Identification of novel antigen candidates for a tuberculosis vaccine in the adult zebrafish (Danio rerio)

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In situ GFP expression in immunized zebrafish.

<p>AB fish were immunized with 12 μg of experimental or control vaccine plasmids, followed by electroporation. Seven days post-injection, the successful vaccinations and expression of the antigen-GFP fusion proteins were verified by fluorescence microscopy. The fluorescence resulting from the expression of the antigen-GFP fusion protein is seen in the dorsal muscle near the injection site. For each antigen, a representative example is shown. Non-immunized AB fish were used as a negative control.</p

Schematic representation of the vaccine antigens.

<p>The <i>M</i>. <i>marinum</i> proteins are represented by bars, different colors indicate cellular location based on the literature and/or Trans Membrane prediction using Hidden Markov Models (TMHMM). The vaccine antigen-GFP fusion proteins are represented by lines, together with their expected molecular weights (See legend for more details). On the right, an immunoblot analysis of antigen-GFP fusion proteins. For the analysis, AB fish were immunized with 12 μg of experimental or control (empty plasmid with GFP only) vaccines, followed by electroporation. Seven days post-injection, fish were dissected under UV light and the dorsal muscles were collected and homogenized, followed by protein extraction. 7.5–15 μg of each protein lysate was run on an SDS-PAGE gel, blotted onto a nitrocellulose membrane followed by immunodetection with a horse radish peroxidase (HRP) conjugated anti-GFP antibody. Non-immunized AB fish were used as the negative control.</p

RpfE, PE5_1, PE31 and cdh antigens reduce bacterial burdens in adult zebrafish infected with a low-dose of <i>M</i>. <i>marinum</i>.

<p>AB fish were immunized intramuscularly with the experimental and control (GFP) antigens, followed by an intraperitoneal infection with ~40 cfu of <i>M</i>. <i>marinum</i>. Five weeks post-infection, the fish were euthanized, and their internal organs were dissected, homogenized and subjected to DNA extraction. The bacterial burden in each fish was determined by qPCR with <i>M</i>. <i>marinum</i> specific primers. The experimental cfu values in each experiment are normalized with the median cfu of the GFP controls of the same experiment. The lines represent median values, and the bars and whiskers the minimum and maximum values for each group, respectively. N = 10–29 per group. * p<0.05, ** p<0.01 (two-tailed Mann-Whitney test).</p

RpfE antigen improves survival of the fish infected with a high dose of <i>M</i>. <i>marinum</i>.

<p>AB fish were immunized intramuscularly with the experimental and control (GFP) antigens, followed by an intraperitoneal infection with ~10.000 cfu of <i>M</i>. <i>marinum</i>. Fish were then followed for 12 weeks for survival. The survival curve for each antigen immunization is shown separately with the GFP control group of the same infection experiment(s). ** p<0.01 (Log-rank (Mantel-Cox) test). N≥19 in each group.</p

The selected antigens and their <i>M</i>. <i>tuberculosis</i> homologs with predicted functions.

<p>The selected antigens and their <i>M</i>. <i>tuberculosis</i> homologs with predicted functions.</p

Expression of the antigens in the <i>M</i>. <i>marinum</i> ATCC 927 strain.

<p>A liquid culture of <i>M</i>. <i>marinum</i> was grown to a log phase, bacteria were harvested by centrifugation and subjected to RNA extraction and DNase treatment. Antigen expression was confirmed by qRT-PCR using primers specific for each antigen (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181942#pone.0181942.s002" target="_blank">S1 Table</a>). The <i>M</i>. <i>marinum</i> transcribed internal spacer (<i>MMITS</i>) was used as a reference gene. The horizontal lines represent medians and the bars and whiskers represent minimum and maximum values. N = 6.</p

Quantification of mycobacterial antigen expression with GFP ELISA.

<p>AB fish were immunized with 12 μg of experimental or control vaccine plasmids, followed by electroporation. Seven days post-injection, fish were dissected under a UV light and the dorsal muscles were collected and homogenized with ceramic beads, followed by protein extraction. 7.5–15 μg of each protein lysate in a 1% SDS buffer was used for a GFP ELISA analysis. A standard curve was used to quantify the absorbance values, which were then normalized with the average of the control values of each experiment before the values were pooled. Non-immunized AB fish were used as the negative control. Mean±SD is shown. N≥4 per group. * p<0.05, ** p<0.01, *** p<0.001 (Two-tailed Mann-Whitney test).</p

Controlled mycobacterial infection is characterized by Th2-type response from 4 weeks post infection.

<p>(A–C) <i>Tbx21</i> (<i>t-bet</i>) and <i>gata3</i> induction was measured in the different subpopulations at 2, 4 wpi and 5 mpi (months post infection). The <i>gata3/tbx21</i> ratio was calculated to determine the dominant Th type. (D–F) The induction of selected type cytokines for Th1 (<i>IFNγ1-2</i>) and Th2 response (<i>IL4b</i>) was measured in the different subpopulations. The <i>il4/IFNγ</i> ratio of induction was calculated. (G–I) <i>Rag1</i> (−/−) mutant zebrafish (n = 20) were infected with 35±18 cfu of <i>M. marinum</i> and analyzed at 4 wpi. (G) Grouping of mutant fish was carried out according to bacterial load similarly to wt fish (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004190#ppat.1004190.s001" target="_blank">Figure S1E</a>). The association of <i>gata3/tbx21</i> with the bacterial load was assessed. (H) The induction of <i>il4</i> at 4 wpi was compared between wt and <i>rag1</i> (−/−) fish. (I) The induction of <i>ifnγ</i> at 4 wpi was compared between wt and <i>rag1</i> (−/−) fish. (J) Semi-quantitative western blots were carried out at 4 wpi from a population of 20 fish. Gata3 antibody was used as the Th2 marker, and CXCR3 as the Th1 marker. The bacterial loads were measured from the corresponding DNA samples to allow grouping to subpopulations. (K) As a control experiment for assessing the effect of initially high bacterial load on <i>gata3/tbx21</i> ratio, WT zebrafish were infected with a high dose (2691±520 cfu) and the <i>gata3/tbx21</i> ratio of this group (n = 25) was compared to those of the group (n = 30) infected with a low dose (21±7 cfu) at 4 wpi. (L) To assess the natural polarization pattern of T cells with regard to <i>gata3/tbx21</i>, WT zebrafish (n = 14) were stimulated by an intraperitoneal injection of heat-killed <i>M. marinum</i>. <i>Gata3/tbx21</i> ratio was determined 10 days post injection.</p