Immune Signature of Chlamydia Vaccine CTH522/CAF®01 Translates from Mouse-to-Human and Induces Durable Protection in Mice

Animals

Female B6C3F1/OlaHsd (H-2b,k)(C57BL/6JOlaHsd inbred female x C3H/HeNHsd inbred male) and C3H/HeNHsd (H-2k) mice, 6–8 weeks of age, were obtained from ENVIGO, The Netherlands. The mice were housed under standard environmental conditions and provided standard food and water ad libitum. Before the initiation of experiments, mice had at least one week of acclimatization in the animal facility. Mice had access to irradiated Teklad Global 16% protein Rodent Diet (Envigo, 2916c) and water ad libitum. Mice were housed at an ambient temperature of 20–23 °C and 45–65% relative humidity on a 12 h/12 h light/dark cycle with 15 min dusk and dawn transition periods under Biosafety Level (BSL) II conditions in individually type III ventilated cages (Scanbur, Denmark) and had access to nesting material (Enviro-Dri) as well as enrichment (aspen bricks, paper house, corn, seed, and nuts, Brogaarden).

Animal experiments were conducted in accordance with regulations of the Danish Ministry of Justice and animal protection committees by Danish Animal Experiments Inspectorate Permit 2018-15-0201-01502 and in compliance with European Union Directive 2010/63/EU. The experiments were approved by a local animal protection committee at Statens Serum Institut, IACUC, headed by DVM Kristin Engelhart Illigen.

Samples from clinical trial participants

Samples from healthy females aged 18–45 years were taken from our first in human clinical trial study². The study was done in accordance with the International Conference on Harmonization’s Good Clinical Practices guidelines, and is registered with ClinicalTrials.gov, number NCT02787109. The study protocol was approved by the London–Chelsea Research Ethics Committee, the Research and Development department at Imperial College Healthcare National Health Service (NHS) Trust, and the Medicines and Healthcare Products Regulatory Agency (EudraCT number 2015-004330-10). All participants gave written informed consent before enrollment. The analyzed human serum samples were collected at baseline and at day 126 post 1st immunization (two weeks post 3rd intramuscular (i.m.) immunization with 85 µg CTH522/CAF®01). The analyzed human PBMC samples were collected at day 140 post 1st immunization (two weeks post 3 x i.m. (3 × 85 µg CTH522/CAF®01) + 1 x i.n. (60 µg CTH522)). PBMC samples from saline immunized humans were used as controls.

Cultivation and harvesting of C.t

C.t. SvD/UW-3/Cx, C.t. SvE/Bour, C.t. SvF/IC-Cal-3, C.t. SvG/UW-57/Cx, SvH/UW-43/Cx, SvI/UW-12/Ur, SvJ/UW-36/Cx, SvK/UW-31,SvA/HAR-13, SvC/TW-3 (all from ATCC), SvIa/sotonIa3/Ia870 (from the Chlamydia Biobank), and SvA/2497 and SvB/Tunis-864 (from LSHTM) were propagated in HeLa-229 cells (ATCC CCL-2.1™) or McCoy cells (ATCC CRL-1696™) for 2–3 days and harvested by repeated centrifugation and sonication steps. Finally, the bacterial suspension was layered on a 30% renografin solution and centrifuged at 40,000 g for 30 min. After centrifugation, the pellet was re-suspended in a sucrose-phosphate-glutamate (SPG) buffer and stored at −80 °C. Serovar typing of the bacteria was confirmed by chromosomal DNA extraction, PCR amplification and sequencing of the gene and flanking regions of ompA. All C.t. serovars were tested negative for mycoplasma (Mycoplasma laboratory, SSI). The concentration (IFU) of the C.t. SvD batch was quantified by titration in McCoy cells. Protein concentrations were determined by bicinchoninic acid protein assay (BCA) (Pierce, Thermo Fisher Sci., Waltham, Massachusetts, US).

