Influence of maternal gestational treatment with mycobacterial antigens on postnatal immunity in an experimental murine model

Abstract

Background: It has been proposed that the immune system could be primed as early as during the fetal life and this might have an impact on postnatal vaccination. Therefore, we addressed in murine models whether gestational treatment with mycobacterial antigens could induce better immune responses in the postnatal life.

Methods/findings: BALB/c mice were treated subcutaneously (s.c.) at the second week of gestation with antigen (Ag)85A or heparin-binding hemagglutinin (HBHA) in the absence of adjuvant. Following birth, offspring mice were immunized intranasally (i.n.) with the same antigens formulated with the adjuvant cholera toxin (CT) at week 1 and week 4. One week after the last immunization, we assessed antigen-specific recall interferon gamma (IFN-gamma) responses by in vitro restimulation of lung-derived lymphocytes. Protection against infection was assessed by challenge with high dose Mycobacterium bovis Bacille Calmette-Guérin (BCG) given i.n. We found that recall IFN-gamma responses were higher in the offspring born to the treated mother compared to the untreated-mother. More importantly, we observed that the offspring born to the treated mother controlled infection better than the offspring born to the untreated mother. Since the gestational treatment was done in absence of adjuvant, essentially there was no antibody production observed in the pregnant mice and therefore no influence of maternal antibodies was expected. We hypothesized that the effect of maternal treatment with antigen on the offspring occurred due to antigen transportation through placenta. To trace the antigens, we conjugated fluorescent nanocrystals with Ag85A (Qdot-ITK-Ag85A). After inoculation in the pregnant mice, Qdot-ITK-Ag85A conjugates were detected in the liver, spleen of pregnant females and in all the fetuses and placentas examined.

Conclusion: The fetal immune system could be primed in utero by mycobacterial antigens transported through the placenta.

Figure 1. In vivo Qdot distribution.

Adult mice (3 animals per group) were injected with Qdot-ITK (120 pmol/mouse) via s.c. Mice were sacrificed at several time points (3 h, 24 h and 48 h) post-injection and skin, kidneys, liver, lungs and spleen were collected and embedded in OCT. Sections were fixed, mounted and observed under green light in a fluorescence microscope. Qdot-ITK fluorescence was detected in skin slides at 3 h (a), 24 h (b) and 48 h (c) post inoculation, and in slides from the spleen (d) and the liver (e) at 48 h post inoculation. Magnification 100×.

Figure 2. Distribution of Qdot-ITK-Ag85A in mother and fetuses.

Pregnant mice (3 animals per group) were injected with Qdot-ITK-Ag85A (120 pmol/mouse) via s.c. Mice were sacrificed 48 h post inoculation and mother organs as well as fetuses and placentas were removed and embedded in OCT. Sections were fixed, mounted and observed under green light in a fluorescence microscope. Qdot-ITK-Ag85A fluorescence was detected in the skin (a), placenta (b) and fetuses (c, d). Magnification 100×. Prior to the inoculation, Qdot-ITK-Ag85A were run in a 0.5% agarose gel (e) to check conjugation. Lane 1, unconjugated Qdot-ITK; lane 2, Qdot-ITK-Ag85A. Recall IFN-γ responses after in vitro restimulation of lung cells with Ag85A were measured in the offspring born to the mother that received Qdot-ITK-Ag85A or Qdot-ITK (f).

Figure 3. Maternal treatment increases postnatal cellular immune responses.

Pregnant mice at 2^nd week of gestation were treated with rHBHA via s.c. Following birth, neonates were immunized twice i.n. with rHBHA formulated with CT at week 1 (W1) and week 4 (W4). One week after the last immunization, lung cells from the rHBHA-immunized animals were re-stimulated in vitro with rHBHA to measure recall IFN-γ responses. Data show mean IFN-γ ± s.d. of triplicate wells prepared from pooled samples (3–4 mice/group). Results were analyzed from two independent experiments. p value (s) was calculated by comparing two groups using t test. * significant when p<0.05.