Animals

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 University holds Animal Welfare Assurance Number A3437-01. The care and use of laboratory animals at UGA 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. Six- to eight-week-old female C57BL/6 mice were purchased from Charles River Laboratories. Standard rodent chow and water were given ad libitum.

Vaccine construction and purification

The NXT-2 vaccine candidate was expressed and purified from E. coli as previously described¹¹. A 6X His-tag and associated linker was incorporated to the NXT-2 construct to allow for purification via TALON (Takara) based metal affinity chromatography¹¹. Prior to immunization, NXT-2 protein preparations were tested for endotoxin using the Pierce^TM chromogenic Endotoxin Quant Kit (Thermo Scientific) following the manufactures instructions.

Immunization of mice

Six- to eight-week-old female C57BL/6 mice were immunized subcutaneously at the base of the tail with 40 μg NXT-2 (n = 28) prepared 1:1 with a water: squalene adjuvant (TiterMax, Sigma-Aldrich, Inc.) according to the manufacturer’s guidelines or were sham immunized (n = 25) with phosphate-buffered saline (PBS) prepared 1:1 with TiterMax in a total volume of 100 μl. Blood was collected in microcentrifuge tubes from the facial vein 1–2 days prior to immunization and 28 days following. No more than 10% of the total blood volume was taken. Blood was centrifuged at 10,000g for 15 min at 20 °C and plasma stored at −80 °C until use. Mice were briefly anesthetized by inhalation of 3% isoflurane, and vaginally lavaged with 100 μl of 1× sterile PBS 1–2 days prior to immunization and 28 days following. Samples were immediately spiked with 1 μl of 100× protease inhibitor cocktail (Sigma-Aldrich, Inc.) before being centrifuged at 600×g for 3 min at 4 °C to remove cellular debris. The supernatant was stored at −80 °C until use.

Murine model of C. albicans vaginitis

Twenty-eight days post immunization, mice (sham n = 25 and NXT-2 n = 28) were anesthetized (as described above) and subcutaneously administered 0.1 mg of β-Estradiol 17-valerate (Sigma-Aldrich, Inc.) dissolved in 0.1 ml of sesame oil (Sigma-Aldrich, Inc.) 3 days prior to challenge. Estrogen was administered weekly throughout the study. To prepare the challenge inoculum, C. albicans SC5314 (ATCC) from glycerol stock stored at −80 °C was spread onto a yeast extract peptone dextrose (YPD) agar plate and incubated for 48 h at 30 °C. A single isolated colony was selected and cultured in 10 ml of YPD broth at 30 °C for 18 h, shaking at 225 rpm. Stationary phase C. albicans cells were washed three times with 1× sterile PBS and counted on a hemocytometer. Mice were briefly anesthetized (as described above) and intravaginally challenged with 5 × 10⁶ blastoconidia in 10 μl of sterile 1× PBS. Following challenge mice were monitored daily for symptoms. At 5 days post challenge mice were euthanized (CO₂ inhalation, followed by cervical dislocation. Euthanasia methods were consistent with the recommendations of the American Veterinary Medical Association (AVMA) guidelines) and vaginal lavages were conducted with 100 μl of 1× sterile PBS before vaginal tissue was excised and fixed in 10% neutral buffer formalin for histological assessment. To assess the effect of lavage procedure on vaginal tissues, additional subsets of mice were analyzed. A group of mice were sham (n = 4), or NXT-2 (n = 4) immunized and challenged (as described above), however, mice were not lavaged during the study and tissue was excised 5 days post challenged and fixed for histological assessment. In addition, samples were harvested from normal mice (non-vaccinated, non-challenged) and either non-lavaged (n = 2) or lavaged (n = 2) and fixed for histological assessment.

Fungal burden

Vaginal lavage fluid was serially diluted 10-fold in sterile 1× PBS and 10 μl was plated in triplicate onto YPD agar plates containing 40 μg/ml chloramphenicol to minimize bacterial outgrowth. Plates were incubated at 37 °C for 24 h and colonies were enumerated. Mean colony forming units (CFU) per milliliter (CFU/ml) values were obtained per group.

PMN infiltration

Ten microliters of vaginal lavage fluid was smeared onto Tissue Path Superfrost Plus Gold Slides (Fisher Scientific), air dried at room temperature, and then fixed with CytoPrep spray fixative (Electron Microscopy Sciences). Slides were stained using the Papanicolaou method (PAP smear)³⁵. Tri-lobed nuclear morphology, along with the staining appearance, were used to identify PMNs. PMNs were enumerated in 5 non-adjacent fields using light microscopy at a 40× objective under blinded conditions and mean values were obtained per sample. To visualize the interaction between PMNs and hyphae, slides were scanned at 40× objective magnification using the Aperio AT2 (Leica Biosystems). Images were reviewed and analyzed using ImageScope ×64 (Leica Biosystems) at 40× digital zoom.

