Published Ahead of Print 12 November 2012. 10.1128/AAC.01152-12.

2013, 57(2):679. DOI:Antimicrob. Agents Chemother. Laer and Lisa Egerer

vonRichter, Heinfried H. Radeke, Jörn E. Schmitz, Dorothee Frances Brauer, Kerstin Schmidt, Roland C. Zahn, Cornelia ImmunogenicityFusion Inhibitor with Greatly Reduced A Rationally Engineered Anti-HIV Peptide

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A Rationally Engineered Anti-HIV Peptide Fusion Inhibitor withGreatly Reduced Immunogenicity

Frances Brauer,a Kerstin Schmidt,b Roland C. Zahn,c* Cornelia Richter,d Heinfried H. Radeke,e Jörn E. Schmitz,c Dorothee von Laer,b

Lisa Egererb

Applied Virology and Gene Therapy, Institute for Biomedical Research Georg-Speyer-Haus, Frankfurt am Main, Germanya; Department of Hygiene, Microbiology andSocial Medicine, Division of Virology, Innsbruck Medical University, Innsbruck, Austriab; Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, HarvardMedical School, Boston, Massachusetts, USAc; Department of Pediatrics, University Clinic Carl Gustav Carus Dresden, Dresden, Germanyd; PharmazentrumFrankfurt/ZAFES, Clinic of the Goethe University, Frankfurt am Main, Germanye

Peptides derived from the C-terminal heptad repeat 2 (HR2) region of the HIV-1 gp41 envelope glycoprotein, so-called C pep-tides, are very efficient HIV-1 fusion inhibitors. We previously developed innovative gene therapeutic approaches aiming at thedirect in vivo production of C peptides from genetically modified host cells and found that T cells expressing membrane-an-chored or secreted C peptides are protected from HIV-1 infection. However, an unwanted immune response against such antivi-ral peptides may significantly impair clinical efficacy and pose safety risks to patients. To overcome this problem, we engineereda novel C peptide, V2o, with greatly reduced immunogenicity and excellent antiviral activity. V2o is based on the chimeric C pep-tide C46-EHO, which is derived from the HR2 regions of HIV-2EHO and HIV-1HxB2 and has broad anti-HIV and anti-simian im-munodeficiency virus activity. Antibody and major histocompatibility complex class I epitopes within the C46-EHO peptide se-quence were identified by in silico and in vitro analyses. Using rational design, we removed these epitopes by amino acidsubstitutions and thus minimized antigenicity and immunogenicity considerably. At the same time, the antiviral activity of thedeimmunized peptide V2o was preserved or even enhanced compared to that of the parental C46-EHO peptide. Thus, V2o is anideal candidate, especially for those novel therapeutic approaches for HIV infection that involve direct in vivo production ofantiviral C peptides.

Peptides and proteins have emerged as potent drugs for thetherapy of various inherent and acquired human diseases, in-

cluding human immunodeficiency virus type 1 (HIV-1) infection(1). However, long-term treatment with therapeutic polypeptidesoften elicits undesirable immune responses that can significantlyimpair clinical efficacy and pose safety risks to patients (2). Amajor problem associated with the chronic administration of ex-ogenously produced recombinant proteins is the generation ofantidrug antibodies (ADAs) (2). The humoral immune responseis mounted upon recognition of antibody epitopes within the pro-tein drug by cognate B cell antigen receptors and subsequent pre-sentation of immunogenic protein fragments on class II HLAmolecules on the surface of professional antigen-presenting cellsto CD4� helper T cells (major histocompatibility complex class II[MHC-II] epitopes).

In gene therapeutic approaches aiming at the direct in vivoproduction of therapeutic proteins from genetically modified hostcells, a cellular immune response mediated by CD8� cytotoxic Tlymphocytes (CTLs) is an additional concern. Protein-specificCTLs could rapidly recognize and eliminate gene-modified cellsdisplaying foreign peptide fragments in the context of MHC-Igene products, the class I HLA molecule HLA-A, HLA-B, orHLA-C (CTL epitopes, MHC-I epitopes).

Peptide fragments presented by class I HLA molecules typicallycomprise between 8 and 11 amino acids (aa), while class II HLAmolecules generally display longer peptides (3, 4). In both cases, acore binding motif of approximately 9 amino acids in length bindswithin the groove of the HLA molecule (3).

However, HLA molecules cannot present all varieties of pep-tide fragments; instead, each isoform has a unique peptide bindingpocket and prefers distinct amino acids at certain positions of the

peptide. This peptide preference is mainly determined by the pri-mary and auxiliary anchor residues, where one particular or aclosely related amino acid is required for efficient peptide binding(5, 6). Consequently, by mutating the anchor amino acids of anantigenic peptide, which is normally bound by a specific HLAtype, the peptide will no longer be presented to the immune sys-tem and is thus rendered nonimmunogenic. A similar strategy canbe applied to delete antibody epitopes preventing recognition by Bcell antigen receptors and the respective antibodies.

Accurate mapping and deletion of all potential antibody andMHC epitopes, while maintaining biotherapeutic activity, may bean elusive goal for larger proteins; however, for small peptide ther-apeutics, complete deimmunization by epitope removal may befeasible. Accordingly, in the present study, we engineered a smallanti-HIV C peptide with reduced immunogenicity while retainingantiviral activity. C peptides are derived from the highly conservedC-terminal heptad repeat 2 region (HR2) of the HIV envelopeglycoprotein gp41 and are very potent inhibitors of virus entry (7,8). Several C-peptide entry inhibitors have been described (8, 9,

Received 1 June 2012 Returned for modification 27 June 2012Accepted 16 October 2012

Published ahead of print 12 November 2012

Address correspondence to Lisa Egerer, lisa.egerer@i-med.ac.at.

* Present address: Roland C. Zahn, Crucell, Leiden, The Netherlands.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01152-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.01152-12

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11–13), and all of these are composed of viral and/or syntheticsequences and thus are potentially immunogenic. The best-known C peptide, T-20 (enfuvirtide [Fuzeon]), is a highly active36-amino-acid peptide derived from the HIV-1HxB2 HR2 whichhas successfully been used in the clinic since 2003. However, T-20therapy induces local immune reactions, and, moreover, T-20-resistant (T-20r) HIV variants develop rapidly.

According to the HIV Molecular Immunology Database at LosAlamos National Laboratory, C-peptide sequences (LANL; e.g.,T-20 or C46, positions 638 to 673 and 628 to 673 of gp160, respec-tively) do not overlap dominant MHC-I or MHC-II epitopes ingp41. In summary, only a few MHC epitopes in the HR2 region ofgp41 have been confirmed experimentally.

