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).

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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).

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.