Prime boost vaccination approaches with different conjugates of a new HIV-1 gp41 epitope encompassing the membrane proximal external region induce neutralizing antibodies in mice

Vaccine 30 (2012) 1911–1916

Contents lists available at SciVerse ScienceDirect

Vaccine journal homepage: www.elsevier.com/locate/vaccine

Short communication

Prime boost vaccination approaches with different conjugates of a new HIV-1 gp41 epitope encompassing the membrane proximal external region induce neutralizing antibodies in mice Mingkui Zhou a,1 , Ioanna Kostoula b,1 , Boris Brill a , Eugenia Panou b , Maria Sakarellos-Daitsiotis b , Ursula Dietrich a,∗ a b

Georg-Speyer-Haus, Institute for Biomedical Research, Paul-Ehrlich-Str. 42-44, 60596 Frankfurt, Germany Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece

a r t i c l e

i n f o

Article history: Received 22 September 2011 Received in revised form 2 December 2011 Accepted 7 January 2012 Available online 23 January 2012 Keywords: HIV-1 Gp41 Epitopes Sequential oligopeptide carriers Immunogens Neutralizing antibodies

a b s t r a c t Peptide mimics of epitopes for pathogen-specific antibodies present in patient sera can be selected based on the phage display technology. Such mimotopes potentially represent vaccine candidates in case they are able to induce neutralizing antibodies upon vaccination. Here we analyze the immunogenicity of different conjugates of epitope EC26-2A4 localizing to the membrane proximal external region (MPER) of the HIV-1 transmembrane protein gp41. The EC26-2A4 epitope, which overlaps with that of the broadly neutralizing monoclonal antibody (mAb) 2F5, was coupled to sequential oligopeptide carriers (SOC) or to palmitoyl acid for better immunogenicity. Upon prime-boost immunizations of mice, the peptide conjugates induced EC26-2A4 specific antibodies in all settings and mice sera neutralized HIV-1SF162.LS in standardized neutralization assays. Thus, the EC26-2A4 MPER epitope represents a promising vaccine candidate for further analysis in larger animals with respect to the breadth of the neutralizing antibodies induced. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction The induction of neutralizing antibodies (nAbs) is a major goal in the development of a preventive HIV-1 vaccine, as several animal studies have shown their decisive role in protective immunity [1–7]. Despite the fact that a handful of broadly nAbs (b12, 2G12, 2F5, 4E10) is known for many years, including structural information of their target epitopes, attempts to induce nAbs upon vaccination have largely failed [8]. Recently, some additional exceptionally broad neutralizing antibodies have been identified from chronically HIV-1 infected patients. These antibodies have been cloned based on sorting and cloning of single B cells from patients with broad neutralizing activity using engineered Env constructs. The new nAbs recognize quaternary structures in the context of the native envelope spike and show features of extensive affinity maturation and adaptation during the course of the infection [9–16].

∗ Corresponding author. Tel.: +49 69 63395 216; fax: +49 69 63395 297. E-mail address: [email protected] (U. Dietrich). 1 These authors contributed equally. 0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2012.01.026

NAbs targeting the well conserved MPER of gp41 are rarely detected in patients [12,17]. This may be due to tolerance mechanisms associated with the polyreactivity of such antibodies with autoantigens [18–20]. Further, some epitopes in the MPER are only transiently exposed due to conformational changes of Env during the viral entry process. Though poorly immunogenic, the few antibodies targeting the MPER were identified to be broadly neutralizing. Among them, mAb 2F5 targeting the core epitope 662 ELDKWA667 neutralizes 80–100% of transmitted HIV1 across the clades [21] and mAb 4E10 recognizing the epitope 671 NWFDIT676 neutralizes close to 100% [22]. So far, only one study described neutralizing antibodies directed against an epitope overlapping with that of mAb 2F5 [23]. However, this mAb as well as 2F5 both show polyreactivity with autoantigens [18,19]. Vaccination with the 2F5 epitope using different strategies failed so far to elicit such bnAbs in vivo [24,25]. In a recent study, we identified an HIV-1 elite controller (EC), who developed antibodies targeting a novel MPER epitope, EC262A4, using an Env tailored phage display library (Zhou et al., unpublished). Fig. 1 shows the location of the EC26-2A4 epitope in the MPER. It overlaps with the 2F5 epitope [24,26], but is shifted toward the N-terminus. Antibodies affinity purified from the EC plasma with the phage EC26-2A4 epitope showed neutralizing