However, antibodies against gp41-derived C peptides arefound in most HIV-infected patients (15–17). Even though theHR2 region of gp41 is not immunodominant, it contains the an-tigenic cluster II region (positions 644 to 663 of gp160), bound byseveral nonneutralizing antibodies (17, 18). Moreover, many C-peptide sequences overlap the membrane-proximal external re-gion of gp41, which is recognized by broadly neutralizing antibod-ies like 2F5 (ELDKWA at positions 662 to 667 of gp160) (19, 20)and 4E10 (NWFDIT at positions 671 to 676 of gp160) (21).

Consequently, reducing or even eliminating the antigenicityand immunogenicity of C peptides (while retaining full function)may significantly promote safety and the antiviral effect. In thisstudy, we successfully generated a highly active therapeutic C pep-tide, designated V2o. This peptide has a greatly reduced immuno-genic potential due to amino acid substitutions in antibodyepitopes and in silico-predicted MHC-I epitopes, while retainingexcellent antiviral activity.

MATERIALS AND METHODSStudy population and sample preparation. Frozen sera from 69 HIV-1-infected individuals were obtained from the HIVCENTER, Goethe Uni-versity Clinic, Frankfurt am Main, Germany, and the University MedicalCenter Hamburg-Eppendorf, Hamburg, Germany. The sera representedall clinical stages of HIV-1 infection. Sera from 10 long-term nonprogres-sors (LTNPs) and 10 HIV-2-infected individuals were provided by theGeorg-Speyer-Haus, Frankfurt am Main, Germany. Frozen Ficoll-puri-fied peripheral blood mononuclear cells (PBMCs) from HIV-1-infectedindividuals were received from the University Medical Center Hamburg-Eppendorf. PBMCs of healthy donors (Georg-Speyer-Haus) were isolatedby density gradient centrifugation using Pancoll (PAN Biotech, Aiden-bach, Germany), cryopreserved with 10% dimethyl sulfoxide in fetal calfserum (PAN Biotech), and stored in liquid nitrogen. Pancoll-purifiedlymphocytes were immunostained using an HLA-A*02-specific mono-clonal antibody (Becton, Dickinson, Heidelberg, Germany) to screen forHLA-A*02-positive donors by flow cytometry (FACSCalibur; Becton,Dickinson). For infection of primary human CD4� T cells with replica-tion-competent HIV-1, CD8� T cells within PBMCs from healthy donorswere depleted using CD8-specific microbeads (Miltenyi Biotec, BergischGladbach, Germany).

Animals. Sera were obtained from nine chronically simian immuno-deficiency virus mac251 (SIVmac251)-infected rhesus macaques (Macacamulatta). All animals were maintained in accordance with the guidelinesof the Committee on the Care and Use of Laboratory Animals under anNIAID-approved animal study protocol, and all studies and procedureswere reviewed and approved by the Institutional Animal Care and UseCommittees of the NIH and Harvard University.

Cells. The human embryonic kidney cell line 293T was obtained fromthe American Type Culture Collection and was maintained in Dulbecco’smodified Eagle’s medium. The T cell line PM1, a subclone of HuT78expressing CD4, CXCR4, and CCR5, was obtained through the AIDS

Research and Reference Reagent Program, Division of AIDS, NIAID,NIH, from Marvin Reitz (22) and was cultured in RPMI 1640 medium. Allmedia were supplemented with 10% fetal calf serum, 4 mM L-glutamine,100 units ml�1 penicillin, and 0.1 mg ml�1 streptomycin (complete me-dia). Human PBMCs were cultured in X-Vivo 15 medium supplementedwith 5% human type AB serum, 4 mM L-glutamine, 100 units ml�1 pen-icillin, 0.1 mg ml�1 streptomycin, and 100 units ml�1 human interleukin2 (Novartis, Nuremberg, Germany) (X5 medium).

Synthetic peptides and monoclonal antibodies. T-20 (enfuvirtide)was obtained from Roche (Grenzach-Whylen, Germany). All other pep-tides were custom synthesized (GeneCust, Dudelange, Luxembourg). Theamino acid sequences of antiviral C peptides are shown in Fig. 1. Cellularimmune responses of HIV-1 PBMCs were investigated either with pep-tides consisting of 9-mers of C46-EHO or V2 or with 15-mers overlappingfor 11 amino acids of C46-EHO or V2o. Using the matrix method, over-lapping peptides were arranged in peptide pools consisting of three pep-tides each, with each peptide being present in two different pools. PBMCsof healthy individuals were incubated with 9-mer peptides derived fromhuman cytomegalovirus (CMV; NLVPMVATV) or Epstein-Barr virus(EBV; GLCTLVAML) or mutated versions of these peptides (CMV-mut;NWVPMVATA) (EBV-mut; GWCTLVAMA). The monoclonal antibody2F5 (recognizing the epitope ELDKWA within the gp41-derived C-pep-tide sequence [20]) was from Polymun Scientific, Vienna, Austria.

MHC-I epitope predictions. Peptide sequences were analyzed for thepresence of epitopes potentially binding class I HLA molecules using thefreely online-accessible algorithms SYFPEITHI (Department of Immu-nology, University of Tübingen, Tübingen, Germany; http://www.syfpeithi.de) (23) and BIMAS (Bioinformatics and Molecular AnalysisSection, National Institutes of Health; http://www-bimas.cit.nih.gov)(24). The SYFPEITHI database reflects epitopes present in natural MHC

FIG 1 Schematic overview of HIV-1 gp41 and of HR2-derived C peptides. (A)The functional domains of the HIV-1 gp41 molecule comprise an N-terminalfusion peptide (FP), two leucine zipper-like heptad repeat domains (HR1 andHR2), the membrane-proximal external region (MPER), the transmembranedomain (TM), and the intracellular domain (Intra). HIV entry-inhibitory Cpeptides C46 and C46-EHO are derived from the HR2 of HIV-1HxB2 andHIV-2EHO/HIV-1HxB2, respectively. Variants V1 to V4 and V2o are based onC46-EHO but contain several amino acid substitutions, as indicated. (B) Flow-chart of V2o peptide development based on C34-EHO.

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ligands, T cell epitopes, or binding peptides. The BIMAS algorithm isbased on experimentally measured �2-microglobulin dissociation half-lives. Both programs provide peptide sequences that are likely to be presentedby the selected HLA molecules as well as a score and ranking for each subse-quence. Peptides were qualified as MHC-I epitopes, if they scored �20 and�10 by the SYFPEITHI and BIMAS algorithms, respectively.

