Post-exposure vaccine protection of CTH522/CAF®01 against reinfection with Chlamydia trachomatis requires Th1/Th17 but not Th2-immunity

Categories: Disease & Virus

April 3, 2025

Ethics statement

Experiments were conducted in accordance with the regulations set forward by the Danish Ministry of Justice and animal protection committees by Danish Animal Experiments Inspectorate Permit 2020-15-0201-00637 and in compliance with European Community Directive 2010/63/EU of the European parliament and of the council of 22 September 2010 on the protection of animals used for scientific purposes, as well as Directive 86/609 and the U.S. Association for Laboratory Animal Care recommendations for the care and use of laboratory animals. The experiments were approved by a local animal protection committee at Statens Serum Institut, IACUC, headed by DVM Kristin Engelhart Illigen.

Animals

Studies were performed with 6- to 8-week-old female B6C3F1 hybrid mice from Envigo, Holland. Animals were housed in appropriate animal facilities at Statens Serum Institut and handled by authorized personnel.

Bacteria preparations and transcervical infection

Chlamydia trachomatis(C.t) Serovar D (SvD) (ATCC) were grown in HeLa cells (ATCC) in RPMI 1640 media (Invitrogen) supplemented with 1%HEPES, 1% of Non-essential amino acids (NEAA) (MP Biomedicals), 1% L-Glutamin (Gibco) and 1% pyruvate (Gibco). Infected HeLa cells were grown for 2–3 days at 37 °C at 5% CO₂. Infected HeLa cells were harvested and C.t. were purified from the cells⁵². Purified C.t. were resuspended in SPG buffer (250 mM Sucrose, 10 mM Na₂HPO₄, 5 mM L-glutamic acid) in aliquots and stored at −80 °C.

All mice were treated 10 and 3 days before infection with 50 mg of medroxyprogesterone (Depo-Provera, Pfizer, Ballerup, Denmark) to synchronize the eostrous cycle and increase susceptibility to chlamydial infection. For transcervical infections, mice were pain relieved with 1μl/g Carprofen (5 mg/ml, Norodyl Vet.) 30 minutes prior to infection and anaesthetized with Zoletil-mix with extra Torbugesic/Buturphanol (2.4 mg/ml Zolazepam, 2.4 mg/ml Tiletamin, 3.8 mg/ml Xylazin, 0.123 mg/ml Buturphanol). 4.5 μl/g of zoletil mix was subcutaneously injected in the neck or at base of the tail. Mice were transcervically (TC) infected within 30 min of anaesthezia using a thin, flexible probe: nonsurgical embryo transfer (NSET) device (Paratechs) to bypass the cervix and to inject bacteria directly into the uterine horn lumen. Mice were expected to wake up within 2–3 h of anesthesia, and their eyes were hydrated with eye gel during this period. Zoletil-mix with extra torbugesic was administered as a TC infection was considered to cause moderate to significant pain development and to ensure the bacteria remained localized, facilitating the establishment of infection.

Antibiotic treatment

Azithromycin dihydrate (Merck) was reconstituted in 99.9% Dimethyl sulfoxide (Merck). For treatment the azithromycin was further diluted to 2.5 mg/ml with 100 mM of Tris-buffer +0.49% glycerol (Merck). Mice were treated for 4 days with 2 × 200 μl subcutaneous injections of Azithromycin (2.5 mg/ml), on each side of the neck or at the base of the tail, each day. The total dose was 4 mg per mouse.

Post-exposure animal model

Mice were TC infected as described above. One week after infection with 1.5 × 10³ inclusion forming units (IFUs) of C.t. SvD, mice were given 400 μl Azithromycin as a subcutaneous injection for 4 consecutive days (4 mg in total). Two weeks after antibiotic treatment, mice were vaccinated 3 times, at two weeks interval. 6 weeks following the last vaccine, the mice were subjected to second TC infection with 1.5 × 10³ IFUs of C.t. SvD.