1912

M. Zhou et al. / Vaccine 30 (2012) 1911–1916

Fig. 1. Schematic location of the EC26-2A4, 2F5 and 4E10 epitopes for neutralizing antibodies in the MPER region. The core epitope of EC26-2A4 corresponding to the minimal domain reactive with antibodies purified from patient EC26 plasma is shown in bold. Numbering is according to HIV-1HXB2. FP, fusion peptide; PR, prolin rich region; HR1, heptad repeat 1; C C, cystein loop; HR2, heptad repeat 2; MPER, membrane proximal external region; and TM, transmembrane domain.

activity against HIV-1 and preliminary immunization studies in mice showed that nAbs can be induced upon immunization with the EC26-2A4 phage. The aim of this study was to compare the immunogenicity of the EC26-2A4 epitope coupled to different carrier systems in mice in order to optimize future immunization studies in larger animal models. This would provide sufficient quantities of sera to analyze the breadth of neutralization of the induced antibodies against a panel of HIV-1 isolates from different subtypes and with different neutralization sensitivity. To further analyze the immunogenicity of the EC26-2A4 epitope we conjugated it to synthetic sequential oligopeptide carriers (SOC) [27] or to a palmitoyl group [28] and performed immunizations of mice. SOC form 310 -helical structures by a repetitive moiety Ac-(Lys-Aib-Gly)n exhibiting well-defined functional sites for peptide conjugation [29]. Structural analysis showed that peptides can retain the original conformations without interfering with each other upon coupling to the SOCn carriers [30]. Thus, SOCn can be used as scaffolds to present immunogenic epitopes with reconstituted native conformations for vaccination. A recent study showed that influenza hemagglutinin A epitopes coupled to a SOC4 carrier can preserve the native conformation and 30–50% of mice immunized with the SOC4 constructs survived from H5N1 influenza virus challenge [31]. In a previous study, we analyzed the effect of a palmitoyl group in conjunction with a SOC-conjugated B cell epitope of the Sm antigen targeted by autoantibodies in patients with systemic lupus erythematosus (SLE), on the immune response in mice. The induced immune response was comparable to that produced with the SOCconjugate administered administered following the conventional immunization protocol with complete/incomplete Freund’s adjuvant showing that the palmitoyl group has adjuvant activity [27]. Therefore we conjugated EC26-2A4 epitopes to either SOC or the palmitoyl group and compared both immunogens in heterologous env DNA prime-epitope boost approaches, which have been shown previously to efficiently focus the immune response on the peptide epitope [32,33].

2. Materials and methods 2.1. Synthesis and characterization of Ac-SOC4-EC26-2A4 and Palm-EC26-2A4 The solid-phase peptide synthesis of all compounds (Table 1) was carried out manually following the Fmoc/tBu methodology [34] on a Rink Amide AM resin (0.67 mmol/g). Fmoc groups were removed using 20% piperidine/DMF. Coupling of each Fmocamino acid (3 mol equiv.) was performed in the presence of HBTU/HOBt/DIEA (2.9/3/6 molar ratio) in a DMF/DCM mixture. Completion of couplings and deprotection reactions were monitored using the Kaiser ninhydrin test. The synthesized compounds were removed from the resin, after side chain deprotection, by treatment with TFA/TIS/H2 O (95/2.5/2.5, v/v/v; 4 h). Crude peptides