Lentiviral packaging. Lentiviral vector supernatants were produced bytransient transfection of 293T cells using the calcium phosphate precipitationmethod as described previously (25). The lentiviral transfer vector plasmidpHR=SIN-cPPT-SEW (26), encoding enhanced green fluorescent protein(eGFP), was packaged using a lentiviral Gag-Pol construct (pCMV-dR8.91[27]) and one of several envelope proteins, either HIV-1HxB2 (kindly pro-vided by G. Melikyan, Atlanta, GA), HIV-1HxB2_T-20r(SIM) (28), HIV-1HxB2_T-20r(DTV) (28), HIV-1Ba-L (29), HIV-1Ba-L_C46r (25), SIVmac251(30), or the glycoprotein of vesicular stomatitis virus (VSV-G) (31). Lentiviralsupernatants were filtered (0.45-�m pore size), concentrated by low-speedcentrifugation (approximately 16 h, 4,000 � g, 4°C), and stored at �80°Cuntil use. All supernatants were titrated on PM-1 cells.

Neutralization assay. A total of 2 � 104 PM-1 cells per well wereseeded in 96-well tissue culture plates. PM-1 cells were transduced at a lowmultiplicity of infection (MOI; �0.2) with lentiviral vector particles en-coding eGFP and packaged with various envelope glycoproteins. Trans-duction was performed in the presence of increasing concentrations ofsynthetic antiviral C peptides in duplicate. The 96-well plate was centri-fuged (1,000 � g) for 1 h at 31°C. Subsequently, cells were incubated in ahumidified CO2 incubator at 37°C with 5% CO2, and transduction effi-cacy (eGFP-positive cells) was determined by flow cytometry 5 days later.Calculation of half-maximal (50%) inhibitory concentrations (IC50s) wasperformed using GraphPad Prism (version 5.00) software (GraphPadSoftware, San Diego, CA); sigmoid dose-response curves were calculatedby nonlinear regression [log(inhibitor) versus normalized response-vari-able slope] after logarithmic transformation of the data.

Infection of primary human CD4� T cells with replication-compe-tent HIV-1. A total of 2 � 105 primary human CD4� T cells were seededas triplicate samples in 48-well plates. Synthetic antiviral peptides wereadded at different concentrations, and cells were infected with differentstrains of replicating HIV-1. After incubation at 37°C for 24 h, cells werewashed three times with phosphate-buffered saline (PBS), resuspended inmedium without peptides, and reseeded in 48-well plates. Cells were cul-tured at 37°C. For quantitative detection of p24 antigen expression, cellculture supernatants were analyzed by p24 enzyme-linked immunosor-bent assay (ELISA) at day 6.

p24 ELISA. ELISA plates (Costar; Corning, Corning, NY) were coatedwith anti-p24 M01 antibody (Polymun) at a concentration of 200 ng/wellovernight at 4°C or at 37°C for 2 h. The plates were washed three timeswith 0.1% Tween 20 –PBS. Samples and the p24 protein standard (Poly-mun) were added at different dilutions in a total volume of 50 �l per well.p24 was detected by a secondary biotin-labeled antibody (37G12-biotin;Polymun) diluted in 0.1% Tween 20 –1% bovine serum albumin (BSA)–PBS (1 h at room temperature). ELISA plates were washed again threetimes and incubated with streptavidin–�-galactosidase (Roche) in 0.1%Tween 20 –1% BSA–PBS with 2 mM MgCl2 for 20 min. Again, the ELISAplates were washed three times and resorufin (Sigma-Aldrich, Steinheim,Germany) was added. The optical density was measured at 550 and 655nm using an ELISA microplate reader (Bio-Rad, Vienna, Austria). Thedetection limit of the assay was �60 pg p24/ml.

Dot blotting. Nitrocellulose membranes (VWR International, Darm-stadt, Germany) were hydrated in 10� phosphate-buffered saline, and 0.5�g of synthetic peptides was spotted onto membranes. Nonspecific siteswere blocked with 5% fat-free milk powder (Sigma-Aldrich) in phos-phate-buffered saline containing 0.1% Tween 20 (MPBST). Membraneswere stained with primary control antibodies (1.25 �g ml�1 in MPBST)or patient sera (5 �l ml�1 in MPBST) and secondary horseradish perox-idase-conjugated human/mouse IgG-specific antibodies from goat (di-luted 1:10,000 in MPBST; Dianova, Hamburg, Germany). Detection was

performed using enhanced chemiluminescence (GE Healthcare, Munich,Germany) according to the manufacturer’s instructions.

Epitope mapping. Nitrocellulose membranes spotted with 15-merpeptides overlapping for 13 amino acids were obtained from JPT PeptideTechnologies GmbH (Berlin, Germany). Membranes were hydrated withmethanol, washed with Tris-buffered saline (TBS), and blocked overnightat 4°C in 1% blocking solution (Roche). Membranes were incubated witheither the primary control antibody 2F5 (2 to 5 ng ml�1 in 0.5% blockingsolution) or HIV-1 patient serum (2 to 5 �l ml�1 in 0.5% blocking solu-tion) for 3 h with rocking and washed with TBS containing 0.05% Tween20. Subsequently, membranes were incubated for 2 h with a secondaryhorseradish peroxidase-conjugated human IgG-specific antibody fromgoat (diluted 1:10,000 in 0.5% blocking solution; Dianova) and washedthree times with TBS. Detection was performed using enhanced chemilu-minescence (GE Healthcare) according to the manufacturer’s instruc-tions.

