Ad35, Ad26, and Ad48 induce more potent antiviral and proinflammatory cytokines and chemokines than Ad5 following vaccination of rhesus monkeys.
We initiated studies by assessing serum cytokine levels in rhesus monkeys following vaccination with 5 different serotype Ad vectors in 26 rhesus monkeys. Rhesus monkeys (n
= 4 to 8 per group) were immunized intramuscularly (i.m.) with 3 × 1010
vp of Ad5, Ad35, Ad26, Ad48, or Ad5HVR48 expressing SIV Env/Gag/Pol (1
). All vectors were replication-incompetent E1/E3-deleted Ad vectors that were prepared similarly and that exhibited comparable characteristics, specific infectivity, and purity (1
). Sera were collected on days 0, 1, 3, 7, 10, 14, and 28 following vaccination, and cytokine levels were assessed by Luminex assays.
Longitudinal analysis of cytokine responses following vaccination with Ad5 revealed only low levels of antiviral and proinflammatory cytokine and chemokine induction on day 1 following vaccination ( and ). In contrast, animals vaccinated with Ad35, Ad26, and Ad48 displayed greater induction of multiple cytokines and chemokines on day 1 following vaccination. Increased induction by Ad35, Ad26, and Ad48 compared with that by Ad5 was observed for the inflammatory markers interleukin 1 receptor antagonist (IL-1RA) (17.7-, 8.2-, and 26.2-fold greater induction than Ad5, respectively; P = 0.006, P = 0.009, and P = 0.02, respectively; Mann-Whitney U test) and IL-6 (8.3-, 4.0-, and 3.4-fold greater induction than Ad5, respectively; P = not significant [NS], P = 0.04, and P = 0.03, respectively). Additionally, significant induction of gamma interferon (IFN-γ) (1.4-, 1.7-, and 1.5-fold greater induction than Ad5; P = NS, P = NS, and P = 0.02, respectively) and its downstream-signaled chemokines 10-kDa gamma interferon-induced protein (IP-10) (8.3-, 6.0-, and 4.9-fold greater induction than Ad5; P = 0.03, P = 0.009, and P = 0.03, respectively) and interferon-inducible T-cell alpha chemoattractant (I-TAC) (2.8-, 1.1-, and 3.6-fold greater induction than Ad5; P = 0.03, P = NS, and P = 0.03, respectively) was observed in animals that received Ad35, Ad26, or Ad48. These data demonstrate that Ad35, Ad26, and Ad48, which utilize CD46 as their cellular receptor, trigger substantially greater antiviral and proinflammatory cytokine responses than does Ad5, which utilizes CAR as its cellular receptor, following vaccination of rhesus monkeys.
Fig 1 Serum concentrations of cytokines and chemokines in rhesus monkeys following vaccination with Ad vectors. Rhesus monkeys (n = 4 to 8 per group) were injected i.m. with 3 × 1010 vp of Ad5, Ad35, Ad26, Ad48, or Ad5HVR48 vector. Sera were collected (more ...)
Interestingly, on day 7 following vaccination, animals that received Ad48 or the chimeric Ad5HVR48 vector also displayed a temporally distinct peak of cytokine induction characterized by the proinflammatory cytokines IL-1RA, IL-6, IL-1β, and TNF-α ( and ). This cytokine peak was not observed with the Ad5, Ad35, or Ad26 vector. Since Ad48 and Ad5HVR48 share only common hexon HVR sequences, we speculate that this day 7 cytokine peak may be triggered by hexons rather than fiber-receptor interactions. These data suggest that both fibers and hexons may contribute to triggering innate immune responses.
Cytokine induction following a heterologous boost immunization.
We next assessed whether innate cytokine responses following an Ad26 boost immunization would be impacted by previous priming with a heterologous Ad vector. Systemic cytokine levels were assessed in rhesus monkeys (n = 4 per group) that were previously primed with Ad35, Ad48, or Ad5HVR48 vector as described above and then boosted 24 weeks later i.m. with 3 × 1010 vp of Ad26 expressing SIV Env/Gag/Pol. Sera were collected and analyzed as described above. Longitudinal analysis of animals revealed cytokine profiles on day 1 following the boost immunization () that appeared similar to the cytokine profile induced following priming with Ad26 (), including induction of IFN-γ, IL-6, IP-10, and IL-1RA (). These data suggest that the innate cytokine profiles following a boost immunization are dictated primarily by the vector utilized for the boost and are not substantially imprinted by the heterologous vector utilized for priming.
