1. Baculovirus GP64-pseudotyped HIV-based lentivirus vectors are stabilized against complement inactivation by codisplay of decay accelerating factor (DAF) or of a GP64– DAF fusion protein 
Ghiabe H. Guibinga, Theodore Friedmann 
Molecular Therapy 
Volume 11, Issue 4, Pages (April 2005)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
Construction and immunoblot detection of native GP64 and the fusion GP64–DAF envelope protein. (A) A full-length GP64 coding sequence synthesized as a 1.6-kb fragment from baculovirus DNA by PCR using previously described primers [9] was ligated into the EcoRI site of the VSV-G expression plasmid from which the full-length VSV-G coding sequence had been excised by digestion with EcoRI. The resulting construct PCMVG64 was further modified by in-frame introduction of a multicloning site containing restriction sites for PstI, KpnI, and SmaI into a position corresponding to amino acid 20 of GP64 [20], to generate PCMVG64-M. PCMVG64-M was further modified to contain the human complement-inhibiting decay accelerating factor (DAF) by ligation of the PCR-amplified fragment of the expression plasmid pCMV-SPORT-DAF (Open Biosystem, Huntsville, AL, USA) encoding a functional domain of human DAF spanning amino acids 1–320 [17] into the PstI site of pCMVG-64-M. The integrity of the DAF domain within PCMVG-64-DAF was confirmed by nucleotide sequencing. (B) Western immunoblotting with anti-gp64 monoclonal antibody. Lane 1, vector pseudotyped with native GP64 envelope protein; lane 2, vector pseudotyped with the multicloning variant of GP64 (PCMVG64-M) in the absence of the DAF insert; lane 3, vector pseudotyped with PCMV64-DAF; and lane 4, vector pseudotyped with both GP64 and GP64–DAF (hybrid envelope protein). Virus pellet containing only GP64 or its M variant demonstrated an immunoreactive band consistent with a molecular weight of approximately 64 kDa. Virus preparations pseudotyped with the fusion protein GP64–DAF demonstrated the larger band consistent with the expected size of approximately 98 kDa. Molecular weights were estimated from the motilities of size markers (Invitrogen, Carlsbad, CA, USA) indicated on the ordinate. The consistency of sample loading was estimated by reblotting of the stripped membrane with antibody to HIV-1 p24. (C) Western immunoblotting of native DAF and pseudotyped vector pellets with anti-DAF antibody. Lane 1, tissue culture supernatant of 293T cells transfected with DAF expression vector pCMV-SPORT-DAF; lanes 2 and 3, vector preparations pseudotyped with fusion protein GP64–DAF alone or native GP64 and fusion protein GP64–DAF together, respectively; lane 4, vector pseudotyped with native GP64 alone. Preparations assembled in the presence of the fusion protein demonstrated a high-molecular-weight DAF-reactive protein consistent with the presence of the fusion protein in the virus particles. All pseudotyped vectors were prepared by established triple transfection protocols [27–30]. Virus pellets were subjected to sodium dodecyl sulfate–15% polyacrylamide gel electrophoresis. GP64, HIV-1 p24, and DAF proteins were detected with anti-GP64 (eBioscience, San Diego, CA, USA), anti-p24 (Biodesign International, Saco, ME, USA), and anti-CD55 (DAF) (Biomeda, Foster City, CA, USA) monoclonal antibodies, respectively.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Infectivity of HIV-1 vectors pseudotyped with GP64 variants and VSV-G. Virus titers were determined by end-point dilution assay on concentrated preparations as previously reported [27–30] and are expressed as means ± standard error of the experiment performed in triplicate. Vectors were pseudotyped with GP64 alone, with the GP64-M variant containing the multicloning site, with the fusion protein GP64-DAF, with both the native GP64 and the fusion protein GP64–DAF, and with VSV-G.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
Stability of HIV-based vector particles in human and nonhuman primate sera. In all assays, virus titers were determined using the end-point dilution method on 293T cells. (A) Vector pseudotyped with native GP64 alone exposed to three human and three nonhuman primate sera. Striped bars indicate preparations incubated with non-heat-inactivated sera while black bars indicate samples incubated with heat-inactivated sera. All titers are normalized to control preparations incubated with DMEM–10% FCS (white bars). (B) Vector preparations pseudotyped with both GP64 and fusion GP64–DAF. The results represent three independent experiments performed in triplicate and are displayed as mean values ± SE. Assays for serum complement inactivation were carried on normal human serum complement obtained from pooled donors (Quidel Corp., San Diego, CA, USA). Also, sera from individual donors, taken from humans and nonhuman primates (baboon, chimpanzee, and rhesus monkey), with normal complement activity were obtained from BioReclamation, Inc. (Hicksville, NY, USA). To evaluate serum inactivation, each of the pseudotyped HIV-1 vector preparations described above was diluted with an equal volume of normal serum, heat-inactivated serum (1 h at 56°C), or 10% FCS in DMEM (control) and incubated for 1 h at 37°C. Following incubation, DMEM was added to the reaction for an additional 10-fold dilution, and the resulting preparations were assayed by end-point titration. The residual infectivity of vector preparations treated with complement was normalized to the titer of vector exposed to control DMEM–10% FCS. (C) Western blot analysis with anti-DAF of HIV-1 vectors pseudotyped by GP64 alone, fusion protein GP64–DAF alone, or GP64 in the presence of DAF protein. Lane 1, pelleted vector pseudotyped with native GP64 in 293T cells also expressing full-length DAF from the plasmid pCMV-SPORT-DAF, demonstrating DAF incorporation into virus particles; lane 2, vector pseudotyped with GP64 alone; lane 3, vector pseudotyped with fusion protein GP64–DAF demonstrating the 100-kDa fusion protein. (D) Complement inactivation assay of HIV-1 vectors pseudotyped with GP64 alone, with fusion protein GP64–DAF, and with GP64 in the presence of full-length DAF expressed from pCMV-SPORT-DAF. The production of GP64-pseudotyped lentivirus preparations containing packaged DAF protein was obtained by triply transfecting the 293T cells as described previously [27–30], but also adding 4 μg of the plasmid pCMV-SPORT-DAF to the transfection mix. All titers were normalized to control preparations incubated with DMEM–10% FCS (white bars) and are shown for vectors incubated with non-heat-inactivated sera (striped bars) and heat-inactivated sera (black bars). The results represent three independent experiments performed in triplicate and the results are displayed as mean values ± SE.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions