Secreted Antiviral Entry Inhibitory (SAVE) Peptides for Gene Therapy of HIV Infection

original article

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Secreted Antiviral Entry Inhibitory (SAVE) Peptides for Gene Therapy of HIV Infection Lisa Egerer1,2, Andreas Volk2, Joerg Kahle3, Janine Kimpel1,2, Frances Brauer2, Felix G Hermann2,3 and Dorothee von Laer1 1 Department of Hygiene, Microbiology and Social Medicine, Division of Virology, Innsbruck Medical University, Innsbruck, Austria; 2Applied Virology and Gene Therapy, Institute for Biomedical Research Georg-Speyer-Haus, Frankfurt am Main, Germany; 3Vision7 GmbH, Frankfurt am Main, Germany

Gene therapeutic strategies for human immunodeficiency virus type 1 (HIV-1) infection could potentially overcome the limitations of standard antiretroviral drug therapy (ART). However, in none of the clinical gene therapy trials published to date, therapeutic levels of genetic protection have been achieved in the target cell population for HIV-1. To improve systemic antiviral efficacy, C peptides, which are efficient inhibitors of HIV-1 entry, were engineered for high-level secretion by genetically modified cells. The size restrictions for efficient peptide export through the secretory pathway were overcome by expressing the C peptides as concatemers, which were processed into monomers by furin protease cleavage. These secreted antiviral entry inhibitory (SAVE) peptides mediated a substantial protective bystander effect on neighboring nonmodified cells, thus suppressing virus replication even if only a small fraction of cells was genetically modified. Accordingly, these SAVE peptides may provide a strong benefit to AIDS patients in future, and, if applied by direct in vivo gene delivery, could present an effective alternative to antiretroviral drug regimen. Received 8 June 2010; accepted 6 February 2011; published online 1 March 2011. doi:10.1038/mt.2011.30

Introduction The limitations of antiretroviral drug therapy (ART), such as toxicity and virus resistance, have become more and more evident in the past years. Most importantly, ART has not had a major impact on the global prevalence of human immunodeficiency virus (HIV)-infection and there is no vaccine in sight that could prevent further spread of the infection. In addition to ART and vaccines, gene therapy approaches for HIV-infection have been under investigation for nearly 20 years now, but have met several technical obstacles so that efficacy data in patients are lacking still.1 The basic problem of antiviral gene therapy strategies has been that the total number of target cells for HIV-1 in the patient is large, >1011, so that direct genetic modification of the entire target cell population, whether by T-cell or stem cell targeting, will not be feasible in the foreseeable future. Sole application of cells containing

an antiviral gene is therefore not expected to lead to a substantial level of gene protection with a significant reduction of nonmodified target cells susceptible to virus infection, unless the genetically protected cells have a substantial selective advantage over the nonmodified cells and accumulate with time.2 Indeed, several antiviral genes have been developed that are expected to confer such a selective advantage, as they have been shown to effectively suppress viral replication and protect cells from the viral cytopathic effect in HIV-infected cell cultures. However, even highly potent antiviral entry inhibitors have failed to show a clear accumulation of geneprotected cells to therapeutic levels in clinical trials so far.3 On the other hand, such a strong selective advantage may not be required for a secreted antiviral gene product, such as an in vivo secreted antiviral entry inhibitor (iSAVE). iSAVE (poly)peptides are expected to exert a bystander effect on neighboring nonmodified cells and thus an overall antiviral effect even at low levels of gene modification. The number of reports on secreted antiviral gene products in gene therapy is still very limited. Examples are neutralizing antibodies, soluble CD4, and interferon β.4–6 Among the known antiviral (poly)peptides that could potentially be engineered into an iSAVE gene therapeutic, we chose a C peptide derived from the C-terminal heptad repeat (HR) 2 region of the HIV-1 glycoprotein (gp) 41, since C peptides are currently the therapeutically most active antiviral proteins/peptides known.7,8 C peptides inhibit fusion of the viral and cellular membranes by competing with the viral gp41 HR2 domains for binding to the coiled-coil formed by the N-terminal HR1 of the gp41 prehairpin structure,9 thereby preventing six-helix bundle formation and subsequent fusion of viral and cellular membranes. We previously developed a membrane-anchored 46 amino acid C peptide, maC46, expressed from a retroviral vector for gene therapy. MaC46 is anchored to the cell surface via a linker and a membrane-spanning domain. However, although maC46 very effectively inhibits virus entry, it is not shed into the surrounding and does not exert a bystander effect.10–12 The major challenge for the development of a C peptide-based iSAVE strategy is the size requirement of at least 50–80 amino acids for efficient entry into the secretory pathway.13,14 This was overcome by linking two therapeutic C46 peptides via a cleavage site recognized by the cellular protein convertase furin. Even a low percentage of cells

Correspondence: Lisa Egerer, Department of Hygiene, Microbiology and Social Medicine, Division of Virology, Innsbruck Medical University, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria. E-mail: [email protected] or Dorothee von Laer, Department of Hygiene, Microbiology and Social Medicine, Division of Virology, Innsbruck Medical University, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria. E-mail: [email protected]

