Modified Vaccinia Virus Ankara-Based Vaccine Vectors Induce Apoptosis in Dendritic Cells Draining from the Skin via both the Extrinsic and Intrinsic Caspase Pathways, Preventing Efficient Antigen Presentation

Abstract

Dendritic cells (DC) are potent antigen-presenting cells and central to the induction of immune responses following infection or vaccination. The collection of DC migrating from peripheral tissues by cannulation of the afferent lymphatic vessels provides DC which can be used directly ex vivo without extensive in vitro manipulations. We have previously used bovine migrating DC to show that recombinant human adenovirus 5 vectors efficiently transduce afferent lymph migrating DEC-205 + CD11c + CD8 − DC (ALDC). We have also shown that recombinant modified vaccinia virus Ankara (MVA) infects ALDC in vitro , causing downregulation of costimulatory molecules, apoptosis, and cell death. We now show that in the bovine system, modified vaccinia virus Ankara-induced apoptosis in DC draining from the skin occurs soon after virus binding via the caspase 8 pathway and is not associated with viral gene expression. We also show that after virus entry, the caspase 9 pathway cascade is initiated. The magnitude of T cell responses to mycobacterial antigen 85A (Ag85A) expressed by recombinant MVA-infected ALDC is increased by blocking caspase-induced apoptosis. Apoptotic bodies generated by recombinant MVA (rMVA)-Ag85A-infected ALDC and containing Ag85A were phagocytosed by noninfected migrating ALDC expressing SIRPα via actin-dependent phagocytosis, and these ALDC in turn presented antigen. However, the addition of fresh ALDC to MVA-infected cultures did not improve on the magnitude of the T cell responses; in contrast, these noninfected DC showed downregulation of major histocompatibility complex class II (MHC-II), CD40, CD80, and CD86. We also observed that MVA-infected ALDC promoted migration of DEC-205 + SIRPα + CD21 + DC as well as CD4 + and CD8 + T cells independently of caspase activation. These in vitro studies show that induction of apoptosis in DC by MVA vectors is detrimental to the subsequent induction of T cell responses.

Subset-Specific Expression of Toll-Like Receptors by Bovine Afferent Lymph Dendritic Cells

“…Distinct subsets of bovine DC have been described draining the skin, which have differential capacities to respond to vaccines (14–16) and to interact with T lymphocytes (1). Whether these subsets also express different profiles of pattern recognition receptors such as TLR was investigated herein.…”

“…Recent evidence suggests that bovine ALDC subsets interact differentially with vaccines (14–16). Phagocytosis of the attenuated vaccine strain Mycobacterium bovis bacillus Calmette–Guerin (BCG) was shown, both in vitro and in vivo , to be predominantly associated with the CD172a + CD206 + CD1b ++ (cDC2_2) subset of ALDC, which was subsequently shown to induce higher expression of interferon (IFN)γ by CD4 + lymphocytes (14–16).…”

“…Recent evidence suggests that bovine ALDC subsets interact differentially with vaccines (14–16). Phagocytosis of the attenuated vaccine strain Mycobacterium bovis bacillus Calmette–Guerin (BCG) was shown, both in vitro and in vivo , to be predominantly associated with the CD172a + CD206 + CD1b ++ (cDC2_2) subset of ALDC, which was subsequently shown to induce higher expression of interferon (IFN)γ by CD4 + lymphocytes (14–16). Similar experiments with a human adenovirus (AdV5) expressing Ag85A, a secreted mycobacterial protein thought to be a promising candidate as a protective antigen, found that the CD172 + (cDC2) population cultured with AdV5-85A induced significantly higher IFNγ expression than either CD172 + (cDC2) cells cultured with AdV5 alone or CD172a − (cDC1) cells cultured with AdV5-85A (14–17).…”

Transduction of skin-migrating dendritic cells by human adenovirus 5 occurs via an actin-dependent phagocytic pathway

“…Ag85A-specific IFN-γ producing lymphocytes were analysed using ELISpot assays as described previously (Guzman et al , 2012; Hope et al , 2012) utilizing purified CD4 + T cells from MHC-matched, BCG-vaccinated animals (Thom et al , 2012). …”

“…Within this population, subpopulations of DC expressing various levels of SIRPα (CD172a), CD11a, CD26 and CD13 have also been described (Brooke et al , 1998; Gliddon et al , 2004; Gliddon & Howard, 2002; Howard et al , 1997). Of these, the SIRPα + DC population is targeted by various vaccine vectors and these cells are more efficient at antigen presentation compared to SIRPα neg/low ALDC (Guzman et al , 2012; Hope et al , 2012). …”