Oral Immunization of Mice with Live Pneumocystis murina Protects against Pneumocystis Pneumonia This information is current as of February 19, 2016.

Derrick R. Samuelson, Nicholas M. de la Rua, Tysheena P. Charles, Sanbao Ruan, Christopher M. Taylor, Eugene E. Blanchard, Meng Luo, Alistair J. Ramsay, Judd E. Shellito and David A. Welsh

Supplementary Material

http://www.jimmunol.org/content/suppl/2016/02/09/jimmunol.150200 4.DCSupplemental.html

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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J Immunol published online 10 February 2016 http://www.jimmunol.org/content/early/2016/02/09/jimmun ol.1502004

Published February 10, 2016, doi:10.4049/jimmunol.1502004 The Journal of Immunology

Oral Immunization of Mice with Live Pneumocystis murina Protects against Pneumocystis Pneumonia Derrick R. Samuelson,* Nicholas M. de la Rua,* Tysheena P. Charles,* Sanbao Ruan,* Christopher M. Taylor,† Eugene E. Blanchard,† Meng Luo,† Alistair J. Ramsay,†,‡ Judd E. Shellito,*,‡ and David A. Welsh*

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neumonia due to the opportunistic human fungal pathogen Pneumocystis jirovecii is an AIDS-defining illness, and there is a direct inverse relationship between CD4+ T cell counts in the blood and the risk for infection (1). Pneumocystis is also a major cause of mortality in patients whose CD4+ T cell number or function are significantly depressed because of malignancy, chemotherapy, or other immunosuppression (1, 2). Animal models of immunodeficiency demonstrate that the loss of CD4+ T cells renders mammals susceptible to Pneumocystis lung infection (2). Despite current treatment strategies for HIV infection Pneumocystis pneumonia remains a common clinical problem (3). Although highly active antiretroviral therapy has reduced the incidence of Pneumocystis infections in HIV+ individuals, the *Section of Pulmonary/Critical Care and Allergy/Immunology, Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA 70112; † Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA 70112; and ‡Louisiana Vaccine Center, Louisiana State University Health Sciences Center, New Orleans, LA 70112 ORCIDs: 0000-0002-5356-1413 (D.R.S.); 0000-0002-7983-5275 (C.M.T.); 0000-00018626-6456 (M.L.); 0000-0002-3506-4544 (A.J.R.); 0000-0001-7729-2708 (J.E.S.); 0000-0002-0008-6161 (D.A.W.). Received for publication September 9, 2015. Accepted for publication January 6, 2016. This work was supported by National Institutes of Health Public Health Service Grant P01-HL076100, the Louisiana State University School of Medicine Microbial Genomics Resource Center, which is funded in part by National Institutes of Health Grant P60-AA009803 and National Institute of General Medical Sciences Grant UG54-GM104940. Address correspondence and reprint requests to Dr. David A. Welsh, Section of Pulmonary/Critical Care and Allergy/Immunology, Department of Medicine, Louisiana State University Health Sciences Center, 1901 Perdido Street, Suite 3205, New Orleans, LA 70112. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: dpi, day postinfection; GI, gastrointestinal; MLN, mesenteric lymph node; OTU, operational taxonomic unit; qPCR, quantitative PCR; ZPS, zwitterionic polysaccharide. Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502004

reduction is not as dramatic as is observed with other opportunistic infections (3). In addition, subpopulations of HIVinfected patients remain at risk despite receiving highly active antiretroviral therapy (3–5). Furthermore, an increasing number of patients are receiving immunosuppressive medical regimens (6). These data indicate that there is a need for vaccination strategies to prevent Pneumocystis infections in the growing number of at-risk patients (6). Several oral vaccines are currently licensed in the United States for the prevention of infectious diseases, including the Sabin polio vaccine, the Ty21 typhoid vaccine, and the rotavirus vaccine (7, 8). Furthermore, there is evidence that the intestinal microbiota may influence the effectiveness of oral vaccines because Lactobacillus spp. has been shown to increase the effectiveness of several oral vaccines (9, 10). Specifically, coupling of oral vaccines with probiotics has led to higher pathogen-specific Ab titers and significantly better protection (10), suggesting that alterations to the intestinal microflora may enhance the efficacy of oral vaccination. In this study, we evaluated the efficacy of oral Pneumocystis murina immunization against respiratory infection with P. murina. In a murine model of Pneumocystis pneumonia, mice orally vaccinated with live P. murina, the mouse-specific strain of Pneumocystis, are protected from a subsequent lung challenge with P. murina, even after CD4+ T cell depletion, and this was associated with enhanced immune responses in the lung. Furthermore, vaccinated animals had increased levels of lung, serum, and fecal P. murina–specific IgG and IgA, and administration of serum from vaccinated mice significantly reduced Pneumocystis lung burden in infected animals. We additionally found that oral immunization with P. murina changes the diversity of the intestinal microbial community. These studies demonstrate for the first time, to our knowledge, an oral vaccination strategy for protection against Pneumocystis pneumonia. The results hold promise for advances in the development of oral vaccines in high-risk hosts with defective CD4+ T cell function.

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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. The Journal of Immunology, 2016, 196: 000–000.

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Materials and Methods Mice Female 6- to 8-wk-old C57BL/6 mice were obtained from Charles Rivers Breeding Laboratories (Wilmington, MA). Animals were housed in filtertopped cages and were provided autoclaved water and chow ad libitum. Animals were kept in the animal care facility at the Louisiana State University Health Sciences Center for $2 d prior to the beginning of any experiment. Animals were handled under a laminar flow hood to maintain specific pathogen-free conditions throughout the course of the experiment. All experiments were approved by the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee.

