A rationally engineered anti-HIV peptide fusion inhibitor with greatly reduced immunogenicity

Abstract

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

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-terminal fusion peptide (FP), two leucine zipper-like heptad repeat domains (HR1 and HR2), the membrane-proximal external region (MPER), the transmembrane domain (TM), and the intracellular domain (Intra). HIV entry-inhibitory C peptides C46 and C46-EHO are derived from the HR2 of HIV-1_HxB2 and HIV-2_EHO/HIV-1_HxB2, respectively. Variants V1 to V4 and V2o are based on C46-EHO but contain several amino acid substitutions, as indicated. (B) Flowchart of V2o peptide development based on C34-EHO.

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 that was predicted for any HLA type is indicated with its respective score. Scores below 20 (SYFPEITHI; A) and 10 (BIMAS; B) are not shown.

Fig 3

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

Fig 4

Mutated peptide V2o has improved antiviral activity in single-round infection assays. PM1 cells were infected with replication-incompetent lentiviral vector particles (packaged with various HIV-1 or SIV envelope glycoproteins and encoding eGFP) in the presence of increasing concentrations of C peptides. eGFP-positive cells were determined by flow cytometry. (A) Data are means from duplicates of two independent experiments; error bars show SDs. (B) Data are means from 2 to 11 independent experiments; error bars show SDs.

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 the presence of increasing concentrations of C peptide V2o or T-20. Virus replication was analyzed by p24 ELISA at day 6 postinfection. Data are means from triplicate samples; error bars show SEMs. The dotted lines represent the detection limit of the p24 ELISA.