Fig 2 Serum concentrations of cytokines and chemokines in rhesus monkeys following a boost vaccination with Ad26 vector. Rhesus monkeys (n = 4 per group) were boosted i.m. with 3 × 1010 vp of Ad26 vector 24 weeks after priming with 3 × 1010 (more ...) Ad35, Ad26, and Ad48 induce higher levels of antiviral and proinflammatory cytokines and chemokines than Ad5 in vitro.
We next sought to probe the mechanism by which different serotype Ad vectors elicit differential innate cytokine profiles. We therefore assessed the capacity of Ad5, Ad35, Ad26, and Ad48 to trigger secretion of innate cytokines in vitro in freshly isolated human PBMC. Human PBMC (n = 8 to 13 per group) were stimulated with Ad5, Ad35, Ad26, or Ad48 at a multiplicity of infection (MOI) of 103 viral particles (vp) per cell. Cytokine and chemokine responses were measured in culture supernatant 24 h postinfection by Luminex assays.
Transduction of PBMC with Ad35 and Ad26 induced substantially higher levels of IFN-α2 (128- and 96-fold higher levels, respectively; P < 0.001 and P < 0.01, respectively; Kruskal-Wallis test with Dunn's correction for multiple comparisons), IFN-γ (38- and 24-fold higher levels, respectively; P < 0.001), IL-1β (10- and 5-fold higher levels, respectively; P < 0.001 and P < 0.01, respectively), and tumor necrosis factor alpha (TNF-α) (10- and 4-fold higher levels, respectively; P < 0.001 and P < 0.01, respectively) than transduction of PBMC with Ad5 (). Ad35 and Ad26 also induced higher levels of macrophage inflammatory protein 1 alpha (MIP-1α) (18- and 8-fold, respectively; P < 0.001), MIP-1β (16- and 9-fold, respectively; P < 0.001), IL-6 (2.4- and 1.4-fold higher levels, respectively; P < 0.001 and P = NS, respectively), and IL-1RA (3-fold higher levels for both; P < 0.001) (). The species D vector Ad48 displayed an intermediate phenotype of cytokine and chemokine induction relative to that of Ad5 and those induced by Ad35 and Ad26. A complete analysis of all cytokines and chemokines measured in the Luminex assays demonstrated that Ad35, Ad26, and Ad48 induced higher levels of multiple antiviral and proinflammatory cytokines as well as chemokines, including MCP-1 and IP-10, than did Ad5 (). These in vitro data are largely consistent with the in vivo data from vaccinated rhesus monkeys with a few notable exceptions, such as the high levels of IFN-α2 induced in vitro compared to those induced in vivo.
Fig 3 Cytokine and chemokine responses induced in vitro in human PBMC following Ad vector stimulation. (A) Individual PBMC (n = 8 to 13 per group) were stimulated with 103 vp/cell of Ad5, Ad35, Ad26, or Ad48 vector, and cytokine responses were measured after (more ...) Both fiber and capsid components contribute to innate immune stimulation by Ad vectors.
We next explored the role of the key structural proteins of the adenovirus capsid on innate immune stimulation. We utilized a panel of chimeric Ad5/Ad35 (34
) vectors and stimulated human PBMC (n
= 8 per group) as described above. Chimeric Ad vectors included Ad5 with the Ad35 fiber (Ad5f35) or Ad35 fiber and penton (Ad5p35f35) and Ad35 with the Ad5 fiber (Ad35f5) or Ad5 fiber knob (Ad35k5). Replacement of the Ad5 fiber with the Ad35 fiber or Ad35 fiber and penton resulted in an increased induction of multiple cytokines, including IFN-α2 (126- and 184-fold higher levels, respectively; P
< 0.01 and P
< 0.001, respectively), IFN-γ (16- and 9-fold higher levels, respectively; P
< 0.05 and P
< 0.01, respectively), IL-1β (2.5- and 1.6-fold higher levels, respectively; P
= NS), TNF-α (12- and 11-fold higher levels, respectively; P
< 0.001), MIP-1α (52- and 40-fold higher levels, respectively; P
< 0.01 and P
< 0.001, respectively), MIP-1β (30- and 25-fold higher levels, respectively; P
< 0.001), IL-6 (7- and 7-fold higher levels, respectively; P
< 0.001), and IL-1RA (4.4- and 3.5-fold higher levels, respectively; P
< 0.05 and P
< 0.01, respectively), relative to that by Ad5 ().
Fig 4 Cytokine and chemokine responses elicited by chimeric Ad5/Ad35 vectors in human PBMC. Normal human PBMC (n = 8 per group) were stimulated for 24 h in the presence of chimeric Ad5/Ad35 vectors, and cytokine and chemokine levels were measured by Luminex (more ...)