were purified by semipreparative RP-HPLC and characterized by analytical RP-HPLC and ESI-MS (Table 2). - Peptide1. Palmitoylated peptide 1 was obtained by treatment of the free N-terminal ␣-amino group with palmitic anhydride in pyridine. - Peptide2. Lysines at the 4th and 10th position of the SOC4 carrier were introduced as Fmoc-Lys(Ac)-OH. The lysines at the 1st and 7th position were introduced as Fmoc-Lys(Mtt)-OH and the Mtt group was removed by 1.8% TFA/DCM. The obtained free LysN␧ H2 groups were coupled to Boc-aminooxy-acetic acid and the Boc protective groups were removed during the cleavage of the compound from the resin [31]. - Peptide3. Oxydation of the 2-amino-alcohol group of serine of IEESQNQQEKNEQELLELDKWASLWNWFDK(S)-NH2 (10 mM) by NaIO4 (12 mM) in imidazole buffer (50 mM, pH 7) resulted in the formation of an aldehyde group. - Peptide 4. Chemoselective ligation [35–37] of peptide 3 to peptide 2 was performed by the formation of an oxime bond between the aldehyde group of peptide 3 and the aminooxy groups of 2. ELISAs were performed with patient IgG affinity purified with EC26-2A4 epitope in order to prove the reactivity of the synthesized peptide conjugates with the original patient antibodies. Briefly, maltose binding protein (MBP) and EC26-2A4 fused to maltose binding protein fusion (EC26-2A4-MBP) were expressed and purified as previously described [38]. 1.5 mg EC26-2A4-MBP proteins were coupled to the resin using AminoLink Plus kit (Thermo). Plasma samples were injected into the fusion protein conjugated column and incubated over night at 4 ◦ C. After washing, the binding fraction was eluted by IgG elution buffer (Thermo). The eluted fractions were combined and concentrated using Amicon 30 centrifugal filter units (Millipore). Plates (Maxisorp, Nunc) were coated with 200 ng/well of Ac-SOC4 -EC26-2A4, Ac-SOC4, Palm-EC26-2A4, BSA, MBP-EC26-2A4 (see below), MBP or 107 cfu/well phages (EC26-2A4 or Hyperphage) at 4 ◦ C over night. Plates were blocked with 5% MPBST for 2 h at room temperature and washed three times before incubation with HRP-conjugated anti-human antibodies for 1 h at room temperature. After washing five times, plates were developed using 100 ␮L/well Sureblue TMB substrate (KPL), stopped with 100 ␮L 1 N HCl and read at 450 nm and 620 nm as reference.

Table 1 Synthesized compounds 1–4. Number

Amino acid sequences/characteristics

1

Palm-IEESQNQQEKNEQELLELDKWASLWNWFDK(S)-NH2 (Palm-EC26-2A4)a Ac–SOC4 [(Ac)2 ,(Aoa)2 ]–NH2 b IEESQNQQEKNEQELLELDKWASLWNWFDK(CHOCO) NH2 Ac–SOC4 {Ac2 , [EC26-2A4 (CH N O)]2 } NH2

2 3 4 a b

Palm: palmitoyl (C15 H31 CO). SOC: (Lys-Aib-Gly)4 ; Aoa: amino-oxy-acetyl (H2 N O CH2 CO).

M. Zhou et al. / Vaccine 30 (2012) 1911–1916

1913

Table 2 Parameters of the synthesis, purification and characterization of the compounds 1–4. Peptide

Yield (%)a

RP-HPLC gradient elutionb

tR (min)

ESI-MS

1

26

19.9

2

38c

3

65

4

51

A/B: 60:40 A/B: 0:100 A/B: 90:10 A/B: 50:50 A/B: 70:30 A/B: 40:60 A/B: 60:40 A/B: 40:60

Calculated [M+3H]3+ : 1369.2 Found [M+3H]3+ : 1369.2 Calculated [M+H]+ : 1371.6 Found [M+H]+ : 1372.4 Calculated [M+3H]3+ : 1279.1 Found [M+3H]3+ : 1279.6 Calculated [M+7H]7+ : 1287.1 Found [M+7H]7+ : 1286.3

a b c

13.0 27.0 11.3

The purity of the final products was analyzed according to HPLC peak integrals at 214 nm on analytical HPLC and was estimated >95%. A, H O/0.1% TFA; B, CH CN/0.1% TFA. Ref [31].

