AbstractChlamydia trachomatis (C.t.) is globally the most common sexually transmitted bacterium with an estimated 131 million new cases occurring every year. There is no licenced vaccine against C.t. Repeated infections are often observed in women, suggesting that natural immunity is only partially protective. It is therefore important to investigate if a vaccine given post exposure, on top of a partially protective natural immunity, can increase protection against reinfection. In mice, an infection leads to robust immunity to subsequent challenges that precludes an investigation of increased protection elicited by a post-exposure vaccine. Therefore, we developed a new animal model where the first infection only provided partial protection against reinfection. Using this model, we show that UV-SvD/CAF®01 and CTH522/CAF®01 as post-exposure parenteral vaccines, but not CTH522/AlOH, protected against reinfection. As CTH522/CAF®01 also reduced the gross pathology score post reinfection, this suggests that CTH522/CAF®01 is both protective and safe as a post-exposure vaccine.
IntroductionChlamydia trachomatis (C.t.) is globally the most common sexually transmitted bacterium with an estimated 131 million new cases occurring every year1. C.t. is an obligate intracellular bacterium infecting both men and women and repeated infections with C.t. are common2. Genital infections are frequently asymptomatic and consequently left untreated. Untreated women can experience serious sequelae such as pelvic inflammatory disease that can lead to fatal ectopic pregnancy and infertility3,4,5. The goal for a preventive vaccine against Chlamydia is to prevent a primary infection. However, it is also important to investigate if a preventive vaccine can be used as a post-exposure vaccine to reduce the risk of subsequent infections in people previously exposed to C.t. This was the focus for the present study.We recently showed that the parenteral vaccine, CTH522/CAF®01, inducing protective systemic Th1/Th17 T cell immunity in animal models6,7, was safe and immunogenic in humans, and that the observed immune responses in animals translated well to humans8,9,10. Here we examine if this vaccine, given as a post-exposure vaccine, was also able to protect against reinfection. However, since infection-induced immunity have been shown to protect efficiently against reinfection11, and previous attempts to boost Chlamydia immunity with vaccines after genital infection led to enhanced immunogenicity, but no increased protection due to the potent immunity elicited by live bacteria12, a new model had to be developed. This model should allow for a window in which additional protection induced by a post-exposure vaccine could be measured. Thus, the model should only induce partial protective immunity after the first infection, which would also better reflect the situation in humans. Furthermore, our objective was to measure post exposure vaccine protection against both infection and pathological changes. Regarding the latter, it has been shown that bypassing the cervix and inoculating C. trachomatis directly in the upper genital tract (uGT) led to efficient colonization of the uGT as well as pathological changes. We therefore decided to use the transcervical route in our infection studies13,14,15. By applying antibiotic treatment one week post the first infection, we achieved partial immunity that showed reduced protection against reinfection. This model was then used to test how post-exposure vaccine-induced immunity was affected by the existing infection-induced immunity and if the vaccine would provide increased protection against reinfection. Moreover, we compared CTH522/CAF®01 with another vaccine, CTH522/AlOH, both previously proven to be safe and immunogenic in humans10. As these vaccines generate different types of immune responses, the results obtained in this study provide important information concerning the design of future Chlamydia vaccines with the ability to protect against both infection in naïve or previously exposed individuals.ResultsDeveloping a