ELISpot assay. The release of gamma interferon (IFN-�) from cyto-toxic CD8� T lymphocytes was measured by enzyme-linked immunospot(ELISpot) assay using an ELISpotPlus kit from Mabtech (Hamburg, Ger-many) according to the manufacturer’s instructions. In short, frozenPBMCs were thawed in X5 medium containing DNase (2 U ml�1) toprevent cell clumping and were incubated in a humidified CO2 incubatorat 37°C and 5% CO2 for 3 h. The cells were then centrifuged, resuspendedin fresh X5 medium, and counted using trypan blue staining. One hun-dred microliters of the cell suspension (5 � 104 to 2 � 105 cells per well)and 50 �l peptide (antigen) solution (final concentration, 5 �g ml�1)were added in duplicate to ELISpotPlus plates precoated with capture an-tibodies specific for IFN-�. PBMCs of HIV-1 patients were stimulatedwith either the CEF peptide pool (Mabtech) (32), HIV-1-derived 9-merpeptides (Gag [77SLYNTVATL85], reverse transcriptase [RT; 309ILKEPVHGV317], Vpr [59AIIRILQQL67]), synthetic 9-mer peptides (C46-EHO,V2), or peptide pools generated by mixing three different 15-mer peptidesof C46-EHO and V2o on the basis of the matrix method. PBMCs ofhealthy donors were incubated with 9-mer peptides of CMV, CMV-mut,EBV, or EBV-mut. After incubation in a humidified CO2 incubator at37°C and 5% CO2 for 20 h, cells were removed and plates were developedusing a biotinylated IFN-�-specific antibody and streptavidin-alkalinephosphatase. Blue spots displayed on the plate membranes were deter-mined with a Zeiss microscope and normalized to a cell number of 1 � 105

PBMCs. The number of IFN-�-secreting cells was obtained by calculatingthe mean value of the duplicates and subtracting the number of spots inunstimulated control wells.

RESULTSSubstitution of primary anchor amino acids reduced the num-ber and scores of in silico-predicted MHC-I epitopes. HIV-1gp41-derived C peptides, e.g., T-20 or C34, are very efficient in-hibitors of HIV-1 entry. The peptide C34-EHO, originating fromthe HIV-2 strain EHO, however, is highly active not only againstHIV-1 but also against SIV (11). This is a useful and desirablecharacteristic, as it permits preclinical efficacy testing in the rhesusmacaque model. However, at least for small HIV-1-derived anti-viral C peptides like T-20 (36 aa), C34 (34 aa), or a membrane-anchored version of T-20, rapid development of resistant virusvariants has been observed (17, 29, 33, 34). On the other hand, wepreviously found that a membrane-anchored version of the elon-gated peptide C46 (46 aa derived from HIV-1HxB2) was highlyactive but less prone to induce the generation of resistant virusstrains (25).

Thus, as a lead structure for the development of a nonim-munogenic antiviral C peptide, we generated the 46-amino-acid peptide C46-EHO, consisting of the HIV-2EHO-derivedC34-EHO elongated at the C terminus by 12 amino acids de-rived from HIV-1HxB2 (Fig. 1). The HIV-1HxB2-derived peptide

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C46 (Fig. 1), which has already been used in preclinical andclinical gene therapy studies (15, 35–37), was used as a controlpeptide in this study.

Putative MHC-I epitopes in C46 and C46-EHO were deter-mined in silico using two matrix-based bioinformatic algo-rithms, SYFPEITHI and BIMAS (23, 24). In both programs, agiven polypeptide sequence is first dissected into overlapping pep-tide fragments between 8 and 11 amino acids in length. Then, thecapacities of the individual peptides to bind to different HLA typesare evaluated and the peptides are ranked according to their scorein silico. Potential MHC-I epitopes in C46 and C46-EHO pre-dicted by SYFPEITHI and BIMAS concentrated mainly in the N-and C-terminal parts of the peptides (Fig. 2). For both therapeuticpeptides, SYFPEITHI issued fewer potential MHC-I epitopes thanthe BIMAS software. C46-EHO epitopes 10 (FLDANITKL) and 34(QELDKWASL) were identified as high-score binders for differ-ent HLA alleles by both algorithms (Table 1). BIMAS predictedthree additional major MHC-I epitopes binding several HLA al-leles with high scores: epitope 8 (VRFLDANIT) overlapping theepitope FLDANITKL, also predicted by SYFPEITHI, as well asepitopes 2 (QQWERQVRF) and 25 (QQEKNMYEL) (Table 1).

We aimed to eliminate as many of these in silico-predictedMHC-I epitopes in C46-EHO as possible, while preserving anti-viral activity. Therefore, the primary anchor amino acids at posi-tions P-2 and P-9 (epitopes 10 and 34) or merely at position P-2

(epitopes 2, 8, and 25) of the five major MHC-I epitopes in C46-EHO were replaced by less preferred residues. In order to main-tain antiviral activity, amino acids were substituted with ones hav-ing similar physical properties or with residues that can be foundat the same position in other antiviral C peptides. Using this ap-proach, four variants of C46-EHO, V1 to V4, were generated (Fig.1). In silico analysis of V1 to V4 using SYFPEITHI and BIMASpredicted a reduced number of MHC-I epitopes and lower scorescompared to those for the original C46-EHO for all peptide vari-ants (an example for V2 is shown in Fig. 2).

The optimized peptide V2o was designed on the basis of theamino acid sequence of V2 (Fig. 1). We selected variant V2 forfurther optimization, as only a few HIV-1 patients had preexistingantibodies to this peptide (see below; Table 2) and its antiviralactivity was very high (see below; Table 3). To generate V2o, theC-terminal membrane-proximal external region (662ELDKWASLWNWF673) originating from HIV-1HxB2 was replaced by the cor-responding amino acid sequence of HIV-2EHO (662KLNQWDIFSNWF673) to further reduce preexisting humoral immunity (seebelow). Moreover, several amino acids in the N-terminal regionwere modified, thus further decreasing immunogenicity accord-ing to SYFPEITHI and BIMAS predictions (Fig. 2; see Fig. S1 in thesupplemental material). Taken together, mutation of primary an-chor residues of MHC-I epitopes considerably decreased the insilico-predicted immunogenicity of C peptides.

FIG 2 Substitution of primary anchor amino acids reduced the number and scores of in silico-predicted MHC-I epitopes. Using the SYFPEITHI (A) and BIMAS(B) software, potential MHC-I epitopes in C peptides C46, C46-EHO, V2, and V2o were predicted for different HLA types in silico. Each 8/9/10- or 11-mer thatwas predicted for any HLA type is indicated with its respective score. Scores below 20 (SYFPEITHI; A) and 10 (BIMAS; B) are not shown.