Antigen cloning and purification

The CTH522 batch was of good manufacturing practice quality produced at Statens Serum Institut^22,29. CTH518 (identical to Hirep2)¹⁰ and CTH523 constructs were based on the amino acid sequences shown in Fig. 1a with addition of six N-terminal histidines. Synthetic DNA constructs were codon-optimized for expression in E. coli followed by insertion into the pJexpress 411 vector (ATUM, Newark, CA, USA). Likewise, VD4_6-22^D/E/F/G is a fusion protein with an N-terminal six histidine tag holding 17 amino acids from VD4 of the serovars D (FDTTTLNPTIAGAGDVK), E (FDTTTLNPTIAGAGDVK), F (VDITTLNPTIAGSGSVA), and G (VDITTLNPTIAGSGSVV). In all constructs cysteines were exchanged with serines to avoid disulphide bridge formation during recombinant production. Purification was done by induction of expression with IPTG in E. coli BL-21 (DE3) cells transformed with the synthetic DNA constructs. Inclusion bodies were isolated and extracts were loaded on a HisTrap column (GE Healthcare, Chicago, Illinois, USA), followed by anion exchange chromatography on a HiTrap Q HP column and dialysis to a 20 mM glycine buffer, pH 9.2. Protein concentrations were determined by BCA assay.

Synthetic peptides

PepSets of 20–21-mer peptides with 10aa overlap covering CTH522 were produced by GeneCust (Boynes, France) (Supplementary Table 1).

Immunization

B6C3F1 mice received a total of three immunizations at two-week intervals subcutaneously (s.c.) at the base of the tail in a total volume of 200 µl. The vaccines consisted of 10–25 ug of antigen diluted in Tris-buffer with 2% glycerol (pH 7.4) and mixed by vortexing with adjuvant consisting of 50 µg/dose of the glycolipid trehalose 6,6′-dibehenate (TDB) incorporated into 250 µg/dose of cationic liposomes composed of dimethyldioctadecylammonium (DDA) (CAF®01). In Exp. 2 (Fig. 5) mice, in addition, received a single dose of 25 µg CTH522 intranasally without adjuvant. Sham-immunized mice were included as controls.

Mouse and human cell preparation

Splenocytes were isolated from individual mice at week 6, 56, 59 (PID10) and 61 (PID21) and GT tissue was isolated at week 59 (PID10). Samples were obtained from 6–9 mice per group in RPMI 1640 (Thermo Fisher Sci, Gibco. cat. #21875-034). 250 µl of anti-CD45.2 FITC (BD Pharmingen, clone 104, cat. #560695, 2.5 µg) was intravenously (iv) injected via the tail vein of each mouse 3–6 min prior to organ harvest to distinguished genital tract-localized (iv-) and vasculature-associated (iv + ) cells. The organs were homogenized through a 100 µm nylon filter (Falcon). In addition, genital tracts (GTs) were incubated before homogenization for 1 h at 37 °C, 5% CO₂ in type IV collagenase (0.8 mg/ml) (Sigma) and 30 min in DNAse I (Roche) (0.08 mg/ml) and processed both before and after incubation with gentleMACS™ Dissociator (Miltenyi Biotec). Washed and centrifuged (700 × g, 5 min.) cell pellets from all organs were resuspended in RPMI-1640 (Thermo Fisher Sci., Gibco, cat. #21875-034) supplemented with 1% (vol/vol) L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 50 µM 2-mercaptoethanol, 1% penicillin-streptomycin, 1% HEPES and 10% heat-inactivated FBS (HI-FBS) (Biowest, South American origin, VWR).

The analyzed human PBMC samples were collected from day 140 post 1st immunization. PBMCs were thawed using prewarmed AIM-V medium (Thermo Fisher Scientific, Gibco # 12055-091) supplemented with 1% penicillin-streptomycin, 20 µg/ml DNase and 10% HI-FBS. After centrifugation at 400 × g for 10 min. at room temperature cells were resuspended in media without DNase and rested overnight in the incubator at 37 °C, 5% CO₂. Subsequently cells were counted by an automated cell counter and number of dead cells subtracted.

