Protective efficacy of a ‘pan-fungal’ vaccination strategy against experimental Pneumocystis infection in drug-immunosuppressed macaques

Introduction: Pneumocystis jirovecii causes life-threatening fungal pneumonia (PJP) and other serious pulmonary sequelae in HIV infected individuals and other immunocompromised populations. In recent years, while the frequency of PJP has declined in HIV infected individuals treated with anti-retroviral therapies, the incidence has increased among non-HIV populations due to the expanding use of corticosteroids and other immunomodulatory agents to treat immune-mediated inflammatory diseases and hematologic and solid malignancies. Despite the success of trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis, patients who are unable to tolerate treatment, take drugs where TMP-SMX is contraindicated, or experience breaks in daily compliance remain at risk. Immunocompromised populations would benefit from vaccine strategies that reduce morbidity and mortality due to acute PJP.

Methods: Herein, we used a newly established non-human primate (NHP) model of Pneumocystis infection in the context of drug-induced immunosuppression to test the immunogenicity and protective efficacy of a vaccine strategy administered prior to and throughout drug-induced immunosuppression using the ‘pan-fungal’ vaccine candidate NXT-2a. Longitudinal blood and bronchoalveolar lavage sampling was performed to monitor anti-NXT-2a antibody titers, lymphocyte populations, and infection status.

Results: Immunization with NXT-2a prior to immunosuppression induced robust humoral immune responses in healthy outbred macaques. Subsequent therapeutic boosting throughout drug-induced immunosuppression prevented protective antibody titer decline. Our collective vaccination strategy provided significant protection against Pneumocystis infection throughout the duration of the study.

Discussion: These studies demonstrate the efficacy and feasibility of an NXT-2a based vaccination strategy in a NHP model with a planned immunosuppressive regimen. This strategy may be further applied toward other opportunistic fungal pathogens, such as Candida spp. and Aspergillus spp. in similarly immunosuppressed populations.

1 Introduction

Pneumocystis jirovecii is an opportunistic fungal pathogen that can cause life-threatening pneumonia (PJP) and has been associated with a number of chronic lung diseases including chronic obstructive pulmonary disease (1), severe asthma (2), cystic fibrosis (3), and interstitial lung disease (4). Transmission is airborne and occurs following exposure to other Pneumocystis carrying individuals (5). Asymptomatic colonization and clearance are common among immunocompetent populations, but in immunosuppressed individuals, persistent colonization can lead to pneumonia. In the pre-anti-retroviral era of the HIV epidemic, PJP was a leading cause of morbidity and mortality in individuals with HIV. In recent years, the incidence of PJP has declined among people with HIV but has steadily increased among non-HIV immunosuppressed individuals (6, 7). Populations at high risk include those receiving corticosteroids and other immunomodulatory therapies for immune-mediated inflammatory diseases, organ transplantation, and cancer (8, 9). PJP in persons without HIV is generally associated with a lower organism burden and a more acute and fulminant disease course than in persons with HIV (10–12), complicating efficient diagnosis and successful treatment. Even with clinical intervention, mortality associated with PJP remains high (25%) and worsens if ICU admission is required (58%) (13). Trimethoprim-sulfamethoxazole (TMP-SMX) is an effective treatment and prophylactic agent against Pneumocystis pneumonia in immunocompromised individuals; however, efficacy is often limited due to drug-drug interactions, treatment-limiting adverse events, and breakthrough infections (14–16). Notably, treatment with TMP-SMX does not prevent re-infection and widespread use has raised concerns over emerging drug resistance (15, 17). Therefore, strategies that can promote anti-Pneumocystis immunity, and are well-tolerated alongside pharmaceutical regimens are necessary to reduce the high rates of morbidity and mortality associated with PJP.

To address this need, our laboratory has previously developed a ‘pan-fungal’ vaccine candidate, NXT-2. NXT-2 is a 90-amino acid consensus peptide, based on a conserved region of the kexin-like protein KEX1 from the pathogenic fungi Pneumocystis jirovecii, Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans (18, 19). The development of NXT-2 was based on extensive prior research demonstrating the importance of antibodies against Pneumocystis KEX1 (PC.KEX1) in the control of Pneumocystis associated disease. Due to natural exposure, most individuals are PC.KEX1-serpositive, and high antibody titers against this antigen correlate with a decreased frequency of Pneumocystis associated disease among HIV infected individuals (20) and in a non-human primate (NHP) model of Pneumocystis and HIV co-infection (21). Kling et al. further demonstrated that immunization with recombinant PC.KEX1 boosts humoral memory in immunocompetent macaques and is protective against subsequent Pneumocystis infection during simian immunodeficiency virus (SIV)- immunosuppression (22). Interestingly, proof-of-concept studies demonstrate that therapeutic immunization with PC.KEX1 during SIV and methylprednisolone/tacrolimus -induced immunosuppression boosts antibody recall responses (23, 24) and can help to maintain immunity in NHP model of Pneumocystis and SIV co-infection (24). Like PC.KEX1, immunization with NXT-2 is highly immunogenic in both mice and NHPs, and induces protective immunity against a range of experimental fungal infections, including systemic candidiasis and pulmonary aspergillosis in immunosuppressed murine models, murine vulvovaginal candidiasis, and in a NHP model of Pneumocystis and SIV co-infection (18, 25).

We recently developed a novel NHP model to study the efficacy of NXT-2-based biologics in the context of severe drug-induced immunosuppression and natural airborne exposure to Pneumocystis (26). Herein, we evaluated the immunogenicity of NXT-2 in healthy macaques and evaluated its protective efficacy against Pneumocystis infection during drug-induced immunosuppression with therapeutic boosting. These studies present a strategy for immunizing individuals who are at risk of developing Pneumocystis pneumonia following immunosuppressive therapies.