Antigens, adjuvant and immunization

Mice were immunized three times at two-week intervals with MOMP-based recombinant antigen CTH522⁴⁵ (5μg per dose) or UV-inactivated SvD bacteria, formulated in CAF^®01 (DDA/TDB 250μg/50μg per dose) or in Aluminium hydroxide (500 μg per dose, Al(OH)3, 2% Alhydrogel, Croda Denmark). 200 μl vaccine were injected subcutaneously at the base of the tail.

Bacterial burden

Quantification of the bacterial load in the infected mice was performed by cultivation of upper GT swab samples. Swab samples were stored at −80 °C in 600 μL SPG buffer (250 mM Sucrose, 10 mM Na2HPO4, 5 mM L-glutamic acid) until analysis. For cell cultivation, 80,000 McCoy cells (ATCC) in McCoy media (RPMI 1640 (Invitrogen), 1% HEPES (Gibco), 1% NEAA (MP Biomedicals), 1% L-Glutamin (Gibco), Sodium Pyruvate (Gibco), 64μM 2-Mercaptoethanol, 5% FBS, 0.1% Gentamicin (Gibco) were seeded in each well of a 48-well plate (Costar) and incubated at 37^o C with 5% CO₂ overnight. At a cell confluence at 85-90% the media was changed to 0.2 ml glucose infection medium (McCoy medium +0.5% glucose). Undiluted and 1:1 diluted samples were added to the wells and the plates were spun at 700 g for 1 h at room temperature before incubation at 37 °C with 5% CO₂ for 2 h. Next, the media was changed to 0.5 mL glucose infection media with 1:1000 Cycloheximide (Sigma) for further incubation for 24 h at 37 °C with 5% CO₂. The cells were then fixed with 96% ethanol for 15 min and kept in 0.4 mL 1xPBS until staining of inclusion-forming units (IFU). Nuclei were stained with 0.1 mg/ml propidium iodid (Sigma) for 10 min and afterwards stained with rabbit anti-CT681 antibody (in house, 1:750) diluted in 1xPBS 1% BSA for 1 h at room temperature. Secondary antibody goat anti-rabbit IgG, conjugated with Alexa Flour 488, (1:1000, Life Technologies) were diluted in 1xPBS 1% BSA and incubated with samples for 1 h at room temperature. IFUs were quantified by fluorescence microscopy either manually or automatic by using ImageExpress^® PICO (Molecular Devices) and the CellReporterXpress^® software (Molecular Devices, San Jose, California, USA) counting 50% of each well. The limit of detection was determined of 4 IFU/mouse. This corresponds to a detection threshold of 1 IFU in the tested swab material, which represents 1/4 of the total swab material collected.

Sample collection and cell preparation

Prior to euthanasia, mice were anaesthetized with 6.9 μl/g of a Zoletil-mix (2.4 mg/ml Zolazepam, 2.4 mg/ml Tiletamin, 3.8 mg/ml Xylazin, 0.095 mg/ml Buturphanol). 3 min before euthanasia, 250 μl of anti-CD45.2 – fluorescein isothiocyanate (BD Pharmingen, clone 104, 1:100 dil.) were intravenously injected into the tail of the mice to label vascular leukocytes. For euthanasia, mice were exposed to CO₂ (3 L/min for 5–10 min).

Samples were obtained from 4 to 12 mice per group (individually or pooled in groups of 2) in RPMI 1640 (Gibco Invitrogen). Single-cell suspensions were created by homogenizing organs through a 100 μm nylon filter (Falcon). In addition. GTs were incubated before homogenization for 45 min. at 37 °C CO₂ with type IV collagenase (0.8 mg/ml) (Sigma) and DNAse I (Roche) (0.08 mg/ml). Before and after incubation GTs were processed with gentleMACS™ Dissociator (Miltenyi Biotec) before mechanical filtering. Cell suspensions were centrifuged (700 × g, 5 min) and washed twice in RPMI 1640. Cell pellets from all organs were resuspended in RPMI-1640 (Gibco Invitrogen) supplemented with 5 ×10⁻⁵ M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES, and 10% FCS (Gibco Invitrogen). An additional filtration step was performed after resuspension of GT samples to further eliminate adhesive cellular debris and mucus.