Cell culturing for cytokine detection in supernatants

Single mouse splenocyte cell suspensions were adjusted to 2 × 10⁵ cells/well and stimulated in triplicates with CTH522, CTH523 and CTH518 at a final concentration of 5 µg/ml and CTH522 overlapping peptides at a final concentration of 10 µg/ml. After 72 h of incubation at 37 °C, 5% CO_2, the culture supernatants were harvested and stored at −20 °C. The amounts of secreted IFN-γ and IL-17A were determined by ELISA.

Human PBMC concentrations were adjusted to 1.25 × 10⁵ cells/well in DNase free AIM-V medium supplemented with 1% penicillin-streptomycin and 10% HI-FBS and stimulated in triplicates with CTH522 (15 µg/ml), CTH523 (5 µg/ml), CTH518 (5 µg/ml) and CTH522 overlapping peptides at a final concentration of 10 µg/ml. After 5 days of incubation at 37 °C, 5% CO_2, the culture supernatants were harvested and stored at −20 C. The amounts of secreted IFN-γ and IL-17A were determined by ELISA. Supernatants were also analysed with the MSD V-plex kit; Proinflammatory panel 1 (See Supplementary Fig. 1 for more details).

Flow Cytometry

Mouse cells were stimulated for 1 h in the presence of CTH522 (5 µg/ml) or without antigen in media containing costimulatory anti-CD28-purified (1 µg/ml) (BD Bioscience, clone: 37.51 cat. #553295) and anti-CD49d-purified (1 µg/ml) (BD Bioscience, clone: 9C10-MFR4.B, cat. #553313). Brefeldin A (SigmaAldrich; B7651-5mg) was added to each well to a final concentration of 10 µg/ml. After 6 h of incubation at 37 °C, the cells were kept at 4 °C until staining.

Surface staining: Cell suspensions were Fc-blocked with anti-CD16/CD32 antibody (BD Bioscience, clone 2.4G2, cat. #553142, 1:100) for 10 min. at 4 °C and stained for surface markers diluted in 50% brilliant stain buffer (BD Horizon, cat. #566349) as indicated and fixable viability dye Viability-eFlour780 (eBioscience, #65-0865-14, 1:500) at 4 °C for 20 min. The cells were then fixed and permeabilized using Cytofix/Cytoperm Solution kit (BD Bioscience, #554714) as per manufactorer’s instructions and intracellular cytokine staining (ICS) for IFN-γ, IL-17A, TNF-α, IL-2 was performed at 4 °C for 30 min. Cells were stained with combinations of the following anti-mouse antibodies conjugated to fluorochromes (company, clone, catalog, dilution): α-CD4-BV510 (BioLegend, cRM4.5, #100559, 1:400), α-CD44-Alexa fluor 700 (Biolegend, IM7, #103026, 1:150), α-CD8-BV421 (Biolegend, 53-6.7, # 100738, 1:200), α-CD3-BV605 (BD Bioscience, 145-2C11, #563004, 1:100), α-CD19-BV786 (BD Bioscience, 1D3, #563333 1:300), α-IL-2-APC (eBioscience, JES6.5H4, #17-7021-82, 1:200), α-IFNγ PE-Cy7 (eBioscience, XMG1.2, #25-7311-82, 1:200), α-TNF-PE (eBioscience, MP6-XT22, #12-7321-82, 1:200), α-IL-17A-PerCP-Cy5.5 (eBioscience, eBio17B7, #45-7177-82, 1:200). The stained cells were analyzed using a Flow cytometer (BD LSRFortessa, BD Bioscience) and FlowJo Software (version 10). Non-specific background cytokine positive events from paired non-CTH522 stimulated cells were subtracted from each Boolean gate individually. See Supplementary Fig. 11 for gating strategies in Spleen and GT.

