Cytokines in lung homogenates of B10.A mice infected i.t. with <i>P. brasiliensis</i> Pb 18 yeasts and submitted to gene immunization for 5 months.

<p>Mice were sacrificed 1 month after the last dose.</p

Immunoprophylactic treatment of PCM.

<p>Gene immunization was initiated 30 days before fungal challenge. CFUs are from lungs of BALB/c mice infected intratracheally with 3×10⁵ yeast cells and subjected to immunization with vectors containing P10 (pP10) and/or IL-12 (pIL-12) DNA insert. Control mice were inoculated with PBS or with vectors without insert. Mice were sacrificed 30 (▪) and 60 (□) days after infection. Each bar represents the average counts and standard deviations of CFUs in lungs from 10 animals in each group. Experiments were carried out in triplicate with similar results. <b>**</b><i>p</i>≤0.0001, comparing vector with and without insert; ^##<i>p</i>≤0.0001, .comparing untreated and other groups.</p

Summary of the treatment protocols used.

<p><b>First protocol</b>: BALB/c mice received 4 weekly injections. Animals were infected and sacrificed 30 or 60 days later. <b>Second protocol</b>: BALB/c and B10.A mice were infected intratracheally. Mice received 4 weekly vaccine doses and animals were sacrificed 1 week after the last injection. <b>Third protocol</b>: B10.A mice were infected i.t. and one month after infection, they were immunized with the DNA vaccine. The animals were sacrificed one month after the last injection, six months after infection.</p

Therapeutic treatment of PCM.

<p>Gene immunization started 30 days after infection. CFUs are from lungs of BALB/c (□) and B10.A(▪) mice infected intratracheally with 3×10⁵ yeast cells and subjected to immunization with vectors containing P10 (pP10) or IL-12 (pIL-12) DNA inserts. Control mice were inoculated with PBS or with vectors without insert. Mice were sacrificed 60 days after infection. Each bar represents the average counts and standard deviations of CFU in lungs from 10 animals in each group. Experiments were performed three times and similar results were achieved. <b>*</b><i>p</i>≤0.05, <b>**</b><i>p</i>≤0.005, comparing vector with and without insert; ^##<i>p</i>≤0.005, comparing untreated and other groups.</p

Histopathology of lungs from intratracheally infected B10.A mice.

<p>Animals were infected with <i>P. brasiliensis</i> for one month, treated with or without vectors carrying P10 or IL-12 DNA inserts according to protocol 3, and sacrificed 6 months after the initial infection. Infected mice treated with (<b>A</b>) control pcDNA3, (<b>B</b>) pP10, (<b>C</b>) pIL-12 DNA, and (<b>D</b>) P10 and IL-12 DNA. Gomori staining; original magnification, 40×.</p

Long term therapeutic treatment of experimental PCM.

<p>Gene immunization started 30 days after infection and mice were sacrificed 6 months after infection. CFUs were counted in lungs of B10.A mice infected intratracheally with 3×10⁵ yeast cells and immunized with vectors containing the insert encoding P10 (pP10) or IL-12 (pIL-12). Control mice were inoculated with PBS or with vector without insert. Each bar represents the average counts and standard deviations of CFU in lungs from 5 to 10 animals in each group. Experiments were carried out in triplicate, with similar results. <b>**</b><i>p</i>≤0.001, comparing vector with and without insert; ^##<i>p</i>≤0.001, comparing untreated and other groups.</p

Development of NH36-specific cellular immune response as disclosed by intracellular staining analysis of splenocytes <i>in vitro</i> cultured with NH36 before and after <i>L. chagasi</i> infection.

<p>Anti-CD4-FITC and anti-CD8-FITC antibodies were used for labeling the cell surfaces and anti-IFN-γ-APC, anti-TNF-α-PE and anti-IL-10-PE for intracellular staining. In order characterize the TH1 response bars represent the ratio of IFN-γ/IL-10 and TNF-α/IL-10 producing cells. Results represent mean + SE of two independent experiments (n = 7–8 mice per treatment). * p<0.05 indicate significant differences from the saline treated controls, from F1sap, ○ from F2sap, and ◆ from the NH36sap vaccine.</p

Development of cell-mediated immune response as disclosed by <i>in vivo</i> depletion with anti-CD4+ and anti-CD8+ monoclonal antibodies.

<p><i>Leishmania chagasi</i> parasite-load (<b>A</b> and <b>B</b>) and percent of liver/corporal relative weight (<b>C</b> and <b>D</b>) in mice vaccinated with NH36sap and F3 sap vaccine and treated with rat serum, anti-CD4+ or anti-CD8+ or the combination of anti-CD4+ and anti-CD8+ MAbs. Maximal parasite load reduction was achieved in mice that received either the NH36sap or the F3sap vaccines and rat serum (rat IgG) as controls for antibody treatment. Bars represent the mean + SD (5 mice per each treatment). The parasite load is expressed in LDU values (number of amastigotes per 600 liver cell nuclei/mg of liver weight) (<i>A</i> and <i>B</i>). Hepatomegaly was assessed by the individual increment in liver relative weight expressed as percent of the body weight. <i>+</i> p<0.05 significant differences between treatments.</p

Development of NH36-specific cellular immune response. ELISA of cytokines in supernatants of mice splenocytes.

<p>The mean of one group of data of vaccinated mice (results of one experiment with n = 7–8 mice per treatment) is or not included in the 95% CI of control groups.</p

Vaccination, challenge and development of NH36-specific humoral immune response.

<p>(<b>A</b>) Study design: Balb/c mice were vaccinated with NH36sap, F1sap, F2sap or F3sap at the indicated time intervals, through the sc route, followed by intravenous challenge with <i>L. chagasi</i> amastigotes<b>.</b> Bars represent the mean ± SE of the absorbance values of anti-NH36 antibodies from 1/100 diluted serum of two independent experiments (n = 11–12 mice per treatment) after immunization (<b>B</b>) and after challenge (<b>C</b>). <b>*</b> p<0.05 different from the saline control. p<0.05 different from F1sap vaccine; <b>○</b> p<0.05 different from the F2sap vaccine; ◆ p<0.05 different from NH36sap vaccine; p<0.05 different from all other vaccines.</p