mouse model for testing post-exposure vaccinesA previous study showed that infection-induced immunity protected efficiently against reinfection11. In agreement with this, mice subjected to a transcervical (TC) infection (Infection 1 (I1)) with 1.5 × 103 inclusion forming units (IFUs) of C.t. serovar D (SvD) and a TC reinfection (Infection 2 (I2)) at week 13 post the first infection showed strong protection against reinfection (Fig. 1a). Since this did not represent a situation with only partial protective immunity, our first objective was to reduce the immunity generated by the first infection. To accomblish this, we used antibiotics. The animals were infected with 1.5 × 103 IFU of C.t. SvD. 1, 2 or 3 weeks after the infection, the animals were then treated with antibiotics (Fig. 1b). 14 weeks after the first infection, the mice were given a second infection with 1.5 × 103 IFU of C.t. SvD. Bacterial levels were then measured at D7 PI2. The data showed that antibiotic treatment given 2 weeks (Antibiotics Week 2,“AW2”) or 3 weeks (AW3) after the first infection had no influence on protection against a second infection (Fig. 1c). In contrast, animals that received antibiotics one week after the first infection (I1-AW1-I2) showed significantly increased bacterial levels. The increased bacterial levels in the animals that received antibiotics one week after the first infection correlated with a reduced serum IgG titer compared to animals not subjected to antibiotic treatment (Fig. 1d, e). Moreover, examining the natural immunity in AW1 animals prior to reinfection (in another independent experiment) also showed a significantly reduced serum IgG titer as well as reduced infection induced CMI response in the GT of AW1 animals (Fig. 1f–h). Thus, the antibiotic treatment at week 1 post the first infection partially reduced, but did not remove, the protective immune response against reinfection, suggesting that this model could be suitable for testing post-exposure vaccines.Fig. 1: Developing a post exposure vaccine testing model.Groups of female B6C3F1 mice were given a transcervical infection (infection 1, I1) 13 weeks after, the animals were given another infection (infection 2, I2). 7 days after the second infection the animals were taken out for analysis. a Bacterial burden in genital tract (GT) swabs is measured (n = 12). Bacterial numbers are calculated as log10 of inclusion-forming units (IFU). Line represents median log10(IFU) levels with 25th and 75th percentiles. b Overview of the model using antibiotics: The animals were infected at week 0 (W0) with 1.5×103 IFU of Chlamydia trachomatis (C.t.) serovar D (SvD). 1, 2 or 3 weeks after the infection, the animals were treated with antibiotics. 13 weeks after the first infection, the mice were given a second infection with 1.5×103 IFU of C.t. SvD. c Bacterial levels measured at day 7 post infection no. 2 (n = 4–12). d, e Total IgG in serum at day 7 post infection 2 shown as titration curve with calculated area under the curve (AUC). f, g, h The effect of week 1 antibiotic treatment on natural immunity 5 weeks post treatment (n = 8). f Total UV-SvD-specific IgG in serum shown as titration curve g with calculated AUC. Points and error bars indicate means ± SD. h the frequency of cytokine-producing CD44high(hi) CD4 T cells (cyt+, expressing combinations of TNFα, IL-2, IFNγ and/or IL-17) among all CD4 T cells in GT (D, n = 8) in response to in vitro UV-SvD stimulation (gating strategy Supplementary Fig. 1). Bars indicate means ± SD. Statistical significance in (a) was evaluated Mann–Whitney test. Statistical significance in (c) and (e) was determined by Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Unpaired t test was used in (g, h) for comparison among groups. *p < 0.05, ***p < 0.001, ****p < 0.0001, ns not significant.Full size imageTo establish this model as a post-exposure vaccine testing model, we initially chose to test a vaccine consisting of the Th1/Th17-inducing adjuvant CAF®01 together with UV-inactivated C.t. Serovar D bacteria (UV-SvD). We have previously shown that