IFN-γ and IL-17A sandwich ELISA

Mouse IFN-γ and IL-17A ELISA

Maxisorp plates (Nunc, Roskilde Denmark) were coated with 100 µl rat anti-mouse IFN-γ (BD Pharmingen cat. #551216, clone R4-6A2) or rat anti-mouse IL-17A (Biolegend, cat. #506902, clone TC11-18H10.1) at a concentration of 1 µg/ml in carbonate buffer (SSI Diagnostics 24203), incubated overnight at 4 °C followed by blocking for 2 h in 1xPBS + 2% skim milk powder (SM) (Natur Drogeriet). After a 1x washing step with 1xPBS + 0.2%Tween-20 (washing buffer), harvested supernatants and either a recombinant IFN-γ standard (BD Pharmingen, cat. #554587) or a recombinant IL-17A standard (Biolegend, cat. #564101) were diluted in 1xPBS + 2%BSA, transferred to ELISA plates and incubated overnight at 4 °C or 2 h at room temperature. Plates were washed 3 times and 100 µl biotin conjugated rat-anti-mouse IFN-γ (BD Pharmingen, cat. #554410, clone XMG1.2, 0.1 µg/ml in 1xPBS + 1%BSA) or 100 µl biotin conjugated anti-mouse IL-17A (BioLegend, cat. #507002, TC11-8H4, 0.25 µg/ml in 1xPBS + 1%BSA) were added to each well and incubated for 1 h at room temperature. Plates were washed 3 times in washing buffer and 100 µl HRP-streptavidin (BD Pharmingen, cat. #554066, 1:5000) solution was added to each well, incubated for 30 min. at room temperature. After 5 washing steps, 100 µl TMB-PLUS (Kem-En-TEC, Taastrup, Denmark) was added to each well and the reaction was stopped with 100 µl 0.2 M H₂SO₄ after 30 min. OD (450-620 nm) was read using an ELISA reader. Mean responses in triplicate control wells were subtracted the mean value of antigen stimulated wells and values below 0 were assigned the value 0.5.

Human IFN-γ ELISA

Maxisorp plates (Nunc, Roskilde Denmark) were coated with 50 µl anti-Human IFN-γ (Thermo Fisher Sci, Invitrogen, cat. #M700A, clone 2 G, 2 µg/ml) and incubated overnight at 4 °C. Next day plates were washed once in 1xPBS + 0.05% Tween-20 (washing buffer) and blocked for 2 h in 1% BSA + 5% Trehalose (Sigma cat. #A8022) in 1xPBS. 100 µl diluted (dilution buffer, 1%BSA + 0.05% Tween-20 in 1xPBS) supernatants and an IFN-γ standard (Thermo Fisher Sci., Invitrogen, cat. #RIFNG50) were transferred to the ELISA plates and incubated overnight at 4 °C. Plates were washed 4 times and 100 µl biotin conjugated anti-human IFN-γ (Thermo Fisher Sci., Invitrogen, cat. #M701B, clone B133.5, 25 ng/ml in dilution buffer) were added to each well and incubated for 2 h at room temperature in the dark. Plates were washed 4 times and 100 µl HRP-streptavidin (BD Pharmingen, cat. #554066, 1:20.000) solution was added to each well and incubated for 30 min. at room temperature. After 6 washing steps, 100 µl TMB-PLUS (Kem-En-TEC, Taastrup, Denmark) was added to each well and the reaction was stopped with 100 µl 0.2 M H₂SO₄ after 30 min. OD (450-620 nm) was read using an ELISA reader. Median responses in control wells were subtracted the median value of antigen stimulated wells and values below 0 were assigned the value 0.5.

Human IL-17A ELISA

Human IL-17A was detected using a Human IL-17A ELISA kit (Thermo Fisher Sci., Invitrogen, cat. #BMS2017) according to the manufacturer’s protocol except for the detection step were 100 µl TMB-PLUS (Kem-En-TEC, Taastrup, Denmark) was added to each well and the reaction was stopped after 10 min. with 100 µl of 0.2 M H₂SO₄. OD (450-620 nm) was read using an ELISA reader. Median responses in control wells were subtracted the median value of antigen stimulated wells and values below 0 were assigned the value 0.5.