2 Materials and methods

2.1 Animals

Eight Japanese macaques (Macaca fuscata) aged 4–9 years were obtained from Oregon National Primate Research Center (ONPRC) and randomly assigned to vaccinated (n=4, 3 females, 1 male) or sham control (n=4, 2 females, 2 males) cohorts. All studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Georgia. All animal studies were performed in the University Research Animal Resources Facility, at the University of Georgia, an American Association for the Accreditation of Laboratory Animal Care (AAALAC) accredited facility. The care and use of laboratory animals at the University of Georgia are in accordance with the principles and standards set forth in the Principles for Use of Animals (NIH Guide for Grants and Contracts), the Guide for the Care and Use of Laboratory Animals, the provision of the Animal Welfare Acts (P.L. 89–544 and its amendments). Compliance is validated by the UGA IACUC and regular inspections by USDA inspecting veterinarians. Prior to admission to the study, all animals underwent physical examination and were screened and found negative for simian retroviruses (SIV, SRV, and STLV).

2.2 Recombinant expression and purification of NXT-2a antigen

The design and expression of NXT-2 was previously reported (18). To generate an affinity tag-free NXT-2 construct, the 90-amino acid pan-fungal consensus sequence first described in Rayens et al. (18) followed by two stop codons (ochre and opal) were cloned into the pET-28b(+) vector (Novagen) using the restriction sites NcoI and BamHI by GenScript. This vector was then used to transform chemically competent Escherichia coli BL21(DE3) pLyS cells (Thermo Fisher) according to the manufacturer’s instructions. This resulted in an affinity tag-free recombinant protein, NXT-2a, expressed as 5’- MGPDDGKTMEGPDILVLRAFINGVQNGRDGKGSIYVFASGNGGGFEDNCNFDGYTNSIYSITVGAIDRKGLHPSYSEACSAQLVVTYSSGSG-3’. Following subculture in BBL Select APS LB Broth base (BD Biosciences) with kanamycin (40µg/ml) and chloramphenicol (34µg/ml), protein expression was induced for four hours at 37°C in a final concentration of 0.5mM IPTG where the protein was found to be expressed primarily in inclusion bodies. Inclusion bodies were isolated by lysing cell pellets with CelLytic B (Sigma) and washing the pellets three times with diluted CelLytic B (1:10 dilution with water) according to the manufacturer’s instructions. Inclusion body pellets were resuspended in buffer A (6M Urea, 20mM Tris-HCl, pH 8.0) and nutated for two hours at room temperature before storing the suspension overnight at 4°C. The next day, the suspension was centrifuged at 10,000g for 15 minutes at 4°C. The supernatant was then collected and filtered over a 0.2µm filter. Supernatants were then run over a HiTrap Capto Q column and NXT-2a was eluted through a gradient of buffer B (1M NaCl 6M urea 20mM Tris-HCl, pH 8.0) using the AKTA Pure FPLC system (Cytiva). Following this initial anion exchange chromatography capture step, NXT-2a enriched fractions were then pooled and concentrated by diafiltration using a 3 kDa MWCO filter (Merck Millipore). Size exclusion chromatography was performed as a final polishing step. Pooled anion exchange fractions were run over a Superdex 75 Increase 10/300 column (Cytiva) and eluted in 6M Urea, 250mM NaCl, 20mM Tris-HCl, pH 8.0 buffer. Protein refolding and buffer exchange were performed by dialyzing the final pooled fractions overnight in 1x PBS using the Pur-A-Lyzer Maxi Dialysis Kit (Sigma-Aldrich) with two changes of buffer. The final protein containing fractions were then analyzed for purity by Coomassie and Western blotting as previously described (18). SDS-PAGE was performed by running 5µg fractions on a 4% stacking/15% resolving polyacrylamide gel. Proteins were then transferred to a 0.2µm nitrocellulose membrane, blocked in 5% nonfat milk in PBS-T (0.05% Tween-20), and incubated with anti-NXT-2 hyperimmune NHP plasma (1:10,000). Detection was performed using goat anti-monkey IgG HRP (ThermoFisher, 1:10,000), SuperSignal PicoWest Plus chemiluminescence substrate (ThermoFisher), and a ChemiDoc (BioRad) imaging system. NXT-2a was used for immunizations and enzyme-linked immunosorbent assays (ELISA). The final purified pool of NXT-2a was endotoxin-tested using the Pierce Chromogenic Endotoxin Kit (ThermoFisher) prior to in vivo use.

2.3 Preparation of NXT-2a vaccine, immunization of macaques, and drug-induced immunosuppression

Seven macaques were intramuscularly immunized with prepared NXT-2a vaccine (n=4) or sham vaccine (n=3) at baseline and boosted 6 weeks later. Each macaque received 500µl prepared vaccine comprised of NXT-2a (100µg) + Alhydrogel 2% (InvivoGen, 0.5mg Al³⁺) diluted in sterile PBS or Alhydrogel 2% (0.5mg Al³⁺) alone in PBS that was rocked overnight at 4°C to encourage antigen and matrix binding. Two weeks after boosting, animals were treated daily with dexamethasone (West-Ward Pharmaceuticals, 1.3-1.6mg/kg/day) until the end of the study to induce immunosuppression. A fourth sham control animal, 39635, received no vaccines prior to and following the start of dexamethasone treatment.

2.4 Pneumocystis challenge

Pneumocystis cannot be reliably cultured in vitro. Immunocompetent individuals and macaques may be transiently or asymptomatically colonized but are only susceptible to Pneumocystis infection when immunosuppressed (26–28). We have previously reported that immunosuppressed macaques may be infected by natural airborne transmission of Pneumocystis from other Pneumocystis positive “seeder” (infected or colonized) macaques by co-housing (22, 24, 26). The “seeder” animal, sham control 39635, used in this experiment was a non-vaccinated macaque that was treated with dexamethasone 4 weeks prior to immunosuppression of the remainder of the cohort. 39635 became persistently colonized by 4 weeks of immunosuppression, coinciding with the start of dexamethasone treatment in the remainder of the cohort.

2.5 Sample collection

Blood and BAL samples were collected and processed as previously described (21, 27). Blood was collected at baseline and then every two weeks until the end of study to monitor anti-NXT-2a IgG antibody levels and lymphocyte kinetics. BAL procedures were performed with 20ml of sterile PBS. BAL samples were collected every 4 weeks for the first 8 weeks of dexamethasone treatment and then every 2 weeks until the end of the study to monitor Pneumocystis infection status and lymphocyte kinetics. In some cases where monitoring results were found to be inconclusive, BAL collection was repeated the following week.