TABLE 1 Major MHC-I epitopes in C46-EHO for different HLA types according to SYFPEITHI and BIMAS predictions

Position MHC-I epitope sequence

Predicted HLA type(s) (score)

SYFPEITHI BIMAS

2 QQWERQVRF B*2705 (500), B*62 (106), B*2702 (100), B*5201 (30)8 VRFLDANIT B*2705 (1000), B*2702 (100), Cw*0401 (20)10 FLDANITKL A*0201 (27) A*0201 (90), B*2705 (30), A*0205 (17), B*3901 (14), Cw*0401 (13)25 QEKNMYEL B*2705 (200), B*3902 (20), B*3901 (14)34 QELDKWASL B*37 (30), B*4402 (22), B*4001 (21),

B*08 (20), B*18 (20)B*60 (320), B*40 (40), B*2705 (30), Cw*0301 (20), A*0205 (18),

B*61 (16), B*4403 (12), A*0201 (11), B*3701 (10)

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Replacement of primary anchor amino acids according to insilico predictions prevented activation of peptide-specific CTLsin vitro. Since in vivo no more than a small amount of predictedMHC-I epitopes is actually processed, bound, and presented byHLA molecules, we intended to (i) verify in silico-identifiedMHC-I epitopes experimentally and (ii) determine the impact ofmutating the primary anchor amino acids of predicted MHC-Iepitopes on the stimulation of cellular immunity.

Strong T cell responses against the HR2 domain of gp41 havenever been measured experimentally (LANL HIV Molecular Im-munology Database), and the scores of the in silico-predictedMHC-I epitopes in C46-EHO were rather low. Therefore, we firstaimed to prove the feasibility of our concept using two strongerMHC-I epitopes derived from human CMV and EBV. We mu-tated the primary anchor amino acids of the viral MHC-I epitopes,both known to be presented by HLA-A*0201, and analyzed theimpact on activation of peptide-specific CTLs in vitro using IFN-�ELISpot assays. The peptide 495NLVPMVATV503 derived from theCMV matrix protein pp65 (SYFPEITHI and BIMAS scores, 30and 160, respectively) was mutated to NWVPMVATA (CMV-mut; where the mutated amino acids are underlined; SYFPEITHIand BIMAS scores, 14 and 0.002, respectively), while the EBVearly lytic protein-derived MHC-I epitope 259GLCTLVAML267

(SYFPEITHI and BIMAS scores, 28 and 149, respectively) was

changed to GWCTLVAMA (EBV-mut; SYFPEITHI and BIMASscores, 12 and 0.002, respectively). The wild-type and mutatedpeptides or control antigens were incubated with HLA-A*02-ex-pressing PBMCs from healthy individuals for 20 h, and IFN-�secretion was quantified by ELISpot assay. The CEF peptide poolconsisting of 23 highly immunogenic peptides from CMV, EBV,and influenza virus was used as a positive control for CTL stimu-lation and caused a specific IFN-� response in the range of 7 to 194spot-forming units (SFUs) (see Fig. S2A in the supplemental ma-terial). C46-EHO-derived MHC-I epitopes 2 (not predicted forHLA-A*0201) and 10 (predicted for HLA-A*0201) were used asnegative controls and, as expected, did not stimulate CTLs fromhealthy donors (2.5 SFUs; see Fig. S2A in the supplemental ma-terial). Incubation of PBMCs with wild-type CMV or EBV pep-tides resulted in low to moderate IFN-� levels (0.25 to 111 SFUsand 0.5 to 74 SFUs, respectively); however, no IFN-� secretion wasdetected for the mutated peptides CMV-mut and EBV-mut (1SFU for both; see Fig. S2A in the supplemental material). Hence,modification of primary anchor amino acids of MHC-I epitopesaccording to in silico predictions impaired activation of peptide-specific CTLs in vitro, indicating successful elimination of theMHC-I epitopes.

Absence of preexisting C-peptide-specific CTL response inHIV-1-infected individuals. In the next step, ELISpot assays were

TABLE 2 Reactivity of sera from HIV-1- or HIV-2-infected individuals and SIVmac251-infected rhesus macaques with therapeutic C peptides

C peptide

Seroreactivitya

HIV-1

HIV-2 (n 10)SIVmac251(n 9)ART (n 35)

No ART(n 34) LTNP (n 10)

Anti-C46-EHOpositive (n 7)

Anti-C46-EHOnegativeb

C46 21 (60.0) 30 (88.2) 8 (80.0) 0 (0.0) 0 (0.0)C46-EHO 3 (8.6) 4 (11.8) 1 (10.0) 7 (100.0) 0 (0.0) 8 (80.0) 7 (77.8)V1 6 (85.7) 5 (33.3) 10 (100.0) NDV2 4 (57.1) 1* (5.0) 10 (100.0) 7 (77.8)V3 7 (100.0) 1 (6.7) 8 (80.0) NDV4 7 (100.0) 4 (26.7) 10 (100.0) NDV2o 0 (0.0) 0 (0.0) 10 (100.0) 8 (88.9)a Seroreactivity is shown as the number (percentage) of serum samples recognizing the respective C peptides in dot blot assays. ART, antiretroviral therapy; LTNP, long-termnonprogressor; ND, not determined.b Fifteen serum samples were tested for all peptides, except as indicated (*), in which case 20 serum samples were tested for V2 reactivity. Anti-C46-EHO-negative samples wererandomly chosen from the ART and no ART groups.

TABLE 3 IC50s for entry inhibition of replication-incompetent lentiviral vectors packaged with various envelope glycoproteins by therapeutic Cpeptides

C peptide

IC50a (nM)

HIV-1

SIVmac251 VSV-GHxB2 (X4tropic)

HxB2_T-20r(DTV)(X4 tropic)

HxB2_T-20r(SIM)(X4 tropic)

BaL(R5 tropic)

BaL_C46r

(R5 tropic)

T-20 1.8 � 0.2 �100 �100 2.5 � 0.8 19.1 � 8.9 �100 �100C46 4.9 � 2.1 6.0 � 1.4 7.0 � 1.3 7.6 � 1.5 27.7 � 6.8 �100 �100C46-EHO 1.9 � 0.6 1.4 � 0.2 1.2 � 0.1 2.1 � 0.8 4.5 � 0.5 11.4 � 2.3 �100V1 1.0 � 0.3 2.0 � 1.0 1.0 � 0.4 1.1 � 0.4 3.7 � 0.8 10.9 � 0.6 �100V2 1.2 � 0.3 2.2 � 0.8 1.4 � 0.3 1.4 � 0.3 3.3 � 0.4 10.8 � 1.4 �100V3 1.7 � 0.4 15.8 � 3.3 15.4 � 10.8 2.4 � 0.4 12.9 � 2.3 30.3 � 23.9 �100V4 1.5 � 0.3 1.5 � 0.9 1.4 � 0.0 1.8 � 0.4 6.7 � 2.5 11.6 � 3.0 �100V2o 0.42 � 0.15 0.26 � 0.13 0.26 � 0.12 0.51 � 0.11 1.1 � 0.2 5.0 � 3.3 �100a IC50s are means � SDs of at least two independent experiments. T-20r, T-20 resistant; C46r, reduced sensitivity to C46; VSV-G, glycoprotein of the vesicular stomatitis virus.