ELISA for antigen-specific antibodies in serum and vaginal washes

Mouse: IgG, IgG1, IgG2a/b/c ELISA

Blood was collected post vaccination and serum was isolated after 10 min. of centrifugation at 10,000 g. Vaginal wash samples were collected by flushing the vagina with 100 µl of sterile 1 x PBS and samples stored at −80 °C until analysis. Before dilution, the vaginal wash samples were treated with 25 µg/ml Bromelain (Sigma-Aldrich). Maxisorp plates (Nunc, Roskilde Denmark) were coated with either recombinant antigens (1 µg/ml), peptides (10 µg/ml) or C.t. serovars (10 µg/ml) overnight at 4 °C, followed by blocking for 2 h in 1 x PBS + 2 % BSA for IgG. The serum and vaginal wash samples were serially diluted in 1 x PBS + 1% BSA for IgG or added to coated plates in a 1:200 dilution (B-cell epitope mapping). After washing, HRP-conjugated rabbit anti-mouse IgG (Thermo Fisher Sci., Invitrogen, cat. #61-6520, 1:2000), HRP-conjugated goat anti-mouse IgG1 (Southern Biotech, cat. #1070-05, 1:16000), HRP-conjugated rabbit anti-mouse IgG2a (Life Technologies, cat. #61-0220, 1:5000), HRP-conjugated goat anti-mouse IgG2b (Thermo Fisher Sci., Invitrogen, cat. #M32407,1:4000) and HRP-conjugated goat anti-mouse IgG2c (Thermo Fisher Sci., Invitrogen, cat. #PA1-29288, 1:20000) were added. Antigen specific antibodies were detected using TMB-PLUS (Kem-En-TEC, Taastrup, Denmark). The reaction was stopped with H₂SO₄ and OD (450–620 nm) was read using an ELISA reader. Results were presented either as titration curves, as absorbance (Abs) at one dilution after background subtraction (values below 0 were assigned the value 0, or as Arbitrary Elisa Units (AEU) calculated from an internal standard (serum pool from CTH522 vaccinated mice) using a five-parameter logistic curve using the package ‘drc’ in R.

Human: Peptide and C.t surface specific IgG ELISA

96-well Maxisorp plates (Nunc, Roskilde, Denmark) were coated with 50 µl of 20–21-mer peptides (10 µg/ml) or C.t. serovars (10 µg/ml) overnight at 4 °C followed by blocking for 2 h in 1 x PBS with 2% SM. Serum samples were either serially diluted in 1 x PBS with 1% SM or added to the plate at a fixed 1:200 dilution (B cell epitope mapping). After washing (1xPBS + 1%SM), HRP-conjugated rabbit anti-human IgG (Agilent, Dako, cat. #P021402-02, 1:8000) were added. Antigen specific antibodies were detected using TMB-PLUS (Kem-En-TEC, Taastrup, Denmark). The reaction was stopped with H₂SO₄ and OD (450-620 nm) was read using an ELISA reader. Results were presented as either absorbance (Abs) at one dilution (1:200, B cell epitope mapping) or as AEU calculated from an internal standard established by combining samples with a positive response to C.t. SvD. The titers were calculated based on a five-parameter logistic curve using the package ‘drc’ in R.

Human: CTH522 specific IgG, IgG1 and IgG3 ELISA

96-well Maxisorp plates (Nunc, Roskilde, Denmark) were coated with 50 µl/well of CTH522 diluted to 0.125 µg/ml in carbonate buffer pH 9.6. The plates were incubated for 2 h at room temperature or overnight at 2–8 °C. After incubation, the plates were washed 8 times with wash buffer (PBS pH 7.2 with 1% Tween 20) using an automated plate washer. The reference serum pool and samples were diluted in dilution buffer (Wash buffer + 1% BSA). The sera from the participants were tested by two-fold serial dilution in parallel with the reference serum pool and the plates were incubated for 2 h at room temperature or overnight at 2–8 °C. The plates were washed and HRP-labelled a) Rabbit anti-human IgG (Agilent, Dako, #P021402-02, 1:3000), b) Mouse anti-human IgG1 (Thermo Fisher Sci., Invitrogen, clone HP6069, cat. #A-10648, 1:200) or c) Mouse anti-human IgG3 (Thermo Fisher Sci., Invitrogen, clone HP6047, cat. #05-3620, 1:200) diluted in dilution buffer was added to wells and plates were incubated for 1 h at room temperature. The plates were washed and substrate (OPD dissolved in a citrus buffer pH 5.5) was added. The plates were incubated in the dark for 30 min before the reaction was stopped with 100 µl 1 M H₂SO₄. OD (492 nm) was read using an ELISA reader. A reference line approach on log-log transformed data was used to calculate the concentration of anti-CTH522 IgG antibodies in the serum samples using the reference serum pool as the calibrator. The samples were repeated if the dilution series had less than 3 points within the OD spectrum confined by the reference.