2.6 Anti-NXT-2a ELISA

ELISA assays were performed using NXT-2a coated plates as previously described (22, 23). Microtiter plates (Immulon 4HBX; Thermo Fisher Scientific) were coated with recombinant NXT-2a (50µl/well at 5µg/ml in 1x PBS) over night at 4°C. After washing twice with PBS-T plates were blocked with blocking buffer (5% non-fat dry milk in PBS) for 1 hour at room temperature. Plates were then washed twice with PBS-T, dried, and then stored at -20°C for up to 6 months prior to use. To measure anti-NXT-2a antibody titers, heat-inactivated plasma samples were initially diluted 1:100 in blocking buffer and two-fold serial dilutions were made prior to adding 50µl of diluted sample in NXT-2a coated plates. Plates were then incubated overnight at 4°C. The next day, plates were washed four times with PBS-T and incubated with 100µl/well of goat anti-monkey IgG-HRP secondary antibody (Nordic Immunology) diluted 1:10,000 in blocking buffer for 1 hour at 37°C. Plates were then washed six times with PBS-T and visualized with 100µl/well TMB (BD Biosciences), the reaction stopped with 50µl/well of 1M H₂SO₄ and read at 450nm. Normal (uninfected, Pneumocystis-negative determined by antibody titer) macaque plasma was used as a negative control, and archival samples from a vaccinated animal with a known titer were used as a positive control as internal controls on all assay plates.

2.7 Evaluation of Pneumocystis infection

BAL samples were processed, and DNA was extracted from BAL pellets as previously described (26). All BAL processing, DNA extraction and PCR steps were performed under sterile conditions in either a biosafety cabinet or a PCR workstation (Fisher Scientific) to prevent ambient Pneumocystis contamination. Pneumocystis infection was defined as detection of the Pneumocystis mitochondrial large subunit rRNA gene (mtLSU) by PCR (26, 27). Pneumocystis colonization was defined as detection of Pneumocystis DNA by nested PCR of the 1st round PCR product only (2nd round PCR positive, +) (21, 26). A β-globin PCR was performed as a control for DNA quality. mtLSU and β-globin PCR reactions were performed using 1µg of BAL template DNA. PCR products were run on a 1.5% agarose gel with SYBR Safe gel stain (ThermoFisher) and visualized with the ChemiDoc (BioRad) imaging system. Semi-quantitative densitometry analysis was performed using Image J (NIH) to compare the ratio of mtLSU: β-globin between longitudinal timepoints. Pneumocystis infected animals with distinctly positive mtLSU gel products displayed a ratio of mtLSU: β-globin greater than 0.25 when analyzed by densitometry.

2.8 Flow cytometry

Blood and BAL samples were collected and processed as previously described (26, 27). Red blood cells were lysed by treating whole blood with red blood cell lysis buffer (150mM NH₄Cl, 10mM NaHO₃, 115µM EDTA). Cells were stained in FACS buffer containing 20% FBS, 2% human sera, 2% goat serum, 5mM EDTA, and 0.05% sodium azide in 1x PBS. FITC anti-CD3 (SP34), PE anti-CD8 (RPA-T8) antibodies were purchased from BD Biosciences. APC anti-CD4 (OKT4), and APC-Cy7 anti-CD20 (2H7) were purchased from BD Biolegend (San Diego, CA). After antibody staining, cells were lysed and fixed in BD FACS Lysing Solution (BD Biosciences) to eliminate residual red blood cells and then stored in 1% paraformaldehyde until sample acquisition. Standard flow cytometric procedures were used to acquire data on a NovoCyte Quanteon flow cytometer (Agilent Technologies, Santa Clara, CA). Analysis was performed using FlowJo analysis software (BD Biosciences). The gating strategy used was as previously described in Rabacal et al. (26).

2.9 Statistical analysis

All statistical analyses were performed using GraphPad Prism (GraphPad Software, La Jolla, CA). Longitudinal changes in lymphocyte populations in all dexamethasone treated animals (NXT-2a immunized and sham controls combined) over time were analyzed using repeated measures mixed modeling and Dunnett’s test for multiple comparisons to identify values that differ significantly from baseline values. Timepoint specific differences in lymphocyte populations between NXT-2a immunized and sham control cohorts were analyzed by multiple Mann-Whitney tests. Log-rank test was used to analyze Pneumocystis infection incidence curves.

3 Results

3.1 Humoral responses in healthy Japanese macaques and following therapeutic boosting during dexamethasone induced immunosuppression

In this study, we examined the immunogenicity and protective efficacy of an NXT-2a based vaccination strategy in the context of drug-induced immunosuppression. The affinity tag-free version of NXT-2, NXT-2a is approximately ~10 kDa and is recognized by sera from an NHP immunized with NXT-2 (Figure 1A). To confirm the immunogenicity of this modified antigen, healthy animals were vaccinated with NXT-2a (100µg) + Alhydrogel 2% (0.5mg Al³⁺) at 8 and 2 weeks prior to immunosuppression, 6 weeks apart (Figure 1B, NXT-2a Immunized). Three sham control animals were vaccinated with PBS + Alhydrogel 2% (0.5mg Al³⁺) at similar intervals and a fourth sham control animal 39635 received no vaccine. In NXT-2a immunized animals mean plasma NXT-2a IgG titers (± SD) peaked at four weeks (31,437 ± 59,060) following initial immunization and achieved robust titers (1,664,000 ± 443,405) two weeks after boosting (Figures 1C, D), demonstrating comparable immunogenicity to NXT-2.