A corresponding decrease in stimulatory capacity was observed with Ad35 vectors containing the Ad5 fiber or fiber knob compared to the stimulatory capacity with Ad35, suggesting the importance of the Ad35 fiber for innate cytokine stimulation. For example, Ad35f5 and Ad35k5 induced lower levels of IFN-γ (4.7- and 7.1-fold lower levels, respectively; P < 0.05 and P < 0.01, respectively), IL-1β (2.7- and 10.6-fold lower levels, respectively; P < 0.05 and 0 < 0.001, respectively), TNF-α (5.5- and 7.1-fold lower levels, respectively; P < 0.01), MIP-1α (13.0- and 23.6-fold lower levels, respectively; P < 0.01 and P < 0.001, respectively), MIP-1β (6.0- and 9.4-fold lower levels, respectively; P < 0.05), and IL-6 (3.4- and 5.2-fold lower levels, respectively; P < 0.05 and 0 < 0.01, respectively) than did Ad35 (). However, Ad35f5 and Ad35k5 still induced most cytokines to a greater level than did Ad5, indicating that other capsid components in addition to fiber also likely contribute to innate cytokine stimulation. Taken together, these results are consistent with the model in which both fibers and hexons contribute to innate immune triggering.
Fiber-receptor interactions are required for Ad35 and Ad26 stimulation of human PBMC.
We next assessed whether fiber interactions with the CD46 receptor were critical for the robust innate immune stimulation by Ad35 and Ad26 vectors. Human PBMC (n
= 4 to 8 per group) were preincubated with 10 μg/ml of the anti-CAR monoclonal antibody RmcB (17
) or the anti-CD46 monoclonal antibodies 13/42 and M177 (43
) and were then stimulated with Ad35 and Ad26 as described above. As expected, IFN-α2 and IFN-γ levels induced by Ad35 and Ad26 vectors were not affected by RmcB. In contrast, Ad35 induced lower levels of IFN-α2 and IFN-γ in the presence of 13/42 (P
< 0.05) and Ad26 elicited dramatically lower levels of IFN-α2 and IFN-γ following preincubation of cells with either 13/42 (P
< 0.05) or M177 (P
< 0.05) (). No interferon responses were observed in control experiments following incubation of cells with the monoclonal antibodies alone (data not shown). These results support previous reports from our laboratory and others showing that CD46 is a primary cellular receptor for Ad35 and Ad26 (1
), and these data demonstrate that fiber-receptor binding contributes substantially to the innate immune stimulation by these vectors. The greater effect of a CD46 blockade on innate responses triggered by Ad26 than on those triggered by Ad35 may reflect subtle differences in primary receptor interactions and/or secondary receptor usage by these vectors.
Fig 5 Interferon induction by Ad35 and Ad26 in the presence of anti-CAR or anti-CD46 blocking MAbs. Normal human PBMC (n = 4 to 6 per group) were preincubated with 10 μg/ml anti-CAR (RmcB) or anti-CD46 (13/42 or M177) for 1 h and then stimulated with (more ...) Multiple cellular subsets are stimulated by Ad35 and Ad26.
To explore the contributions of various cellular subsets to innate cytokine secretion following Ad35 and Ad26 stimulation, we depleted specific cell subsets from healthy human PBMC (n = 6 per group) and repeated the cytokine assays as shown in . T cells, B cells, NK cells, monocytes/macrophages (MonoMac), myeloid dendritic cells (mDC), or plasmacytoid dendritic cells (pDC) were depleted by magnetic bead separation, and effective depletion was confirmed by flow cytometry (data not shown). PBMC depleted of these various cell subsets were then incubated with Ad35 or Ad26, and cytokine and chemokine responses were assessed. Depletion of T cells resulted in complete abrogation of IFN-γ induction and a partial reduction of IL-1RA, whereas depletion of pDC led to a substantial reduction of IFN-α2, TNF-α, and IL-1RA (). Depletion of monocytes and macrophages resulted in a pronounced reduction of the proinflammatory markers IL-1RA, MIP-1α, and MIP-1β (). These data suggest that multiple cellular subsets contribute to the overall milieu of innate cytokines triggered by Ad vectors.
Fig 6 Cytokine and chemokine responses elicited by Ad vectors in normal human PBMC depleted of various cell populations. Normal human PBMC (n = 6 per group) were depleted of the indicated cell subsets by magnetic bead separation, and depletion was confirmed (more ...)