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Results Enhanced secretion of elongated or multimerized C peptide Peptides of <50–80 amino acids are generally not secreted efficiently.13,14 The simplest version of the C46 SAVE peptide consisting of the 46 amino acid fusion inhibitory peptide C46 fused to a signal peptide for endoplasmic reticulum translocation (construct C46, 69 amino acids, Figure 1a) is therefore not expected to enter the secretory pathway efficiently. Accordingly, although considerable amounts of intracellular C46 peptide were detected by western blotting in lysates of 293T cells transfected with a retroviral vector expressing C46, the peptide was hardly detectable in the cell culture supernatant (Figure 1b). To enhance secretion, 12 amino acids from the membrane proximal region of gp41 (here referred to as hinge (H); amino acids 674–685 of HIV-1HxB2 Env) were added to the C terminus of C46. In addition, a glycine–alanine repeat (GAr; 18 amino acids) and a c-myc tag (M; 10 amino acids) were added resulting in construct C46-H-GAr (Figure 1a). The GA-repeat motif derived from Epstein-Barr virus nuclear antigen 1 is known to prevent MHC class I presentation and was added in order to potentially reduce the immunogenicity of the SAVE peptide.15 A further even longer construct, L-C46-H-GAr, contained additional 23 amino acids derived from the gp41 loop (L; amino acids 605–627 of HIV-1HxB2 Env) N-terminal of C46, resulting in a total protein length of 145 amino acids (Figure 1a). The loop sequence contains three potential N-glycosylation sites, which presumably augment protein export.16 Two additional N-glycosylation sites are located within the C46 sequence and the hinge region, respectively. Western blot analysis of cell lysates and cell culture supernatants revealed that secretion increased with peptide length (Figure 1b). We then analyzed the impact of glycosylation on secretion levels. The five N-glycosylation sites in L-C46-H-GAr were mutated individually or in combination. While the loss of individual glycosylation sites had little effect on the efficacy of peptide secretion, the elimination of two or more sites led to a dramatic reduction of peptide secretion. No secretion of the fusion protein was detected if all five N-glycosylation sites were mutated (Supplementary Figure S1), substantiating the significance of N-glycans for efficient protein secretion. Finally, we analyzed the impact of dimerization on secretion. Substitution of the GAr motif by a linker derived from human IgG2, containing four cysteines for dimerization (L-C46-H-IgC; IgC = 19 amino acids), resulted in a five- to tenfold increase in peptide expression and secretion (Figure  1b, compare lanes 3 and 4). This increase was found to be dependent on dimerization since point mutation of the cysteines to serines (L-C46-H-IgS) resulted in monomeric peptides with an expression and secretion level comparable to that of L-C46-H-GAr (Figure  1b, compare lanes 3 and 5). Taken together, these data show that secretion of peptides can be effectively enhanced by fusion to a protein scaffold, by introducing N-glycosylation sites and by homodimerization via disulfide bridges. Molecular Therapy vol. 19 no. 7 july 2011

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engineered to release C46 SAVE peptides inhibited virus replication in cell culture effectively. SAVE C ­peptides thus hold considerable promise for future treatment or even prophylaxis of HIV-1 infection.

SAVE Peptides for Gene Therapy of HIV Infection

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Figure 1  Multimerization and addition of scaffold enhances C peptide secretion. (a) Schematic overview of retroviral vector constructs encoding elongated C peptides. The amino acid sequences of the depicted domains are given in the Supplementary Materials and Methods section. (b) C46 fusion peptides were analyzed by western blot using a human monoclonal antibody to glycoprotein subunit 41 (gp41) (C heptad repeat, mab2F546). Deglycosylated supernatants and cell lysates of 293T cells transfected with the indicated retroviral vector constructs are shown. *IgC supernatant was diluted 1:5 before loading. Defined amounts of synthetic (syn) C46 peptide were analyzed in parallel (10, 20, and 50 nmol/l, respectively). (c) Single-round infection assay. PM1 cells were infected with lentiviral vector particles packaged with HIV-1JRFL Env and encoding enhanced green fluorescent protein (eGFP) in the presence of increasing concentrations of peptide-containing supernatants from 293T cells transfected with the indicated constructs. Peptide concentrations of 293T supernatants were estimated as shown in b. eGFP-positive cells were determined by fluorescence-activated cell sorting analysis. 100% relative infection corresponds to 6.5% eGFP-positive cells. Data are means from duplicates; error bars show SEM. aa, amino acid; GAr, glycine–alanine repeat; H, hinge (HIV gp41 membrane proximal region); IgC, linker from human IgG2 containing four cysteine residues; IgS, linker from human IgG2 with cysteine residues mutated to serines; L, loop derived from gp41; M, c-myc tag; S, signal peptide from human tissue type plasminogen activator; SPase, cleavage site for signal peptidase.