2.2. Immunization of mice Monophosphoryl lipid A (MPL, Sigma) aqueous dispersion was prepared as previously described [39]. Six-week old NMRI mice were primed with codon optimized HIV-1 JR-FL env DNA (pJRFLsyngp140, NIH, 50 ␮g in 100 ␮L PBS; intramuscular injection) and boosted monthly following two distinct protocols. - Protocol I: Ac-SOC4 -EC26-2A4 conjugate was dissolved in PBS (100 ␮g in 100 ␮L PBS) and MPL aqueous dispersion was added to 10 ␮g/mL before subcutanous injection. - Protocol II: Palm-EC26-2A4 conjugate was dissolved in sterile water (50 ␮g in 100 ␮L) for subcutanous injection without adjuvants. Blood samples were collected 1 week after the third boost. Serum samples isolated from whole blood were heat inactivated at 56 ◦ C for 30 min and analyzed for binding and neutralizing antibodies. Pooled preimmune sera were used as control. All animal experiments were performed according to the institutional guidelines. 2.3. Detection of antigen-specific IgG in mouse immune sera by ELISA For titering immune sera, plates were coated in duplicates with 200 ng/well MBP-EC26-2A4 or MBP in PBS at 4 ◦ C over night. After washing three times, plates were blocked with 5% MPBST for 4 h at room temperature and incubated over night with 2 fold serial dilutions of mice immune sera (1:100–1:102,400). Plates were washed three times and incubated with HRP-conjugated anti-mouse antibodies (Jackson ImmunoRes) for 1 h at room temperature. After washing five times, plates were developed using 100 ␮L/well Sureblue TMB substrate (KPL), stopped with 100 ␮L 1 N HCl and read at 450 nm and 620 nm as a reference. The titers refer to the highest dilution at which EC26-2A4-MBP specific binding is detectable above the binding to MBP. ELISAs to determine the reactivity of purified IgG to cardiolipin were performed on plates precoated with cardiolipin. Monoclonal neutralizing antibody controls and purified EC26-2A4-IgGs were tested at 100 ␮g/mL.

After preincubation of the pseudovirus stock with serial dilutions of serum for 1 h at 37 ◦ C, TZM-bl cells were infected in a 96-well plate for 48 h in duplicates. After cell lysis relative luminescence units (RLU) were determined using a luminometer (BMG). The 50% inhibitory dose (IC50 ) was defined as the reciprocal serum dilution that caused 50% reduction in RLU compared to virus controls.

3. Results 3.1. Synthesis and characterization of EC26-2A4 epitope conjugates To analyze the immunogenicity of the EC26-2A4 epitope outside the original phage context, we synthesized the epitope and conjugated it to SOC4 and to the palmitoyl group (Table 1). SOC4 EC26-2A4 was synthesized in three steps: (i) solid phase synthesis of SOC4 bearing two amino-oxy-acetyl (NH2 OCH2 CO) groups on the first and third Lys-N␧ H2 residues, (ii) solid phase synthesis of each epitope and creation of an aldehyde group and (iii) chemoselective ligation of the aldehyde-epitope to the carrier through the formation of an oxime bond. Palm-EC26-2A4 was synthesized in one step on the resin. All final products were obtained in sufficient yields and high purity as confirmed by HPLC and ESI-MS (Table 2). To prove the correct antigenicity of the conjugated epitopes, we analyzed their reactivity with antibodies affinity-purified from the EC26 patient plasma with the original epitope in the phage context. The purified antibodies (referred to as EC26-2A4-IgG) showed strong reactivity with both peptide conjugates in ELISA (Fig. 2). The reactivity was even stronger than with the original phage or with the EC26-2A4-MBP fusion proteins, although this may be due to avidity effects due to different copy numbers of epitopes in the constructs.

2.4. Neutralization assays on TZM-bl cells Neutralization studies were performed using HIV-1 pseudoviruses on TZM-bl cells [40]. All serum samples were heat-inactivated at 56 ◦ C for 30 min prior to use. All assays were performed with the HIV-1 reference subtype B strain SF162.LS [40]. Briefly, pseudoviruses were produced by cotransfection of 293T/17 cells with an HIV-1 env-expressing vector and an envdeficient backbone vector (pSG3env) and titered on TZM-bl cells.

Fig. 2. Reactivity of different epitope conjugates and controls (conjugates alone) with antibodies affinity purified from patient EC26 plasma. Hyperphage corresponds to a wildtype phage without peptide inserts.

