Multi-component Prime-boost Chlamydia Trachomatis Vaccination Regimes Induce Antibody And T Cell Responses And Accelerate Clearance Of Infection in A Non-human Primate Model Ⅱ

Materials and methods

Vaccine antigens and adjuvants

Different vaccine antigens and adjuvants were used in this study: CTH522 vaccine antigen, based on MOMP (11) was administered through the intramuscular route (IM) with CAF01 adjuvant (42) or Aluminium hydroxide (AlOH) adjuvant, or through the intranasal route (IN) without adjuvant. Plasmid DNA vaccine, pcDNA3.1-MOMP, was made by cloning consensus MOMP into the plasmid vector pcDNA3.1 (14) and administrated by intra-dermal (ID) route with electroporation (EP) in the back. Recombinant Human Adenovirus serotype 5 (rHuAd5) vaccine expressing MOMP (HuAd5-MOMP) was made by homologously recombining consensus MOMP (AAC45154.1) into an E1 and E2 deleted HuAd5 plasmid pAL1112 vector and administered through intramuscular route (14). Modified vaccinia Ankara-MOMP vaccine made by recombining consensus MOMP (AAC45154.1) into the p3186 plasmid and attenuated MVA vector (14), and administered through the intramuscular route.

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C. trachomatis serovar D stock

Chlamydia trachomatis serovar D (Ct SvD; UW-3/Cx, ATCC® VR-885™) was obtained from American Type Culture Collection (ATCC, Manassas, VA) and propagated in HeLa-229 cells, harvested, and purified as described by Caldwell et al. (43). The C. trachomatis SvD stock was stored at -80°C in 0.2 M sucrose, 20 mM sodium phosphate (pH 7.4), and 5 mM glutamic acid (SPG) and the concentration of inclusions forming units (IFUs) was determined in HeLa-229 cells and McCoy cells.

Non-human primates 

Thirty cynomolgus macaques (Macaca fascicularis) weighing 3 to 5 kg were included in the study handled in biosafety level 3 (ABSL3) NHP facilities of IDMIT (“Infectious Disease Models and Innovative Therapies” at the CEA “Commissariat à l’Energie Atomique,” Fontenay-aux-Roses, France; accreditation no. #D92- 032-02). The protocols were approved by the institutional ethical committee “Comité d’Ethique en Expé rimentation Animale du Commissariat à l’Energie Atomique et aux Energies Alternatives” (CEtEA #44) under statement number A14-026. The study was authorized by the “Research, Innovation, and Education Ministry” under registration number APAFIS#720-201505281237660. All animals were tested negative for SIV, SHIV, STLV (simian Tlymphotropic virus), herpes B virus, filovirus, SRV-1, SRV-2 before the study and confirmed to be seronegative for Chlamydia infection. All experimental procedures were conducted according to European guidelines for animal care (“Journal Officiel de l’Union Europé enne”, directive 2010/63/ UE, September 22, 2010).

Vaccination protocols

The 30 macaques were randomly divided into 6 groups with 5 animals in each. The different vaccination prime-boost regimes for the 6 groups are illustrated in detail in Figure 1. During all vaccinations, the macaques were sedated with ketamine chlorhydrate (10 mg/kg body weight intramuscularly). CTH522 (85 µg per animal) was administered by the intramuscular (IM) route with either CAF01 adjuvant (625 µg/125 µg) or AlOH adjuvant (0.425 mg), as shown in Figure 1. IM immunizations were performed in 0.6 ml volume in the right thigh (M. quadriceps) of the animal. CTH522 Intranasal (IN) immunizations (30 µg per animal) were performed with a Vaxinator device connected to a 1 ml syringe with two successive administrations of 0.25 ml, one in each nostril. The DNA-MOMP vaccine was administered by intradermal (ID) route with electroporation (EP) in the skin of the back. 6 repeated volumes of 0.1 ml were injected ID (in total 1 mg) followed by EP using the Nepagen™ system (settings: 6 square-ware pulses of 10 ms with 90 ms intervals at 110V, (300- 700mA)). The hAd5-MOMP vaccine was administered IM with a dose of 1011 viral particles in 1 ml per animal. The MVA-MOMP vaccine was administered IM with a dose of 4*108 PFU per animal in 0.5 ml volume **. The naïve group was not given any vaccination and was considered to be the control group.

Sample collection

During all sampling procedures, the macaques were sedated with ketamine chlorhydrate (10 mg/kg body weight IM). Blood was collected via femoral venipuncture at week 0, 2, 4, 6, 8, 10, 16, 18, 20, 22, 24. Cervicovaginal fluid for antibody detection was collected with Weck-Cel®spears (Medtronic Ophthalmics, Jacksonville, FL, USA) at week 4, 8, 16, 18, 20, 22 and 24. Cervicovaginal samples for chlamydial load detection were collected with regular flatlock swabs (Mast Diagnostic - ref.: 552C/ 10). Following contact with the vagino-cervical mucosa, the swab was placed in a vial containing UTM-RT medium (Mast Diagnostic – ref.: 330C/6) and kept on ice.