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performed with PBMCs of 23 HIV-1-infected individuals incu-bated with peptides corresponding to the five predicted MHC-Iepitopes of C46-EHO or the mutated versions of these epitopesthat can be found in the V2 variant of C46-EHO (Fig. 3). The CEFpeptide pool used as a positive control caused a specific IFN-�response in the majority of patient samples (89.5%); however,incubation of the patient cells with the HIV-1-derived control pep-tide Vpr (59AIIRILQQL67) induced only marginal IFN-� levels (1to 9 SFUs), while for the HIV-1 peptides Gag (77SLYNTVATL85)and RT (309ILKEPVHGV317), IFN-� responses were near back-ground levels (4 SFUs), indicating a poor general immune statusof the patients. Correspondingly, neither the five predictedMHC-I epitopes of C46-EHO nor the corresponding mutatedepitopes of V2 induced IFN-� secretion (Fig. 3). Consequently,the HIV-1 patients did not have a preexisting CTL responseagainst the in silico-predicted MHC-I epitopes of C46-EHO oragainst the potentially less immunogenic C-peptide variant V2.

Furthermore, we analyzed whether any additional MHC-Iepitopes that had not been predicted in silico were present in theC46-EHO and V2o sequences. Overlapping peptides (15-mersoverlapping by 11 amino acids) covering the whole amino acidsequence of C46-EHO or V2o were arranged in peptide pools bythe matrix method (three peptides per pool, each peptide presenttwice) and used to stimulate PBMCs of 20 HIV-1-infected indi-viduals. However, no significant stimulation of IFN-� secretionwas detected by ELISpot assay (see Fig. S2B in the supplementalmaterial). These results were further confirmed by stimulatingPBMCs of one HIV-2-infected individual as well as PBMCs ofseven SIVmac251-infected rhesus macaques with overlappingpeptides of V2o (data not shown). In conclusion, neither the orig-inal peptide C46-EHO nor modified variant V2 or V2o inducedany MHC-I epitope-dependent IFN-� T cell response in vitro.

Preexisting antibodies against C46, but not C46-EHO, aredetectable in most HIV-1-infected patients. Although broadly

neutralizing antibodies are rarely found in HIV patient sera, anti-bodies binding the viral envelope proteins are frequently detectedin infected individuals (15–17). Since C peptides are derived fromthe viral gp41 sequences, preexisting IgG antibody responses inserum samples of 89 HIV-infected patients and nine SIV-infectedrhesus macaques were investigated in dot blot assays. The HIVgroup comprised 10 HIV-2-infected individuals as well as 79 HIV-1-infected patients, which were subdivided into 35 patients whoobtained antiretroviral therapy, 34 therapy-naïve patients, and 10long-term nonprogressors. The C peptides were spotted on nitro-cellulose membranes and incubated with the serum samples, andC-peptide-specific antibodies were detected using a peroxidase-conjugated human IgG-specific secondary antibody.

As expected, the majority of the HIV-1-infected patient serareacted with C46; however, neither the HIV-2-infected patientsnor the SIV-infected rhesus macaques had preexisting antibodiesto this peptide (Table 2). Within the HIV-1 group, most of theuntreated patient samples contained C46-specific antibodies(88%), whereas in patients on antiretroviral therapy, this fractionwas considerably smaller (60%), probably due to the decreasedviral antigenic load. C46-EHO, which is mainly derived fromHIV-2 sequences, was recognized only by about 10% of all HIV-1-infected patient sera, while in most of the HIV-2 and SIV sam-ples, C46-EHO-specific antibodies could be detected (Table 2).Hence, C46-specific antibodies were mainly preexisting in HIV-1-infected individuals, while antibodies to C46-EHO were pri-marily found in HIV-2- and SIV-infected sera.

Preexisting antibodies in HIV-1-infected patient sera bindthe HIV-1-derived C terminus of C46 and C46-EHO. In the nextstep, we aimed to map the epitopes within the C46 and C46-EHOsequences recognized by preexisting antibodies of HIV-1-infectedpatients. Cellulose membranes spotted with overlapping peptidefragments (15-mers, overlapping for 13 amino acids) spanningthe whole length of C46 and C46-EHO were used for epitopemappings. Upon incubation with patient sera containing C-pep-tide-specific antibodies, a positive reaction occurred at one ormore peptide spots and the relevant epitope was determined. Thewell-known monoclonal antibody 2F5 that binds the linearepitope 662ELDKWA667 (20, 21, 38) present in C46 as well as inC46-EHO was used as a positive control. In accordance with pub-lished results, 2F5 recognized peptide fragments 15, 16, and 17 ofC46- and C46-EHO-spotted membranes, corresponding to theamino acid sequence 659ELLELDKWASLW670 of C46 and 659ELQELDKWASLW670 of C46-EHO (see Fig. S3 and Table S1 in thesupplemental material).

We performed epitope mappings for C46 using serum samplesfrom 14 randomly chosen HIV-1-infected patients, all of whichreacted with the C-terminal part of C46, with 659ELLELDKW666

being the core epitope (see Fig. S3 and Table S1 in the supplemen-tal material). In addition, two of the patient serum samples recog-nized the amino acid sequence 628WMEWDREINNYT639 at the Nterminus of C46. Out of seven HIV-1-infected patient serum sam-ples that contained C46-EHO-specific antibodies, three were usedfor epitope mapping of C46-EHO. None of these serum samplesreacted with the HIV-2-derived part of C46-EHO, but instead, theC terminus, which is almost completely derived from HIV-1 andthus is identical to the C terminus of C46, except for a single aminoacid substitution (L661Q), was recognized. In conclusion, the ma-jor epitope for antibody binding in C46 and C46-EHO is locatedin the HIV-1-derived C-terminal part of the peptides.

FIG 3 Absence of preexisting C-peptide-specific CTL response in HIV-1-infected individuals. PBMCs from HIV-1-infected patients (Pt.) were incu-bated with peptides representing the five major MHC-I epitopes predicted insilico for C46-EHO (2-, 8-, 10-, 25-, and 34-wt [wild type]) or with the respec-tive peptides from V2 with mutated primary anchor residues (2-, 8-, 10-, 25-,and 34-mut). After 20 h, IFN-� secretion from activated peptide-specific CTLswas quantified as the number of SFUs by ELISpot assay. A CEF peptide pooland the HIV-1-derived peptides Gag, RT, and Vpr were used as positive con-trols for CTL activation. The dotted line represents the detection limit of theELISpot assay. Data represent the means of duplicate samples reduced by thenumber of spots detected for PBMCs incubated with medium.