Total IgG, IgG subclasses and IgA-ELISA

Nunc MaxiSorp 96-well plates (Sigma-Aldrich) were coated with CTH522 antigen (1μg/ml) diluted in carbonate buffer overnight at 4 °C. For detection of IgG antibodies, the plates were blocked with 1xPBS (made from 10x PBS, Gibco Invitrogen) with 2% BSA for 2 h. For detection of IgA antibodies, the plates were blocked with 1% skim milk and 0.05% Tween (Merck). The plates were afterwards washed 3 times with washing buffer (PBS + 0.2% Tween). The samples were titrated with 1% BSA as indicated in each figure, and incubated for 2 h at room temperature. The samples were then incubated for 1 h at room temperature with secondary HRP-conjugated against IgG antibodies: rabbit anti-mouse IgG (H + L) (AH Diagnostics), goat anti-mouse IgG1 (Southern Biotech), rabbit anti-mouse IgG2a (AH Diagnostics), rabbit anti-mouse IgG2b (AH Diagnostics) or goat anti-mouse IgG2c (Southern Biotech). For IgA detection the samples were incubated with goat anti-mouse IgA-biotin for 1 h at room temperature followed by incubation with Streptavidin-HRP antibody for 30 min at room temperature. The samples were developed by adding 3, 3’, 5, 5’-tetramethylbenzidine (TMB PLUS2^®, Kementec). After 5–15 min the reactions were stopped by adding 0.5 M H₂SO₄ sulfuric acid (Honeywell Fluka™). Plates were read at 450 nm and with a background correction at 620 nm by using SunriseTM Absorbance Reader (Tecan Life Sciences).

Neutralization assay

Neutralization assay was performed essentially as described in ref. ⁹. Briefly, Hamster kidney cells (HaK) (ATCC® CCL-15) were grown in 96-well flat-bottom microtiter plates (Nunc) in RPMI 1640 supplemented with 1% (vol/vol) L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 70 μM 2-mercaptoethanol, 10 μg/ml gentamicin, 1% HEPES and 5% heat inactivated fetal bovine serum at 37 °C, 5% CO₂. Heat-inactivated (56 °C for 30 min) and serially diluted serum were mixed with a pre-determined concentration of C.t. SvD in SPG buffer. The mixtures were incubated for 45 min. at 37 °C, inoculated onto HaK cells in duplicates and incubated for 2 h at 35 °C on a rocking table. The mixtures were removed and the cells were further incubated in culture media containing 0.5% glucose and cycloheximide (1μg/ml) for 24 h at 37 °C, 5% CO₂. Visualization of the inclusions was done by staining fixed (96% ethanol) and propidium iodide (Thermo Fisher Sci., Invitrogen) stained cells with polyclonal rabbit anti-rCT110 serum (produced in our lab), followed by Alexa 488-conjugated goat anti-rabbit immunoglobulin (1:1000) (Thermo Fisher Sci., Invitrogen, cat #A11008). IFUs were enumerated by fluorescence microscopy using an automated cell imaging system, ImageXpress Pico and CellreporterXpress software (Molecular Devices, San Jose, California, USA) counting 25% of each well. Percentage neutralization was calculated as percentage reduction in mean IFU relative to control serum.

MSD analysis

MSD U-plex was performed to quantify levels of cytokine expression. Harvested supernatants from CTH522 stimulated cells (stimulated for 72 h) were diluted 1:4 and added to 96-well multi-SPOT plate (MSD). The cytokine levels were quantified according to the manufactor’s instructions. The standard and samples were measured in duplicate and blank values were subtracted from all readings. The plates were read and analyzed by SECTOR^® Imager (MSD).