P. murina inoculation

Adoptive-transfer and passive immunization studies C57BL/6 mice were orally immunized with P. murina as described above. Animals were then sacrificed, and CD4+ T cells were isolated from mesenteric lymph nodes from both immunized and naive mice, as described elsewhere (16). CD4 + T cells were purified using the commercially available CD4+ T Cell Isolation Kit (Miltenyi Biotec. San Diego, CA). Approximately 1 3 105 isolated CD4+ T cells from immunized or naive mice were then administered via i.p. injection. In parallel, the remaining MLN leukocytes (2CD4+ T cells) were isolated, and ∼1 3 105 cells were given via i.p. injection. Mice were then infected with 2 3 105 P. murina cysts intratracheally and sacrificed 7 d later. P. murina lung burden was assessed, as described above. Passive immunization experiments were performed by giving i.p. injections of 200 ml pooled serum from immunized or naive mice 1 d prior to infection with P. murina and 4 d postinfection. Mice were then sacrificed 7 d postinfection, and lung P. murina burden was assessed, as described above.

Determining the population structure of the microbiota in C57BL/6 mice

Total RNA was isolated from lung tissue of infected mice by the TRIzol method (Invitrogen, Grand Island, NY), reverse transcribed, and real-time quantitative PCR (mitrochondrial small ribosomal subunit RNA) was used to determine P. murina lung burden. qPCR has been previously validated against microscopic enumeration and was performed as described elsewhere (12, 13).

Sequencing and bioinformatics were performed by the Louisiana State University School of Medicine Microbial Genomics Resource Group (http://metagenomics.lsuhsc.edu/). The intestinal contents were flash frozen, and genomic DNA extraction was performed using the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA) modified to include bead-beating and 16S metagenomic sequencing was performed as described previously (17). Briefly, 16S ribosomal DNA hypervariable regions V3 and V4 were amplified using gene-specific sequences, Illumina adapters, and molecular barcodes primers. Samples were then sequenced on an Illumina MiSeq (Illumina, San Diego, CA) using the 2 3 300-bp V3 sequencing kit. The read files were processed through the UPARSE pipeline (drive5, Tiburon, CA) using forward reads that covered the V3 region truncated to a uniform length of 250 bp, discarding reads with quality score less than 15 remaining in the first 250 bp. Unique reads were clustered into operational taxonomic units (OTU) at 97% similarity. Chimeric OTUs were removed as identified by UCHIME by drive5. Finally, the original filtered reads were mapped to the OTUs using USEARCH by drive5 at 97% identity. QIIME 1.9.1 (open source, www.qiime.org) was used to pick and align a representative set (18). Uclust by drive5 was used to assign a taxonomic classification to each read in the representative set (19). Relative abundance of each OTU was examined at phylum, class, order, family, genus, and species levels. b and a diversity metrics as well as taxonomic community assessments were produced using QIIME 1.9.1 scripts.

Flow cytometric analysis of lymphocytes from lung tissue

Statistical analysis

Lung tissue of each animal was minced, suspended in 10 ml homogenization buffer consisting of RPMI 1640 medium with 1 mg/ml Collagenase type 1 (Worthington Biochemical, Lakewood, NJ) and 30 mg/ml DNase I (Roche Diagnostics, Indianapolis, IN), and incubated at 37˚C with shaking for 30 min. Cell suspensions were further disrupted by passing through a 70-mm nylon mesh. RBCs were lysed using RBC lysis buffer (BioLegend, San Diego, CA) prior to staining. After washing with PBS, viable cells were counted on a hemocytometer using the trypan blue–exclusion method. One million viable cells were stained with the Live/Dead Fixable Dead Cell Stain Kit (Invitrogen Eugene, OR), followed by immunological staining with various combinations of fluorochrome-conjugated Abs specific for murine CD45, CD3e, CD4, CD8a, CD44, CD69, CD19, CD11b, CD11c, 33D1 (BioLegend), CD73, CD80, and CD273 (Novus Biologicals, Littleton, CO), suspended in FACS buffer at predetermined concentrations for 30 min at 4˚C. All cells were pretreated with TruStain FcX Anti-mouse CD16/32 Ab (BioLegend). Wells were then washed with FACS buffer and fixed with PBS + 1% formalin. For all experiments, cells were acquired using an LSR II flow cytometer (BD Biosciences, San Jose, CA), and analyses were performed using FlowJo software (version 9.4; Tree Star, Ashland, OR). Gating strategies are described in Supplemental Fig. 1.

Results are presented as mean 6 SEM. Statistical analyses were performed using GraphPad Prism 5 (La Jolla, CA), and statistical significance was measured at p # 0.05. The nonparametric Kruskal–Wallis one-way ANOVA, followed by post hoc Dunn’s multiple comparisons of the means was used for all in vivo assays.

CD4 depletion Mice were depleted of CD4+ T cells by i.p. injection of 0.1 mg anti-CD4+ mAb (hybridoma GK1.5; National Cell Culture Center) in 100 ml PBS 3 d prior to infection. Depletion was maintained by i.p. injection every 6 d. This treatment protocol results in .97% sustained depletion of CD4+ lymphocytes from blood and lymphoid tissue for up to 14 wk (11).

