Evaluation of cross-reactive cell-mediated immune responses among human, bovine and porcine adenoviruses

Abstract

The absence of preexisting immunity against porcine adenovirus (Ad) serotype 3 (PAd3) and bovine Ad serotype 3 (BAd3) in humans makes them attractive alternatives to human Ad serotype 5 (HAd5) vectors. To determine whether there is significant cross-reactivity among HAd5, BAd3 and PAd3 at the level of cell-mediated immune responses, BALB/c mice were inoculated intraperitoneally with wild-type (WT) or replication-defective (RD) HAd5, BAd3 or PAd3. After 35 days of the first inoculation, cross-reactive CD8+ cytotoxic T cells, as well as CD4+ Th1- and Th2-helper T cells, in the spleen were analyzed by enzyme-linked-immunospot, flow cytometry and cytotoxic T lymphocyte assays. Virus-neutralization assays were used to evaluate humoral cross-reactivity. CD8+ or CD4+ T cells primed with WT or RD HAd5, PAd3 or BAd3 showed significant (P<0.005) reactivity with homologous Ad antigens, whereas only minimal cross-reactivity was observed on stimulation with heterologous Ad antigens. Ad-neutralizing antibodies were found to be homologous Ad specific. Overall, these results suggest that there is no significant immunological cross-reactivity among HAd5, BAd3 and PAd3, thereby supporting the rationale for the use of BAd3 and PAd3 as alternative HAd vectors to circumvent anti-HAd immunity in humans.

Figure 1. Cross-reactivity of positively selected CD4+ splenocytes from HAdΔE1E3-, PAdΔE1E3-, BAdΔE1E3-, HAd-WT-, PAd-WT-, BAd-WT-, or mock-inoculated mice

Splenocytes were positively selected with anti-CD4 monoclonal antibody-coated magnetic beads and were stimulated with HAdΔE1E3-, PAdΔE1E3-, BAdΔE1E3-, or mock-infected cell lysate for 20 h. The number of cells expressing IFNγ (A) or IL-4 (B) was measured by enzyme-linked immunospot (ELISPOT) assay. (C) The percentage of CD4+ cells expressing IFNγ was measured by flow cytometry assay. Values are reported as the average ± standard deviation for five animals per group. *P < 0.005 versus values at mock stimulation within each treatment group. †P < 0.005 for homologous stimulation versus heterologous stimulation within each treatment group.

Figure 2. Cross-reactivity of positively selected CD8+ splenocytes from HAdΔE1E3-, PAdΔE1E3-, BAdΔE1E3-, HAd-WT-, PAd-WT-, BAd-WT- , or mock-inoculated mice

(A) Splenocytes were positively selected with anti-CD8 monoclonal antibody-coated magnetic beads and were stimulated with HAdΔE1E3-, PAd3ΔE1E3-, BAd3ΔE1E3-, or mock-infected and chemically inactivated syngeneic NIH3T3 stimulator cells. The number of cells expressing IFNγ was measured by enzyme-linked immunospot (ELISPOT) assay. (B) The percentage of CD8+ cells expressing IFNγ was measured by flow cytometry. (C) Neutral red uptake cytotoxic T lymphocyte (CTL) assay. Positively selected CD8+ cells from HAdΔE1E3-, PAdΔE1E3-, BAdΔE1E3-, HAd-WT-, PAd-WT-, BAd-WT-, or mock-inoculated mice were stimulated with HAdΔE1E3-, PAd3ΔE1E3-, BAd3ΔE1E3-, or mock-infected and chemically inactivated syngeneic NIH3T3 stimulator cells for 7 days. The target cells were HAdΔE1E3-, PAd3ΔE1E3-, BAd3ΔE1E3-, or mock-infected NIH3T3 cells. The effector to target cell ratio was 5:1. Values are reported as the average ± standard deviation for five animals per group. *P < 0.005 versus values at mock stimulation within each treatment group. †P <0.005 for homologous stimulation versus heterologous stimulation within each treatment group.