Flow cytometry

For intracellular cytokine staining, cells were stimulated for 1 h in the presence of CTH522 antigen or UV-SvD and 1 μg/ml of costimulatory antibodies CD28 (BD Pharmingen,clone: 37.51) and CD49d (BD Pharmingen, clone: 9C10 (MFR4.B)). Brefeldin A was added afterwards at a concentration of 200 μg/ml to each sample and were subsequently incubated at 37 °C for 5 h and kept at 4 °C until staining. Cell suspensions were Fc-blocked with anti-CD16/CD32 antibody (BD Pharmingen, clone 2.4G2, 1:100 dil.) for 10 min. at 4 °C. CD4 T cells were stained with combinations of the following anti-mouse antibodies conjugated to fluorochromes (company, clone, dilution): Viability-eFluor506 (Invitrogen, 1:500), α-CD4-BV786 (BD Horizon, GK 1.5, 1:600), α-CD44-Alexa fluor 700 (Biolegend, IM7, 1:150), α-CD8-BV421 (Biolegend, 53-6.7, 1:200). To stain intracellular proteins, cells were fixed for 30 minutes with BD Cytofix™ Fixation Buffer (BD Biosciences), washed with BD Perm/Wash™ Buffer (BD Biosciences) and incubated with 50μl/well antibody mix diluted in BD Perm/Wash™ Buffer: αmu-CD3e-BV650 (17A2, Biolegend, 1:200), α-IL-2-APC-Cy7 (BD Pharmingen, JES6-5H4, 1:200), α-IFNγ-PE (BD Pharmingen, XMG1.2, 1:200), α-TNFα-APC (BD Pharmingen, MP6-XT22, 1:200), α-IL-17-PerCP-Cy5.5 (Invitrogen, eBIO17B7, 45-7177-82, 1:200).

To stain innate cells Viability-eFluor506 (Invitrogen, 1:500), samples were stained with the following antibody mix: αmu-CD11b-APC-Cy7 (Biolegend, M1/70, 1:100), αmu-CD11c-APC (BD Pharmingen, HL3, 1:100), αmu-Ly6G-PE (BD Pharmingen, HL3, 1:100), αmu-F4/80-BV650 (BD, T45-2342, 1:100), αmu-CD86-PE-Cy7 (Biolegend, PO3, 1:100), αmu-MHC II-PerCP-Cy5.5 (Biolegend, M5/114, 1:200). The samples were analyzed using a Flow cytometer (BD LSRFortessa, BD Bioscience) and FlowJo Software (version 10). To analyze the cells in the organs we excluded doublets on forward scatter height (FSC-H) and FSC area (FSC-A) plot as well as side scatter height (SSC-H) and area (SSC-A), excluded cell debris on SSC-H and FSC-H and last excluded dead cells using the viability marker. In the GT vascular leukocytes was excluded from the analysis by the intravenous staining of CD45.2, and the remaining i.v. anti-CD45.2 negative cells were defined as tissue cells. Leukocytes were divided into CD4 T cells (CD4⁺), CD8 T cells (CD8⁺), neutrophils (Ly6G⁺ CD11b⁺), macrophages (Ly6G^–CD11b⁺CD11c^–) and dendritic cells (Ly6G^–CD11b⁺CD11c⁺MHC-II⁺).

Statistical analysis

Cell percentages among groups were presumed to meet the assumptions of parametric testing based on previous studies. Accordingly, one-way ANOVA analyses were applied, followed by Tukey’s multiple comparison test, when more than two groups were compared. If only two groups were compared an unpaired Student’s t test was used to determine significance among cell percentages. Bacteria numbers (log10(IFU)) among groups were not presumed to meet the assumptions of parametric testing based on previous studies. Therefore, to determine statistical differences in bacteria numbers (log₁₀IFU) among groups non-parametric tests were performed. Statistically significant difference between bacteria counts among two groups was determined by Mann–Whitney test. Among more than two groups, Kruskal–Wallis test was done followed by Dunn’s multiple comparisons test. All statistical tests were two-sided. A p-value of ≤0.05 was considered a significant difference. Prism version 8 software (GraphPad) was used for analysis.