RNA isolation and real-time RT-PCR for P. murina rRNA quantification

Lung, serum, and fecal IgG/IgA analysis P. murina whole-cell lysate and P. murina–specific IgG and IgA ELISA were generated and performed as described previously (14, 15). Fecal lavages were performed by resuspending the intestinal content of each individual mouse in 1.0 ml sterile PBS. Samples were then vortexed

Results Vaccination strategy To evaluate oral vaccine approaches for P. murina infection, we tested the protective efficacy of two vaccination strategies, which used oral administration of P. murina in a murine immunization Pneumocystis pneumonia model. A three-dose vaccination strategy and a homologous prime-boost vaccination strategy were each used to determine the efficacy of oral vaccination. To investigate the three-dose strategy, C57BL/6 mice were orally gavaged with live P. murina (∼2 3 105 cysts) three times every other day, followed by 2 wk of rest. Prime-boost vaccination relies on the restimulation of Ag-specific immune cells generated following vaccination. Homologous prime-boost immunizations use readministration of the same vaccine, whereas heterologous prime-boost vaccination uses Ag delivered in one vector and then administered as the same Ag in the context of a different vector (20). In our homologous

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P. murina organisms for inoculation were obtained from lung homogenates from chronically infected C57BL/6/NCr (C57BL/6 background) mice and purified as described previously (2, 11). Respiratory challenge: the number of P. murina cysts was quantified microscopically, and the inoculum concentration was adjusted to 2 3 106 cysts/ml. Recipient mice were lightly anesthetized with isoflurane (1–4% to effect). Animals were suspended by their front incisors, the tongue was gently extended out with forceps, and 100 ml inoculum (2 3 105 cysts) was injected into the trachea using a P200 pipette. After inhalation of inoculum was observed, the tongue was released, and the animal was allowed to recover from anesthesia. Oral gavage: P. murina cysts were quantified microscopically, the inoculum concentration was adjusted to 1 3 107 or 2 3 106 cysts/ml, and 100 ml inoculum (1 3 106 or 2 3 105 cysts) was orally gavaged into the stomach using a 24-gauge 25-mm animal feeding needle (Fine Science Tools, Foster City, CA). Heat-killed P. murina was generated by incubating for 1 h at 100˚C. No viable P. murina organisms were detected following treatment as determined by quantitative PCR (qPCR; data not shown). Control immunized and sham-infected animals received a naive lung homogenate.

thoroughly and centrifuged at 10,000 3 g to pellet organic matter and bacteria. Supernatants were collected and used immediately in ELISA or stored at 280˚C for later use. ELISA data are presented as the mean 6 the SEM and blank wells (background values) are subtracted from each sample value prior to mean calculation.

The Journal of Immunology prime-boost protocol, C57BL/6 mice were orally gavaged with live P. murina (∼1 3 106 cysts) three times, with the first two doses given 2 d apart followed by 2 wk of rest (prime). The mice then received the third oral gavage of live P. murina (homologous boost). In addition, both vaccination strategies were tested in groups of mice that were depleted of CD4+ T cells with the anti-CD4 mAb GK 1.5 every 6 d beginning prior to respiratory infection. This treatment protocol causes a sustained .97% depletion of CD4+ cells from blood and lymphoid tissue for up to 14 wk (1). All mice were then challenged by intratracheal inoculation with P. murina (∼2 3 105 cysts) 2 wk after the last immunization. We also confirmed oral and respiratory delivery of P. murina only to the target sites (Supplemental Fig. 2). Specifically, mice were orally gavaged and intratracheally inoculated with 1 M methylene blue. Methylene blue staining was not detected in the lung of orally gavaged mice. Oral immunization prevents subsequent lung infection

patient experiences immunosuppression. Therefore, a vaccine that could be given prior to immunosuppression would be a valuable addition to supplement/enhance natural immunity to Pneumocystis. Prior to respiratory infection, P. murina lung burden and intestinal load were determined. P. murina was not detected in the lungs or in the intestinal tract of mice that received oral vaccination 3 d prior to lung infection (Fig. 1B, 1C, respectively). P. murina lung burden at 2 d (Fig. 1D), 7 d (Fig. 1E), and 14 d (Fig. 1F) postinfection was determined by qPCR. P. murina was not recovered from any of the mice 3 d prior to respiratory challenge, indicating that the oral vaccination did not deposit organisms in the lungs and that P. murina does not colonize the intestinal tract. Oral vaccination significantly reduced both P. murina lung burden and the number of CD4-intact animals infected at 7 and 14 d postinfection (dpi) compared with the unvaccinated control animals. However, no significant protection was observed in CD4-depleted animals. These results indicate that mice orally vaccinated using a threedose vaccination are protected from a subsequent P. murina lung infection only in CD4-intact animals. We then went on to test whether oral vaccination could be enhanced by alternative vaccination strategies to provide protection to animals even after loss of CD4+ T cells. Oral immunization increases the level of serum P. murina– specific IgG and enhances cellular lung immune responses following respiratory infection We measured P. murina–specific IgG in the serum (Fig. 2A) of mice that received three-dose oral vaccination. At all time points

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 3 105 cysts in 100 ml PBS) every other day for a total of three immunizations. Control immunized mice received a naive lung homogenate. Following immunization mice were infected intratracheally with live P. murina (∼2 3 105 cysts in 100 ml PBS). Sham-infected animals were given a naive lung homogenate. Two groups of mice were depleted of CD4+ T cells every 6 d 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 d post lung infection (D), 7 d post lung infection (E), and 14 d post lung infection (F) via qPCR. Mice with no detectable P. murina in the lung were given a count of 1 for visualization on a log scale. Each dot represents an individual mouse; bars, mean 6 SEM; n = 4 at indicated time points; *p , 0.05 for the indicated comparison by ANOVA with Dunn’s posttest.

