In vivo attenuation and genetic evolution of a ST247-SCCmecI MRSA clone after 13 years of pathogenic bronchopulmonary colonization in a patient with cystic fibrosis: implications of the innate immune response.

Methicillin-resistant Staphylococcus aureus (MRSA) causes chronic pulmonary infections in patients with cystic fibrosis (CF). This study tracks the 13-year evolution (1996-2009) of a single MRSA clone in a male patient with CF, evaluating both the host immunogenic response and the microbial variations. Whole-genome sequencing was performed for the initial (CF-96) and evolved (CF-09) isolates. The immunogenicity of CF-96 and CF-09 was evaluated by incubation with innate immune cells from healthy volunteers. We also studied the patient's innate immune response profile, cytokine production, expression of triggering receptor expressed on myeloid cells-1 (TREM-1), and phagocytosis. A total of 30 MRSA ST247-SCCmecI-pvl(-) isolates were collected, which evidenced a genome size reduction from the CF-96 ancestor to the evolved CF-09 strain. Up to six changes in the spa-type were observed over the course of the 13-year evolution. Cytokine production, TREM-1 expression, and phagocytosis were significantly lower for the healthy volunteer monocytes exposed to CF-09, compared with those exposed to CF-96. Patient monocytes exhibited a reduced inflammatory response when challenged with CF-09. Genetic changes in MRSA, leading to reduced immunogenicity and entry into the refractory state, may contribute to the attenuation of virulence and efficient persistence of the bacteria in the CF lung.

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.

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.

Migratory sub-populations of afferent lymphatic dendritic cells differ in their interactions with Mycobacterium bovis Bacille Calmette Guerin.

Understanding how pathogens or vaccine antigens are targeted to dendritic cell (DC) subsets is important for disease pathogenesis studies and vaccine design. We characterised the sub-populations of migrating bovine DC with functional and phenotypic diversity present in pseudoafferent lymph draining the skin. These skin draining DC exist as a series of maturation dependent subsets with differential capacities for antigen uptake and cytokine expression, and include both Langerhans' cells (LC) and dermal derived cells. Furthermore, Mycobacterium bovis Bacille Calmette Guerin, a vaccine which is administered by the intradermal route, was only taken up by a small number of the migrating DC, which were SIRPα(+) and expressed the mannose receptor and CD1b. This was evident following in vitro infection and also in vivo following inoculation of green fluorescent BCG over the lymphatic cannulation site. Only the SIRPα(+) DC were able to present antigen to T cells isolated from BCG vaccinated calves. Furthermore, presentation of BCG antigens by DC to T lymphocytes was ineffective compared to mycobacterial proteins. However, mycobacterial antigen 85 was delivered more effectively to DC via an adenoviral vector and the magnitude of the subsequent antigen-specific T cell response was significantly increased. This study further extends our understanding of the biology of migrating DC, identifies potential explanations for the modest success of BCG vaccination and demonstrates that targeted delivery of antigens via adenoviruses to DC can improve antigen presentation.

Differential effects of viral vectors on migratory afferent lymph dendritic cells in vitro predict enhanced immunogenicity in vivo.

Targeting dendritic cells (DC) is key to driving effective immune responses. Lymphatic cannulation provides access to the heterogeneous populations of DC draining peripheral sites in rodents and ruminants. Afferent lymph DEC-205(+) CD11c(+) SIRPα(+) DC were preferentially infected ex vivo with three vaccine viral vectors: recombinant human replication-defective human adenovirus 5 (rhuAdV5), recombinant modified vaccinia virus Ankara (rMVA), and recombinant fowlpox virus (rFPV), all expressing green fluorescent protein (GFP). The rhuAdV5-infected cells remained viable, and peak GFP expression was observed 16 to 24 h posttransduction. Increasing the incubation period of DC with rhuAdV5 enhanced GFP expression. In contrast, DC infected with rMVA-GFP or rFPV-GFP became rapidly apoptotic and GFP expression peaked at 6 h postinfection. Delivery of foot-and-mouth disease virus (FMDV) A(22) antigen to DC by rhuAdV5-FMDV-A(22) ex vivo resulted in significantly greater CD4(+) T cell proliferation than did delivery by rFPV-FMDV-A(22). Delivery of rhuAdV5-GFP in oil adjuvant in vivo, to enhance DC-vector contact, resulted in increased GFP expression in migrating DC compared to that with vector alone. Similarly, CD4(+) T cell responses were significantly enhanced when using rhuAdV5-FMDV-A(22) in adjuvant. Therefore, the interaction between viral vectors and afferent lymph DC ex vivo can predict the outcome of in vivo immunization and provide a means of rapidly assessing the effects of vector modification.