ELISA assay

ELISA: NXT-2: Microtiter plates were coated with 5 μg/ml of purified NXT-2 in sterile 1× PBS, as previously described¹¹. Briefly, plasma samples obtained prior to NXT-2 immunization and sham immunization, along with sham samples from 28 days post immunization were diluted 1:100 in 5% non-fat milk in sterile 1× PBS (blocking buffer) and a 1:2 serial dilutions were conducted to determine the endpoint titers as previously described⁴⁵. Plasma samples from NXT-2 immunized animals obtained 28 days post immunization were initially diluted 1:1000 in blocking buffer before additional 1:2 serial dilutions were conducted. All vaginal lavage samples were diluted 1:20 in blocking buffer before 1:2 serial dilutions. All samples were run in duplicate. Immunoglobulin G: Goat anti-mouse IgG conjugated with horseradish peroxidase (Southern Biotech) was used for detection of total IgG. Goat anti-mouse IgG2C and IgG1 conjugated with horseradish peroxidase (Southern Biotech) were used for detection of Th1 and Th2 responses respectively. TMB was added to develop the plates and 1 M sulphuric acid was added to stop the reaction which was subsequently read at an absorbance of 450 nm. On a subset of lavage samples (sham n = 13 and NXT-2 n = 14) the following ELISAs were performed: IL-1β: Utilizing the Ready Set Go ELISA kit (Ebioscience), samples were diluted 1:50 and the manufacturer’s instructions were followed. Samples were plated in duplicate. S100A8: Utilizing the Mouse S100A8 ELISA kit (Rockland Immunochemicals), samples were diluted 1:100 and the manufacturer’s instructions were followed. Samples were plated in duplicate.

Lactase dehydrogenase assay

To assess the levels of lactase dehydrogenase (LDH) in the vaginal lavage the CytoTox 96 nonradioactive cytotoxicity assay kit (Promega) was utilized. Vaginal lavage samples (sham n = 13 and NXT-2 n = 14) were diluted 1:100 in sterile 1× PBS and the manufacturer’s instructions were followed. Samples were plated in duplicate and 1× PBS was used as the negative control, while purified LDH was used as a positive control. Samples were read at an absorbance of 492 nm and duplicate values were averaged.

Histological analysis of inflammation

Fixed vaginal tissue was embedded in paraffin before 4 μm traversal sections were cut and stained with hematoxylin and eosin (H&E). All slides were scanned using the Aperio AT2 (Leica Biosystems) at 40× objective magnification. Images were reviewed and analyzed using ImageScope ×64 (Leica Biosystems). For evaluation of inflammation, vaginal tissue was assessed in eight equally sized areas and 2 fields per area were scored at 20× digital zoom, with the score averaged over the eight areas. Inflammation scoring (0–3) was based on methods previously described with some modifications⁴³. Tissue was scored as the following: 0—no inflammation, 0 neutrophils detected in the squamous epithelial layer; 1— mild inflammation, 1–10 neutrophils or 1–3 micro abscesses in the squamous epithelial layer; 2—moderate inflammation, 11–20 neutrophils or 4–6 micro abscesses in the squamous epithelial layer; 3—severe inflammation, >20 neutrophils, >6 micro abscesses or large abscesses in the squamous epithelial layer. Inflammation scoring was conducted under blinded conditions by two readers.

Immunohistochemistry

Ly6G has been established as a marker for neutrophils in murine vaginal tissues⁴⁶. Vaginal tissue was stained by immunohistochemistry using anti-Ly6G antibodies to conclusively establish the presence of neutrophils in tissue. Vaginal tissue was fixed and processed as described above. Paraffin-embedded tissue was deparaffinized in xylene for 30 min and rehydrated before antigen retrieval under pressure in a Tris/EDTA buffer. To block endogenous avidin/biotin and non-specific binding of primary antibody, sections were pre-treated with avidin/biotin solution diluted 1:1 with 10% normal serum (NS) in tris-buffered saline (TBS). Tissues were then incubated with biotinylated rat anti-mouse Ly6G (clone 1A8 Biolegend, 0.5 mg/ml) at 1:500 in TBS with 10% NS. Biotin rat IgG2a, κ (Biolegend, 0.5 mg/ml) 1:500 was used as an isotype control to confirm antibody specificity. Tissue was then treated with 0.6% H₂O₂ for 1 h to block endogenous peroxidase activity and washed with TBS. The anti-mouse Ly6G antibody was visualized using VECTASTAIN ELITE ABC kit and DAB Peroxidase (HRP) Substrate Kit (Vector Labs). Tissue sections were counterstained with Mayer’s Hematoxylin (Sigma-Aldrich, Inc) and treated with 0.037 M ammonium hydroxide. Tissue was then dehydrated and mounted with VectaMount Permanent Mounting Medium (Vector Labs). All slides were scanned using the Aperio AT2 (Leica Biosystems) at 40× objective magnification. Images were reviewed and analyzed using ImageScope ×64 (Leica Biosystems) and captured at 20× digital zoom.