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Mice orally vaccinated with live P. murina using a three-dose vaccination strategy with a vaccine dose of 2 3 105 cysts/dose were protected from a subsequent lung infection with P. murina in CD4-intact animals (Fig. 1). Significant levels of protection were not observed in CD4-depleted animals. A schematic outline of the three dose vaccination strategy is shown in Fig. 1A. CD4-intact animals were also included in our vaccination strategy because they allow us to evaluate the normal immune responses to vaccination. More importantly, we choose to vaccinate immune-intact animals because most humans are exposed to Pneumocystis early in life (2), yet naturally acquired immunity is not protective if the

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Oral prime-boost immunization prevents subsequent lung infection even after loss of CD4+ T cells Mice orally vaccinated with live P. murina using a prime-boost vaccination strategy with a vaccine dose of 1 3 106 cysts/dose

were protected from a subsequent lung infection with P. murina in both CD4-intact and CD4-depleted animals (Fig. 3). A schematic outline of the prime-boost vaccination strategy is depicted in Fig. 3A. As described above, P. murina lung burden and intestinal load were determined prior to respiratory infection. P. murina was not detected in the lungs or in the intestinal tract of mice that received oral vaccination 3 d prior to lung infection (Fig. 3B, 3C, respectively), indicating that oral vaccination did not deposit organisms in the lungs and that P. murina does not colonize the intestinal tract. P. murina lung burden at 2 d (Fig. 3D), 7 d (Fig. 3E), 14 d (Fig. 3F), and 28 d (Fig. 3G) postinfection was determined by qPCR. Prime-boost vaccination significantly reduced both P. murina burden and the number of CD4-intact animals infected at 2 dpi. Similarly, the lung burden and the number of infected animals in both CD4-intact and CD4-depleted animals were significantly reduced at 7 and 14 dpi compared with unvaccinated control animals. This remained the case at 28 dpi for CD4-depleted mice. These results indicate that mice orally vaccinated using prime-boost vaccination are protected from a subsequent P. murina lung infection. In addition, the data suggest that oral vaccination generates a systemic and pulmonary immune response, which are still protective after loss of CD4+ T cells. P. murina oral immunization increases the level of serum, lung, and fecal P. murina–specific IgG/IgA We measured P. murina–specific IgG and IgA in the serum (Fig. 4A, 4B), in the lung (Fig. 4C, 4D), and in the intestinal tract (Fig. 4E, 4F) of mice that received prime-boost vaccination. At all time points assessed in the serum, lung and fecal (IgA only), P. murina–specific IgG and IgA levels were significantly higher in the vaccinated animals as compared with control animals, regardless of CD4 depletion. Fecal P. murina–specific IgG levels

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 postimmunization (respiratory infection is the 0 time point). (A) P. murina–specific IgG, in the serum was assayed by ELISA. Lung immunological responses: CD4+ T cells (B), CD8+ T cells (C), CD11b+ macrophage (D), 33D1+ dendritic cells (E), and CD19+ B cells (F) were quantified via flow cytometry. Arrow indicates the time of P. murina respiratory challenge. Dots and bars, mean 6 SEM; n = 4 at each time points; *p , 0.05 comparing immunized mice to control mice (without CD4 depletion), +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.

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assessed, P. murina–specific IgG was significantly higher in the vaccinated animals as compared with control animals, regardless of CD4 depletion. We further assessed immune responses in the lung, spleen, and intestinal tract of orally vaccinated and control animals. We did not observe significantly different immune cell populations in the spleen or intestinal tract between orally immunized and control mice (Supplemental Fig. 3). However, significant differences in responses between immunized and control mice were observed in the lungs following respiratory infection with P. murina. The percentage of CD4+ T cells in the lung 7 dpi in CD4-intact animals was significantly increased compared with control animals. Likewise, the percentage of lung CD4+ T cells was significantly depleted compared with control animals in CD4depleted groups (Fig. 2B). Conversely, oral immunization did not significantly increase the percentage (or absolute numbers) of CD8+ T cells in the lung 2, 7, or 14 dpi in CD4-intact or CD4depleted animals (Fig. 2C). We next evaluated the immune responses of APCs following vaccination. CD4-intact animals had a significant increase in the percentage of CD11b+ macrophages [CD11b is a cell surface integrin of macrophages (21)] in the lung 2 dpi (Fig. 2D). No significant differences were observed in the percentage of 33D1+ dendritic, or CD19+ B cells in the lung following respiratory infection (Fig. 2E, 2F, respectively) in orally immunized mice compared with control animals in both CD4intact and CD4-depleted mice. These results suggest that both systemic and pulmonary immune responses are augmented following oral vaccination.

