Adeno-associated virus vectors serotype 2 induce prolonged proliferation of capsid-specific CD8+ T cells in mice

Abstract

Using adoptive transfer models we determined that an adeno-associated viral vector of serotype 2 (AAV2) induces in mice proliferation of CD8(+) T cells that recognize an epitope within the viral capsid. Proliferation to an endogenous epitope within viral protein (VP)3 could be observed for at least 3 weeks while a foreign epitope placed at multiple copies within VP2 elicited CD8(+) T cell expansion for at least 10 weeks. These data show that capsid antigens of AAV2 degrade slowly over a period of weeks and during this period provide targets to CD8(+) T cells.

Figures

Figure 1

Adeno-associated viral vector of serotype 2 (MOVA) expressing hF.IX under a hepatocyte-specific promoter [AAV2(MOVA)- hF.IX]. (a) AAV-helper plasmids expressing AAV2(MOVA) capsid consist of one plasmid expressing the AAV2 viral protein 1 (VP1) and VP3 protein with a mutated VP2 start codon and another expressing the AAV2 VP2 protein with mutated VP1 and VP3 start codons. Five copies of SIINFEKLAA sequences were inserted into the VP2-expressing plasmid right after the VP2 start codon. (b) The expression of VP1, VP2, and VP3 proteins of the AAV2(MOVA)-hF.IX vector and the AAV2-hF.IX vector was tested for by western blot. (c) C57BL/6 mice were intramuscularly injected with 2 × 1011 vector genomes of AAV2(MOVA)-hF.IX or 1 × 109 virus particles of AdC68-NP.OVA.GFP. After 14 and 10 days respectively, peripheral blood mononuclear cells were isolated and tested by intracellular cytokine staining for interferon-γ (IFN-γ).

Figure 2

Proliferation of adeno-associated viral vector of serotype 2 (AAV2) capsid-specific memory CD8+ T cells in AAV2 vectors expressing hF.IX under a hepatocyte-specific promoter (AAV2-hF.IX) transduced mice. Thy1.1 BALB/c mice were intravenously injected with 2 × 1011 vector genomes of AAV2-hF.IX. Mice then received 5 × 107 carboxyfluorescein succinimidyl ester-labeled splenocytes from the Thy1.2 BALB/c mice, which had been immunized with 1 × 1011 virus particles of adenovirus human serotype 5 vectors expressing capsid antigens of AAV2 under a cytomegalovirus promoter (Ad-AAV2cap) at least 4 months earlier, at day 2, week 3 and week 6 following AAV2 transfer. (a) The proliferation of Thy1.2+CD8+AAV2-tet+ cells was tested 10 days later from the spleens of the recipient mice. Control mice did not receive AAV2-hF.IX before the adoptive transfer of splenocytes. Each group contains two to three control mice, results for one of those are shown. Similar results were obtained with the other control mice. (b) The average number of division cycle (proliferation index) and the percentage of cells of the original sample which divided (% divided) were calculated. Error bars represent standard deviation (SD) for 2–3 mice per group.

Figure 3

Proliferation of splenic CD8+ T cells from OT-1 mice in adeno-associated viral vector of serotype 2 (MOVA) vectors expressing hF.IX under a hepatocyte-specific promoter [AAV2(MOVA)-hF.IX]transduced mice. Thy1.1 C57BL/6 mice were intravenously injected with 5 × 1011 vector genomes of AAV2(MOVA)-hF.IX. Mice then received 5 × 106 CFSE-labeled splenocytes from OT-1 mice at day 2, week 2, week 10 and week 16 following AAV2 transfer. (a) The proliferation of Thy1.2+CD8+Vα2+ cells in spleens of individual mice and pooled lymphocytes isolated from livers was tested 10 days later. Control mice did not receive AAV2(MOVA)-hF.IX before splenocytes were adoptively transferred. Results are shown for one of the three control mice; similar results were obtained with the other control mice. (b) The average number of division cycle (Proliferation Index) and the percentage of cells of the original sample which divided (% divided) were calculated. Error bars represent SD for two to three mice per group. The proliferation of cells from liver was tested with pooled samples; thus p values were not calculated for the liver samples.

Figure 4

The stability of adeno-associated viral vector of serotype 2 (MOVA) vectors expressing hF.IX under a hepatocyte-specific promoter [AAV2(MOVA)-hF.IX] vector. Thy1.1 BALB/c mice were intravenously injected with 2 × 1011 vector genomes of AAV2(MOVA)-hF.IX or AAV2-hF.IX. On day 2, mice received 5 × 107 CFSE-labeled splenocytes from Thy1.2 BALB/c mice, which had been immunized with 1 × 1011 virus particles of adenovirus human serotype five vectors expressing capsid antigens of AAV2 under a cytomegalovirus promoter (Ad-AAV2cap) at least 4 months earlier. (a) Proliferation of Thy1.2+CD8+AAV2-tet+ cells was tested 10 days later using lymphocytes isolated from spleens. The control mice were intravenously injected with phosphate-buffered saline before splenocytes were adoptively transferred. One control mouse is shown here and similar results were obtained in other control mice. (b) The average number of division cycle (proliferation index) and the percentage of cells of the original sample which divided (% divided) were calculated. Error bars represent SD for two to three mice per group.

Figure 5

Proliferation of splenic CD8+ T cells from OT-1 mice in dendritic cell depleted adeno-associated viral vector of serotype 2 (MOVA) vectors expressing hF.IX under a hepatocyte-specific promoter [AAV2(MOVA)-hF.IX]-injected recipient mice. (a) B6.FVB.TG mice were injected with DT and splenocytes were isolated the next day. CD11c+ dendritic cells were tested from spleens of mice that were or were not treated with diphtheria toxin (DT). (b) B6.FVB.TG mice were injected with 5 × 1011 vector genomes of AAV2(MOVA)-hF.IX. Two days later, some mice were injected with 4 ng/g DT to deplete dendritic cells. 4–5 hours after DT treatment, mice received 5 × 106 CFSE-labeled splenocytes from OT1-Thy1.1 mice. Proliferation of Thy1.1+CD8+Vα2+ cells in mouse spleens was measured 3 days later. (c) The percentage of cells of the original sample which divided (% divided) and the average number of division cycle (proliferation index) were calculated. Error bars represent SD for two to three mice per group. Negative control mice were injected with splenocytes from OT1-Thy1.1 mice but did not receive AAV2(MOVA)-hF.IX.

Figure 6

Proliferation of splenic CD8+ T cells from OT-1 mice in mice transplanted with hepatocytes from AAV2(MOVA)-hF.IX transduced mice. Thy1.1 C57BL/6 mice were injected with 5 × 106 CFSE-labeled splenocytes from OT-1 mice. The following day, mice then received 1 × 107 hepatocytes from Thy1.2 C57BL/6 mice, which had been injected with either 5 × 1011 vector genomes of AAV2(MOVA)-hF.IX or AAV-hF.IX, or 1 × 1011 virus particles of AdC68-NP.OVA.GFP or AdC68-rab.gp 1 week earlier. Proliferation of Thy1.2+CD8+Vα2+ cells was tested 10 days later. (a–c) The pooled results from two to three mice. (d–f) The results for three individual mice, which received hepatocytes from mice transduced with AAV2(MOVA)-hF.IX. (g) The average number of division cycle (proliferation index) and (h) the percentage of cells of the original sample which divided (% divided) were calculated. Error bars represent SD for two to three mice per group.