Type I interferon-dependent activation of NK cells by rAd28 or rAd35, but not rAd5, leads to loss of vector-insert expression

Highlights

• rAd28 and rAd35 vectors, but not rAd5 vectors, induce monocyte apoptosis.

• Monocyte apoptosis leads to rapid loss of vector-insert expression.

• Vector-induced monocytes apoptosis is, at least in part, due to NK cell activation and killing.

• NK cells are activated by vector-induced interferon alpha production.

Abstract

Vaccines constructed from rare-serotype recombinant adenovirus vectors (rAd) such as rAd serotype 28 (rAd28) and rAd35 are currently being explored as alternatives to rAd5-based vaccines because they circumvent the problems with pre-existing immunity that complicate the effectiveness of rAd5 vaccines. However, previous work has demonstrated that the immunogenicity of rAd28 and rAd35 is substantially lower than rAd5. Here we show that rAd28 and rAd35 increase apoptosis of antigen presenting cells (APCs), such as monocytes, relative to rAd5 and mock infected controls. APCs undergoing apoptosis showed an increased loss of vector-insert expression. Loss of vector-insert expression correlated with activation of NK cells, which resulted in apoptosis of co-cultured monocytes. Finally, we show that activation of NK cells is dependent on IFNα which is produced by exposure to rAd28 or rAd35, but not to rAd5. Taken together, these data demonstrate that IFNα-induced activation of NK cells leads to increased monocyte apoptosis and subsequent vector-insert loss. This may be a possible mechanism that results in reduced immunogenicity of rAd28 and rAd35-based vectors.

Introduction

Adenoviruses (Ad) have been well studied as vectors for recombinant vaccines because of their ability to generate strong, insert-specific memory immune responses [1], [2]. Vectors constructed from Ad serotype 5 (rAd5) are the most well characterized, but their use as a clinical vaccine product is limited by high preexisting immunity [3], [4]. As a result vectors constructed from rarer serotypes, such as rAd28 or rAd35, are under consideration. The differing immunogenicity of vectors constructed from different adenovirus serotypes is well documented, with rAd5 being the most immunogenic, rAd35 being the least immunogenic, and other serotypes, including rAd28, being moderately immunogenic [1], [3], [4], [5], [6]. The mechanistic causes of this differential immunogenicity are incompletely understood. The serotypes differ in infectivity, trophism, cellular receptor usage, intracellular trafficking routes, and genome CpG content but these factors have not been conclusively shown to be directly responsible for the differing immunogenicity [1], [7], [8], [9], [10].

We have previously shown that rAd5 vectors induce large insert-specific CD8 T cell populations with a high proportion producing both IFNγ and TNF and that the CD8 T cell population resulting from exposure to vectors constructed from rare-serotypes, such as rAd28 and rAd35, is lower in magnitude, but contain a greater frequency of triple positive IFNγ-, TNF-, and IL2-producing cells and a higher frequency of long-lived CD127+ cells [4], [11]. These differences are largely attributable to the induction of type I interferon (IFNα) by rAd28 and rAd35, but not by rAd5 [11].

Here we further examined the impact of innate immunity on vector-insert expression. Specifically we examined the impact of cell death and apoptosis on the duration of vector-insert expression and the contribution of NK cells. We show that human CD14+ monocytes are lost during infection with rAd28 or rAd35, but not rAd5. rAd28 and rAd35, but not rAd5, induced IFNα-dependent activation of NK cells and these activated NK cells were capable of inducing monocyte apoptosis. This provides a possible mechanism for the loss of CD14+ monocytes after infection with rAd28 and rAd35, but not rAd5. Collectively, these data suggest that duration of the vector-insert expression, APC apoptosis, and NK cell activation differs greatly between vectors constructed from different Ad serotypes and should be taken into consideration when designing rAd vaccines.