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Absence of preexisting humoral immunity against V2o inHIV-1-infected patients. The HIV-1-infected patient sera con-taining C46-EHO-specific antibodies as well as 15 to 20 anti-C46-EHO antibody-negative HIV-1-infected patient sera were used ina dot blot to screen for preexisting antibodies binding the C46-EHO-derived peptide variants V1 to V4 and V2o. Peptides V1, V3,and V4 were recognized by most of the anti-C46-EHO-positivesera and, in addition, by some sera that lacked C46-EHO-specificantibodies (Table 2). However, antibody reactivity for V2 wasclearly reduced within the anti-C46-EHO-positive sera, and only1 out of 20 anti-C46-EHO-negative HIV-1-infected patient serumsamples was able to detect V2. In peptide variant V2o, the HIV-1-derived C terminus had been replaced by HIV-2-derived se-quences, and thus, preexisting antibodies to V2o were not foundin any of the HIV-1-infected patient sera tested. However, all ofthe HIV-2 and most of the SIV samples reacted with V2o (Table2). Consequently, antibodies to the C46-EHO peptide variantswere primarily present in HIV-2- and SIV-infected sera but virtu-ally absent in the HIV-1-infected patient sera tested. In particular,no preexisting humoral immunity against V2o was observed inany of the HIV-1-infected patients analyzed.

C46-EHO and its variants are highly active HIV-1 andSIVmac251 entry inhibitors. The antiviral activity of the peptidesC46 and C46-EHO and the C46-EHO variants was analyzed bysingle-round infection assays using replication-incompetent len-tiviral vectors encoding eGFP and PM-1 cells as target cells. Thelentiviral vectors were packaged with various HIV-1 envelope gly-coproteins, the SIVmac251 envelope glycoprotein, or VSV-G. The36-amino-acid C peptide T-20 (enfuvirtide), the only HIV-1 entryinhibitor approved for clinical application, was used as a controlin these assays.

All C peptides inhibited entry of wild-type HIV-1 envelopeglycoproteins HxB2 (X4 tropic) and Ba-L (R5 tropic) in a dose-dependent manner, with IC50s being in the low-nanomolar range(Fig. 4 and Table 3). In addition, lentiviral vectors packaged eitherwith HxB2-derived envelope glycoproteins bearing mutations inthe gp41 HR1 547GIV549 motif that confer resistance to T-20[HIV-1HxB2_T-20r(SIM) and HIV-1HxB2_T-20r(DTV)] or with a Ba-L-derived envelope glycoprotein (Ba-L_C46r [where C46r indi-cates reduced sensitivity to C46] carrying three mutations ingp120 [I184T, N302Y, E351K] and two mutations in gp41[A582T, N637K]) (25) with reduced sensitivity to C46 were usedin the single-round infection assays. As expected, T-20 was notable to prevent entry of the T-20-resistant pseudoviruses at thehighest concentration tested (100 nM) and had a clearly increasedIC50 for the inhibition of Ba-L_C46r-mediated entry (Table 3).C46 was active against T-20-resistant HIV-1HxB2 strains butshowed an increased IC50 for the inhibition of Ba-L_C46r, in ac-cordance with results previously published by our group (25).However, neither T-20 nor C46 was able to inhibit SIVmac251-mediated entry. In contrast, C46-EHO and the less immunogenicpeptide variants V1 to V4 and V2o were highly efficient inhibitorsof all HIV-1 and SIV strains analyzed. Interestingly, the mutationsin peptide variants V1, V2, V4, and V2o that were introduced toreduce immunogenicity resulted in even improved antiviral activ-ity compared to that of the original C46-EHO peptide (Table 3).The optimized peptide V2o especially demonstrated superior en-try-inhibitory properties, with IC50s in the picomolar range formost envelope glycoproteins tested (Fig. 4). Entry of VSV-G-

pseudotyped virus particles was not affected by any of the C pep-tides, indicating HIV/SIV-specific entry inhibition.

Taken together, the HIV-1-derived C peptide C46 was able toprevent entry of pseudoviruses with HIV-1 envelope glycopro-teins efficiently, while HIV-2EHO-derived peptides and variantsthereof had superior antiviral activity not only against variousHIV-1 strains (including T-20-resistant strains) but also againstSIVmac251.

The deimmunized peptide V2o efficiently inhibits HIV-1replication. In addition to the single-round infection experimentsusing replication-incompetent pseudoviruses, we performed neu-tralization assays using primary human CD4� T cells infectedwith various strains of replication-competent HIV-1 in the pres-ence of different concentrations of V2o. T-20 peptide was used asa control in these experiments. We found that V2o inhibitedHIV-1 replication at least as efficiently as T-20 for all virus strainstested, including dual-tropic D117II, X4-tropic NL4-3, and thetwo R5-tropic strains Ba-L and Ba-L_C46r (Fig. 5).

DISCUSSION

In the present work, we engineered a highly active anti-HIV pep-tide fusion inhibitor with greatly reduced immunogenicity. Thenovel peptide V2o was developed by identification and removal ofantibody and MHC-I epitopes in the prototypic peptide C46-EHO, which is derived from HR2 sequences of HIV-2EHO and

FIG 4 Mutated peptide V2o has improved antiviral activity in single-roundinfection assays. PM1 cells were infected with replication-incompetent lenti-viral vector particles (packaged with various HIV-1 or SIV envelope glycopro-teins and encoding eGFP) in the presence of increasing concentrations of Cpeptides. eGFP-positive cells were determined by flow cytometry. (A) Data aremeans from duplicates of two independent experiments; error bars show SDs.(B) Data are means from 2 to 11 independent experiments; error bars showSDs.

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HIV-1HxB2. Using rational design, we have introduced amino acidsubstitutions into this C peptide to minimize antigenicity and immu-nogenicity, while preserving or even enhancing antiviral activity.

Over the past years, several C peptides with improved pharmaceu-tical properties, such as enhanced solubility, helical structure, six-helix bundle stability, and antiviral activity, have been engineered (9,12, 13, 39). However, to our knowledge this is the first description ofa deimmunized anti-HIV C peptide. Unwanted clinical immunoge-nicity can reduce the biotherapeutic activity of protein drugs and posesafety risks to patients. Epitope removal by amino acid substitutionhas been successfully used in the past to deimmunize therapeuticproteins, even though complete deimmunization of larger proteindrugs probably remains out of reach. However, the intrinsic antige-nicity and immunogenicity of recombinant therapeutic proteins suchas erythropoietin (40), clotting factor VIII (41), and staphylokinase(43, 44) have been significantly reduced by identification and re-moval of a limited number of immunodominant epitopes.

