The immunological impact of adenovirus early genes on vaccine-induced responses in mice and nonhuman primates

Abstract

Adenovirus (Ad) is being explored for use in the prevention and treatment of a variety of infectious diseases and cancers. Ad with a deletion in early region 3 (ΔE3) provokes a stronger immune response than Ad with deletions in early regions 1 and E3 (ΔE1/ΔE3). The ΔE1/ΔE3 Ads are more popular because they can carry a larger transgene and because of the deleted E1 (E1A and E1B), are perceived safer for clinical use. Ad with a deletion in E1B55K (ΔE1B55K) has been in phase III clinical trials for use in cancer therapy in the US and has been approved for use in head and neck tumor therapy in China, demonstrating that Ad containing E1A are safe for clinical use. We have shown previously that ΔE1B55K Ad, even while promoting lower levels of an inserted transgene, promoted similar levels of transgene-specific immune responses as a ΔE3 Ad. Products of the Ad early region 4 (E4) limit the ability of cells to mount an innate immune response. Using this knowledge, we deleted the Ad E4 open reading frames 1-4 (E4orf1-4) from the ΔE1B55K Ad. Here, we show that innate cytokine network genes are elevated in the ΔE4 Ad-infected cells beyond that of ΔE3 Ad-infected cells. Further, in immunized mice the IgG2a subclass was favored as was the IgG1 subclass in immunized nonhuman primates. Thus, Ad E4 impacts immune responses in cells, in immunized mice, and immunized nonhuman primates. These Ad may offer advantages that are beneficial for clinical use.Importance: Adenovirus (Ad) is being explored for use in the prevention and treatment of a variety of infectious diseases and cancers. Here we provide evidence in cells, mice, and nonhuman primates supporting the notion that Ad early gene-products limit specific immune responses. Ad constructed with deletions in early genes and expressing HIV envelope protein was shown to induce greater HIV-specific cellular immune responses and higher titer antibodies compared to the parental Ad with the early genes. In addition to eliciting enhanced immunity, the deleted Ad possesses more space for insertion of additional or larger transgenes needed for targeting other infectious agents or cancers.

FIG 1

Characterization of Ad5 HIV vaccine candidates in infected human lung epithelial cells. (A) Adenovirus genome, with “X” indicating deleted genes and ITR inverted terminal repeat. (B to F) A549 cells were infected at an MOI of 5 or 50 for 24, 48, and 72 h. (B) Previously described E4orf1, 1-2, 1-3, E4orf4, and E1B55K primers (24, 37) were used in PCR experiments to reveal the presence or absence of the indicated genes. (C) PCR for a segment of the Ad fiber gene was performed. (D) Plaques were counted, and mean values ± standard errors of the means (SEM) are plotted. (E and F) The static levels of Ad DNA binding protein (DBP), actin, and Ad late proteins (hexon, penton, and fiber) (E) and levels of gp120 (F) were assessed by Western blotting. All experiments were repeated at least 3 times. Representative examples are shown in panels B, C, E, and F. P values were obtained by ANOVA after Bonferroni correction.

FIG 2

Expression levels of innate cytokine network genes in Ad-infected human epithelial cells. A549 cells were infected at an MOI of 50 with ΔE3 or ΔE4 Ad for 48 h. Total RNA was isolated and reverse transcribed to cDNA. The fold change was determined using the 2^−ΔΔCT method. (A) P values were obtained using multiple t tests. (B and C) The mean values of all the innate cytokines measured from ΔE4- and ΔE3-infected A549 cells (B) as well as those above zero from both vectors (C) were compared. P values were obtained using the Wilcoxon matched-pairs signed-rank test. For each experiment, 3 biological replicates were performed and analyzed. *, P < 0.05; **, P < 0.01.

FIG 3

Levels of Env-specific serum binding antibodies in immunized mice. (A) Randomly assigned experimental groups of 5 mice were inoculated intraperitoneally (IP) at weeks 0 and 4 with 5.0 × 10⁸ PFU ΔE3 or ΔE4 Ad per mouse. Five naive mice served as controls. Blood was collected at week 6. (B) Titers of Ad-, rhFLSC-, and gp120-specific IgG antibodies were measured by an ELISA. (C) Titers of Ad-, rhFLSC-, and gp120-specific serum IgG2a antibodies were measured by an ELISA. The values for the control mice were below the background and are not shown. Results are presented as means ± SEM. P values were obtained using 2-way ANOVA.

FIG 4

Levels of Env-specific cytokine-producing mucosal memory CD4 and CD8 cells in immunized rhesus macaques. (A) Three macaques per group were immunized at week 0 intranasally (IN) and orally (O) and at week 12 intratracheally (IT) with ΔE3, ΔE1B55K, or ΔE4 Ad as described in Materials and Methods. One control macaque, C, received empty Ad5hr. The rhFLSC boost in alum was administered intramuscularly (IM). (B and C) The mean percentages ± SEM of cytokine (TNF-α, IL-2, and IFN-γ)-producing rectal CM and EM CD4 and CD8 T cells prior to immunization (pre) and at week 14 for each immunization group are shown. (D to G) The mean percentages ± SEM of cytokine (TNF-α, IL-2, and IFN-γ)-producing T cells on week 14 are compared across immunization groups.

FIG 5

Levels of Env-specific IgG antibody in immunized rhesus macaques. (A and B) Serum levels of rhFLSC (A)- and gp120 (B)-specific IgG antibodies were measured by an ELISA. Results are displayed as log₁₀ geometric means. (C) Mean values of week 38 ADCC for gp120- and rhFLSC-coated target cells are shown. (D) Geometric mean values of SHIV_BaL-P4 week 38 serum neutralization titers are shown. P values were obtained using repeated-measures ANOVA and the Wilcoxon-Mann-Whitney test.

FIG 6

Levels of Env-specific IgG1 antibody in immunized rhesus macaques. Serum levels of rhFLSC (A)- and gp120 (B)-specific IgG1 antibodies were measured by an ELISA. Results are displayed as log₁₀ geometric means. P values were obtained using repeated-measures ANOVA and the Wilcoxon-Mann-Whitney test.