Bacterial load and IFN expression during the course of primary infection.

<p><b>A.</b> Course of primary infection in mice with the wild type <i>Lm</i> (EGD-e) and the recombinant <i>L. inn</i>::vgc strain. Mice were infected i.v. with 10³ cfu <i>Lm</i>, 10⁷ cfu <i>L.inn</i>, or 10⁷ cfu <i>L.inn::vgc</i> strains. At different time intervals after the infection, mice were sacrificed and the number of viable bacteria in the organs was enumerated. <b>B.</b> Quantitative measurement of IFNα2 and IFNb1 expression in bone marrow-derived macrophages using RT-PCR at 2 h and 8 h following infection with <i>Lm</i> , <i>L.inn</i>, or the <i>L.inn</i>::<i>vgc</i> strains. *P<0.05 (<i>L.inn</i> vs. <i>Lm</i> and <i>L.inn::vgc</i> strains).</p

Expression levels of CD62L on CD8⁺ splenocytes following primary and recall infection with <i>Lm</i>, <i>L.inn</i> and the <i>L.inn::vgc</i> strain.

<p>Flow cytometry was performed on spleen cells, isolated from mice on day 60 after the primary infection or day 5 after the challenge. Cells were stained with FITC-labelled anti-Lyt-2 and biotinylated anti-CD62L. The binding of anti-CD62L on the cell surface was detected with PE-conjugated streptavidin. Numbers shown are gated CD8⁺CD62^lo T cells and analyzed with CELLQuest software.</p

Protective immunity and cellular immune response after infection with <i>Lm</i> and the <i>L.inn::vgc</i> strain.

<p><b>A.</b> Induction of protective immunity conferred after infection with the <i>L.inn::vgc strain</i>. Groups of 15 mice were infected i.v. as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035503#pone-0035503-g001" target="_blank">Fig. 1</a>. Two months later all mice were challenged with a lethal dose (20×LD₅₀) of the wild type <i>Lm</i>. As a control, a group of uninfected normal mice was included. Survival of mice after the challenge was monitored up to 8 days. <b>B.</b> Number of antigen-specific IFN-gamma producing CD8+ T cells in spleens of mice infected i.v. with the wild type <i>Lm</i>, <i>L.inn</i> and <i>L.inn::vgc</i> strain determined by ELISPOT. Spleen cells from infected mice were isolated either on day 9 after the primary infection or day 5 after challenge infection and stimulated with the immunodominant MHC class I peptide LLO_91–99 in triplicates in nitrocellulose based 96-well culture plates. Number of specific IFN-gamma producing cells against the dominant H-2K^d restricted LLO_91–99 epitope were determined by counting the number of spots under the microscope. *P<0.05 (<i>L.inn</i> vs. <i>Lm</i> and <i>L.inn::vgc</i> strains).</p

Measurement of proinflammatory cytokine levels in serum.

<p>Sera was obtained from mice on days 1, 2, 3, and 4 post-infection after inoculation with 10³ cfu <i>Lm</i>, 10⁷ cfu <i>L.inn</i>, or 10⁷ cfu <i>L.inn::vgc</i>. Levels of IL-1ß, IL-6, IL-12(p70), and TNF-alpha were quantified using a multiplex cytokine assay kit. *P<0.05 (EGD-e vs. <i>L.inn</i> and <i>L.inn::vgc</i> strains).</p

Examination of spleens and DTH response after infection with <i>Lm</i> and the recombinant <i>L.inn::vgc</i> strain.

<p><b>A.</b> Morphological examination of spleens from mice inoculated i.v. with the wild type <i>Lm</i> and the recombinant <i>L.inn::vgc</i> strain. Spleens of mice infected i.v. as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035503#pone-0035503-g001" target="_blank">Fig. 1</a> were isolated on day 3 after infection. Shown is a spleen from mice infected with the wild type <i>Lm</i>, the wild type <i>L.inn</i> and its recombinant mutant strain <i>L.inn::vgc</i>. Infiltration of monocytic cells and granulomatous lesions are only detectable in the spleens isolated from mice infected with the wild type <i>Lm</i>. <b>B.</b> Spleen sections were stained with HE and examined. Granulomas with massive leukocyte aggregates can only be detected in spleens of mice infected with <i>Lm</i>. <b>C.</b> DTH response to listerial antigen 9 days after primary infection. Mice were infected with 10³ CFU of <i>Lm</i>, 10⁷ CFU of <i>L.inn</i>, or 10⁷ CFU of <i>L.inn::vgc</i> strain. 9 days after infection, DTH was triggered through injection of soluble somatic listerial antigen. Twenty-four hours later, the specific skin response was determined. The mean value ± S.E. of five animals of a representative experiment is shown.*P<0.05 (EGD-e vs. <i>L.inn::vgc</i> strain).</p