Antiviral activity of the peptides secreted from transfected 293T  cells was then analyzed by single-round infection assays with a replication-incompetent lentiviral vector expressing enhanced green fluorescent protein (eGFP) and pseudotyped with the HIV-1JRFL envelope glycoprotein. The half maximal inhibitory concentration of synthetic C46 was 5 nmol/l in this assay. Although concentrations of the secreted peptides were above this value (~250 nmol/l for IgC, and 50 nmol/l for the other 1237

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SAVE Peptides for Gene Therapy of HIV Infection

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Figure 2 Secreted peptide concatemers with cleavable linkers efficiently block HIV-1 entry. (a) Schematic overview of the concatemeric C peptides encoded by retroviral vectors. Two C46 peptides were connected by the indicated linker sequences. (b) To test the antiviral activity of the secreted C46, PM1 cells were challenged with lentiviral vector particles packaged with HIV-1JRFL Env and encoding enhanced green fluorescent protein (eGFP) in the presence of increasing concentrations of supernatant from 293T cells previously transfected with the indicated constructs. The concentration of peptide in the cell culture supernatants was determined in a fluorescence-activated cell sorting (FACS)-based assay as described in Materials and Methods section. eGFP-positive cells were determined by FACS analysis. 100% relative infection corresponds to 11% eGFP-positive cells. Data are means from duplicates; error bars show SEM. (c,d) Western blots of deglycosylated cell culture supernatants and cell lysates of 293T cells transfected with the indicated constructs are shown. A furin expression plasmid was cotransfected were indicated in d. D, noncleaved dimeric concatemers; M, cleaved monomeric peptides; S, signal peptide from human tissue type plasminogen activator; SPase, cleavage site for signal peptidase.

constructs as determined by western blotting, Figure 1b), a clear antiviral activity of these SAVE C peptide-containing cell culture supernatants was not detected (Figure  1c and Supplementary Figure S1). Thus, despite substantial secretion levels of the elongated C46 SAVE peptides, antiviral activity was lost if C46 was fused to a scaffold.

Efficient secretion of cleavable C peptide concatemers Since all tested scaffolds interfered with antiviral activity, we aimed to release the C46 peptide from its scaffold. Therefore, we generated concatemers consisting of two antiviral C46 peptides connected by a furin cleavable linker. The ubiquitously expressed protein convertase furin is active in the constitutive secretory pathway, typically operating in the trans-Golgi network.17 Furinmediated processing of polypeptide precursors occurs at the C-terminal side of the multibasic consensus recognition motif R-X-K/R-R ↓.18 The construct Fur contains the minimal furin consensus site RAKR ↓ D. The control construct Furm contains the mutated, noncleavable sequence RAKVD. In a second control construct, the two C46 domains were connected by a flexible 13 amino acid GS linker (GS, Figure 2a). Transfected 293T cells secreted the concatemeric peptides efficiently; however, unexpectedly, no cleavage of the Fur precursor protein into monomeric C46 peptides was detectable by western blotting (Figure  2c). Accordingly, neither of the concatemeric peptides showed antiviral activity in a single-round infection assay (Figure 2b), again demonstrating that elongated C peptides are inactive. 1238

Since the amino acids flanking the minimal four amino acids recognition motif can critically determine furin processing,18–23 the cleavage site was modified. Using the prediction software ProP23 (available at http://www.cbs.dtu.dk/services/ProP) the furin cleavage site was optimized to RSRAKR ↓ SV (Furo, Figure 2a). This modification resulted in partial cleavage of the Furo concatemer into C46 monomers (Figure 2c) and clear antiviral activity in a single-round infection assay (Figure 2b). This result encouraged us to further optimize the protease cleavage site, particularly focusing on the spatial accessibility to the enzyme. Several natural furin substrates are cleaved within a flexible loop24–26 and further data suggest that a helix-loop-helix structural motif is the target for furin cleavage.27,28 Therefore, the Furo cleavage site was further elongated on one or both sides by a flexible GAr sequence of eight amino acid length (Figure 2a). C-terminal (FuroGA) and particularly N-terminal (GAFuro) addition of GA led to enhanced precursor cleavage and considerably improved inhibitory activity (Figure 2b,c). Moreover, flanking the Furo sequence on both sides with GA repeats (GAFuroGA) further improved furin processing and resulted in almost complete cleavage and high antiviral activity of the secreted peptides (Figure 2b,c). Coexpression of recombinant furin additionally increased the cleavage of these concatemers (Figure 2d), indicating saturation of furin-mediated processing in transfected 293T cells. Insertion of the N- and C-terminal GAr obviously increased the accessibility of the cleavage site for furin, and thus improved cleavage efficacy, despite limited endogenous furin levels. To further verify that processing of the concatemers is solely accomplished by furin, we produced peptide supernatants by transfection of LoVo www.moleculartherapy.org vol. 19 no. 7 july 2011

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Figure 3 Efficient cleavage of the C peptide precursor with two GA repeats flanking the furin cleavage site. (a) 293T  cells were transiently transfected with plasmid DNA coding for Furo sequence on both sides with GA repeats (GAFuroGA) (see Figure  2a). Twenty-four hours post-transfection cells were pulse-labeled with 35S-L-methionine for 30 minutes and chased with an excess of unlabeled methionine (Dulbecco’s modified Eagle’s medium) for the indicated period of time. C46 peptides in the cell lysates and cell culture supernatants were immunoprecipitated with an antibody specific for the HIV-1 glycoprotein subunit 41 (gp41) C heptad repeat (mab2F546) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by phosphorimaging. Western blot for actin was performed as a loading control. Immunoprecipitated secreted peptides were digested with PNGase F to discriminate between monomeric C46 and the concatemeric precursor. (b) The signals were quantified (ImageQuant) and a graphic representation is shown for duplicate experiments; error bars show SEM. Values are given relative to the initial amount of labeled C46 precursor. (c) The amount of C46 monomers in the cell culture supernatant relative to the total amount of secreted FuroGA (in 293T cells, n = 9) and GAFuroGA (in 293T cells, n = 16; and peripheral blood lymphocytes (PBL), n = 5) was calculated. Error bars indicate SEM. D, noncleaved dimeric concatemers; GAFuroGA, Furo which contains two GA repeats flanking the cleavage site; M, cleaved monomeric peptides.