Vaginal challenge infection with 5*107 IFUs of C. trachomatis SvD 

The animals were sedated with ketamine chlorhydrate (10 mg/kg IM), placed in ventral recumbency with their hips elevated and 1 ml of the inoculum was traumatically applied directly to the vaginal mucosa at the vagino-cervical transition using a 1 ml syringe. A dose of 5*107 IFUs was given to each animal. The animal was allowed to stay in ventral recumbency with their hips elevated for a short time after the inoculation.

Enzyme-linked immunosorbent assay 

CTH522 specifific antibodies in serum and swab samples were detected with an indirect ELISA. Maxisorp® plates (NUNC A/S, Roskilde, Denmark) were coated with CTH522 (0.5 µg/ml) overnight at 4°C. The isotypes IgG and IgA were detected with HRPconjugated antibodies specifific against non-human primate IgG (43R-IG020HRP, Fitzgerald, Acton, MA, USA, 1:75.000) and IgA (43C-CB1631, Fitzgerald, USA, 1:50.000). The reactions were visualized with TMB PLUS substrate (KemEnTec, Taastrup, Denmark) and stopped with 0.5 M sulphuric acid. The plates were read on an ELISA reader at 450 nm with correction at 650 nm. A positive control (serum from a previous study) was used as an internal standard to correct for plate-to-plate variation. Two wells were run without substrate as a negative control on each plate. The antibody titers were calculated as the reciprocal of the highest dilution with an optical density (OD) value higher than the cut-off. The cut-off was determined from the day 0 sample mean + 2*SD.

Peptide array and analysis 

The CTH522 peptide array was designed with triplicates of immobilized 15mer peptides overlapping by 14 amino acids which were printed on functionalized glass slides and incubated with serum samples by JPT Peptide Technologies, Berlin. Briefly, the array was incubated with serum samples collected at week 24 and IgG peptide complexes were visualized using biotinylated goat anti–monkey IgG and Alexa Fluor® 647 Streptavidin. Slides were scanned with a high-resolution scanner at 635 nm and data was reported as arbitrary fluorescence units. The mean of triplicate values was determined, if the coefficient of variation was larger than 0.5, only the 2 closest values were used. 10% of the maximum intensity signal (65,535) was used as a cut-off for a positive response, and mean signals below this threshold were assigned a value corresponding to half the threshold value (3277).

In vitro neutralization assay 

The in vitro neutralization assay was performed to test the vaccine-induced serum antibodies’ capacity to neutralize Chlamydia infection of HaK cells in vitro. The assay principally followed the protocol previously described (44). First, serum samples from individual animals were incubated at 56°C for 30 minutes to inactivate the complement. Chlamydia stock (SvD, SvE, and SvF) was mixed and incubated with serial dilutions of inactivated serum from each of the vaccinated animals for 30 minutes and then inoculated onto a monolayer of HaK cells in 96-well flat-bottom plates in duplicates. Following 30 hours of incubation, cells, and inclusions were fixated and visualized with polyclonal rabbit anti-Ct043 serum and fluorescence-labeled secondary antibody (Alexa Flour 488, goat-anti-rabbit IgG, A11008, Life Technologies) in the dilution 1:500. Counting of inclusions was performed manually in 20 fields of view at 40x magnification (Olympus IX71 inverted fluorescence microscope). Neutralization was calculated as the percentage reduction in the mean number of IFUs compared to a pool of sera from control group animals (naïve).

Intracellular PBMC cytokine staining and fellow cytometry 

PBMCs were isolated and (1-2 x 106 ) were resuspended in 150 µl of complete medium containing 0.2 µg of each costimulatory antibody CD28 and CD49b. Stimulation was performed in 96 wells/plates using 2 µM of each peptide pool or SEB (as positive control) or medium alone (as negative control). Brefeldin A was added to each well at a final concentration of 10 µg/ml and the plate was incubated at 37° C, 5% CO2 overnight. The cells were then washed, stained with a viability dye (violet, fluorescent reactive dye, Invitrogen), fixed, and permeabilized with the BD Cytofifix/Cytoperm reagent. Permeabilized cell samples were then stored at -80°C before the staining procedure. Antibody staining was performed in a single step following permeabilization. All the used antibodies are listed in Table 1. After 30 min of incubation on ice in the dark, cells were washed in BD Perm/Wash buffer. Cells were counted with an LSR II (BD) immediately after the staining procedure and FlowJo software was used for data analysis.

TABLE 1 Antibodies used for staining of immune cell markers and intracellular cytokines

Detection of vaginal chlamydial DNA by qualitative PCR 

The Abbott RealTime CT/NG assay (ABBOTT, Rungis, France) is an in vitro polymerase chain reaction (PCR) assay for the direct, qualitative detection of the plasmid DNA for CT (C.trachomatis) and NG (N. gonorrhoeae) genomes. Nucleic acid was extracted from UTM (universal transport media) samples on M2000SP and amplification was performed on M2000SP, according to the manufacturer’s instructions. Results were expressed as cycle threshold (Ct).

