Effect of preexisting immunity on an adenovirus vaccine vector: in vitro neutralization assays fail to predict inhibition by antiviral antibody in vivo

Abstract

A major obstacle to the use of adenovirus vectors derived from common human serotypes, such as human adenovirus 5 (AdHu5), is the high prevalence of virus-neutralizing antibodies in the human population. We previously constructed a variant of chimpanzee adenovirus 68 (AdC68) that maintained the fundamental properties of the carrier but was serologically distinct from AdC68 and resisted neutralization by AdC68 antibodies. In the present study, we tested whether this modified vector, termed AdCDQ, could induce transgene product-specific CD8(+) T cells in mice with preexisting neutralizing antibody to wild-type AdC68. Contrary to our expectation, the data show conclusively that antibodies that fail to neutralize the AdCDQ mutant vector in vitro nevertheless impair the vector's capacity to transduce cells and to stimulate a transgene product-specific CD8(+) T-cell response in vivo. The results thus suggest that in vitro neutralization assays may not reliably predict the effects of virus-specific antibodies on adenovirus vectors in vivo.

FIG. 1.

CD8⁺ T-cell responses to Gag in mice immunized with AdC68- and AdCDQ-Gag. Mice were immunized i.m. with 1 × 10⁹, 1 × 10¹⁰, or 1 × 10¹¹ vp/mouse, and splenocytes were isolated 10 days later. Frequencies of CD8⁺ T cells specific for Gag were measured by intracellular cytokine staining as described in Materials and Methods. Results are shown as the means (and standard deviations [SD]) for samples from three individual mice.

FIG. 2.

Preexisting immunity to AdC68 inhibits immunization with AdCDQ. (A) Mice were preexposed to AdC68-GFP or AdCDQ-GFP (1 × 10¹¹ vp/mouse) 2 weeks before immunization with AdC68-Gag or AdCDQ-Gag (1 × 10¹¹ vp/mouse). Mice were sacrificed 10 days later, and Gag-specific CD8⁺ T-cell responses are described in the legend of Fig. 1. Results are shown as the means (and SD) for samples from four individual mice. (B) T-cell responses to a prime-boost regimen. Mice were immunized with 1 × 10¹¹ vp on day 0 and boosted on day 60 (1 × 10¹¹ vp/mouse). Some mice were not primed in order to measure responses to a single dose (indicated as −/boost). T-cell responses were measured on days 70 and 120. Results are shown as the means (and SD) for samples from five individual mice.

FIG. 3.

AdCDQ is inhibited by passively transferred polyclonal anti-AdC68 serum in vivo but not in vitro. (A) Mice were intravenously injected with rhesus macaque serum and bled at 24 h for measurement of neutralizing titers as described in Materials and Methods. (B) Immediately after bleeding, mice were immunized with AdC68-Gag or AdCDQ-Gag, and numbers of Gag-specific T cells were measured at day 10. Results are shown as the means (and SD) for samples from five individual mice.

FIG. 4.

Passive transfer of an AdC68 neutralizing MAb inhibits AdCDQ in vivo but not in vitro. (A) Mice were injected intravenously with an AdC68 neutralizing antibody (4C1) and bled 1 day later for measurement of neutralizing titers. (B) Immediately after bleeding, mice were immunized i.m. with 1 × 10¹¹ vp/mouse AdC68-Gag or AdCDQ-Gag, and numbers of Gag-specific CD8⁺ T cells were measured 10 days later. Results are shown as the means (and SD) for samples from five individual mice.

FIG. 5.

Passive transfer of MAb 4C1 reduces GFP transgene delivery by both AdC68 and AdCDQ. (A) GFP expression in muscle. Mice were injected intravenously with an AdC68-neutralizing antibody (4C1) or a control antibody (502). Twenty-four hours later, mice were injected in the leg muscle with 1 × 10¹¹ vp of AdC68-GFP or AdCDQ-GFP. After an additional 24 h, GFP expression was visualized. (B) GFP expression in local dendritic cells (DCs). Mice were injected with MAbs 4C1 and 502 and immunized with GFP vectors as described above (A). Twenty-four hours later, popliteal lymph nodes (LN) were isolated from the injected leg, and GFP expression in CD11c-positive cells was measured. Results are shown as the means (and SD) for samples from four individual mice.

FIG. 6.

Plasma does not enhance antibody-mediated neutralization of AdCDQ. AdCDQ-GFP and AdC68-GFP were incubated with medium or with MAb 502 or 4C1 in the presence or absence of 10% murine serum or 10% murine plasma, and GFP delivery to HeLa cells was observed, as described in Materials and Methods.

FIG. 7.

MAb binding to immobilized AdC68 and AdCDQ. ELISA plates coated with AdC68 (A) and AdCDQ (B) were exposed to twofold serial dilutions of MAb 4C1, 4D1, or 502 or to PBS, and bound antibodies were detected as described in Materials and Methods.

FIG. 8.

Fc receptors are not required for antibody-mediated inhibition of AdCDQ in vivo. (A and B) GFP expression in muscle. Wild-type (A) or Fc-receptor-deficient (B) mice were injected intravenously with an AdC68-neutralizing antibody (4C1) or a control antibody (502). Twenty-four hours later, mice were injected in the leg muscle with 1 × 10¹¹ vp of AdC68-GFP or AdCDQ-GFP. After an additional 24 h, GFP expression was visualized. (C and D) GFP expression in local dendritic cells. Mice were injected with MAbs 4C1 and 502 and immunized with GFP vectors as described above (A and B). Twenty-four hours later, popliteal lymph nodes (LN) were isolated from the injected leg, and GFP expression in CD11c-positive cells was measured. Results are shown as the mean numbers of GFP-expressing dendritic cells (DCs) (and SD) per lymph node for three individual mice.