 at day 0 and day 168, as well as at day 898 (Ab titer v10). This result attests for an inability of these HIVz patients to develop Abs to the p17 functional epitope AT20 in the absence of AT20-KLH immunization (Figure 1). Finally, it is worth to note that at day 898 all patients showed high-avidity AT20 Abs (avidity grade i3). Although the number of patients in the study was small, we also sought to evaluate if Ab titers detected at day 898 in AT20-vaccinated patients correlate with the laboratory parameters evaluated at the same time point. When data were analyzed by Pearson test, no correlation was observed between Ab titer and absolute CD4zT-cell count, CD4% and/or CD4/CD8 ratio. As shown in Figure 2, sera obtained from vaccinated patients – but not sera obtained from control patients – neutralized the capability of p17 to promote human endothelial cell migration, which occurs following the interaction between viral protein and the chemokine receptors CXCR1 and CXCR2.3 This study points to a strong immunogenicity of the AT20-KLH molecule. This is probably due, at least in part, to the characteristic of the synthetic peptide. The AT20 peptide has been designed as a long (20-mer) peptide to be degraded by proteolytic enzymes and presented exclusively by professional APCs, thereby ensuring sufficient co-stimulation. In fact, shorter peptides can be directly loaded on any MHC molecule, also on non-professional APCs, which may lead to the induction of tolerance.13 Also, the modality of cross-linking the non

Figure 1 Changes in Ab titers over time after AT20-KLH immunization. Levels of anti-AT20 Abs were evaluated by ELISA, using plates coated with unconjugated AT20 peptide (A) or with the entire recombinant p17 protein (B). Ab titers detected in sera obtained at day 0, at the end of immunization protocol (day 168) and 2 years later (day 898) are shown. Each sign represents data obtained from a single AT20-KLH-immunized subject or from a control patient. A value of 1 was arbitrarily assigned to each Ab titer of