Human: CTH522 specific IgG2 and IgG4 ELISA

Due to low content of both anti-CTH522 IgG2 and CTH522 IgG4 in the internal reference serum pool, the reference line approach was changed to a fixed dilution assay testing samples from same donor on the same plate. Plates were coated with 50 µl/well of CTH522 diluted to 0.125 µg/ml in carbonate buffer pH 9.6. The plates were incubated for 2 h. at room temperature or overnight at 2–8 °C. After incubation, the plates were washed 8 times with Wash buffer (PBS pH 7.2 with 1% Tween 20). All sera from the participants were tested as triplicates in a 1:10 dilution. Plates were incubated for 2 h. at room temperature or overnight at 2–8 °C. The plates were washed and HRP-labelled (a) Mouse anti-human IgG2 (Thermo Fisher Sci., Invitrogen, clone HP6014, cat. #050520, 1:25) or (b) Mouse anti-human IgG4 (Thermo Fisher Sci., Invitrogen, clone HP6025, cat. #A10564, 1:50) was added to wells. Hereafter the plates were incubated for 1 h at room temperature and the substrate reaction was run as describe above. The mean of triplicates values was multiplied with the dilution factor to obtain an AEU.

In vitro and in vivo neutralization

In vitro neutralization assay

The assay was performed in Hamster kidney cells (HaK) (ATCC CCL-15^TM) and was done essentially as published by ref. ⁴⁶. Briefly, HaK cells were maintained 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. Cells were grown to confluence in 96-well flat-bottom microtiter plates (Nunc). The different C.t. stocks were diluted to a pre-determined concentration in SPG buffer and mixed with heat-inactivated (56 °C for 30 min) and serially diluted serum. The mixture was incubated for 45 min. at 37 °C and inoculated onto HaK cells in duplicates or triplicates. After 2 h of incubation at 36 °C on a rocking table the mixture was removed and the cells were and further incubated 24 h at 37 °C, 5 % CO₂ in culture media containing 0.5% glucose and Cycloheximide (1:1000). The cells were fixed with 96% ethanol and inclusions were visualized by staining with polyclonal rabbit anti-rCT043 or rabbit anti-rCT110 serum (produced in our lab), followed by Alexa 488-conjugated goat anti-rabbit immunoglobulin (1:500-1:1000) (Thermo Fisher Sci., Invitrogen, cat #A11008). Cell staining was done with Propidium Iodide (Thermo Fisher Sci., Invitrogen). IFU were enumerated by fluorescence microscopy using an automated cell imaging system (ImageXpress Pico automated Cell imaging system (Molecular Devices, San Jose, California, USA and CellreporterXpress software) counting 25–90% of each well or by manual counting. The neutralization was calculated as percentage reduction in mean IFU relative to control serum. A serum dilution giving a 50% or greater reduction in IFU relative to the control was defined as neutralizing. The serum dilution giving a 50% reduction in IFU was named reciprocal 50% neutralization titer (NT₅₀). NT₅₀ values were calculated based on a five-parameter logistic curve using the package ‘drc’ in R. For samples where no titer could be calculated, the samples were assigned a titer of half the lowest dilution tested; for experiments with human serum: 10, for experiments with mouse serum Fig. 2f: 16, Fig. 2h:12.5 or 25 and Fig. 7b: 8.

