Oral Immunization of Mice with Live Pneumocystis murina Protects against Pneumocystis Pneumonia

Abstract

Pneumocystis pneumonia is a major cause of morbidity and mortality in immunocompromised patients, particularly those infected with HIV. In this study, we evaluated the potential of oral immunization with live Pneumocystis to elicit protection against respiratory infection with Pneumocystis murina. C57BL/6 mice vaccinated with live P. murina using a prime-boost vaccination strategy were protected from a subsequent lung challenge with P. murina at 2, 7, 14, and 28 d postinfection even after CD4(+) T cell depletion. Specifically, vaccinated immunocompetent mice had significantly faster clearance than unvaccinated immunocompetent mice and unvaccinated CD4-depleted mice remained persistently infected with P. murina. Vaccination also increased numbers of CD4(+) T cells, CD8(+) T cells, CD19(+) B cells, and CD11b(+) macrophages in the lungs following respiratory infection. In addition, levels of lung, serum, and fecal P. murina-specific IgG and IgA were increased in vaccinated animals. Furthermore, administration of serum from vaccinated mice significantly reduced Pneumocystis lung burden in infected animals compared with control serum. We also found that the diversity of the intestinal microbial community was altered by oral immunization with P. murina. To our knowledge, our data demonstrate for the first time that an oral vaccination strategy prevents Pneumocystis infection.

Figure 1. Oral immunization with live P. murina prevents subsequent lung infection

(A) Schematic of the immunization protocol. C57BL/6 mice were orally gavaged with live P. murina (~2 × 10⁵ cysts in 100ul of PBS) every other day for a total of three immunizations. Control immunized mice received a naïve lung homogenate. Following immunization mice were infected intratracheally with live P. murina (~2 × 10⁵ cysts in 100 µl of PBS). Sham infected animals were given a naïve lung homogenate. Two groups of mice were depleted of CD4+ T cells every 6 days with CD4 depletion beginning prior to respiratory challenge. (B) P. murina lung burden following immunization. (C) Levels of P. murina in the intestinal tract following immunization. P. murina lung burden was assessed at the following time points: 2 days post lung infection (D), 7 days post lung infection (E), and 14 days post lung infection (F) via qPCR. Each dot represents an individual mouse, bars represent mean ± SEM, N=4 at indicated time points, * indicates P < 0.05 for the indicated comparison, by ANOVA with Dunn’s posttest. Mice with no detectable P. murina in the lung were given a count of 1 for visualization on a log scale.

Figure 2. Oral immunization stimulates the production of P. murina specific IgG and increases the number of macrophages and CD4 T cells in the lung

C57BL/6 mice were gavaged with live P. murina, as described previously. Serum and lung homogenates for flow cytometric analysis were collected and processed at the indicated time points post-immunization (respiratory infection is the 0 time point). (A) P. murina-specific IgG, in the serum was assayed by ELISA. Lung immunological responses: (B) CD4+ T cells, (C) CD8+ T cells, (D) CD11b+ macrophage, (E) 33D1+ dendritic cells, and (F) CD19+ B cells were quantified via flow cytometry. Dots and bars represent mean ± SEM, N=4 at each time points, * indicates P < 0.05, comparing immunized mice to control mice (without CD4 depletion) and + indicates P < 0.05, comparing immunized mice to control mice (with CD4 depletion) for each of the indicated time point, by ANOVA with Dunn’s posttest. Arrow indicates the time of P. murina respiratory challenge.

Figure 3. Oral immunization with live P. murina prevents subsequent lung infection independent of CD4+ T cells

(A) Schematic of the immunization protocol. Groups of C57BL/6 were immunized by gavage with live P. murina (~1 × 10⁶ cysts in 100 µl of PBS) at day 0, 2, and 14, following a prime-boost vaccination strategy. Control immunized mice received a naïve lung homogenate. Mice were then challenged by intratracheal inoculation with (~2 × 10⁵ cysts in 100 µl of PBS) 2 weeks after the last immunization. Sham infected animals were given a naïve lung homogenate. Two groups of mice were depleted of CD4+ T cells every 6 days with CD4 depletion beginning prior to respiratory challenge. (B) P. murina lung burden following immunization. (C) Levels of P. murina present in the intestinal tract following immunization. P. murina lung burden was assessed at the following time points: 2 days post lung infection (D), 7 days post lung infection (E), 14 days post lung infection (F), and 28 days post lung infection (G) via qPCR. Each dot represents an individual mouse, bars represent mean ± SEM, N=10 at indicated time points, * indicates P < 0.05 for the indicated comparison, by ANOVA with Dunn’s posttest. Mice with no detectable P. murina in the lung were given a count of 1 for visualization on a log scale.