cells, which are devoid of functional furin. Transfected LoVo cells exclusively secreted noncleaved concatemers, which could be cleaved subsequently with recombinant, purified furin in vitro (data not shown). Several additional natural furin cleavage motifs with their flanking sequences were tested in the C46 concatemer context (Supplementary Figure S2), but none of these were cleaved (data not shown). Supplementary data show that glycosylation of the peptide not only considerably enhances secretion (Supplementary Figure S1) but also enhances furin processing (Supplementary Figure S3). These data also indicate that this effect is most likely achieved by increasing the solubility of the hydrophobic C46 and thereby preventing aggregation of peptide in the secretory pathway and an unfolded protein response (Supplementary Figure S4).

Kinetic studies of concatemeric SAVE production and turnover The SAVE peptide, in which two GA repeats frame the furin cleavage site (GAFuroGA), was most efficiently cleaved (see again Figure 2c) and therefore studied further. Initially, the kinetics of intracellular processing, cleavage, and secretion were analyzed by pulse-chase experiments with transfected 293T cells. Pulse-labeled GAFuroGA migrating at ~18 kDa was found to leave the endoplasmic reticulum at a high rate and to appear concurrently in the cell culture supernatant (Figure 3a), showing similar kinetics as Molecular Therapy vol. 19 no. 7 july 2011

FuroGA (for comparison, see Supplementary Figure S3a). After 3 hours, nearly all (>90%) the 35S-L-methionine-labeled GAFuroGA had egressed from the cell. No labeled Golgi-modified fully glycosylated form of GAFuroGA was detected in the cell lysates indicating fast trafficking through the late secretory pathway. Comparison of the signal intensities of the initial amount of cellular precursor and the maximum amount of secreted monomer revealed an 80% loss of protein within the secretory pathway (Figure 3b). However, relative to the FuroGA precursor, furin-mediated cleavage into monomers was drastically increased for GAFuroGA, from 14 to 72% (Figure 3c). As the absolute amount of peptide in the supernatant was in the range of 1 µmol/l, this indicates a concentration of ~700 nmol/l of the active monomeric peptide. Further, kinetic studies showed that secreted C46 monomers in the cell culture supernatant were very stable (even in the presence of cells) with a half-life of >30 hours (Supplementary Figure S5). In addition, the processing of GAFuroGA was analyzed in stimulated peripheral blood lymphocytes, which represent potential target cells for gene therapy. GAFuroGA was also efficiently processed in peripheral blood lymphocytes (77% cleavage, Figure 3c). Relative to the membrane-anchored C46 described by us in earlier studies, the SAVE C46 showed similar kinetics of egress but a much longer half-life of the extracellular, active form of the peptide (Supplementary Figure S6). 1239

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SAVE Peptides for Gene Therapy of HIV Infection

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Figure 4  Monomeric secreted peptides show broad and specific neutralization. (a) In a single-round infection assay, PM1 cells were challenged with lentiviral vector particles packaged with vesicular stomatitis virus (VSV)-G as control or Envs from the HIV-1 strains JRFL, HxB2, or HxB2-SIM. The latter contains a mutation in the GIV motif that confers resistance to T-2011. The vectors encode enhanced green fluorescent protein (eGFP). Infection was performed in the presence of supernatants from 293T  cells expressing GAFuroGA or different concentrations of synthetic T-20. The concentration of secreted antiviral entry inhibitory peptide in the supernatants was determined in a fluorescence-activated cell sorting (FACS)-based assay as described in Materials and Methods. eGFP-positive cells were measured by FACS analysis. 100% relative infection corresponds to ~20% and 10% eGFP-positive cells for HIV-1 Envs and VSV-G, respectively. Data are means from duplicates; error bars show SEM. (b) In a single-round-infection assay Jurkat T cells transduced to 3% with GAFuroGA or mock-transduced cells were infected with replication incompetent eGFP-encoding lentiviral vectors packaged with HIV-1HxB2 Env. eGFP-positive cells were determined by FACS. (c) Jurkat T cells transduced to 3% with GAFuroGA or mock-transduced cells were infected with replicating CXCR4-tropic HIV-1NL4–3 and p24 antigen was measured by enzyme-linked immunosorbent assay (ELISA). The dotted line shows the detection limit. n = 3; error bars show SEM. (d) Primary human CD4+ T cells transduced to 37% with GAFuroGA or mock-transduced were infected with replicating CCR5-tropic HIV-1JR-CSF and p24 antigen was measured by ELISA. The dotted line shows the detection limit. n = 3; error bars show SEM.