Figure 1

Figure 1. Humoral responses in NXT-2a immunized macaques prior to and following dexamethasone induced immunosuppression. (A) Coomassie stained gel (left) and Western blot of recombinant NXT-2a (~10kDa) used for immunization and ELISA assays. Immunoblotting was performed using polyclonal sera from a macaque immunized with NXT-2 (affinity tagged construct). (B) Study design of immunization, immunosuppression, and Pneumocystis infection in Japanese macaques. Purple arrows indicate the primary vaccine series administered prior to immunosuppression. Green arrows indicate the secondary vaccine series administered and therapeutically throughout immunosuppression. A non-vaccinated animal, sham control 39635 initiated dexamethasone treatment ~4 weeks prior to the remainder of the cohort. 39635 became Pneumocystis colonized (Pc+) by 4 weeks of immunosuppression when the remainder of the cohort initiated dexamethasone treatment and functioned as a “seeder” to facilitate the spread of Pneumocystis to other co-housed animals (orange arrow). Cross transmission of Pneumocystis among co-housed macaques is indicated by the double arrow. (C) Kinetics of NXT-2a specific reciprocal endpoint titers (RET) in the plasma of NXT-2a immunized and sham control animals expressed as (C) mean titers ± SD and in (D) longitudinal plots of individual animals. The red arrows and vertical fine dashed lines indicate the start of dexamethasone treatment.

To induce immunosuppression and render animals susceptible to Pneumocystis infection, vaccinated animals were then treated with dexamethasone (1.3-1.6mg/kg/day) beginning two weeks after the first boost. Sham control, 39635, initiated dexamethasone treatment ~4 weeks prior to the remainder of the cohort to function as a “seeder” to hasten infection within the co-housed experiment. Titers declined to 29,500 ± 34,307 within 4 weeks of dexamethasone treatment (Figures 1C, D). To determine if anti-Pneumocystis humoral immunity can be boosted in the context of drug-induced immunosuppression, NXT-2a immunized macaques were therapeutically immunized at 4 and 8 weeks following the start of dexamethasone treatment with NXT-2a (100µg) + Alhydrogel 2% (0.5mg Al³⁺) whereas sham controls received PBS + Alhydrogel 2% (0.5mg Al³⁺) or no vaccine (39635 only). After a third vaccination at 4 weeks of immunosuppression, 2 of 4 macaques responded to therapeutic boosting with NXT-2a (Figure 1D). Titers in NHP 40056 (Figure 1D, solid orange box) increased 5.9-fold between 4 and 6 weeks from 4,000 to 24,000 RET. Titers in NHP 40053 (Figure 1D, solid green box) showed a 1.2-fold increase during the same period from 80,000 to 96,000 RET. After a fourth vaccination at 8 weeks of immunosuppression, 4 of 4 animals responded to therapeutic boosting. Therapeutic vaccination increased mean titers approximately 6.9-fold between 8 and 10 weeks of immunosuppression from 37,250 ± 51,090 to 256,000 ± 78,383 RET. Mean NXT-2a IgG antibody titers remained above 10⁴ RET until the end of the study, 12 weeks after the start of immunosuppression (58,666 ± 40,266). Throughout our immunogenicity studies we did not observe any significant swelling, redness, or adverse reactions associated with NXT-2a immunization. These data demonstrate that NXT-2a is safe, highly immunogenic in healthy macaques, and a repeated boosting strategy maintains mean titers above 10⁴ RET for 12 weeks during dexamethasone-induced immunosuppression.

3.2 Effects of dexamethasone treatment on lymphocyte kinetics in NXT-2a and sham immunized macaques

To confirm the immunosuppressive effects of dexamethasone throughout the course of this study, we monitored changes in immune subsets in the peripheral blood and BAL in all animals (Figures 2A–D). We observed significant longitudinal declines in lymphocytes (Figure 2A, P = 0.0002) and in the frequency (Figure 2B, P<0.0001) and cell number (Figure 2C, P<0.0001) of CD4 T cells in the peripheral blood. We also observed a decline in the frequency of CD4 T cells at the site of Pneumocystis infection in BAL samples (Figure 2D, P = 0.04). These data are consistent with previous observations reported in Japanese and Rhesus macaques similarly treated with dexamethasone (26). We did not observe significant differences in lymphocyte numbers, CD4 frequency, and CD4 T cell numbers between NXT-2a immunized or sham control animals (Figures 2E–H), indicating that both cohorts were similarly immunosuppressed throughout dexamethasone treatment. Longitudinal plots for individual animals are displayed in Supplementary Figure 1.

Figure 2

Figure 2. Lymphocyte and CD4 T cell populations in the blood and bronchoalveolar lavage (BAL) throughout dexamethasone treatment. Kinetics of lymphocyte depletion in (A-D) all dexamethasone treated macaques (grey circles) and in (E-H) NXT-2a immunized (closed box) vs Sham control (open box) animals. (A, E) Lymphocyte count, (B, F) CD4 T cell frequency and (C, G) cell number in the peripheral blood. (D, H) CD4 T cell frequency in the bronchoalveolar lavage (BAL). (A-D) Whole population kinetics were analyzed using repeated measures mixed modeling analysis (P-values are indicated) and Dunnett’s test for multiple comparisons. *P<0.05, indicates values that differ significantly from baseline (BL) in Dunnett’s post-hoc analyses. (E-H) Multiple Mann-Whitney tests were performed to evaluate the differences between NXT-2a immunized and Sham controls. Data represents the mean ± SD.

3.3 NXT-2a immunization is protective against Pneumocystis infection during drug induced immunosuppression

We have previously established that Pneumocystis infection and colonization can be reliably diagnosed in dexamethasone and SIV immunosuppressed macaques through the detection of Pneumocystis DNA in PCR amplified BAL samples (22, 24, 26). To monitor Pneumocystis infection, throughout the course of this study, longitudinal BAL sampling was performed at baseline and throughout immunosuppression. At the start of the study, we confirmed that all animals were uninfected with Pneumocystis. Following the start of dexamethasone treatment, we observed Pneumocystis infection in sham control macaques 39558, 39570, 39635, and 4007 at 12, 9, 10, and 8 weeks of immunosuppression, respectively (Figure 3A; Table 1) and in NXT-2a immunized macaque 39584 at 10 weeks (Figure 3B second row; Table 1). At study termination, only 25% (1 of 4) NXT-2a immunized animals became Pneumocystis infected compared to 100% (4 of 4) of sham controls (Figure 4, P = 0.03, Table 1), despite being comparably immunosuppressed (Figure 2).