The Journal of Immunology

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were increased at 2, 7, and 14 dpi in immunized animals compared with control animals. These data suggest that both systemic and pulmonary immune responses are augmented following vaccination. In addition, the quantity of P. murina–specific IgG or IgA may be critical for effective clearance of P. murina in the context of immunosuppression. Oral immunization enhances cellular immune responses in the lungs following respiratory infection We then assessed immune responses in the lung of orally vaccinated and control animals. We first evaluated T cell responses following prime-boost vaccination. The percentage of CD4+ T cells in the lungs at 23, 2, 7, and 14 dpi in immunized CD4-intact animals was significantly increased compared with control animals. Likewise, the percentage of CD4+ T cells in the lungs at 7 and 14 dpi was significantly increased compared with control animals in the CD4-depleted group (Fig. 5A). Although CD4depleted vaccinated animals still had significantly more CD4+ T cells then unvaccinated CD4-depleted mice, they were markedly depleted compared with intact animals. Conversely, oral immunization did not significantly increase the percentage (or absolute

numbers) of CD8+ T cells in the lungs at 2, 7, 14, or 28 dpi in either CD4-intact or CD4-depleted animals (Fig. 5D). We further evaluated T cell responses by examining CD4+ and CD8+ T cell activation and memory using the activation marker CD44 (22) and the mucosal memory marker CD69 (23). Prime-boost vaccination significantly increased the percentage of CD4+CD44+ and CD4+ CD69+ T cells in the lungs at 7 and 14 dpi compared with control animals for both CD4-intact and CD4-depleted mice (Fig. 5B, 5C). Vaccinated mice also had a significantly higher percentage of activated CD8+CD44+ and CD8+CD69+ T cells in the lungs at 7 and 14 dpi compared with control animals for both CD4-intact and CD4-depleted mice (Fig. 5E, 5F). These results indicate that oral vaccination activates CD4+ and CD8+ T cells and induces memory response in the lung following respiratory infection. We next evaluated the immune responses of APCs following vaccination. CD4-intact animals had a significant increase in the absolute number of CD11b+ macrophages in the lungs 2 dpi. In addition, immunized CD4-depleted mice also had a significant increase in the absolute numbers of CD11b+ macrophages in the lungs at 7 and 14 dpi (Fig. 5G). We also examined absolute numbers of CD11c + macrophage/dendritic cells in the lungs

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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 3 106 cysts in 100 ml PBS) at days 230, 228, and 214, following a prime-boost vaccination strategy. Control immunized mice received a naive lung homogenate. Mice were then challenged by intratracheal inoculation with (∼2 3 105 cysts in 100 ml PBS) 2 wk after the last immunization. Sham-infected animals were given a naive lung homogenate. Two groups of mice were depleted of CD4+ T cells every 6 d 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 d post lung infection (D), 7 d post lung infection (E), 14 d post lung infection (F), and 28 d post lung infection (G) via qPCR. Mice with no detectable P. murina in the lung were given a count of 1 for visualization on a log scale. Each dot represents an individual mouse; bars, mean 6 SEM; n = 10 at indicated time points; *p , 0.05 for the indicated comparison by ANOVA with Dunn’s posttest.

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following respiratory infection. CD11c is a type I transmembrane protein expressed at high levels on most dendritic cells but can also be detected on monocytes and macrophages (24). Vaccination significantly increased the absolute numbers of CD11c + macrophage/dendritic cells in the lungs at 2 and 7 dpi in CD4intact animals and 7 and 14 dpi in CD4-depleted mice (Fig. 5H). Dendritic cell responses were then determined using the mouse dendritic cell–specific surface marker 33D1 (25). The absolute number of 33D1+ dendritic cells in orally immunized mice were significantly increased in the lung 7 dpi compared with control animals in both CD4-intact and CD4-depleted mice (Fig. 5I). These results indicate that oral vaccination significantly increases numbers of APCs in the lung following respiratory infection. Finally, B cell responses in the lung following prime-boost vaccination were assessed. CD4-intact immunized mice had significantly more lung CD19+ B cells at 7 dpi [CD19 is a cell surface molecule that assembles with the AgR of B cells (26)]. We also found a significant increase in CD19+ B cell numbers at 14 dpi in CD4-depleted animals (Fig. 5J). Furthermore, we determined the absolute numbers of memory B cells in the lung following respiratory infection, using the murine B cell memory markers CD73, CD80, and CD273 (27). Prime-boost vaccinated CD4intact mice had significantly more memory B cells in the lungs at 2 and 7 dpi. Likewise, CD4-depleted animals had significantly increased numbers of CD19 + memory B cells in the lungs at 7 and 14 dpi (Fig. 5K). These results indicate that oral vaccination significantly increases populations of CD19+ B cells and memory B cells in the lungs following respiratory infection. Immune sera from orally immunized mice reduces P. murina lung burden Three possible mechanisms for the induced protection against respiratory infection were examined: 1) production of protective Abs, 2) induction of MLN-associated CD4+ T cells, or 3) induction of MLN-associated (non-CD4+) leukocytes. We first assessed the capacity of vaccine-induced Abs to protect naive mice from respiratory infection via passive immunization. We measured P. murina–specific IgG and IgA in the serum prior to passive immunization (Fig. 6A, 6B, respectively). In these experiments,