Statistical analysis

All statistics were performed with GraphPad Prism software (version 8.1.2). The not-normally distributed data were analyzed with non-parametric tests; Kruskal–Wallis test and Dunn’s multiple comparison tests to determine the significance of the difference between groups. Curves showing percent PCR negative animals were compared with a Log-rank (Mantel-Cox) statistical test. The comparisons were considered statistically signifificant if the P value was < 0.05 (P < 0.05). Further levels of significance are indicated with asterisks *P < 0.05, **P < 0.01, ***P < 0.001.

Data availability statement 

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author. 

Ethics statement

The animal study was reviewed and approved by “Comité d’Ethique en Expérimentation Animale du Commissariat à l’Energie Atomique et aux Energies Alternatives” (CEtEA #44) under statement number A14-026. The study was authorized by the “Research, Innovation and Education Ministry” under registration number APAFIS#720-201505281237660. 

Author contributions 

Conceived and designed experiments: JD, FF, EL, PA, RG, RS. Performed experiments: VC, DD, BD, SL, CD, CJ, ND-B, CB, BB, AT. Produced vaccines and antigen constructs: IR, PM, AO. Analyzed the data: JD, EL, FF, IR, HJ. Drafted and edited the paper: JD, EL, FF. Each of the listed co-authors made substantial contributions to the work through design and conception, and/or acquisition, analysis, and interpretation of the data. All authors contributed to the article and approved the submitted version.

Acknowledgments 

We thank the Fondation Dormeur, Vaduz for the donation to Viral Evolution and Transmission Unit for laboratory instruments relevant to this project.

Conflflict of interest FF, IR, PA and AWO are co-inventors on a patent application on vaccines against chlamydia [WO2014146663A1]. All rights have been assigned to Statens Serum Institut, a Danish not-for-profit governmental institute. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 

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.

References 

1. Newman L, Rowley J, Vander Hoorn S, Wijesooriya NS, Unemo M, Low N, et al. Global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012 based on systematic review and global reporting. PLoS One (2015) 10(12):e0143304. doi: 10.1371/journal.pone.0143304

2. Longbottom D, Coulter LJ. Animal chlamydioses and zoonotic implications. J Comp Pathol (2003) 128(4):217–44. doi: 10.1053/jcpa.2002.0629

3. Haggerty CL, Gottlieb SL, Taylor BD, Low N, Xu F, Ness RB. Risk of sequelae after chlamydia trachomatis genital infection in women. J Infect Dis (2010) 201 Suppl 2:S134–55. doi: 10.1086/652395

4. Dodet B. Current barriers, challenges, and opportunities for the development of effective STI vaccines: point of view of vaccine producers, biotech companies and funding agencies. Vaccine (2014) 32(14):1624–9. doi: 10.1016/ j.vaccine.2013.08.032 

5. Brunham RC, Kuo CC, Cles L, Holmes KK. Correlation of host immune response with quantitative recovery of Chlamydia trachomatis from the human endocervix. Infect Immun (1983) 39(3):1491–4. doi: 10.1128/iai.39.3.1491- 1494.1983

6. Cotter TW, Meng Q, Shen ZL, Zhang YX, Su H, Caldwell HD. Protective efficacy of major outer membrane protein-specific immunoglobulin a (IgA) and IgG monoclonal antibodies in a murine model of chlamydia trachomatis genital tract infection. Infect Immun (1995) 63(12):4704–14. doi: 10.1128/iai.63.12.4704-4714.1995

7. Johansson M, Schon K, Ward M, Lycke N. Studies in knockout mice reveal that anti-chlamydial protection requires TH1 cells producing IFN-gamma: is this true for humans? Scand J Immunol (1997) 46(6):546–52. doi: 10.1046/j.1365- 3083.1997.d01-167.x 

8. Gondek DC, Olive AJ, Stary G, Starnbach MN. CD4+ T cells are necessary and sufficient to confer protection against chlamydia trachomatis infection in the murine upper genital tract. J Immunol (2012) 189(5):2441–9. doi: 10.4049/ jimmunol.1103032 

9. Armitage CW, O’Meara CP, Harvie MC, Timms P, Wijburg OL, Beagley KW. Evaluation of intra- and extra-epithelial secretory IgA in chlamydial infections. Immunology (2014) 143(4):520–30. doi: 10.1111/imm.12317

10. Armitage CW, O’Meara CP, Harvie MC, Timms P, Blumberg RS, Beagley KW. Divergent outcomes following transcytosis of IgG targeting intracellular and extracellular chlamydial antigens. Immunol Cell Biol (2014) 92(5):417–26. doi: 10.1038/icb.2013.110

11. Olsen AW, Follmann F, Erneholm K, Rosenkrands I, Andersen P. Protection against Chlamydia trachomatis infection and upper genital tract pathological changes by vaccine-promoted neutralizing antibodies directed to the VD4 of the major outer membrane protein. J Infect Dis (2015) 212(6):978–89. doi: 10.1093/infdis/jiv137 

12. Nguyen N, Guleed S, Olsen AW, Follmann F, Christensen JP, Dietrich J. Th1/Th17 T cell tissue-resident immunity increases protection but is not required in a vaccine strategy against genital infection with chlamydia trachomatis. Front Immunol (2021) 12:790463. doi: 10.3389/fifimmu.2021.790463

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