Figure 3

Figure 3. Pneumocystis-specific PCR of bronchoalveolar lavage samples throughout dexamethasone induced immunosuppression in NXT-2a immunized and sham control macaques. Gel analysis of mtLSU and β-globin PCR reaction products of (A) Sham control and (B) NXT-2a Immunized cohorts. Asterisks (*) indicate gel products in which β-globin was not detected. Positive control (Ctrl) reactions were performed in tandem at the indicated timepoints. Bar graphs represent densitometry analysis of PCR gel products expressed as a ratio of mtLSU:β-globin. Dashed lines on graphs indicate the threshold of Pneumocystis infection (+) at 0.25.

Table 1

Table 1. Summary of Pneumocystis BAL PCR results.

Figure 4

Figure 4. Incidence of Pneumocystis infection throughout dexamethasone induced immunosuppression in NXT-2a immunized and Sham control Japanese macaques. A diagnosis of Pneumocystis infection was made through the detection of the mtLSU gene in PCR amplified samples and subsequent densitometry analysis in samples with a ratio of mtLSU:β-globin greater than 0.25. Significance was determined by Log-rank test.

We have previously reported that low antibody titers against below 10⁴ IgG RET against PC.KEX1 are predictive of Pneumocystis susceptibility following SIV immunosuppression (21). In the single NXT-2a immunized macaque that became Pneumocystis infected, NHP 39584, plasma NXT-2a IgG antibody titers dipped below 10⁴ RET after 8 weeks of dexamethasone treatment, approximately 2 weeks prior to infection. At this same timepoint, 39584 also experienced a decline in CD4 T cell numbers to <200 cells/µl (Figure 5A left panel) and the CD4 frequency in the BAL was < 10% (Figure 5A right panel), reflective of a severe state of immunosuppression. In contrast, NXT-2a immunized NHP 40056, maintained plasma NXT-2a IgG titers above 10⁴ RET when similarly immunosuppressed between 8–12 weeks of dexamethasone treatment (Figure 5B) and remained uninfected. These data indicate that our vaccination strategy with therapeutic boosting during immunosuppression is antibody mediated and protective against Pneumocystis infection.

Figure 5

Figure 5. NXT-2a antibody responses in animals with severe CD4 T cell depletion. CD4 T cell depletion profiles and NXT-2a antibody responses throughout dexamethasone treatment and therapeutic boosting. (A) Pneumocystis infected NHP 39584. (B) Pneumocystis protected NHP 40056. (Left panels) Number of CD4 T cells in the peripheral blood and plasma NXT-2a IgG RET throughout immunosuppression. (Right panels) Frequency of CD4 T cells in the bronchoalveolar lavage and plasma NXT-2a IgG RET throughout immunosuppression. Thick dashed lines indicate 100 CD4 T cells/µl or 10% CD4+ T cells. Dotted dashed lines indicate 10⁴ RET. Green arrows indicate therapeutic boosting at 4 and 8 weeks of immunosuppression. Plus sign (+) indicates the timepoint of Pneumocystis infection in NHP 39584.

4 Discussion

The expanding use of corticosteroids and immunomodulatory drugs in solid-organ and hematopoietic transplant recipients, cancer patients, and persons with inflammatory autoimmune disorders likely contributes to the increase the number of persons at risk of Pneumocystis associated pulmonary disease (8, 9, 29). Pneumocystis pneumonia risk is especially problematic in individuals receiving high dose corticosteroids for greater than 4 weeks, have CD4 T cell counts of <200cells/µl, and are TMP-SMX prophylaxis non-adherent (9, 30–32). Using a highly relevant pre-clinical model with natural transmission by airborne exposure (26), we sought to evaluate the immunogenicity and protective efficacy of a pan-fungal vaccination strategy in a NHP model of Pneumocystis infection in the context of drug-induced immunosuppression.

In the current study, healthy macaques were immunized with an affinity tag-free variant of NXT-2 (18), NXT-2a, 8 and 2 weeks prior to immunosuppression. Immunization with NXT-2a during immunocompetency induced robust NXT-2a IgG titers above 10⁶ RET. These results were consistent with prior studies with NXT-2 in healthy rhesus macaques (18) and confirmed the immunogenicity of our modified antigen. To test the hypothesis that NXT-2a immunization could boost titers in the context of drug-induced immunosuppression, we administered a third and fourth vaccine at 4 and 8 weeks of dexamethasone treatment, respectively. During immunosuppression, NXT-2a antibody titers were boosted in 2 of 4 NXT-2 immunized macaques after a 3^rd immunization and 4 of 4 after 4^th immunization. Mean NXT-2a IgG antibody titers remained above 10⁴ RET until the end of the study. NXT-2a immunization afforded significant protection against Pneumocystis infection when compared with sham controls (1 of 4 (25%) NXT-2a immunized vs. 4 of 4 (100%) Sham controls). This vaccine strategy was even protective in a profoundly immunosuppressed animal with only 100–200 cells/µl (NHP 40056).

We have previously reported that antibody titers above 10⁴ IgG RET against a similar antigen PC.KEX1, in healthy macaques correlate with protection against Pneumocystis co-infection following SIV-induced immunosuppression (21). In the single NXT-2a immunized macaque that became infected (NHP 39584) plasma NXT-2a antibody levels subsequently declined below 10⁴ RET approximately two weeks prior to a positive diagnosis. This animal eventually responded to a second therapeutic immunization, but due to the gap in coverage this delayed response was not sufficient to prevent infection. Interestingly, when we re-evaluated this animal by PCR a week after diagnosis and boosting, the ratio of mtLSU:β-globin declined from 0.90 to 0.30. We speculate that the rise in NXT-2a antibody levels induced by therapeutic boosting may have prolonged control of Pneumocystis infection. These results demonstrate effective boosting and protection by our NXT-2a based vaccine strategy in drug-immunosuppressed macaques.

