Fig 1.
DNA and MVA constructs and their expression.
(A) Schematic for the expression cassette of the DNA vaccine expressing 505 T/F sequences. CMVIE+IA, CMV-immediate early promoter plus intron A; BGHpA, bovine growth hormone polyadenylations sequences; gp120 and gp41, HIV sequences encoding the receptor binding and transmembrane subunits of Env; gag, HIV sequences encoding the group-specific antigens of HIV; X, inactivating point mutations in the zinc fingers for packaging of HIV RNA. (B) Schematic of MVA expressing MVA-T/F sequences. I8R and G1L, conserved vaccinia sequences flanking the insertion site for the T/F env; A50R and B1R, conserved sequences flanking the insertion site for the T/F gag; PmH5, a modified immediate early H5 vaccinia promoter; gp41t, gp41 truncated at amino acid 36 of the endodomain. Numbers indicate positions in the MVA genome which is abbreviated and not to scale. (C) Western blot of MVA-expressed Gag and Env proteins. Supernatants and cell lysates were collected from 293T cells infected with the indicated recombinant MVA vaccines or the parental MVA (P), separated by SDS-PAGE, and western blotted for detection of Gag and Env.
Table 1.
Immunization schema1.
Fig 2.
Electron micrographs of VLPs expressed by the DNA and MVA vaccines.
Thin section electron micrographs were immunogold stained for Env using the PGT145 and PGT151 Ab that bind native trimers (see methods). The DNA vaccine is expressed in transiently transfected 293T cells and the MVA vaccine in infected DF1 cells. The VLPs being analyzed and nanometer (nm) size markers are indicated in the panels. Arrows, indicate examples of immunogold staining on VLPs. The triangles point to examples of immunogold staining on the plasma membranes of vaccine infected cells.
Fig 3.
Antigenicity of Env expressed by the 505 vaccines.
The vaccines being tested are indicated at the top of the schematic and the recombinant Ab and their specificities on the left side. See Methods for procedures.
Fig 4.
(A) Patterns of binding Ab for the 3 immunization groups. Vertical dotted lines indicate immunizations. The horizontal dashed line compares troughs across immunizations. (B) Temporal peak titers of neutralizing Ab compared to peak titers of binding Ab (gray dashed line) in Group 1 animals. The patterns for A11R011 and A10L002 are typical for all animals that did not develop nAb to the CD4bs. (C). Mapping of neutralizing activity using mutants that knock out the V1V2 target for bnAb (N160A and N160A.N173A); the CH0505 target for bnAb to the CD4bs (N280D and G458Y); and the V3-glycan target for bnAb (N301A and N334A).