Bystander inhibition of HIV-1 by monomeric secreted peptides We observed potent anti-HIV activity of monomeric secreted GAFuroGA peptides in a single-round infection assay using viral vector particles pseudotyped with the CCR5-tropic HIV-1JRFL envelope glycoprotein (Env) (Figure 2b). To further characterize the antiviral effect of SAVE peptides, we performed single-round infection assays with replication-incompetent lentiviral particles pseudotyped with the Env from the CCR5-tropic strain HIV-1JRFL, the CXCR4-tropic strain HIV-1HxB2 and a T-20 resistant variant of the HIV-1HxB2 Env, which carries a mutation in the GIV motif (SIM)11. SAVE peptides secreted from transfected 293T  cells specifically inhibited the different HIV-1 Envs with half maximal inhibitory concentration values in the low nanomolar range 1240

(7–35 nmol/l). The entry of vesicular stomatitis virus (VSV)-G pseudotyped virus particles, used as a control, was not affected (Figure  4a). Deglycosylation of the secreted peptides increased the inhibitory efficacy by twofold (Supplementary Figure S7). To analyze SAVE peptides in more relevant cell types, Jurkat T  cells or primary human CD4+ T  cells were transduced with the GAFuroGA retroviral vector (3% or 37% GAFuroGA positive, respectively, as determined by intracellular immunostaining). The mixed Jurkat culture was resistant to HIV infection, as determined in a single-round challenge with lentiviral HIV-1HxB2 Env pseudotype vectors at high multiplicity of infection (Figure 4b). In addition, replication of HIV-1NL4–3 virus was completely suppressed (Figure 4c). Finally, replication of HIV-1JR-CSF was clearly suppressed in the mixed primary CD4+ T-cell culture compared to the nontransduced control (Figure 4d). However, suppression of virus replication was not complete. This was obviously due to a low level of secretion, which was below the detection limits of our assays. In experiments using exogenously added GAFuroGA peptide, we found that the half maximal inhibitory concentration for inhibition of virus entry into the primary T cells was somewhat higher than for the Jurkat T-cell line, but in a comparable range (25–90 nmol/l, Supplementary Figure S8a, as compared to 7–35 nmol/l for Jurkat T cells, Figure 4a). Accordingly, in the presence of ~850 nmol/l of exogenous SAVE peptide, infection of primary human CD4+ T cells with HIVJR-CSF was completely suppressed (Supplementary Figure S8b). These results demonstrate a strong inhibitory effect provided by the C46 SAVE peptide, although the secretion levels in primary T cells are low. Thus, further optimization of the peptide or the use of a different producer cell type is required to achieve efficacy in vivo.

Discussion In this study, we present a secreted version of the HIV-1 fusion inhibitory C peptide, C46, which is capable of providing a strong antiviral bystander protective effect on neighboring cells. Since the C46 peptide was found to be too short for efficient secretion it was linked to different scaffolds, which supported secretion. However, these fusion proteins had no antiviral activity. Activity was only regained by expression of peptide concatemers consisting of two C46 units connected by a unique furin cleavage motif. Proteolytic cleavage of the C peptide concatemer, which was critical for antiviral activity, was only achieved upon extensive engineering of the cleavage site. For several natural furin target proteins, cleavage is found to occur within a flexible loop.24–26 In the concatemeric SAVE C46, the GAr introduced on both sides of the cleavage motif were essential for efficient concatemer cleavage and most likely provided the required flexibility. Mammalian cells contain only relatively low levels of endogenous proprotein convertases such as furin.29 Accordingly, furin overexpression considerably increased the cleavage of concatemeric precursors in transfected cells, suggesting that the availability of furin is rate limiting. Therefore, optimization of the furin cleavage site was crucial in order to compensate for the limited endogenous furin levels. As also shown by others, N-glycosylation was found to enhance peptide secretion dramatically.16,30–32 In addition and unexpectedly, N-glycans near the furin target site did not inhibit SAVE www.moleculartherapy.org vol. 19 no. 7 july 2011

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concatemer processing by masking the cleavage site, but instead even seemed to favor peptide conformations required for efficient cleavage. While the synthesis rate of single N-glycosylation site mutants N33Q and N95Q was comparable to that of fully glycosylated FuroGA, the lack of either C46 glycosylation substantially reduced precursor cleavage. Chemical crosslinking experiments suggested that the hydrophilicity of N-glycans prevented the aggregation of hydrophobic peptide regions, thereby facilitating furin cleavage (Supplementary Figure S4). Interestingly, although glycosylation was previously found to considerably reduce the activity of C peptides,33 the anti-HIV activity of secreted monomeric SAVE C46 improved only moderately after deglycosylation in vitro. On the other hand, the sugar moieties of SAVE C peptides might significantly prolong in vivo serum half-life due to protection from proteolysis and reduced clearance. The latter occurs rapidly for synthetically produced and nonglycosylated C peptides like T-208. Slightly decreased antiviral activity and protection from proteolysis in vitro were indeed observed by others when N-glycans were attached to the synthetically produced C peptide C34.34,35 Taken together, glycosylation is expected to improve the therapeutic efficacy of iSAVE C peptides. We have previously shown that HIV-1 infected individuals have pre-existing antibodies to the membrane-anchored C46 peptide, as it is derived from HIV-1 sequences. However, in a clinical gene therapy trial using the maC46 peptide no de novo immune reaction to the antiviral peptide was observed.3 Accordingly, for the secreted version of C46, we could also detect pre-existing antibodies in patient sera. Furthermore, in silico models predict potential major histocompatibility complex epitopes in the peptide sequence. To minimize the risk of mounting a de novo immune reaction, we have recently developed nonimmunogenic variants of the C46 peptide that are devoid of immunodominant epitopes, but retain their antiviral properties (F. Brauer, L. Egerer and D. Von Laer, unpublished results). Finally, N-glycosylation might reduce the immunogenicity of iSAVE C peptides as N-glycans were shown to impair class I MHC presentation of potentially antigenic peptides.36 Compared to the membrane-anchored C peptide, maC46, described by us in several previous reports, iSAVE peptides have the crucial advantage of protecting nontransduced neighboring cells and thereby the potential to suppress viral load in the patient at relatively low levels of gene modification. In addition, we found that the pharmacokinetics are more favorable for iSAVE peptides than for maC peptides. Although the transport process of maC46 might differ mechanistically and energetically from the secreted SAVE C46, both variants showed similar intracellular export kinetics. The FuroGA and GAFuroGA variant SAVE peptides were transported along the secretory pathway and released from the cell surface within 30 minutes, similar to other secreted proteins37,38 and similar to the kinetics of maC46 transport to the cell surface. However, turnover of maC46 was found to be rapid, with half-lives of 1–3 hours (Supplementary Figure S6). Such a high turnover rate would require a constantly high expression of maC46 in order to achieve sustained protection of the cell and suppression of viral load. However, the level of gene expression in T lymphocytes depends on the activation status of the cell and quiescent T lymphocytes generally only support low levels Molecular Therapy vol. 19 no. 7 july 2011