Immunosuppressive therapies such as dexamethasone and other glucocorticoids induce acute dysfunction in both innate and adaptive immune compartments that may hinder the performance of traditional vaccine strategies in these populations even more so than in persons with CD4 targeted depletion due to HIV infection. Due to the assumed strain on vaccine durability in immunosuppressed individuals in general, current guidelines for Pneumocystis susceptible non-HIV populations, such as solid organ transplant recipients, recommend patients be up to date on vaccinations prior to transplantation and receive booster doses between 3–12 months post-transplant, except in the case of viral or live attenuated vaccines and the influenza vaccine (33). A multi-center cohort study found that administration of inactivated influenza vaccine within the first three months of transplantation is safe and effective (34). Due to seasonal exposure, inactivated influenza vaccines are now routinely recommended as early as one-month post-transplant with consideration of repeating additional doses at two and three months if disease transmission continues (33, 35).

In contrast to the seasonality of influenza exposure, Pneumocystis is ubiquitous, and disease has been demonstrated to arise through exposure to other Pneumocystis carrying hosts (5, 36). Pneumocystis pneumonia clusters have been frequently found within transplant centers due to nosocomial exposure (37, 38). Given the safety of the influenza and many other inactivated vaccines administered post-transplant, the ubiquitous nature of Pneumocystis, and our observations that NXT-2a immunization limits Pneumocystis infection, we argue that the vaccine strategy used in this study is safe and highly relevant. The data presented herein accurately mimics the challenges to immunization faced by immunosuppressed clinical populations. We hypothesize that NXT-2a could be administered to patients before and after the start of immunosuppression, as well as to close contacts of patients, to limit Pneumocystis infection and exposure. Responses to therapeutic immunization administered after induction of immunosuppression therapy would also likely increase in amplitude and durability as regimens are tapered to maintenance levels. Furthermore, as a ‘pan-fungal’ vaccine candidate, this NXT-2a based immunization strategy may provide additional coverage against other invasive fungal pathogens such as Aspergillus spp., Candida spp., and more with demonstrated anti-NXT-2 antibody cross-reactivity (18).

There are limitations to our study. We did not evaluate the protective efficacy of the primary vaccine series (first and second vaccine) alone in the absence of therapeutic boosting during immunosuppression. In addition, we did not test the ability of our vaccine to generate a de novo memory response during immunosuppression. Studies to improve memory responses against NXT-2a and antibody titer durability in both immunocompetent and immunosuppressed conditions in larger cohorts are ongoing. Further studies investigating de novo memory response against NXT-2a in the context of drug-induced immunosuppression will help to establish the potential and limitations of NXT-2a based vaccination strategies in patients who are already severely immunosuppressed.

In summary, we report a novel vaccination strategy for the prevention of Pneumocystis infection in a NHP model of drug-induced immunosuppression. Our data demonstrates the importance of anti-NXT-2a antibodies in the control of Pneumocystis infection in the context of drug-induced immunosuppression. These data warrant future investigation of passive transfer studies with anti-NXT-2a antibodies in populations that are similarly immunosuppressed as well as in populations who are less likely to benefit from active immunization. These data may be of interest to investigators and clinicians interested in immunization strategies for vaccine preventable diseases in immunocompromised populations.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement

The animal study was approved by University of Georgia Institutional Animal Care and Use Committee. The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

WR: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. AH: Data curation, Investigation, Methodology, Project administration, Validation, Writing – review & editing, Formal analysis. GK: Data curation, Investigation, Validation, Writing – review & editing, Formal analysis. TC: Investigation, Writing – review & editing, Data curation, Project administration. DW: Investigation, Writing – review & editing. KO: Investigation, Writing – review & editing. KAN: Conceptualization, Funding acquisition, Resources, Supervision, Writing – original draft, Writing – review & editing, Validation.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This work was supported by the National Institute for Allergy and Infectious Diseases at the National Institutes of Health [Grant Number R01AI148365 to KAN].

Acknowledgments

We thank Gina Kim, Matthew Doster, Cheryl Paladino, and Caitlan Dutton of University Research Animal Resources at UGA for veterinary support. We acknowledge Jamie Barber at the UGA College of Veterinary Medicine Cytometry Core Facility for cytometry assistance.

Conflict of interest

KAN has a financial interest in a biotech company. KAN and WR are co-inventors of the vaccine candidate used in this paper. An approved plan is in place with the University of Georgia for managing potential conflicts.

The remaining author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author KAN declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Generative AI statement

The author(s) declare that no Generative AI was used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2025.1729080/full#supplementary-material

References

1. Morris A, Netravali M, Kling HM, Shipley T, Ross T, Sciurba FC, et al. Relationship of Pneumocystis antibody response to severity of chronic obstructive pulmonary disease. Clin Infect Dis. (2008) 47:e64–8. doi: 10.1086/591701

PubMed Abstract | Crossref Full Text | Google Scholar

2. Rayens E, Noble B, Vicencio A, Goldman DL, Bunyavanich S, and Norris KA. Relationship of Pneumocystis antibody responses to paediatric asthma severity. BMJ Open Respir Res. (2021) 8:e000842. doi: 10.1136/bmjresp-2020-000842

PubMed Abstract | Crossref Full Text | Google Scholar

3. Green HD, Bright-Thomas RJ, Mutton KJ, Guiver M, and Jones AM. Increased prevalence of Pneumocystis jirovecii colonisation in acute pulmonary exacerbations of cystic fibrosis. J Infect. (2016) 73:1–7. doi: 10.1016/j.jinf.2016.05.001

PubMed Abstract | Crossref Full Text | Google Scholar

4. Liu L, Ji T, Chen R, Fan L, Dai J, and Qiu Y. High prevalence of Pneumocystis pneumonia in interstitial lung disease: a retrospective study. Infection. (2024) 52:985–93. doi: 10.1007/s15010-023-02148-y

PubMed Abstract | Crossref Full Text | Google Scholar

5. Vargas SL, Ponce CA, Gigliotti F, Ulloa AV, Prieto S, Muñoz MP, et al. Transmission of Pneumocystis carinii DNA from a patient with P. carinii pneumonia to immunocompetent contact health care workers. J Clin Microbiol. (2000) 38:1536–8. doi: 10.1128/JCM.38.4.1536-1538.2000

