Safety and efficiency modifications of SIV-based integrase-defective lentiviral vectors for immunization

Abstract

Integrase-defective lentiviral vectors (IDLVs) represent an attractive platform for vaccine development as a result of the ability to induce persistent humoral- and cellular-mediated immune responses against the encoded transgene. Compared with the parental integrating vector, the main advantages for using IDLV are the reduced hazard of insertional mutagenesis and the decreased risk for vector mobilization by wild-type viruses. Here we report on the development and use in the mouse immunogenicity model of simian immunodeficiency virus (SIV)-based IDLV containing a long deletion in the U3 region and with the 3′ polypurine tract (PPT) removed from the transfer vector for improving safety and/or efficacy. Results show that a safer extended deletion of U3 sequences did not modify integrase-mediated or -independent integration efficiency. Interestingly, 3′ PPT deletion impaired integrase-mediated integration but did not reduce illegitimate, integrase-independent integration efficiency, contrary to what was previously reported in the HIV system. Importantly, although the extended deletion in the U3 did not affect expression or immunogenicity from IDLV, deletion of 3′ PPT considerably reduced both expression and immunogenicity of IDLV.

Different configurations of SARS-CoV-2 spike protein delivered by integrase-defective lentiviral vectors induce persistent functional immune responses, characterized by distinct immunogenicity profiles

“…We hypothesized that presence of different Spike conformations on lentiviral particles impacted on ensuing immune response after immunization, similarly to what has been shown with Ad26 vector ( 63 ). For immunogenicity studies in the mouse model, we engineered a series of lentiviral transfer vectors (pGAE) ( 44 ) expressing different membrane-tethered configurations derived from Wuhan-Hu-1 SARS-CoV-2 Spike protein coding sequence (GenBank: NC_045512.2) ( Table 1 and Supplementary Figure 1A ), including native full-length wild-type Spike (aa 1-1273, pGAE-Spike), full-length Spike with mutated FCS (RRAR to GSAS) and 2P substitutions (pGAE-S2PF), delta21 wild-type Spike with D614G mutation and 2P substitutions (aa 1-1252, pGAE-S2PGC) and delta21 wild-type Spike with D614G mutation, 2P substitutions and mutated FCS (pGAE-S2PFGC). The Beta (B.1.351) Spike sequence with delta21 and 2P substitutions (pGAE-betaS2PGC) was also generated to assess cross-neutralizing responses compared to the related Wuhan-based vaccine.…”

“…Spike sequences were removed from pUC57 plasmids with AgeI/SalI restriction sites and cloned into the corresponding restriction sites of pGAE-GFP self-inactivating lentiviral transfer vector plasmid by substituting the GFP coding sequence ( 43 ) ( Supplementary Figure 1A ). Plasmid pGAE-Spike encodes the codon optimized full-length wild type SARS-CoV-2 Spike protein open reading frame (ORF) (Wuhan-Hu-1, GenBank: NC_045512.2); plasmid pGAE-S2PF encodes the Spike ORF, stabilized by the introduction of 2 prolines (2P, K986P and V987P) and by functional mutation of RRAR into GSAS at the furin cleavage site (FCS); plasmid pGAE-S2PGC encodes the Spike ORF and contains a 21 amino acid (aa) deletion in the cytoplasmic tail (delta21) with 2P and D614G mutations; plasmid pGAE-betaS2PGC encodes the delta21 codon optimized B.1.351 (beta) Spike ORF with the 2P stabilizing mutation; plasmid pGAE-S2PFGC encodes the delta21 Spike ORF with the 2P, D614G and FCS mutations; plasmids pGAE-JR, pGAE-Luc and pGAE-GFP encode the HIV-1 JR-FL gp120 envelope, the luciferase and the GFP ORFs, respectively ( 44 ). Plasmids pAdSIVD64V and pAdSIV3+ and are the Integrase defective and Integrase competent packaging vectors, respectively ( 43 ).…”

“…Spike variants pseudotyped lentiviral vectors expressing Luciferase (LV-Luc) were generated by transient transfection of 293T Lenti-X cells as previously described ( 40 , 45 , 46 ). In brief, 293T Lenti-X cells (3.5x10 6 cells) were plated on 10 cm Petri dishes (Corning) and transfected with the lentiviral transfer vector plasmid pGAE-Luc expressing the luciferase coding sequence ( 44 ), the packaging plasmid pAd-SIV3+ and each of the pseudotyping plasmid expressing Spike protein described above ( Supplementary Figure 1B ) or control VSV.G (phCMV-VSV.G) utilizing the JetPrime transfection kit (Polyplus Transfection, Illkirch, France) using a plasmid ratio of 1:2:1 (transfer vector: packaging plasmid: Spike/VSV.G plasmid). Forty-eight hours post transfection, the supernatants containing the LV-Luc pseudoviruses were collected, filtered with a 0.45 μm pore size filter (Millipore) and stored in 0.5 mL aliquotes at -80°C.…”

Intranasal Lentiviral Vector-Mediated Antibody Delivery Confers Reduction of SARS-CoV-2 Infection in Elderly and Immunocompromised Mice

“…Another benefit achieved with rSIV.F/HN vectors is the longterm transgene expression due, in part, to the integration of its transgene expression cassette within the host genome safe harbor (10,14). Where required, the rSIV.F/HN vector can be engineered and developed to be integrase defective (IDLVs) to reduce the potential risk of insertional mutagenesis (33). Uniquely, rSIV.F/HN vectors can be effectively administered repeatedly (10,12) allowing therapeutic antibody levels to be boosted if required and the addition of alternate ultrapotent single antibodies or antibody cocktails to counter newly emerging pathogenic variants and to counter immune escape.…”

Integrase deficient lentiviral vector: prospects for safe clinical applications

“…Another investigation of the extent of deletion in the transfer vector U3 region showed no influence in the integrase-mediated or -independent integration efficiency while removing such viral elements ( Bona et al, 2021 ). Novel strategies to inactivate the integration reaction, such as new mutations affecting the involving elements are essential to further improve its safety of usage in clinical application, thus corroborating little to no side effects ( Banasik & McCray, 2009 ).…”