Optimal DNA vaccine efficacy requires circumventing several obstacles, including low immunogenicity, a need for adjuvant, and the costs of purifying injection grade plasmid DNA. Bacterial delivery of plasmid DNA may provide an efficient and low-cost alternative to plasmid purification and injection. Also, the bacterial vector may exhibit potential as an immune adjuvant in vivo. Thus, we elected to examine the use of cell-wall-deficient Listeria monocytogenes as a DNA delivery vehicle in vitro. First, the D-alanine-deficient (Deltadal-dat) L. monocytogenes strain DP-L3506, which undergoes autolysis inside eukaryotic host cells in the absence of D-alanine, was transformed with a plasmid encoding green fluorescent protein (GFP) under control of the CMV promoter (pAM-EGFP). Then COS-7 and MC57G cell lines were infected with the transformed DP-L3506 at various multiplicities of infection (MOI) in the presence or absence of D-alanine. Subsequent GFP expression was observed in both cell lines by 24 h post-infection with DP-L3506(pAM-EGFP). Notably, no GFP positive cells were observed when D-alanine was omitted. Although transfection efficiency initially increased as a result of D-alanine supplementation, high concentration or long-term supplementation led to sustained bacterial growth that killed the infected host cells, resulting in fewer GFP-expressing cells. Thus, efficient DNA delivery by transformed bacteria must balance bacterial invasion and survival with target cell health and survival.
Chlamydia pneumoniae (Cpn) infection has been associated with graft chronic rejection (CR) in human heart and liver transplant recipients, though the high frequency of human Cpn infection confounds such studies. Therefore, we tested whether Cpn infection accelerates CR in a rat cardiac allograft model. F344 rat hearts were heterotopically transplanted into Lewis recipients, either treated with CsA or previously made chimeric with F344 bone marrow cells. Cpn administration resulted in disseminated infection. The allografts were palpated daily until pulsation was no longer detectable (graft failure), or post‐operative day (POD) 120 (maximum study length). F344 allografts in Lewis recipients infected with Cpn on POD 1, POD 7, or PODs 1 and 19 failed significantly earlier than those in the control group. Preexisting recipient infection also significantly accelerated CR, while preexisting donor infection did not accelerate CR. Accelerated CR required infectious Cpn. Chimeric Lewis rat recipients rejected third party allografts, but did not reject F344 allografts regardless of recipient Cpn infection. Therefore rat cardiac allograft recipient Cpn infection, either prior to, at the time of, or a week following transplantation, accelerates CR but does not impact graft survival in the absence of alloreactivity. Supported by Merit Review Funds, Dept. of Veterans Affairs.
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