1914

M. Zhou et al. / Vaccine 30 (2012) 1911–1916

3.2. Peptide conjugates induce epitope specific and HIV-1 neutralizing antibodies upon vaccination of mice After priming with a codon-optimized HIV-1 env DNA plasmid, monthly boosts were performed with SOC4 -EC26-2A4 in MPL adjuvant [39] or with the palmitoylated Palm-EC26-2A4. One week after the third boost, serum antibody titers against the EC26-2A4-MBP fusion protein were determined by ELISA. Five out of six mice from the SOC4 -EC26-2A4 group and four out of six from the Palm-EC262A4 group developed anti-EC26-2A4 antibodies. The medium titers of sera from mice boosted by SOC4-EC26-2A4 or Palm-EC26-2A4 were about 50 and 24, respectively (Fig. 3A). We further analyzed the mice sera from both groups for neutralizing activity against the HIV-1 subtype B isolate SF162.LS (Tier 1) in standardized single round neutralization assays on Tzm-bl cells. Compared to the pooled preimmune sera, four out of the six SOC4 -EC26-2A4 boosted mice and four out of six from the PalmEC26-2A4 boosted mice developed neutralizing antibodies against HIV-1 SF162.LS (Fig. 3B). Thus, the EC26-2A4 epitope indeed represents a target for neutralizing antibodies able to induce in turn neutralizing antibodies upon vaccination.

4. Discussion Eliciting broadly nAbs against HIV-1 is a key issue in the development of a successful prophylactic vaccine [41,42]. Therefore, HIV-1 neutralizing antibodies were intensely studied in the past and a handful of potent broadly neutralizing mAbs have been well characterized in terms of their neutralization breadth and the structures of their target epitopes [12,13,24,43–47]. Some of these very potent mAbs neutralize 90% of the viruses of a panel representing all major globally circulating subtypes at less than 0.1 ␮g/mL [13]. Among them, mAbs 2F5 and 4E10 target an epitope located in the conserved MPER of gp41. Recently, a number of new even more potent mAbs have been identified based on new methodological approaches like single B cell sorting and cloning [9–15]. However, these mAbs mostly target gp120 epitopes in the quaternary context of the envelope spike and broadly neutralizing mAbs against gp41, especially the MPER, are extremely rare [4,48–50]. In previous studies, we identified HIV-1 controllers with broadly neutralizing antibodies against HIV-1 [51] and Zhou et al. (unpublished). The new MPER epitope EC26-2A4 potentially represents an interesting vaccine candidate as we can induce neutralizing antibodies with different epitope conjugates in mice. Furthermore, antibodies purified from the EC26 patient serum with the EC262A4 epitope do not show reactivity with cardiolipin, like mAbs 2F5 and 4E10 antibodies (Fig. 3C). To further characterize this promising epitope we took it out of the phage context and conjugated it to a synthetic SOC carrier and palmitic acid, which were previously used as scaffolds for the reconstitution of antigenic/immunogenic proteins and as adjuvant for the induction of immune response, respectively [27,52,53]. Based on the reactivity of the synthetic epitope constructs with antibodies affinity purified with the phage EC26-2A4 epitope from the patient’s plasma, we could prove the antigenic integrity of the conjugated EC26-2A4 epitopes. This is in line with previous studies, which have shown that anchoring of epitopes with various specificities to SOC resulted in the antigenic reconstitution of the cognate protein [28,30]. Furthermore, both epitope conjugates induced epitope-specific antibodies upon immunization of mice, as shown by ELISA. More interestingly, induced antibodies showed neutralizing activity against HIV-1 SF162.LS. Thus, although neutralizing titers were low, we could clearly prove that the EC26-2A4 epitope per se is able to induce neutralizing antibodies if coupled to different carrier systems. Thus, the EC26-2A4 MPER epitope is a

Fig. 3. (A and B) Analysis of sera from mice immunized with Palm-EC26-2A4 or SOC-EC26-2A4. Bars indicate the mean of one experiment in duplicates. (A) AntiEC26-2A4 titers. Reciprocal serum dilutions are shown for each mouse. Pooled preimmune sera were used as control. (B) Neutralization titer against HIV-1 SF162.LS. The reciprocal serum dilution giving 50% neutralization (IC50) is shown for each mouse serum. Pooled preimmune sera were used as control. (C) Reactivity of affinity purified EC26-2A4-IgG from EC26 plasma with cardiolipin by ELISA. MAbs 2F5 and 4E10 were included as positive control, mAb 447-52 (V3 region) as negative control. All antibodies were used at 100 ␮g/mL. The bars indicate means of three independent experiments with standard deviation.

promising candidate in view of vaccine development, in particular, due to the absence of crossreactivity with cardiolipin associated with other potent MPER mAbs like 2F5. Immunization studies in larger animals have now to be performed in order to obtain sufficient amounts of sera to analyze the breadth of neutralization with a panel of different HIV-1 isolates.