PubMed Abstract | Crossref Full Text | Google Scholar

6. Kolbrink B, Scheikholeslami-Sabzewari J, Borzikowsky C, von Samson-Himmelstjerna FA, Ullmann AJ, Kunzendorf U, et al. Evolving epidemiology of Pneumocystis pneumonia: Findings from a longitudinal population-based study and a retrospective multi-center study in Germany. Lancet Reg Health Eur. (2022) 18:100400. doi: 10.1016/j.lanepe.2022.100400

PubMed Abstract | Crossref Full Text | Google Scholar

7. Pates K, Periselneris J, Russell MD, Mehra V, Schelenz S, and Galloway JB. Rising incidence of Pneumocystis pneumonia: A population-level descriptive ecological study in England. J Infect. (2023) 86:385–90. doi: 10.1016/j.jinf.2023.02.014

PubMed Abstract | Crossref Full Text | Google Scholar

8. Limper AH. In Search of Clinical Factors that Predict Risk for Pneumocystis jirovecii Pneumonia in Patients without HIV/AIDS. Am J Respir Crit Care Med. (2018) 198(12):1467–8. doi: 10.1164/rccm.201807-1358ED

PubMed Abstract | Crossref Full Text | Google Scholar

9. Lecuyer R, Issa N, Camou F, Lavergne RA, Gabriel F, Morio F, et al. Characteristics and prognosis factors of Pneumocystis jirovecii pneumonia according to underlying disease: A retrospective multicenter study. Chest. (2024) 165:1319–29. doi: 10.1016/j.chest.2024.01.015

PubMed Abstract | Crossref Full Text | Google Scholar

10. Bienvenu AL, Traore K, Plekhanova I, Bouchrik M, Bossard C, and Picot S. Pneumocystis pneumonia suspected cases in 604 non-HIV and HIV patients. Int J Infect Dis. (2016) 46:11–7. doi: 10.1016/j.ijid.2016.03.018

PubMed Abstract | Crossref Full Text | Google Scholar

11. Limper AH, Offord KP, Smith TF, and Martin WJ 2nd. Pneumocystis carinii pneumonia. Differences in lung parasite number and inflammation in patients with and without AIDS. Am Rev Respir Dis. (1989) 140:1204–9. doi: 10.1164/ajrccm/140.5.1204

PubMed Abstract | Crossref Full Text | Google Scholar

12. Alanio A, Desoubeaux G, Sarfati C, Hamane S, Bergeron A, Azoulay E, et al. Real-time PCR assay-based strategy for differentiation between active Pneumocystis jirovecii pneumonia and colonization in immunocompromised patients. Clin Microbiol Infect. (2011) 17:1531–7. doi: 10.1111/j.1469-0691.2010.03400.x

PubMed Abstract | Crossref Full Text | Google Scholar

13. Schmidt JJ, Lueck C, Ziesing S, Stoll M, Haller H, Gottlieb J, et al. Clinical course, treatment and outcome of Pneumocystis pneumonia in immunocompromised adults: a retrospective analysis over 17 years. Crit Care. (2018) 22:307. doi: 10.1186/s13054-018-2221-8

PubMed Abstract | Crossref Full Text | Google Scholar

14. Faul JL, Akindipe OA, Berry GJ, Doyle RL, and Theodore J. Recurrent Pneumocystis carinii colonization in a heart-lung transplant recipient on long-term trimethoprim-sulfamethoxazole prophylaxis. J Heart Lung Transplant. (1999) 18:384–7. doi: 10.1016/s1053-2498(98)00038-2

PubMed Abstract | Crossref Full Text | Google Scholar

15. Masters PA, O'Bryan TA, Zurlo J, Miller DQ, and Joshi N. Trimethoprim-sulfamethoxazole revisited. Arch Intern Med. (2003) 163:402–10. doi: 10.1001/archinte.163.4.402

PubMed Abstract | Crossref Full Text | Google Scholar

16. Zieneldien T, Kim J, and Greene J. Breakthrough Pneumocystis jirovecii pneumonia in an allogeneic hematopoietic stem cell transplant recipient. Cureus. (2024) 16:e61890. doi: 10.7759/cureus.61890

PubMed Abstract | Crossref Full Text | Google Scholar

17. Arend SM and van't Wout JW. Editorial response: Prophylaxis for Pneumocystis carinii pneumonia in solid organ transplant recipients–as long as the pros outweigh the cons. Clin Infect Dis. (1999) 28:247–9. doi: 10.1086/515127

PubMed Abstract | Crossref Full Text | Google Scholar

18. Rayens E, Rabacal W, Willems HME, Kirton GM, Barber JP, Mousa JJ, et al. Immunogenicity and protective efficacy of a pan-fungal vaccine in preclinical models of aspergillosis, candidiasis, and pneumocystosis. PNAS Nexus. (2022) 1:pgac248. doi: 10.1093/pnasnexus/pgac248

PubMed Abstract | Crossref Full Text | Google Scholar

19. Lee LH, Gigliotti F, Wright TW, Simpson-Haidaris PJ, Weinberg GA, and Haidaris CG. Molecular characterization of KEX1, a kexin-like protease in mouse Pneumocystis carinii. Gene. (2000) 242:141–50. doi: 10.1016/S0378-1119(99)00533-8

PubMed Abstract | Crossref Full Text | Google Scholar

20. Gingo MR, Lucht L, Daly KR, Djawe K, Palella FJ, Abraham AG, et al. Serologic responses to Pneumocystis proteins in HIV patients with and without Pneumocystis jirovecii pneumonia. J Acquir Immune Defic Syndr. (2011) 57:190–6. doi: 10.1097/QAI.0b013e3182167516

PubMed Abstract | Crossref Full Text | Google Scholar

21. Kling HM, Shipley TW, Patil SP, Kristoff J, Bryan M, Montelaro RC, et al. Relationship of Pneumocystis jiroveci humoral immunity to prevention of colonization and chronic obstructive pulmonary disease in a primate model of HIV infection. Infect Immun. (2010) 78:4320–30. doi: 10.1128/IAI.00507-10