SAVE Peptides for Gene Therapy of HIV Infection

of transgene expression.39 In contrast, secreted SAVE C peptides were highly stable (half-life of >30 hours). This is expected to correspond to a longer and more stable availability of the active peptide in vivo. Synthetic peptides delivered systemically (intravenously or subcutaneously) generally have a very short half-life and are cleared rapidly.8 Here, the in vivo secreted iSAVE peptides could have a great advantage as they are delivered continuously and, if produced locally in the relevant tissues, are expected to be cleared slower than peptides delivered via the circulation to their site of action. Lymphatic tissue is the major site of HIV replication and thus T and B cells for instance may be an ideal target cell for an iSAVE gene therapy approach.40 Secretion of the antiviral gene product in the lymphatic tissues is likely to lead to high and stable local peptide concentrations and substantially suppress virus replication. However, our experiments with primary human CD4+ T cells indicate that T cells may not be the optimal target cell for the iSAVE strategy. In contrast to T-cell lines secretion of the antiviral peptide from the primary cells failed to entirely inhibit virus replication even in vitro. Thus, expression from T cells may result in therapeutically insufficient peptide levels in vivo. On the other hand, B cells, which are much more prone to high-level secretion, are expected to be a highly promising in vivo producer cell for the iSAVE peptide. Finally, in contrast to maC46, iSAVE peptides no longer depend on expression from HIV-1 target cells, but instead several other cell types or secretory tissues (e.g., liver) could serve as producer cells in the body. Here, systemic direct application of a vector expressing the iSAVE peptide even becomes feasible. Such direct in vivo approaches make gene therapy less complex and costly and thus feasible also for treatment of patients in the developing world. Furthermore, if proven to be safe, the iSAVE strategy has the potential for application as a gene transfer vaccine in a prophylactic setting. In this regard, the antiviral peptide could for instance be used as a genetic topical microbicide to prevent HIV mucosal transmission. High-level secretion of the antiviral peptide from gene-modified cells in the vagina or rectum has the potential to confer local sterilizing immunity, prevent HIV genital transmission and thus act as a passive genetic vaccine.

Materials and Methods Retroviral vectors. All gammaretroviral vectors encoding secretable C

peptides were derivatives of MP91.41 Detailed cloning procedures are given in the Supplementary Materials and Methods. The vector M87o encoding membrane-anchored C46 has been described previously.11

Cells. The human embryonic kidney cell line 293T was obtained from the American Type Culture Collection (ATCC, Manassas, VA), and was maintained in Dulbecco’s modified Eagle’s medium (DMEM). The T-cell line PM1, a subclone of HuT78 expressing CD4, CXCR4, and CCR5, was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, from Dr Marvin Reitz.42 The T-cell line Jurkat (Clone E6-1) was obtained from ATCC. Both T-cell lines were cultured in RPMI 1640 medium. All media were supplemented with 10% fetal calf serum, 4 mmol/l l-glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin (complete media). Peripheral blood mononuclear cells were isolated from the blood of healthy donors by density gradient centrifugation, using Pancoll (PAN Biotech, Aidenbach, Germany). After depletion of CD8+ cells, using CD8-specific microbeads (Miltenyi Biotec, Bergisch

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Gladbach, Germany), the CD4+ T  cells were cultured in X-Vivo 15 medium supplemented with 5% human AB-Serum, 4 mmol/l l-glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 100 units/ml human interleukin-2 (Novartis, Nuremberg, Germany). Peptides. The peptides C46 (WMEWDREINNYTSLIHSLIEESQNQQEK

NEQELLELDKWASLWNWF) and C46-Furo (WMEWDREINNYTSL IHSLIEESQNQQEKNEQELLEL DKWASLWNWFRSRAKR) were custom synthesized (Thermo Fisher Scientific, Ulm, Germany). T-20 (Enfuvirtide, Fuzeon, YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF) was obtained from Roche (Grenzach-Whylen, Germany).

