Sm29, but Not Sm22.6 Retains its Ability to Induce a Protective Immune Response in Mice Previously Exposed to a <i>Schistosoma mansoni</i> Infection

<div><p>Background</p><p>A vaccine against schistosomiasis would have a great impact in disease elimination. Sm29 and Sm22.6 are two parasite tegument proteins which represent promising antigens to compose a vaccine. These antigens have been associated with resistance to infection and reinfection in individuals living in endemic area for the disease and induced partial protection when evaluated in immunization trials using naïve mice.</p><p>Methodology/principals findings</p><p>In this study we evaluated rSm29 and rSm22.6 ability to induce protection in Balb/c mice that had been previously infected with <i>S</i>. <i>mansoni</i> and further treated with Praziquantel. Our results demonstrate that three doses of the vaccine containing rSm29 were necessary to elicit significant protection (26%–48%). Immunization of mice with rSm29 induced a significant production of IL-2, IFN-γ, IL-17, IL-4; significant production of specific antibodies; increased percentage of CD4+ central memory cells in comparison with infected and treated saline group and increased percentage of CD4+ effector memory cells in comparison with naïve Balb/c mice immunized with rSm29. On the other hand, although immunization with Sm22.6 induced a robust immune response, it failed to induce protection.</p><p>Conclusion/significance</p><p>Our results demonstrate that rSm29 retains its ability to induce protection in previously infected animals, reinforcing its potential as a vaccine candidate.</p></div

Hepatic granuloma area in mice immunized with rSm29, previous infected and treated.

<p>Representative picture of granuloma reaction (A) and the measurement of granuloma area of each group (B). To determine granuloma area, approximately 100 granulomas from IT/rSm29 group and from its control group (IT/Saline), with a single well-defined egg (exudative stage), were measured. Total area of the granulomas was expressed in square micrometers (μm²). Scale bar = 100 μm (x100).</p

Experimental design.

<p>The assessment of the protection (A) and the immune response (B) triggered by rSm22.6 or rSm29 immunization in previously infected and treated Balb/c mice were performed as indicated in the figure.</p

Frequency of memory cells in mice immunized with rSm29 or rSm22.6, or inoculated with saline.

<p>One week after the last immunization, spleen cells from IT/Saline, IT/rSm22.6, IT/rSm29, rSm22.6 and rSm29 groups were labeled to assess the frequency of memory lymphocytes and were acquired in flow cytometer. Data analysis was carried out as follows (A): within singlet cells/lymphocyte population, CD4+CD44^high or CD8CD44^high cells were selected and, within that population, the percentage of CD127⁺CD62^low cells representing CD4⁺ or CD8⁺ T effector memory cells, CD127⁺CD62^high population representing the CD4⁺ or CD8⁺ T central memory cells were determined. Furthermore, within the lymphocytes population, CD19⁺ cells were selected and the percentage of CD19⁺CD27⁺ cells was determined. (B): The results are expressed in bars as median with interquartile range of the percentage of memory cells. Statistically significant difference is pointed in the graphic. Statistical analyses were performed by the Mann-Whitney test.</p

Kinetics of specific anti-rSm22.6 or anti-rSm29 antibodies production induced by immunization.

<p>Sera from 10 animals/group were collected forty-five days after infection, fifteen days after treatment and two weeks after each immunization dose. The levels of specific IgG, IgG1, IgG2a, IgE against rSm22.6 or against rSm29 were determined by ELISA. Sera dilution was: 1:1.000 (IgG and IgG1—rSm29 tests), 1:100 (IgG2a—rSm29 test), 1:600 (IgG—rSm22.6 test), 1: 1.000 (IgG1—rSm22.6 test), 1:400 (IgG2a—rSm22.6 test) or 1:40 (IgE—rSm22.6 and rSm29 test). Bars represent the mean absorbance values measured at 450 nm ± SD (Standard Deviation). Arrows indicate the timing of infection, treatment and immunization. Statistically significant differences between rSm22.6 or rSm29 group and saline control group is denoted in the graphic by one asterisk (p<0.05), two asterisks (p<0.01) or three asterisks (p<0.001). Statistically significant difference between the immunization doses is pointed in the graphic. &: difference compared to infected mice; #: difference compared to treated mice.</p

Protection level induced by immunization with rSm22.6 plus Freund’s adjuvant in Balb/c mice previously infected/treated.

<p>^aWorm burden recovered from mice immunization with one, two or three doses. For each vaccinated group: n = 10 mice.</p><p>Comparison between total worm burden recovered^b or numbers of eggs per gram of intestine^c from IT/Sm22.6 group and IT/Saline control group, which received the same number of immunization doses and were challenged with 50 <i>S</i>. <i>mansoni</i> LE strain cercariae. NS = not significant. ND = not determined.</p><p>Protection level induced by immunization with rSm22.6 plus Freund’s adjuvant in Balb/c mice previously infected/treated.</p

Antibody titer in mice immunized with rSm22.6 and rSm29 after the third immunization.

<p>Antibody titer in mice immunized with rSm22.6 and rSm29 after the third immunization.</p

Protection level induced by immunization with rSm29 or rSm22.6 plus Freund’s adjuvant in Balb/c naïve mice.

<p>^aWorm burden recovered from mice infected with 50 cercariae.</p><p>^bWorm burden recovered from mice infected with 100 cercariae. For each vaccinated group: n = 10 mice.</p><p>^cComparison between total worm burden recovered from rSm29 or rSm22.6 group and Saline control group.</p><p>Protection level induced by immunization with rSm29 or rSm22.6 plus Freund’s adjuvant in Balb/c naïve mice.</p

Protection level induced by immunization with rSm29 plus Freund’s adjuvant in Balb/c mice previously infected/treated animals.

<p>^aWorm burden recovered from mice immunization with one, two or three doses. For each vaccinated group: n = 10 mice.</p><p>Comparison between total worm burden recovered^b or numbers of eggs per gram of intestine^c from IT/rSm29 group and IT/Saline control group, which received the same number of immunization doses and were challenged with 100 <i>S</i>. <i>mansoni</i> LE strain cercariae. NS = not significant. ND = not determined.</p><p>*Statistically significant (p<0.05)</p><p>Protection level induced by immunization with rSm29 plus Freund’s adjuvant in Balb/c mice previously infected/treated animals.</p

Recognition of native Sm22.6 and Sm29 in schistosomula surface by sera from immunized mice.

<p>Twenty newly-transformed schistosomula (A) were incubated with sera from mice inoculated with saline + CFA/IFA, immunized with rSm29 + CFA/IFA or immunized with rSm22.6 + CFA/IFA. Antibody reactivity to Sm29 and Sm22.6 surface proteins were detected by a secondary anti-mouse IgG-FITC-conjugated antibody. As an experimental control, parasites were incubated with anti-mouse IgG-FITC-conjugated antibody. The fluorescence in the schistosomula tegument, after incubation, was observed by fluorescence microscopy. Scale bar = 25 μm (x400). A significant fluorescence measurement was detected (B) in the schistosomulum incubated with sera from rSm22.6 and rSm29 immunized mice compared to parasite incubated with sera from saline inoculated animals. Fluorescence intensity in the schistosomula tegument was measure using ImageJ software. The graphic represent box plot with whiskers from minimum to maximum values of relative fluorescence (Arbitrary unit). Significant differences were denoted in the graphic.</p