In vitro neutralization assay using the endocervical cell line

The cell line End1/E6E7 (ATCC CRL-2615^TM)³⁶ was maintained in Complete Keratinocyte-Serum Free Medium (K-SFM) (Keratinocyte-Serum Free Medium (Thermo Fisher Sci., cat. #17005-042) supplemented with 0.4 mM CaCl₂, 0.05 mg/ml Bovine Pituitary Extract (BPE), 0.1 ng/ml human recombinant Epidermal Growth Factor (rEGF), 10 µg/ml Gentamicin and 1% vol/vol HEPES. Cells were incubated at 37 °C, 5% CO₂ and the neutralization assay was performed as described above with the only exception that cells were incubated 24 h at 37 °C, 5% CO₂ in 100 µl Complete K-SFM containing 0.5% glucose and Cycloheximide (1 µg/ml). Participants with NT₅₀ > 200 in HaK cells were tested using End1/E6E7 cells.

Competitive inhibition of neutralization

In the competitive inhibition of neutralization assay purified serum from CTH522 vaccinated mice and control mice were diluted 1:200 in SPG buffer and pre-incubated for 45 min. at 37 °C with 0.5 mg/ml CTH522, CTH523, CTH518 and VD4_6-22^D/E/F/G or SPG buffer alone prior to 45 min. of incubation at 37 °C with equal volume of C.t. SvD, E, F, and G. The mixtures were inoculated onto a HaK cell monolayer in duplicate and the assay was performed as described above.

In vivo neutralization

Human (a serum pool of 7 CTH522 vaccinated participants with C.t. SvD NT₅₀ > 200, and a serum pool of 5 placebo participants) and mouse (a serum pool of CTH522 vaccinated mice and a serum pool of control mice) serum pools were heat-inactivated, sterile-filtered and diluted 1:2 and 1:4 with a fixed concentration of C.t. SvD. After 30 min. at 37 °C, depo-provera treated C3H/HeN mice were infected with 10 µl of the inoculum (a total of 1.5 × 10³ IFU/mouse), swabbed at day 3 and 7 post infection, and IFU were determined as described below.

Vaginal challenge and bacterial burden

Ten and three days before C.t. SvD challenge, the oestrous cycle was synchronized by injection of 2.5 mg Medroxyprogesteronacetat (Depo-Provera, Pfizer, Ballerup, Denmark), increasing mouse susceptibility to chlamydial infection by prolonging dioestrus. C.t. SvD in concentrations of 1.5 × 10³–1 × 10⁵ IFU/mice C.t. SvD in 10 µl SPG buffer was inoculated i.vag. At several days post infection (PID) mice were swabbed vaginally. To determine protection against ascending infection in immunocompetent B6C3F1 mice, the GTs were removed at PID21-22 and the uterine horns (middle GT; mGT) and upper genital tract tissue (uGT) comprising the fallopian tubes and ovaries were homogenized (in one experiment the horns were swabbed, Fig. 6g, Exp. 3) using a gentleMACS™ Dissociator (Miltenyi Biotec). The homogenates were placed in Eppendorf tubes with glass-beads and stored at −80 °C until analysis. Before use they were vortexed for 1 min. Swab samples were collected in 0.6 ml SPG buffer with glass-beads, vortexed for 1 min. and stored at –80 °C until analysis. The infectious load was assessed in GT tissue or swab material by infecting 48 plate wells seeded with McCoy cells with the GT tissue or swab material. The McCoy cells were maintained in RPMI 1640 supplemented with 1% 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. Cells were grown to confluence in 48-well flat-bottom microtiter plates (Costar, Corning, NY, USA). The cells were infected by 1 h of centrifugation at 750 g. After 2 h of incubation at 37 °C the wells were aspired and incubated 24 h at 37 °C, 5 % CO₂ in 100 µl culture media containing 0.5% glucose and Cycloheximide (1 µg/ml). After 24 h of incubation wells were aspired. Inclusions were visualized by staining with an in-house polyclonal rabbit anti-MOMP serum, followed by an Alexa 488-conjugated goat anti-rabbit immunoglobulin (Thermo Fisher Sci., Invitrogen, cat. #A11008, 1:500–1:1000). Background staining was done with Propidium iodide (Invitrogen). IFU were enumerated by fluorescence microscopy either manually or by using an automated cell imaging system (ImageXpress Pico automated Cell imaging system) (Molecular Devices) counting 50% of each well. If no IFU were detected in the counted area, 100% of each well was counted manually. For the swab samples culture-negative mice were assigned a limit of detection of 4 IFU/mouse representing one IFU in the tested swab material (1/4 of the total swab material). To investigate the ability of the vaccine to protect against upper genital infection, all tissue homogenates as well as all swab materials were analysed and the results presented as culture positive samples out of total.