mice that received serum (100 ml given i.p. on 0 and 4 dpi) from mice orally vaccinated with P. murina (either three-dose vaccination or prime-boost vaccination) had significant reductions in Pneumocystis lung burden compared with mice that received serum from control animals (Fig. 6C, 6D). We further evaluated potential mechanisms of vaccine induced protection by performing two adoptive transfer experiments. First, we isolated CD4+ T cells from the MLN of orally immunized and control animals. The isolated CD4+ T cells were quantified and 1 3 105 CD4+ T cells were adoptively transferred to naive mice by i.p. injection. In parallel, the remaining MLN leukocytes (minus CD4 + T cells) were quantified, and 1 3 10 5 total cells were adoptively transferred to naive mice. Mice were then infected with P. murina and sacrificed 7 d later, and P. murina lung burden was determined via qPCR. No protection was observed in mice given immune CD4+ T cells (Fig. 6E) or MLN cells (Fig. 6F). Viable P. murina are not required for effective oral vaccination To further investigate the efficacy of prime-boost oral vaccination, we investigated a heat-killed P. murina vaccine. Mice orally vaccinated with heat-killed P. murina using a prime-boost vaccination strategy were protected from a subsequent lung infection with P. murina in CD4-intact animals (Fig. 7). P. murina lung burden at 7 dpi (Fig. 7A) was determined by qPCR. Prime-boost vaccination significantly reduced both P. murina burden and the number of CD4-intact animals infected 7 dpi compared with the unvaccinated control animals. In addition, P. murina–specific serum IgG and IgA levels were significantly increased in animals vaccinated with heat-killed P. murina (Fig. 7B, 7C). Similar to oral vaccination with live P. murina, vaccination with a heat-killed P. murina lead to a significant increase in the percentage of lung CD4+ T cells (Fig. 7D), Ag-experienced CD4+CD44+ T cells (Fig. 7F), mucosal memory CD4+CD69+ T cells (Fig. 7H), CD8+ T cells (Fig. 7E), Ag-experienced CD8+CD44+ T cells (Fig. 7G), and mucosal memory CD8+CD69+ T cells (Fig. 7I) as judged by flow cytometry. However, heat-killed P. murina was intermediate in protection compared with live P. murina, suggesting that peptide folding, structure, and/or live organisms maybe required for optimal efficacy.

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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 postimmunization (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. Arrow indicates the time of P. murina respiratory challenge. Dots represent mean 6 SEM; n = 10 at each time points; *p , 0.05 comparing immunized mice to control mice (without CD4 depletion) and +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.

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The intestinal microbiota is altered by oral P. murina vaccination The intestinal microbiota participates in the development of immune function, both in the gut and in other organs, and is involved in the metabolism of certain drugs and toxins, suggesting that the intestinal microbiota could significantly affect how individuals respond to oral vaccines. Therefore, we sought to determine whether the intestinal microbiota is altered by our P. murina oral vaccination strategies. We first assessed the differences in the intestinal microbiota of mice vaccinated using the three-dose method. Specifically, we assessed the gastrointestinal (GI) microbial communities in mice 10 d after the third gavage with P. murina or a naive lung control using 16s rDNA deep sequencing. Analysis of the Unifrac metric showed that the microbial community b diversity was significantly impacted by oral immunization with P. murina (p = 0.0001 for unweighted analysis and p = 0.0123 for weighted analysis) (Supplemental Fig. 4A). Furthermore, immunized mice had significant increases in the genus Akkermansia (p = 0.009) and the family Coriobacteriaceae (p = 0.005). In addition, we observed a significant decrease in the family Lachnospiraceae (p = 0.04) in orally immunized animals compared with control mice (Supplemental Fig. 4B). We also assessed differences in the intestinal microbiota of mice vaccinated using a prime-boost vaccination strategy. We assessed the GI microbial communities of mice 11 d after the second gavage

(Fig. 8A), 1 d after the third gavage (Fig. 8B), 4 d after the third gavage (Fig. 8C), 7 d after the third gavage (Fig. 8D), and 11 d after the third gavage (Fig. 8E) with P. murina, or a naive lung control, using 16s rDNA deep sequencing. Analysis of the Unifrac metric showed that the microbial community b diversity was significantly impacted by oral immunization with P. murina (p = 0.0001 for unweighted analysis and p = 0.0001 for weighted analysis) (Fig. 8). Furthermore, immunized mice had significant increases of the genre Akkermansia (p , 0.0001), Ruminococcus (p , 0.0001), Bacteroides (p , 0.0001), Parabacteroides (p , 0.0001), Mucispirillum (p , 0.05), and Oscillospira (p , 0.01), the families Ruminococcaceae (p , 0.0001) and Lachnospiraceae (p , 0.0001), and the orders RF39 (p , 0.01) and Clostridiales (p , 0.0001). In addition, we observed a significant decrease in the genus Turicibacter (p , 0.0001) and in the families Lachnospiraceae (p , 0.001) and S24-7 (p , 0.0001) in orally immunized animals compared with control mice (Fig. 8). These data demonstrate that P. murina oral vaccination alters the intestinal microbiota, suggesting that vaccine efficacy may be influenced by differences in microbial diversity. Whether or not the microbiota changes participate in the induction of P. murina immune memory will require further investigation.

Discussion Immunological cross-talk occurs between the GI tract and the respiratory tract (28–30). Fluids, particles, or even microorganisms

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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 wk after the last immunization. Two groups of mice were depleted of CD4+ T cells. Mice were then sacrificed at the following time points: 3 d prior to lung infection, 2 d post lung infection, 7 d post lung infection, 14 d post lung infection, and 28 d post lung infection and lung immunological responses were determined. Lung CD4+ T cells (A), Ag-experienced CD4+ CD44+ T cells (B), mucosal memory CD4+CD69+ T cells (C), CD8+ T cells (D), Ag-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+, and CD273+ memory B cells (K) were quantified via flow cytometry. Bars, mean 6 SEM; n = 10 at each time points; *p , 0.05, for each of the indicated comparisons by ANOVA with Dunn’s posttest. N.C., not collected at the indicated time point.

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can be found in the GI tract a short time after inhalation into the respiratory tract (31, 32). Thus, the GI tract may frequently be exposed to respiratory system pathogens. It is also possible that

the mucosal immune system of the GI tract may serve as a primary sensor of foreign organisms from the environment. Importantly, disturbances in intestinal homeostasis could have drastic effects

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 postimmunization. (A) P. murina lung burden was assessed 7 d 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, mean 6 SEM; n = 10 at each time points; *p , 0.05 for each of the indicated comparison by ANOVA with Dunn’s posttest.