M. Zhou et al. / Vaccine 30 (2012) 1911–1916

Acknowledgments The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pSyngp140JR-FL from Dr. Eun-Chung Park and Dr. Brian Seed. This project was supported by the Deutsche Forschungsgemeinschaft (GRK1172). The Georg-Speyer-Haus is supported by the Federal Ministry of Health and the Ministry of Higher Education, Research and the Arts from the state of Hessen.Contributors: M.Z., I.K., B.B., E.P. performed experiments; U.D., M.S.-D. designed research; M.Z., M.S.-D. and U.D. wrote paper.

References [1] Baba TW, Liska V, Hofmann-Lehmann R, Vlasak J, Xu W, Ayehunie S, et al. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat Med 2000;6(2):200–6. [2] Ferrantelli F, Hofmann-Lehmann R, Rasmussen RA, Wang T, Xu W, Li PL, et al. Post-exposure prophylaxis with human monoclonal antibodies prevented SHIV89.6P infection or disease in neonatal macaques. AIDS 2003;17(3):301–9. [3] Hessell AJ, Poignard P, Hunter M, Hangartner L, Tehrani DM, Bleeker WK, et al. Effective, low-titer antibody protection against low-dose repeated mucosal SHIV challenge in macaques. Nat Med 2009;15(8):951–4. [4] Mascola JR, Lewis MG, Stiegler G, Harris D, VanCott TC, Hayes D, et al. Protection of Macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies. J Virol 1999;73(5):4009–18. [5] Parren PW, Marx PA, Hessell AJ, Luckay A, Harouse J, Cheng-Mayer C, et al. Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro. J Virol 2001;75(17):8340–7. [6] Ruprecht RM. Passive immunization with human neutralizing monoclonal antibodies against HIV-1 in macaque models: experimental approaches. Methods Mol Biol 2009;525:559–66, xiv. [7] Shibata R, Igarashi T, Haigwood N, Buckler-White A, Ogert R, Ross W, et al. Neutralizing antibody directed against the HIV-1 envelope glycoprotein can completely block HIV-1/SIV chimeric virus infections of macaque monkeys. Nat Med 1999;5(2):204–10. [8] Mascola JR, Montefiori DC. The role of antibodies in HIV vaccines. Annu Rev Immunol 2010;28:413–44. [9] Burton DR, Weiss RA. AIDS/HIV. A boost for HIV vaccine design. Science 2010;329(5993):770–3. [10] Corti D, Langedijk JP, Hinz A, Seaman MS, Vanzetta F, Fernandez-Rodriguez BM, et al. Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals. PLoS One 2010;5(1):e8805. [11] Moore PL, Gray ES, Sheward D, Madiga M, Ranchobe N, Lai Z, et al. Potent and broad neutralization of HIV-1 subtype C by plasma antibodies targeting a quaternary epitope including residues in the V2 loop. J Virol 2011;85(7): 3128–41. [12] Scheid JF, Mouquet H, Feldhahn N, Seaman MS, Velinzon K, Pietzsch J, et al. Broad diversity of neutralizing antibodies isolated from memory B cells in HIVinfected individuals. Nature 2009;458(7238):636–40. [13] Walker LM, Huber M, Doores KJ, Falkowska E, Pejchal R, Julien J-P, et al. Broad neutralization coverage of HIV-1 by multiple highly potent antibodies. Nature 2011;477(7365):466–70. [14] Walker LM, Phogat SK, Chan-Hui PY, Wagner D, Phung P, Goss JL, et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 2009;326(5950):285–9. [15] Wu X, Yang ZY, Li Y, Hogerkorp CM, Schief WR, Seaman MS, et al. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 2010;329(5993):856–61. [16] Johnston MI, Fauci AS. HIV vaccine development – improving on natural immunity. N Engl J Med 2011;365(10):873–5. [17] Pietzsch J, Scheid JF, Mouquet H, Seaman MS, Broder CC, Nussenzweig MC. Antigp41 antibodies cloned from HIV-infected patients with broadly neutralizing serologic activity. J Virol 2010;84(10):5032–42. [18] Haynes BF, Fleming J, St Clair EW, Katinger H, Stiegler G, Kunert R, et al. Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies. Science 2005;308(5730):1906–8. [19] Haynes BF, Moody MA, Verkoczy L, Kelsoe G, Alam SM. Antibody polyspecificity and neutralization of HIV-1: a hypothesis. Hum Antibodies 2005;14(34):59–67. [20] Zhu Z, Qin HR, Chen W, Zhao Q, Fouda G, Schutte R, et al. New broadly neutralizing human monoclonal antibodies targeting the 2F5 epitope. AIDS Res Hum Retroviruses 2010;26(10):A14–5. [21] Mehandru S, Wrin T, Galovich J, Stiegler G, Vcelar B, Hurley A, et al. Neutralization profiles of newly transmitted human immunodeficiency virus type 1 by monoclonal antibodies 2G12, 2F5, and 4E10. J Virol 2004;78(24): 14039–42.