Production of gammaretroviral and lentiviral pseudotypes. Pseudotyped

gammaretroviral and lentiviral vector particles were generated by transient transfection of 293T cells as described previously.43

Transduction of cells with gammaretroviral vectors. T-cell lines were transduced with gammaretroviral vector supernatants by spinocculation (1 hour, 1,000g, 31 °C) and then cultured under normal cell culture conditions. Transduction of primary human CD4+ T cells was performed as described previously.44 Transduction efficacies were determined 3 days after transduction by flow cytometry. Staining of transduced cells was performed with a phycoerythrin-conjugate of the human monoclonal antibody 2F5 (Polymun, Vienna, Austria), recognizing an epitope within the gp41-derived C peptide sequence.45 For cells expressing secretable C peptides intracellular immunostaining was performed using the Fix&Perm Cell Permeabilization Kit (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s instructions. Collection of secreted C peptides and preparation of cell lysates. Transient

transfection of 293T cells with retroviral vectors was performed by the calcium phosphate precipitation method as described previously.43 10 µg of retroviral vector plasmid were used for each transfection. For overexpression of furin 10 µg of a furin expression plasmid were cotransfected. The medium was exchanged for chloroquine-free medium after incubation for 6–8 hours. Cell culture supernatants containing the secreted peptides were collected from 24 to 48 hours post-transfection, filtered (0.22 µm pore size) and stored at −80 °C until use. Cells were harvested, washed with phosphate-buffered saline (PBS), and subsequently lysed in ice-cold lysis buffer (50 mmol/l HEPES, pH 7.5; 150 mmol/l NaCl; 1% Triton X-100; 2% aprotinin; 2 mmol/l EDTA, pH 8.0; 50 mmol/l sodium fluoride; 10 mmol/l sodium pyrophosphate; 10% glycerol; 1 mmol/l sodium vanadate; and 2 mmol/l Pefabloc SC). After 30 minutes incubation on ice with regular vortexing, cell debris was removed by centrifugation in a microcentrifuge (13,000 rpm for 10 minutes). Quantification of secreted C peptides by FACS. To quantify the amount of secreted C peptides in cell culture supernatants a FACS assay was established. Cell culture supernatant or the synthetic C peptide C46-Furo was incubated with the human monoclonal antibody 2F5 (Polymun) for 30 minutes at 4 °C allowing binding of the antibody to all available C peptides, thereby reducing the amount of free unbound antibody. Peptide-antibody mixtures were added to PM1 cells stably expressing membrane-anchored (ma) C4644 and incubated for 30 minutes at 4 °C, to enable binding of unbound 2F5 to the membrane-anchored C peptide. Cells were washed and afterwards stained with a phycoerythrin-labeled human IgG-specific secondary antibody from goat (Dianova, Hamburg, Germany) for 30 ­minutes at 4 °C. Finally cells were washed and analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany). Preincubation of the 2F5 antibody with different concentrations of synthetic C peptides allowed the generation of a standard curve, where increasing C peptide concentrations resulted in reduced staining of maC46 on the cell surface and thus weaker fluorescence signals. Referring to the standard curve C peptide concentrations in cell culture supernatants were determined.

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Deglycosylation of proteins. The removal of oligosaccharides from N-linked glycoproteins was performed with Endoglycosidase H (Endo H; New England Biolabs, Schwalbach/Ts, Germany) and N-Glycosidase F (PNGase F; New England Biolabs) according to manufacturer’s instructions. Native samples were deglycosylated with 100 NEB units of PNGase F for 16 hours at 37 °C. Denatured samples were incubated for 2 hours at 37 °C with 10 µl deglycosylation mix (containing 2 µl 10× G7 buffer, 2 µl 10% NP-40 solution, and 100 NEB units of PNGase F or 2 µl 10× G5 buffer, and 500 NEB units of Endo H, respectively). Mock samples were treated under identical conditions without PNGase F and Endo H. Western blotting. Western blotting was performed under reducing con-

ditions following standard procedures. Proteins were electrophoretically transferred to nitrocellulose membranes (Bio-Rad, Munich, Germany). Membranes were blocked with 5% dry milk in PBS containing 0.1% Tween20 (MPBST), stained with a gp41-specific human monoclonal antibody (2F5; Polymun; 1 µg/ml in MPBST), and a horseradish peroxidase-conjugated human IgG-specific antibody from goat (Jackson ImmunoResearch, Newmarket, UK; diluted 1:10,000 in MPBST). Detection was performed using enhanced chemiluminescence (GE Healthcare, Munich, Germany) according to the manufacturer’s instructions. After stripping and blocking of membranes, actin was stained with an actin-specific antibody from rabbit [(I-19)-R; Santa Cruz Biotechnology, Heidelberg, Germany; diluted 1:1,000 in MPBST], and a horseradish peroxidase-conjugated rabbit IgGspecific antibody from goat (Jackson ImmunoResearch; diluted 1:40,000 in MPBST).