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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 3 106 cysts in 100 ml PBS) following a prime-boost vaccination strategy, as described previously. Control animals received a naive lung homogenate following the same vaccination schedule and protocol. Two weeks following the last immunization, mice were sacrificed and serum, 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 was assessed via ELISA (A and B, respectively). The serum was then used to passively immunize mice (C and D). Mice received two doses (200 ml serum via i.p. injection), with the first does 1 d prior to intratracheal inoculation with P. murina and the second dose 4 d following P. murina challenge. MLN leukocytes were adoptively transferred to naive mice (E and F). Mice received ∼1 3 105 cells in 200 ml PBS via i.p. injection and were then intratracheal inoculation with P. murina. Mice were then sacrificed 7 dpi, and P. murina lung burden was assessed via qPCR. Each dot represents an individual mouse; bars, mean 6 SEM; n = 5 for treatment group; *p , 0.05 for the indicated comparison by ANOVA with Dunn’s posttest.

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on systemic (e.g., lung) immune responses. Following from this, GI mucosal vaccination against respiratory pathogens may be a valuable method for the prevention of infection. Mucosal vaccination provides several benefits over conventional systemic vaccination, such as higher levels of Abs and protection at mucosal surfaces (7, 33–36). Although mucosal vaccines may target a specific mucosal surface (e.g., respiratory or intestinal mucosa), it is noteworthy that vaccination at one mucosal surface often confers resistance at other sites. Specifically, it is possible to immunize against respiratory pathogens via a gut immunization strategy (37–40). Several studies have addressed gut-mediated lung immunity (7, 37–40). Izadjoo et al. (39) found that orally administered live attenuated Brucella melitensis elicited a cellular and humoral immune response and mice were protected against intranasal challenge with virulent B. melitensis. Optimal protection following inoculation with the attenuated bacteria was dose dependent and enhanced by a booster vaccination (39, 40), which is consistent with our findings showing that vaccine efficacy was increased with boosting and a higher dose. Additional studies using oral vaccination have shown protective immune responses in the lungs of mice against Mycobacterium tuberculosis and Francisella tularensis (38, 40). Both of these studies demonstrated induction of Ag-specific Ab responses in serum and bronchoalveolar lavage fluids, proliferation of Ag-specific IFN-g– producing cells, and an overall reduction in pathogen burdens (38, 40). These data are consistent with our data showing production of P. murina–specific Abs in the serum, lung, and intestine following oral immunization. Our current experiments demonstrate that mice orally vaccinated with live P. murina using a prime-boost vaccination strategy are protected from a subsequent lung challenge with P. murina as compared with control animals in both CD4-intact and CD4depleted animals. Vaccinated mice exhibited a significant reduction in both the lung burden of P. murina and the numbers

of animals infected 2, 7, 14, and 28 dpi. These results suggest that oral vaccination mediates a systemic immune response that remains protective even after loss of CD4+ T cells. To our knowledge this represents the first paper on the effectiveness of an oral vaccine in a CD4-deficient model. We observed a systemic response characterized by increases in P. murina–specific IgG and IgA in the serum, lung, and intestinal tract as well as significant increases in CD4+ T cells, activated CD4+ T cells, memory CD4+ T cells, activated CD8+ T cells, memory CD8+ T cells, APCs (macrophages and dendritic cells), and both CD19+ B cells and memory CD19+ B cells. Also of interest, we found that at 28 dpi the unvaccinated CD4-depleted mice infected with P. murina had an increased numbers of DCs and B cells and an increased proportions of activated CD8 T cells compared with the vaccinated CD4-depleted mice infected with P. murina, which we believe could be due the inability to clear the infection, thus driving a more intense immune response. Although we have demonstrated associated changes in overall numbers of relevant T and B cells, work is under way to correlate immunization with P. murina– specific T and B cells. Although we observed significant immunological responses in the lung of orally vaccinated animals, no significant differences in immune responses were observed between orally immunized or control mice in the spleen or intestinal tract. We suspect that memory immune responses may have been generated in the spleen and/or the intestinal tract, but given that the pathogenic insult was in the lung, all cellular responses were only detectable in the respiratory tract, possibly as a result of trafficking from the intestine and/or spleen to the site of Ag deposition. We examined three possible mechanisms for the protective effect observed with oral immunization: 1) production of protective Abs, 2) MLN-associated CD4+ T cell memory, and 3) MLN-associated, non-CD4+ leukocyte memory. Administration of serum from vaccinated mice significantly reduced the Pneumocystis lung

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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 naive 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 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 .1% total representation.