Anti-NXT-2 antibody functionality: biofilm inhibition and opsonophagocytic killing

For in vitro biofilm and opsonophagocytic killing neutrophil and macrophage assays, hyperimmune plasma was obtained 28 days post immunization from NXT-2 or sham immunized animals. To assess the ability of anti-NXT-2 antibodies to inhibit C. albicans biofilm formation, 5% (v/v) pooled heat-inactivated plasma from NXT-2 or sham groups was added to 1 × 10⁶ blastoconidia of C. albicans (100 μl) cultured in yeast nitrogen base (YNB) medium supplemented with 5% glucose. The samples were incubated at 37 °C for 24 h in a non-humidified chamber. Samples were then washed twice with 200 μl sterile 1× PBS and biofilm formation was assessed through a XTT assay, where 100 μl of XTT (Thomas Scientific) was added to each sample and incubated at 37 °C for 2 h in darkness, before 80 μl of the supernatant was read at an absorbance measured at 490 nm¹¹. Sixteen technical replicates were conducted for each group and the mean OD₄₉₀ values per group were reported.

To assess opsonophagocytic killing in the presence of NXT-2 or sham plasma, murine neutrophils and macrophages were utilized. Neutrophils were isolated from peritoneal fluid of C57BL/6 mice as previously described with slight modifications⁴⁷. Briefly, C57BL/6 mice were intraperitoneally administered 1 ml of 10% casein sodium (Sigma-Aldrich, Inc)-PBS. This process was repeated 16 h after the first administration. Three hours after the second administration mice were euthanized (as described above) and the peritoneal cavity was washed twice with 3 ml of sterile 1× PBS with 0.02% EDTA (Promega). The peritoneal fluid was centrifuged at 200g for 10 min at 20 °C. The supernatant was removed, and the peritoneal exudates were washed three times, each time cells were resuspended in 10 ml of sterile 1× PBS and centrifuged at 200g for 10 min at 20 °C. Peritoneal exudate cells were resuspended in 1 ml sterile 1× PBS and overlayed on top of a discontinuous Percoll gradient (55%, 65%, 75% in the order of top to bottom in 1× PBS) (Cytiva) and centrifuged at 20,000 rpm for 20 min at 20 °C with no break. The neutrophil layer was collected, and the cells were washed with 10 ml of sterile 1× PBS and centrifuged at 200g for 5 min 20 °C before being resuspended in 1 ml of RPMI-1640 (Cytiva). To evaluate neutrophil purity, 20 μl of the cell suspension was added to 180 μl of RPMI-1640 with 50% fetal bovine serum (FBS) (Biowest) and the sample was centrifuged onto a slide at 1000 rpm for 5 min in a Cytospin cytocentrifuge and subsequently stained with Diff-Quick stain (Hema 3 Stat Pack Fisher). Murine macrophages (RAW-264, Sigma-Aldrich, Inc) were cultured and activated with 1 ng/ml of lipopolysaccharides (LPS) (Sigma-Aldrich, Inc) 24 h prior to the experiment as previously described¹¹. All cells were counted using trypan blue dye exclusion. A single C. albicans SC5314 (ATCC) colony was cultured in 10 ml of YPD broth at 30 °C for 18 h, shaking at 225 rpm. C. albicans cells were washed three times with 10 ml 1× PBS and counted on a hemacytometer. Ten percent (v/v) pooled plasma from NXT-2 or sham immunized groups was added to 2 × 10⁵ blastoconidia and incubated at 37 °C for 30 min. Neutrophils or activated macrophages were then added to each sample at a ratio of 1:1 (neutrophils or macrophages: C. albicans) and incubated 37 °C for 2 h with gentle shaking. Eight replicates were conducted for each group. Samples were 10-fold serially diluted and 10 μl was plated in triplicate onto YPD agar plates and incubated at 37 °C for 24 h and colonies were enumerated.

Statistical analysis

All data was plotted and statistically analyzed using GraphPad Prism (GraphPad Software). IgG titers, fungal burden, PMN infiltration, biofilm inhibition and inflammation scoring were analyzed by unpaired student t-test. IL-1β concentrations, S100A8 concentrations and opsonophagocytic killing was analyzed using the Mann-Whitney test. LDH was analyzed via a two-way ANOVA with pairwise comparisons between sham and NXT-2 immunized cohorts assessed using Tukey’s multiple comparisons test. Mean values per vaccine group were reported ± standard error of the mean (SEM).