PubMed Abstract | Crossref Full Text | Google Scholar

22. Kling HM and Norris KA. Vaccine-induced immunogenicity and protection against Pneumocystis pneumonia in a nonhuman primate model of HIV and Pneumocystis coinfection. J Infect Dis. (2016) 213:1586–95. doi: 10.1093/infdis/jiw032

PubMed Abstract | Crossref Full Text | Google Scholar

23. Cobos Jimenez V, Rabacal W, Rayens E, and Norris KA. Immunization with Pneumocystis recombinant KEX1 induces robust and durable humoral responses in immunocompromised non-human primates. Hum Vaccin Immunother. (2019) 15:2075–80. doi: 10.1080/21645515.2019.1631135

PubMed Abstract | Crossref Full Text | Google Scholar

24. Rabacal W, Schweitzer F, Kling HM, Buzzelli L, Rayens E, and Norris KA. A therapeutic vaccine strategy to prevent Pneumocystis pneumonia in an immunocompromised host in a non-human primate model of HIV and Pneumocystis co-infection. Front Immunol. (2022) 13:1036658. doi: 10.3389/fimmu.2022.1036658

PubMed Abstract | Crossref Full Text | Google Scholar

25. Wychrij DA, Chapman TI, Rayens E, Rabacal W, Willems HME, Oworae KO, et al. Protective efficacy of the pan-fungal vaccine NXT-2 against vulvovaginal candidiasis in a murine model. NPJ Vaccines. (2025) 10:112. doi: 10.1038/s41541-025-01171-4

PubMed Abstract | Crossref Full Text | Google Scholar

26. Rabacal W, Hu A, Khalife S, Kim G, and Norris KA. Experimental Pneumocystis infection in dexamethasone immunosuppressed macaques. Med Mycology. (2025) 63:myaf083. doi: 10.1093/mmy/myaf083

PubMed Abstract | Crossref Full Text | Google Scholar

27. Board KF, Patil S, Lebedeva I, Capuano S 3rd, Trichel AM, Murphey-Corb M, et al. Experimental Pneumocystis carinii pneumonia in simian immunodeficiency virus-infected rhesus macaques. J Infect Dis. (2003) 187:576–88. doi: 10.1086/373997

PubMed Abstract | Crossref Full Text | Google Scholar

28. Morris A and Norris KA. Colonization by Pneumocystis jirovecii and its role in disease. Clin Microbiol Rev. (2012) 25:297–317. doi: 10.1128/CMR.00013-12

PubMed Abstract | Crossref Full Text | Google Scholar

29. Rayens E and Norris KA. Prevalence and healthcare burden of fungal infections in the United States, 2018. Open Forum Infect Dis. (2022) 9:ofab593. doi: 10.1093/ofid/ofab593

PubMed Abstract | Crossref Full Text | Google Scholar

30. Yale SH and Limper AH. Pneumocystis carinii pneumonia in patients without acquired immunodeficiency syndrome: associated illness and prior corticosteroid therapy. Mayo Clin Proc. (1996) 71:5–13. doi: 10.4065/71.1.5

PubMed Abstract | Crossref Full Text | Google Scholar

31. Fishman JA and Gans H. Practice ASTIDCo. Pneumocystis jiroveci in solid organ transplantation: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clin Transplant. (2019) 33:e13587. doi: 10.1111/ctr.13587

PubMed Abstract | Crossref Full Text | Google Scholar

32. Quigley N, d'Amours L, Gervais P, and Dion G. Epidemiology, risk factors, and prophylaxis use for Pneumocystis jirovecii pneumonia in the non-HIV population: A retrospective study in quebec, Canada. Open Forum Infect Dis. (2024) 11:ofad639. doi: 10.1093/ofid/ofad639

PubMed Abstract | Crossref Full Text | Google Scholar

33. Vigano M, Beretta M, Lepore M, Abete R, Benatti SV, Grassini MV, et al. Vaccination recommendations in solid organ transplant adult candidates and recipients. Vaccines (Basel). (2023) 11:1611. doi: 10.3390/vaccines11101611

PubMed Abstract | Crossref Full Text | Google Scholar

34. Perez-Romero P, Bulnes-Ramos A, Torre-Cisneros J, Gavalda J, Aydillo TA, Moreno A, et al. Influenza vaccination during the first 6 months after solid organ transplantation is efficacious and safe. Clin Microbiol Infect. (2015) 21:1040 e11–8. doi: 10.1016/j.cmi.2015.07.014

PubMed Abstract | Crossref Full Text | Google Scholar

35. Radcliffe C and Kotton CN. Vaccination strategies for solid organ transplant candidates and recipients: insights and recommendations. Expert Rev Vaccines. (2025) 24:313–23. doi: 10.1080/14760584.2025.2489659

PubMed Abstract | Crossref Full Text | Google Scholar

36. Choukri F, Menotti J, Sarfati C, Lucet JC, Nevez G, Garin YJ, et al. Quantification and spread of Pneumocystis jirovecii in the surrounding air of patients with Pneumocystis pneumonia. Clin Infect Dis. (2010) 51:259–65. doi: 10.1086/653933

PubMed Abstract | Crossref Full Text | Google Scholar

37. Sassi M, Ripamonti C, Mueller NJ, Yazaki H, Kutty G, Ma L, et al. Outbreaks of Pneumocystis pneumonia in 2 renal transplant centers linked to a single strain of Pneumocystis: implications for transmission and virulence. Clin Infect Dis. (2012) 54:1437–44. doi: 10.1093/cid/cis217

PubMed Abstract | Crossref Full Text | Google Scholar

38. Veronese G, Ammirati E, Moioli MC, Baldan R, Orcese CA, De Rezende G, et al. Single-center outbreak of Pneumocystis jirovecii pneumonia in heart transplant recipients. Transpl Infect Dis. (2018) 20:e12880. doi: 10.1111/tid.12880

PubMed Abstract | Crossref Full Text | Google Scholar