this vaccine is highly protective as a preventive vaccine (Nguyen et al., unpublished observations). After antibiotic treatment, the AW1 animals were vaccinated at week 3, 5 and 7 post the first infection. At week 13 post the first TC infection, the animals were subjected to another TC challenge with 1.5 × 103 bacteria, and 7 days after we measured the bacterial levels (Log10 IFUs) in the GT (Fig. 2a). Vaccinated animals showed a significant reduction in bacterial levels in the upper genital tract (Fig. 2b). Compared to non-vaccinated animals, vaccinated animals also showed an increase in the percentage of cytokine positive(Cyt+) CD4 T cells in the GT tissue from 4.49 +/− 2.18% to 12.15 +/− 3.47% of all CD4 T cells (Fig. 2c), and a significantly increased humoral response (Fig. 2d, e). The increased recruitment of antigen-specific CD4 T cells to the GT correlated with an early increase in innate cells in the GT, such as neutrophils (CD11b + Ly6G+), monocytes (CD11b + Ly6G−) and inflammatory dendritic cells (CD11b + CD11c + Ly6G−) (Supplementary Fig. 2).Fig. 2: Protection with UV-SvD/CAF®01 vaccine.a Overview of the model: After the first infection and antibiotics treatment, groups of female B6C3F1 mice were vaccinated three times as indicated with two weeks intervals. 13 weeks after the first infection, the mice were given a second infection with 1.5 ×103 IFU of C.t. SvD. b Bacterial levels were measured at day 7 post infection 2 (n = 16). Line represents median log10(IFU) levels with 25th and 75th percentiles. c Percentage of Cyt+ CD44hi CD4 T cells out of all CD4 T cells in the GT at day 7 post infection 2 (n = 8, gating strategy in Supplementary Fig. 1). Bars indicate means ± SD. d, e Total UV-SvD-specific IgG in serum (n = 8) shown as titration curve with calculated AUC. Points and error bars indicate means ± SD. Statistical significance in (b) was evaluated Mann–Whitney test. In (c, e) unpaired t test was used for comparison among groups. *p < 0.05, ns not significant.Full size imageTaken together, our data demonstrate that the reinfection animal model can be used to measure post-exposure vaccine protection against reinfection.Testing the CTH522/CAF®01 vaccine as a post-exposure vaccineHaving established the reinfection animal model, we next tested the CTH522/CAF®01 vaccine as a post-exposure vaccine. The mice were infected as described above. Following antibiotic treatment, a group of mice received a post-exposure CTH522/CAF®01 vaccine at week 3, 5, and 7. 21 days after the last vaccination, the vaccine-induced immune response was analyzed. In the spleen, the percentage of antigen-specific CD4 T cells out of the CD4 T cell population increased to 2.45% (Fig. 3a). These CD4 T cells represented both multifunctional Th1 T cells (CD4 + IL17- T cells expressing combinations of IFNγ, IL-2, and TNFα) and Th17 CD4 T cells CD4 + IL17 + T cells expressing combinations of IFNγ, IL-2, and TNFα (Fig. 3b, c). The serum IgG titer was also significantly increased in the vaccine group (Fig. 3d, e), but in the vaginal fluid, no changes were observed in the IgG or IgA titer (Fig. 3f, g).Fig. 3: Day 21 response after CTH522/CAF®01 post exposure vaccination.Female B6C3F1 mice were infected, treated with antibiotics one week post infection and vaccinated three times with CTH522/CAF®01. At day 21 post vaccination the vaccine-induced immunity was analyzed. a Percentage of Cyt+ CD44hi CD4 T cells out of all CD4 T cells in the spleen (n = 8 pooled in pairs). b Percentages of IL-17 negative CD4 T cells (Th1) and IL-17 positive CD4 cells (Th17) out of all CD4 T cells in the spleen were analyzed by flow cytometry for the frequency of cytokine subsets (expression of TNFα, IL-2, IFNγ, and/or IL-17) in the spleen. c Percentage of IL17+ Cyt+ CD44 + CD4 T cells (expressing combinations of IL-17 and TNFα, IL-2 and/or IFNγ) out of all CD4 T cells in the spleen stimulated with CTH522 in vitro (n = 4, gating strategy in Supplementary Fig. 1). d, e CTH522-specific IgG titer in serum (n = 8) shown as titration curve with calculated AUC. f, g IgA and IgG in the