Persisting presence of vector sequences. (A) Adult female outbred (ICR) mice received 10¹¹v.p.AdC68rab.gp vector in saline, given once intramuscularly. On days 4, 30, 90, and 360 after vector application, mice were killed and perfused with cold PBS. Tissues were harvested from individual mice. DNA was isolated and the Gapdh sequences were amplified by a real-time PCR. The samples were adjusted to 10³ copies of Gapdh and the rabies virus glycoprotein gene was amplified by a real-time nested PCR. (B) Mice were vaccinated intramuscularly in the lower leg with 10¹¹ particles of an Ad vector expressing green fluorescent protein. Mice were killed 1 day and 39 days after vaccination and legs were illuminated with an Illumatool Lighting System. Digital photographs were taken using a Kodak DCS14N SLR camera with a 60-mm Micro Nikkor lens (Nikon). (C) DNA and RNA were isolated from spleens of mice immunized at different times previously with 1011 vp of AdC8gag37. RNA was reverse-transcribed. Gapdh was quantified from each sample by real-time PCR.¹⁷  Samples were adjusted before amplification to 6 × 10⁷ or 1.5 × 10⁹ copies of Gapdh DNA or cDNA, respectively, and amplified by a nested PCR. After amplification by the internal real-time PCR, each sample was analyzed by gel electrophoresis. The lower graph shows results for Gag DNA or cDNA. The arrow indicates the anticipated size of the Gag amplicon. These results are from a single experiment in which we ran multiple gels to accommodate the samples. Lines were added to show where the lanes were cut. (D) Mice were immunized with AdC68gag37. Twenty months later they were boosted with 10⁶ pfu of a vaccinia virus vector expressing Gag to increase frequencies of Gag-specific CD8⁺ T cells. In pre-experiments it was shown that Gag sequences from the vaccinia virus vector could not be amplified from spleens as of 1 week after inoculation. Five weeks after the boost, splenocytes were isolated. Cells were stained with a Gag-specific tetramer (tet) and an antibody to CD8. Cells were sorted into CD8⁺tet⁻ cells, CD8⁻tet⁻ cells and CD8⁺tet⁺ cells. Total cellular RNA was isolated and purified from each cell fraction. Complementary DNA was synthesized. The HIV Gag gene in each cell fraction was amplified first by regular PCR. The amplicon from the first PCR product was then used as template for a second real-time PCR to quantify the Gag gene in different cell fractions. The copy numbers of Gag in each cell fraction were normalized in comparison to Gapdh sequences quantified by a real-time PCR from the same samples. (E) Monkeys 18, 48, 140, and 145 were immunized intramuscularly with 10¹² vp of AdC7gag37 vector and boosted 8 months later with 10¹² vp of AdC6gag37 vector. Animal 164 was injected at the same time with the same vector backbone expressing the rabies virus glycoprotein.²¹  DNA from peripheral blood mononuclear cells harvested 99 days after the boost was tested for gag DNA as described in panel A on adjustments of samples to 3 × 10⁴ β-actin molecules. These results are from a single experiment; the lanes were rearranged to change the order of the samples. Lines were added to show where the lanes were cut. (F) The graph shows the gels from a hexon-specific nested PCR that was used to amplify vector sequences from peripheral blood mononuclear cells of vaccinated rhesus macaques. Lanes 1 and 2 show results from peripheral blood mononuclear cells harvested 6 (1) and 17 (2) weeks after intramuscular vaccination of a monkey with 10¹¹ vp of a mixture of 4 AdC68 vectors expressing HIV-1 Gag, gp140, 5′pol, or 3′pol⁺nef. Lanes 3 and 4 show results from peripheral blood mononuclear cells from a monkey harvested 6 (3) and 17 (4) weeks after intramuscular immunization with 10¹¹ vp of 4 AdHu5 vectors expressing the same antigens as used for results in lanes 1 and 2. Lanes 5 through 8 show results from peripheral blood mononuclear cells from 2 animals harvested 2 (5,7) and 14 (6,8) weeks after immunization with 10¹¹ vp of an AdHu5 vector expressing Gag. Lanes 9 and 10 show the negative control of the PCR reactions.