Neutralization assay. PM1 cells were transduced at low multiplicity of

infection (0.1–0.2) with lentiviral vector particles encoding eGFP and pseudotyped with the envelope glycoproteins of HIV-1HxB2, HIV-1JRFL or VSV. Transduction was performed in the presence of increasing concentrations of 293T cell culture supernatant containing secreted peptides to determine the inhibitory activity of the secreted peptides. For single-round infection of Jurkat T cells expressing SAVE peptides, cells were seeded in conditioned media at day 4 post-transduction and lentiviral vector particles were added. The percentage of transduced cells (eGFP-positive) was determined by flow cytometry after culture of cells for 5 more days at 37 °C. Infection of cells with replication competent HIV-1. Native Jurkat T cells, primary human CD4+ T  cells or cells expressing SAVE peptides were seeded in conditioned medium, and replication competent HIV-1 was added. After incubation at 37 °C for 4 hours cells were washed four times with PBS, resuspended in fresh preconditioned medium, and seeded as triplicate samples in 48-well plates. Cells were kept at high density and cultured at 37 °C. For quantitative detection of p24 antigen expression, cell culture supernatants were analyzed using the Innotest HIV Antigen mAB kit (Innogenetics, Gent, Belgium) according to the manufacturer’s instructions. Pulse-chase experiments. Twenty-four hours post-transfection, 1.6 × 107 293T cells in a 10 cm dish were rinsed twice with 10 ml PBS and starved for 30 minutes at 37 °C in DMEM starvation medium (methionine and cysteine free DMEM, 5% dialyzed fetal calf serum, 4 mmol/l l-glutamine, 1 mmol/l sodium pyruvate, 0.4 mmol/l l-cysteine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 25 mmol/l HEPES-NaOH, pH 7.4). Cells were pulsed for 30 minutes at 37 °C with 200 µCi 35S-L-methionine per dish (10 mCi/ml; PerkinElmer, Waltham, MA). Medium was removed and cells were rinsed with 10 ml prewarmed PBS to stop metabolic labeling. Cells were chased with 6 ml DMEM containing 4 mmol/l methionine. At the end of the chase, cell culture supernatant was collected, filtered (0.22 µm), and stored at −20 °C. The cells were rinsed with 10 ml ice-cold PBS, scraped off, and lysed for 1 hour with 1 ml of nonyl phenoxypolyethoxylethanol (NP-40) buffer (150 mmol/l NaCl, 1% NP-40, 50 mmol/l Tris–HCl, pH 8.0, 1 mmol/l phenylmethanesulfonylfluoride, protease inhibitor cocktail). Lysates were stored at −20 °C. www.moleculartherapy.org vol. 19 no. 7 july 2011

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Immunoprecipitation. Defrosted cell lysates were incubated with

protamine sulfate (0.03%) for 15 minutes on ice and subsequently centrifuged (10 minutes at 15,000 rpm, BioFuge15R; Heraeus, Hanau, Germany) to precipitate chromosomal DNA and cell debris. 166 µl of the resulting cell supernatant (adjusted to 1 ml with NP-40 buffer) and 1 ml medium was used for immunoprecipitation with 1 µg of human monoclonal antibody to gp41 (2F5; Polymun). After 6 hours of incubation at 4 °C, the immunocomplexes were precipitated overnight using equilibrated protein A-agarose beads (Santa Cruz Biotechnology). The matrix-bound proteins were washed three times with different buffers: NP-40 buffer, Neufeld buffer [10 mmol/l Tris–HCl, pH 8.5, 0.6 mol/l NaCl, 0.1% sodium dodecyl sulfate, and 0.05% NP-40], and Tris–HCl buffer (50 mmol/l Tris–HCl, pH 8.0). The matrix-bound proteins were denatured by boiling (10 minutes) in sodium dodecyl sulfate sample buffer (20 µl) or denaturing buffer (1 µl; New England Biolabs) in case of subsequent deglycosylation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were dried followed by phosphorimaging (GE Healthcare). Data were quantified using the ImageQuant software (GE Healthcare).

SUPPLEMENTARY MATERIAL Figure  S1.  N-glycosylation is required for efficient secretion of C peptides. Figure  S2.  Nonfunctional cleavage site constructs. Figure  S3.  N-glycosylation of the secretable C46 precursor favors furin-mediated cleavage into monomers. Figure  S4.  N-glycosylation counteracts complex formation of the FuroGA precursor. Figure  S5.  Secreted GAFuroGA peptides are very stable in the cell culture supernatant with a half-life of >30 hours. Figure  S6.  Rapid turnover of membrane-anchored C46 (maC46). Figure  S7.  N-glycosylation reduces inhibitory efficacy of secreted C peptides. Figure  S8.  GAFuroGA peptide efficiently prevents virus entry into primary human CD4+ T cells. Materials and Methods.

ACKNOWLEDGMENTS The study was sponsored by the European Commission project PoxGene (LSHP CT 2005 018680) and the project TreatID funded by the German Federal Ministry for Research BMBF. Ja.K. and A.V. were supported by scholarships from Deutsche Forschungsgemeinschaft (DFG, GRK1172) and Schering Stiftung, respectively. D.v.L. is an inventor of membrane-anchored C peptides and participator in the biotech company Vision7 GmbH, which holds intellectual property on membraneanchored anti-HIV C peptides. L.E. and D.v.L. are listed as inventors on a patent application related to secreted C peptides. For the other authors no competing financial interests exist.

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