Depletion of CD4⁺ and CD8⁺ T cells

C3H/HeN mice were depleted of CD4⁺ and CD8⁺ T cells by i.v. administration of monoclonal rat anti-mouse CD4 (Bio X Cell clone GK1.5, cat. #BE0003-1) and rat anti-mouse CD8 (Bio X Cell clone YTS169, cat. # BE0117) diluted in Dilution buffer pH 7.0 (Bio X Cell, cat. #IP0070). At depletion injections days PID-11, −6, −3 and 1 mice received 200 µg CD8 and 400 µg CD4 antibodies and at depletion injections PID 8, 15, and 22 they received 100 µg CD8 and 200 µg CD4 antibodies. The depletion status in blood and GT tissues were verified by flow cytometry (Supplementary Fig. 6).

Purification of antibodies and adoptive transfer of IgG to C3H/HeN mice

Blood was collected from CTH522 vaccinated and control B6C3F1 mice (Exp.1 n = 100, Exp 2. n = 150) and serum isolated by centrifugation at 10,000 g for 10 min. In Exp. 1 mice were vaccinated 3 times with 14 days interval with 25 µg CTH522/CAF®01 and serum were isolated and pooled 14 days post last vaccination. In Exp. 2 mice were vaccinated 3 times with 14 days interval with 10 µg CTH522/CAF®01 followed by a boost with 25 µg CTH522 without adjuvant. Sera were isolated and pooled 10 days post last vaccination. The pools of serum were filtered through a 0.45 μm Minisart® syringe filter (Sartorius), diluted 1:2 by PBS and IgG were purified on a 5 mL HiTrap™ Protein G HP column (GE-healthcare Bio-Sciences AB) according to the manufacturers protocol. Collected fractions were dialyzed overnight against PBS using a Slide-A-Lyzer™ Dialysis Cassette (Thermo Fisher Scientific), filtered through a 0.22 μm Minisart® syringe filter (Sartorius), IgG concentration quantified by NanoDrop™ 2000, and the purified IgG stored at −20 °C until use. (Supplementary Fig. 8; SDS page of purified IgG from CTH522 vaccinated and control mice and CTH522 specific IgG1 and IgG2c responses of purified IgG). Purified IgG were transferred to recipient C3H/HeN mice by the intraperitoneal (i.p.) route. In Exp. 1 each recipient mice received either a total of 1 mg purified IgG from CTH522 vaccinated mice or 0.43 mg IgG from control mice at PID-3. In Exp.2 each recipient mice received either a total of 4.6 mg purified IgG from CTH522 vaccinated mice or 1.43 mg IgG from control mice at PID-3 and at PID15 they received an additional 1.44 mg IgG from CTH522 vaccinated or 0.06 mg IgG from control mice.

Immunohistochemical staining

Genital tracts from mice were removed following euthanasia and fixed at room temperature in 4% formaldehyde (VWR chemicals) and paraffin embedded. Processing, sectioning and staining were done by the technical staff at BioSiteHisto (Finland). Four µm thick sections were collected on TOMO adhesive coated slides for immunohistochemical (IHC) staining. Detection of C.t. was performed with an in-house rabbit anti-MOMP polyclonal antibody and an HRP based detection system and a brown DAB chromogen. The IHC slides were counterstained with Mayer Hematoxylin (Merck). Slides were digitalized as WSI in Mirax format with 3DHistech Panoramic MIDI scanner (3DHistech Ltd.). Slides were visualized and analyzed using CaseViewer version 2.3. (3Dhistech Ltd.).

Statistical analysis

GraphPad Prism v 9.3.1. and 10.0.2 was used for data handling, analysis and graphical visualizations. The statistical tests used are described in the relevant figure legends and p-values are shown either in the figure or in figure legends. A p-value above 0.05 was considered not significantly different. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, non-significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.