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. 2021 Jun 21;12:702705. doi: 10.3389/fimmu.2021.702705

Figure 1.

Figure 1

(A) Schematic diagram of Recombinant OPVs: orange boxes illustrate the SHIV recombinant antigens that are ultimately produced by OPV. The SIVmac239CA-p6 sequence was inserted between two 2A protease cleavage sites in pSabin2-eGFP, replacing gfp to generate SIVmac239CA-p6-OPV. The corresponding protein becomes expressed intracellularly once the cleavage of the OPV polyprotein occurs. The HIVBG505 Env C1-V2 region was cloned linked to tPA signal peptide that permits its secretion to generate HIVBG505C1-V2-OPV. The recombinant fragment amino acid sequence, starting and ending with polio protease cleavage sequences, with polio protease TTY/G and T2A NPG/P cleavage indicated by a bar, and with HIV Env sequence underlined, is the following: GLTTY/GFGHGGGGGGSRLEGSGEGRGSLLTCGDVEENPG/PMDAMKRGLCCVLLLCGAVFVSASAENLWVTVYYGVPVWKDAETTLFCASDAKAYETEKHNVWATHACVPTDPNPQEIHLENVTEEFNMWKNNMVEQMHTDIISLWDQSLKPCVKLTPLCVTLQCTNVTNNITDDMRGELKNCSFNMTTELRDKKQKVYSLFYRLDVVQINENQGNRSNNSNKEYRLINCNTSATQACPKVSFHHHHHHVDGLTTY/GFGH. (B) Stability of passaged recombinant OPVs: RT-PCR to detect recombinant OPV expression in RNA of infected cells after virus passage. Selective passages from 0 to 12 are reported. (C) Left top panel: Western blot of cell lysates from 293T cells: non-infected (lane 2), infected with Sabin2-eGFP (lane 3), transfected with SIVmac239 DNA (lane 4), infected with SIVmac239CA-p6-OPV (lanes 5-9, harvested at 12 to 48 hrs after infection). A SHIV-infected monkey serum was used as primary antibody. Left bottom panels: flow cytometric analysis of 293T infected cells stained with an anti-SIV p27 antibody (unstained, DAPI stained, DAPI+anti-p27 panels). Right top panel: Detection of the HIVBG505C1-V2 fragment by Western blot, probed with anti-HIS mAb: mAb PG16 (lane 2); mAb PG9 (lane 3), Protein A (lane 4), HIVBG505C1-V2 fragment, purified from tissue culture supernatant after HIVBG505C1-V2-OPV infection and immunoprecipitated with NAb PG9 (lane 5) or with NAb PG16. (lane 6), purified HIVBG505C1-V2 (lane 7). Right bottom panels: flow cytometric analysis of 293T infected cells stained with an anti-HIS mAb (unstained, DAPI alone staining, DAPI+anti-HIS staining panels). (D) Growth curve of recombinant OPVs in 293T cells. After 293T cell infection at 0.1 MOI with Sabin2-eGFP, SIVmac239CA-p6-OPV and HIVBG505C1-V2-OPV, supernatants were harvested at time points indicated on the X axis and the corresponding titer, obtained by TCID50 evaluation, is reported on the Y axis.