10

ORAL IMMUNIZATION PROTECTS AGAINST PNEUMOCYSTIS PNEUMONIA alterations to the intestinal microbiota following vaccination occurred rapidly and lasted for 2 wk following the last vaccination, which may suggest that changes in the microbiota occur quickly and remain altered longer to facilitate the generation of host immunity. Differences in the composition of the intestinal microbiota are an area of interest in the field of mucosal immunology as a plethora of data demonstrating that changes in the intestinal microbiota are crucial for normal mucosal immune responses have emerged (30, 49–53). Evaluation of the differences between the taxonomic structures between immunized animals and control immunized mice revealed that the taxa that are increased in immunized animals are involved in regulation of normal metabolism, protection against inflammation, and stimulation of a normal immune response. Conversely, taxa that were decreased in immunized animals have been associated with obesity, production of butyric acid, and inflammation, which suggests that the changes in the microbiota could influence the host immune response. For example, Bacteroides fragilis produces a zwitterionic polysaccharide (ZPS) that activates CD4+ T cells. Normal polysaccharides are considered activators of B cells. However, ZPS binds to MHC class II on APCs and can be presented to CD4+ T cells in the same way as a peptide or glycopeptide. In addition, ZPS are required for the development of CD4+ T cells because splenocytes from germfree mice showed a lower proportion of splenic CD4+ cells compared with conventionally colonized mice or mice colonized with ZPS producing B. fragilis (even in the absence of all other gut microflora) (54). Taken together, the microbiota data suggest an association between P. murina oral vaccination and changes in the GI microbiota and hints at the possibility the P. murina oral vaccination may be improved with codelivery of probiotics. It is noteworthy that Lactobacillus spp. (recognized probiotics) have been demonstrated in other studies to increase the effectiveness of several oral vaccines (9, 10). In these studies, oral vaccines coupled with probiotics generate higher pathogen-specific Ab titers and provide significantly better protection, when compared with vaccine alone (10). These data also raise many additional questions regarding the role of the intestinal microbiota in the generation of the host immune response to oral vaccination and whether the microbiota changes participate in the induction of P. murina immune memory will require further investigation. Oral vaccination, coupled with probiotic therapy, may represent a unique and beneficial approach to vaccinate against infection with the human AIDS-associated pathogen P. jirovecii. In this study, we evaluated the potential of oral immunization with live Pneumocystis to elicit protection against respiratory infection with Pneumocystis murina. To our knowledge, this is the first report of an effective oral vaccination strategy for the prevention of Pneumocystis infection.

Acknowledgments We thank the members of the Section of Pulmonary/Critical Care and Allergy/Immunology for advice and discussions. In addition, we thank Connie Porretta for expert flow cytometry assistance.

Disclosures The authors have no financial conflicts of interest.

References 1. Beck, J. M., M. L. Warnock, H. B. Kaltreider, and J. E. Shellito. 1993. Host defenses against Pneumocystis carinii in mice selectively depleted of CD4+ lymphocytes. Chest 103(Suppl. 2): 116S–118S. 2. Shellito, J., V. V. Suzara, W. Blumenfeld, J. M. Beck, H. J. Steger, and T. H. Ermak. 1990. A new model of Pneumocystis carinii infection in mice selectively depleted of helper T lymphocytes. J. Clin. Invest. 85: 1686–1693.

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burden compared with control serum. However, we found no differences between immunized and control mice after either adoptive transfer of CD4+ T cells or total MLN cells. The lack of cell-mediated protection could be due to several different possibilities and limitations of the model system. First, the route of adoptive transfer could have been suboptimal and/or the transferred cells were unable to migrate to the site of infection. In addition, it is possible that the quantity and/or viability of transferred cells was below the threshold required to observe a protective effect. Finally, adoptive transfer into immune intact animals may mask or limit the cellular immune response because adoptive transfer of immune CD4+ T cells has been shown to induce pathogen clearance in Rag12/2 or scid mice. However, on the basis of our experimental data, mucosal vaccination with P. murina does lead to a protective humoral response as evidenced by the production of P. murina– specific Abs and the protection afforded by immune sera. Furthermore, the protection observed with our model in CD4deficient mice is consistent with humoral immunity. In contrast to our results, adoptive transfer of immune CD4+ T cells have been shown to facilitate pathogen clearance in Rag12/2 or scid mice (41, 42). Specifically, CD4 T cells from Pneumocystisinfected mice are able to mediate clearance of Pneumocystis infection upon adoptive transfer into Rag1 2/2 hosts (41). In addition, adoptive transfer of lymphocyte subsets or hyperimmune serum has been shown to be mediate effective immunity to Pneumocystis by the action of CD4+ (in the absence of Ab) or by humoral immunity (in the absence of T cells) (42). Furthermore, Gigliotti and colleagues have shown that immunocompetent mice immunized against Pneumocystis by intratracheal inoculations with Pneumocystis are protected from subsequent lung infection following depletion of CD4+ T cells with anti-CD4 mAbs (43), which suggests that immunization of an immunocompetent host against Pneumocystis can protect against Pneumocystis pneumonia even after the host is depleted of CD4+ cells. In addition, the results are consistent with those presented by Harmsen et al. (43), which suggest that Abs are responsible for the observed protection against P. carinii in the absence of CD4+ T cells. Our results are consistent with prior vaccination and immunization studies by several research groups that demonstrate that direct intratracheal or i.p. administration of whole organisms to CD4 intact mice confers immunity when they are CD4 depleted and rechallenged (1, 43–48). Although several other groups have described sterilizing immunity following intratracheal immunization, we do not observe sterilizing immunity at the time points tested, which indicates that oral vaccination may be improved with further optimization. However, our results are consistent with previous immunization studies, as we observed; a reduction in pathogen burden, the generation of Pneumocystis-specific Abs, activation of specific immune cell populations, and adoptive transfer of immune sera confers protection, all of which are consistent with previous reports. Although currently it would be virtually impossible to use live Pneumocystis organisms as a vaccine in humans, we believe this study provides the needed “proof-of-concept” for oral vaccination and will facilitate the translation work for the development of a human vaccine. Taken together, these data suggest that oral vaccination may represent an attractive and unexplored method for vaccination against Pneumocystis. We further examined the intestinal microbial community following oral immunization because changes in the GI microbiota have been linked to differential immune response. We found that the diversity of microbial community in the GI tract was significantly impacted by oral immunization with P. murina. In addition,

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