1915

[22] Binley JM, Wrin T, Korber B, Zwick MB, Wang M, Chappey C, et al. Comprehensive cross-clade neutralization analysis of a panel of antihuman immunodeficiency virus type 1 monoclonal antibodies. J Virol 2004;78(23):13232–52. [23] Shen X, Parks RJ, Montefiori DC, Kirchherr JL, Keele BF, Decker JM, et al. In vivo gp41 antibodies targeting the 2F5 monoclonal antibody epitope mediate human immunodeficiency virus type 1 neutralization breadth. J Virol 2009;83(8):3617–25. [24] Muster T, Steindl F, Purtscher M, Trkola A, Klima A, Himmler G, et al. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J Virol 1993;67(11):6642–7. [25] Zwick MB, Labrijn AF, Wang M, Spenlehauer C, Saphire EO, Binley JM, et al. Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41. J Virol 2001;75(22):10892–905. [26] Muster T, Guinea R, Trkola A, Purtscher M, Klima A, Steindl F, et al. Crossneutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS. J Virol 1994;68(6): 4031–4. [27] Kargakis M, Zevgiti S, Krikorian D, Sakarellos-Daitsiotis M, Sakarellos C, PanouPomonis E. A palmitoyl-tailed sequential oligopeptide carrier for engineering immunogenic conjugates. Vaccine 2007;25(37-38):6708–12. [28] Alexopoulos C, Sakarellos-Daitsiotis M, Sakarellos C. Synthetic carriers: sequential oligopeptide carriers SOCn-I and SOCn-II as an innovative and multifunctional approach. Curr Med Chem 2005;12(13):1469–79. [29] Sakarellos-Daitsiotis M, Tsikaris V, Sakarellos C, Vlachoyiannopoulos PG, Tzioufas AG, Moutsopoulos HM. A new helicoid-type sequential oligopeptide carrier (SOC(n)) for developing potent antigens and immunogens. Vaccine 2000;18(3–4):302–10. [30] Sakarellos-Daitsiotis M, Alexopoulos C, Sakarellos C. Sequential oligopeptide carriers, SOCn, as scaffolds for the reconstitution of antigenic proteins: applications in solid phase immunoassays. J Pharm Biomed Anal 2004;34(4): 761–9. [31] Skarlas T, Zevgiti S, Droebner K, Panou-Pomonis E, Planz O, Sakarellos-Daitsiotis M. Influenza virus H5N1 hemagglutinin (HA) T-cell epitope conjugates: design, synthesis and immunogenicity. J Pept Sci 2011;17(3):226–32. [32] Humbert M, Rasmussen RA, Ong H, Kaiser FM, Hu SL, Ruprecht RM. Inducing cross-clade neutralizing antibodies against HIV-1 by immunofocusing. PLoS One 2008;3(12):e3937. [33] Zolla-Pazner S, Cohen S, Pinter A, Krachmarov C, Wrin T, Wang S, et al. Cross-clade neutralizing antibodies against HIV-1 induced in rabbits by focusing the immune response on a neutralizing epitope. Virology 2009;392(1): 82–93. [34] Giralt E, Albericio F. Synthesis of peptides on solid supports. In: Galovich J, editor. Synthesis of Peptides and Peptidomimetics Stuttgard. New York: Georg Thieme Verlag; 2003. p. 665–824. [35] Rose K, Zeng W, Brown LE, Jackson DC. A synthetic peptide-based polyoxime vaccine construct of high purity and activity. Mol Immunol 1995;32(1415):1031–7. [36] Spetzler JC, Tam JP. Unprotected peptides as building blocks for branched peptides and peptide dendrimers. Int J Pept Protein Res 1995;45(1):78–85. [37] Papas S, Strongylis C, Tsikaris V. Synthetic Approaches for Total Chemical Synthesis of Proteins and Protein-Like Macromolecules of Branched Architecture. Curr Org Chem 2006;10(14):1727–44. [38] Zwick MB, Bonnycastle LL, Noren KA, Venturini S, Leong E, Barbas 3rd CF, et al. The maltose-binding protein as a scaffold for monovalent display of peptides derived from phage libraries. Anal Biochem 1998;264(1):87–97. [39] Terry Ulrich J. MPL® immunostimulant: adjuvant formulations. Vaccine adjuvants: preparation methods and research protocols; 2000. p. 273–82. [40] Montefiori DC. Measuring HIV neutralization in a luciferase reporter gene assay. Methods Mol Biol 2009;485:395–405. [41] Burton DR, Desrosiers RC, Doms RW, Koff WC, Kwong PD, Moore JP, et al. HIV vaccine design and the neutralizing antibody problem. Nat Immunol 2004;5(3):233–6. [42] Humbert M, Dietrich U. The role of neutralizing antibodies in HIV infection. AIDS Rev 2006;8(2):51–9. [43] Burton DR, Pyati J, Koduri R, Sharp SJ, Thornton GB, Parren PW, et al. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science 1994;266(5187):1024–7. [44] Stiegler G, Kunert R, Purtscher M, Wolbank S, Voglauer R, Steindl F, et al. A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 2001;17(18):1757–65. [45] Ofek G, Tang M, Sambor A, Katinger H, Mascola JR, Wyatt R, et al. Structure and mechanistic analysis of the anti-human immunodeficiency virus type 1 antibody 2F5 in complex with its gp41 epitope. J Virol 2004;78(19): 10724–37. [46] Saphire EO, Parren PW, Pantophlet R, Zwick MB, Morris GM, Rudd PM, et al. Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design. Science 2001;293(5532):1155–9. [47] Cardoso RM, Zwick MB, Stanfield RL, Kunert R, Binley JM, Katinger H, et al. Broadly neutralizing anti-HIV antibody 4E10 recognizes a helical conformation of a highly conserved fusion-associated motif in gp41. Immunity 2005;22(2):163–73. [48] Binley JM, Lybarger EA, Crooks ET, Seaman MS, Gray E, Davis KL, et al. Profiling the specificity of neutralizing antibodies in a large panel of plasmas from