Figure 4. Oral immunization with live P. murina stimulates the production of serum, lung, and intestinal P. murina specific IgG and IgA

C57BL/6 mice were gavaged with live P. murina, as described previously. Serum, lung homogenate, and fecal lavage were collected and processed at the indicated time points post-immunization (respiratory infection is the 0 time point). The presence of P. murina-specific IgG and IgA, in the serum (A and B, respectively), lung homogenate (C and D, respectively), and fecal lavage (E and F, respectively) was assayed by ELISA. Dots represent mean ± SEM, N=10 at each time points, * indicates P < 0.05, comparing immunized mice to control mice (without CD4 depletion) and + indicates P < 0.05, comparing immunized mice to control mice (with CD4 depletion) for each of the indicated time point, by ANOVA with Dunn’s posttest. Arrow indicates the time of P. murina respiratory challenge.

Figure 5. Oral immunization with live P. murina stimulates the proliferation and/or recruitment and activation of immune cells to the lung following respiratory infection

C57BL/6 mice were immunized by gavage with live P. murina following a prime-boost vaccination strategy, as described previously. Mice were then infection by intratracheal inoculation 2 weeks after the last immunization. Two groups of mice were depleted of CD4+ T cells. Mice were then sacrificed at the following time points: 3 days prior to lung infection, 2 days post lung infection, 7 days post lung infection, 14 days post lung infection, and 28 days post lung infection and lung immunological responses were determined. Lung CD4+ T cells (A), antigen experienced CD4+ CD44+ T cells (B), mucosal memory CD4+ CD69+ T cells (C), CD8+ T cells (D), antigen experienced CD8+ CD44+ T cells (E), mucosal memory CD8+ CD69+ T cells (F), CD11b+ macrophages (G), CD11c+ dendritic cells/macrophages (H), 33D1+ dendritic cells (I), CD19+ B cells (J) and CD19+, CD73+, CD80+, CD273+ memory B cells (K) were quantified via flow cytometry. Bars represent mean ± SEM, N=10 at each time points, * indicates P < 0.05, for each of the indicated comparisons, by ANOVA with Dunn’s posttest. N.C. = not collected at the indicated time point.

Figure 6. Passive immunization with P. murina specific IgG reduces P. murina lung burden

C57BL/6 were immunized by gavage with live P. murina (~1 × 10⁶ cysts in 100 µl of PBS) following a prime-boost vaccination strategy, as described previously. Control animals received a naïve lung homogenate following the same vaccination schedule and protocol. Two weeks following the last immunization mice were sacrificed and serum, mesenteric lymph node (MLN) CD4 T cells, and MLN non-CD4 leukocytes from each mouse was collected and pooled. Serum level of P. murina-specific IgG and IgA were assessed via ELISA (A and B, respectively). The serum was then used to passively immunize mice (C and D). Mice received two doses (200 µl of serum via intraperitoneal injection), with the first does one day prior to intratracheal inoculation with P. murina and the second dose four days following P. murina challenge. MLN leukocytes were adoptively transferred to naïve mice (E and F). Mice received approximately 1 × 10⁵ cells in 200 µl of PBS via intraperitoneal injection, and were then intratracheal inoculation with P. murina. Mice were then sacrificed 7 post infection and P. murina lung burden was assessed via qPCR. Each dot represents an individual mouse, bars represent mean ± SEM, N=5 for treatment group, * indicates P < 0.05 for the indicated comparison, by ANOVA with Dunn’s posttest.

Figure 7. Viable P. murina are not required for effective oral vaccination

C57BL/6 mice were gavaged with heat-killed P. murina. Serum and lung homogenates for P. murina burden and flow cytometric analysis were collected and processed at the indicated time points post-immunization. (A) P. murina lung burden was assessed 7 days post lung infection. P. murina-specific IgG (B) and IgA (C), in the serum was assayed by ELISA. Lung immunological responses: CD4+ T cells (D), CD8+ T cells (E), CD4+ CD44+ T cells (F), CD8+ CD44+ T cells (G), CD4+ CD69+ T cells (H), and CD8+ CD69+ T cells (I) were quantified via flow cytometry. Dots and bars represent mean ± SEM, N=10 at each time points, * indicates P < 0.05, for each of the indicated comparison, by ANOVA with Dunn’s posttest.

Figure 8. Oral vaccination alters the intestinal flora

Taxonomy summary and microbial diversity of OTUs from intestinal luminal samples of C57BL/6 mice orally immunized with P. murina or a naïve lung control following a homologous prime-boost immunization strategy. DNA extracted from luminal samples was used for 16S rRNA PCR amplification, sequenced and clustered into operational taxonomic units (OTUs). Principal coordinate analysis of the Unifrac metric and taxonomy (A–E) were analyzed using QIIME software to determine differences in microbial community structures for mice either receiving oral vaccination or control vaccination. The sequences from 10 animals per treatment are shown in each analysis. The taxonomy summary (A–E) represents OTUs with greater than 1% total representation.