Both MHC-I epitope prediction programs used in this studyare known to predict many more epitopes than are relevant in vivo(23, 45, 46). Therefore, we concentrated on epitopes predicted byboth algorithms for several HLA alleles and with a high score inorder to engineer the deimmunized C peptide. It is well-knownthat the immunogenicity of peptides strongly correlates with theability to induce a stable MHC-peptide complex (47). Amino acid

substitutions to less preferred residues are expected to destabilizethe MHC-peptide complex due to enhanced dissociation and/ordecreased binding affinity of the modified peptide (47). Conse-quently, anchor amino acids of the predicted major MHC-Iepitopes were exchanged for residues which fit less well into thebinding pockets of the respective HLA molecules in order to pre-vent efficient presentation of the peptide to the immune system.

Although the cellular immunogenicity of the optimized pep-tide V2o could be significantly reduced compared to that of theparental peptide C46-EHO in silico, successful removal of theMHC-I epitopes could not be confirmed experimentally, as noteven the parental peptide C46-EHO induced a strong CTL re-sponse in ELISpot assays with PBMCs from HIV-1-infected indi-viduals. Nevertheless, we clearly demonstrated the feasibility ofour strategy using two dominant viral class I MHC epitopes. Here,amino acid substitutions at the peptide anchor positions P-2 andP-9 led to dramatically reduced CTL activation in vitro, consistentwith the in silico calculations.

The removal of antibody epitopes, however, had a striking effecton the seroreactivity of V2o. While about 10% of HIV-1-infectedpatients had preexisting antibodies to C46-EHO, the engineered pep-tide V2o was no longer recognized by any of the HIV-1-infected pa-tient sera tested, making it a much safer protein drug. Even though itcontains several amino acid exchanges, the V2o peptide sequence is

FIG 5 The deimmunized C peptide V2o is an efficient HIV-1 entry inhibitor. Primary human CD4� T cells were infected with different HIV-1 strains in thepresence of increasing concentrations of C peptide V2o or T-20. Virus replication was analyzed by p24 ELISA at day 6 postinfection. Data are means fromtriplicate samples; error bars show SEMs. The dotted lines represent the detection limit of the p24 ELISA.

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still closely related to HIV-2 sequences. Consequently, all HIV-2-infected patients and most of the SIVmac251-infected rhesus ma-caques had preexisting antibodies to V2o.

Mapping and destruction of antibody epitopes are a sensibleapproach to deimmunize protein therapeutics. However, they willnot necessarily prevent the generation of novel antidrug antibod-ies. The estimated human antibody repertoire diversity is in therange of 1010 to 1011 (48, 49), making it impossible to mutate allpotential antibody epitopes in a therapeutic protein. Here, re-moval of MHC-II epitopes is a more reasonable strategy, as thediversity of HLA molecules comprises only several thousand al-leles (51). However, bioinformatic calculation of MHC-IIepitopes is not as straightforward as prediction of MHC-Iepitopes, as peptides extending the 9-amino-acid core bindingmotif can also be presented by class II HLA molecules. As a result,currently available algorithms for prediction of MHC-II bindingpeptides perform much worse than those for MHC-I epitope pre-diction and are rather unreliable (52–54).

The optimized peptide V2o contains a substantial number ofamino acid substitutions compared with the sequence of parentalpeptide C46-EHO. Nevertheless, using rational design, we were ablenot only to preserve its antiviral activity but also even to enhance itstherapeutic potential: in particular, V2o is active against differentHIV-1 strains (including T-20-resistant strains and strains with re-duced sensitivity to C46) but also inhibits entry of SIVmac251 with anIC50 in the low-nanomolar range. The same efficacy profile was ob-served for the C46-EHO peptide, however, with 2- to 6-fold lowerantiviral activity. In contrast, neither of the HIV-1-derived peptidesT-20 (data not shown) and C46 prevented cell entry mediated by theSIVmac251 envelope glycoprotein at the highest concentrationtested. Yet, efficient inhibition of SIV entry is also a highly desirablefeature of viral fusion inhibitors, as this enables preclinical efficacystudies in the rhesus macaque model system.

The failure of HIV-1-derived C peptides to inhibit HIV-2 orSIV entry has previously been observed by others (55) and is dueto mechanistic differences in the fusion process of HIV-1 andHIV-2/SIV. Gallo and colleagues (55) have shown that C peptideshave a much smaller window of opportunity for binding theirtarget structure during the HIV-2 and SIV fusion process com-pared with that with HIV-1 entry. Thus, inhibition of HIV-2 orSIV fusion by C peptides is generally much harder to achieve (55).

The C46-EHO and V2o peptides are based on the amino acidsequence of the HIV-2EHO strain, which is a highly pathogenicisolate (11, 56). It has previously been shown that the gp41 HR2domain is responsible for enhanced fusion kinetics and the patho-genicity of a highly pathogenic HIV-1 envelope glycoprotein (58).Consequently, the C peptides derived from HIV-2EHO might bind theprehairpin structure more rapidly and thus have the chance to effi-ciently interfere even with six-helix bundle formation of SIV and po-tentially also HIV-2 glycoproteins. In addition, enhanced C-peptidebinding could impede the generation of resistant virus strains.

In summary, we generated a highly and broadly active, deimmu-nized C peptide for therapy of HIV infection. Its superior antiviralactivity along with the decreased immunogenicity makes V2o an at-tractive antiviral peptide for further clinical development.

ACKNOWLEDGMENTS

This study was sponsored by the project iGene funded by the GermanFederal Ministry for Research (BMBF). F.B. was supported by a scholar-ship from the Deutsche Forschungsgemeinschaft (DFG; GRK1172).

We thank Janine Kimpel, Catherine Dold, Felix G. Hermann, and Chris-toph Leder for support and critical discussions, Jan van Lunzen for providingHIV-1 patient sera and PBMC samples, as well as Ursula Dietrich andSchlomo Staszewski for providing HIV-1 and HIV-2 patient sera.

D.V.L. is inventor of membrane-anchored C peptides and participatorin the biotech company Vision7 GmbH, which holds intellectual propertyon membrane-anchored anti-HIV C peptides. L.E. and D.V.L. are listed asinventors on a patent application related to secreted C peptides. For theother authors, no competing financial interests exist.

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