1916

M. Zhou et al. / Vaccine 30 (2012) 1911–1916

patients chronically infected with human immunodeficiency virus type 1 subtypes B and C. J Virol 2008;82(23):11651–68. [49] Dhillon AK, Donners H, Pantophlet R, Johnson WE, Decker JM, Shaw GM, et al. Dissecting the neutralizing antibody specificities of broadly neutralizing sera from human immunodeficiency virus type 1-infected donors. J Virol 2007;81(12):6548–62. [50] Sather DN, Stamatatos L. Epitope specificities of broadly neutralizing plasmas from HIV-1 infected subjects. Vaccine 2010;28(Suppl 2): B8–12.

[51] Humbert M, Antoni S, Brill B, Landersz M, Rodes B, Soriano V, et al. Mimotopes selected with antibodies from HIV-1-neutralizing long-term non-progressor plasma. Eur J Immunol 2007;37(2):501–15. [52] Sakarellos-Daitsiotis M, Krikorian D, Panou-Pomonis E, Sakarellos C. Artificial carriers: a strategy for constructing antigenic/immunogenic conjugates. Curr Top Med Chem 2006;6(16):1715–35. [53] Matoba N, Shah NR, Mor TS. Humoral immunogenicity of an HIV-1 envelope residue 649–684 membrane-proximal region peptide fused to the plague antigen F1-V. Vaccine 2011;29(34):5584–90.