GT (n = 8). Figure show a 1:25 dilution of vaginal wash. Bars indicate means ± SD. Points and error bars indicate means ± SD. Statistical significance was evaluated by an unpaired t-test. **p < 0.01, ****p < 0.0001 ns not significant.Full size imageNext, the animals received a reinfection 13 weeks post the first infection, whereafter the immune response and bacterial levels 7 days post infection 2 were analyzed. The data showed that CTH522/CAF®01, given as a post-exposure vaccine, protected against reinfection. The bacterial level was reduced by 85% in the vaccine group (Fig. 4a). Moreover, depleting CD4 T cells prior to, and during, reinfection abrogated CTH522/CAF®01-induced protection (Supplementary Fig. 3, where CTH522/CAF®01 vaccination reduced bacterial levels by 87%). Protection correlated with an increase in cytokine (IFNγ, IL2, IL17, and/or TNFα) producing Th1/Th17 T cell subsets in the GT, ILN and spleen, most of which co-expressed multiple cytokines (Fig. 4b–f). In the GT, the frequency of low quality Th1 subsets (defined by Th1 T cells only expressing IFNγ16), was reduced in vaccinated mice to 10.17 +/− 3.06% compared to 49.03% +/- 10.58 in non-vaccinated mice (Fig. 4g). Protection also correlated with a significantly increased IgG titer against CTH522 in serum (Fig. 5a, b), consisting of antibodies that were able to neutralize C.t. SvD (Fig. 5c). Finally, we observed an increased IgA and IgG recall response in the GT in the vaccine group (Fig. 5d, e), the latter representing another independent experiment).Fig. 4: CTH522/CAF®01 post exposure vaccination protects against re-infection.Following the first infection and antibiotics treatment, groups of female B6C3F1 mice were vaccinated three times with two weeks intervals with CTH522/CAF®01. 13 weeks after the first infection, the mice were given a second infection with 1.5 ×103 IFU of C.t. SvD. a Bacterial levels were measured at day 7 post infection no 2 (n = 12/16) Line represents median value with 25th and 75th percentiles. b Percentage of Cyt+ CD44hi CD4 T cells out of all CD4 T cells in the GT stimulated with CTH522 in vitro (n = 8). c, d Percentage of Cyt+ CD44hi CD4 T cells out of all CD4 T cells in the iliac lymph node (n = 6/8 pooled in pairs) and spleen (n = 8 pooled in pairs) stimulated with CTH522 in vitro (gating strategy in Supplementary Fig. 1). e, f Percentages of IL-17 negative CD4 T cells (Th1) and IL-17 positive CD4 cells (Th17) out of all CD4 T cells in the GT were analyzed by flow cytometry for the frequency of cytokine subsets (expression of TNFα, IL-2, IFNγ and/or IL-17) (n = 8). g Percentage of IFNγ single-producing T cells (“low quality”) out of the total pool of Cyt+ Th1 cells in non-vaccinated and vaccinated mice. Bars indicate means ± SD. Statistical significance in (a) was evaluated Mann–Whitney test. In (b, e, d, g) unpaired t test was used for comparison among groups. *p < 0.05, **p < 0.01, ****p < 0.0001.Full size imageFig. 5: CTH522/CAF®01 increases the local IgG and IgA recall response.a, b CTH522-specific IgG in serum (n = 16) day 7 post infection 2 shown as titration curve with calculated area under the curve. Points indicate means ± SD. c Serum neutralization titers (n = 6). Points indicate medians with 25th and 75th percentiles and dotted lines show reciprocal 50% neutralization titers. d, e IgA and IgG titer in a 1:25 dilution of vaginal wash (n = 16) from two experiments. Bars indicate means ± SD. Statistical significance was evaluated by an unpaired t test. *p < 0.05, **p < 0.01, ****p < 0.0001.Full size imageIn summary, the clinically relevant CTH522/CAF®01 vaccine generated protective immunity against reinfection. Protection correlated with recruitment of Th17 T cells and with Th1 T cells of higher quality than those induced by the infection itself. Protection also correlated with a local IgA/IgG recall response at day 7 post reinfection.Testing the CTH522/AlOH vaccine as a post exposure vaccineWe next addressed whether protective immunity could be achieved with CTH522 in another clinically relevant adjuvant, the Aluminium hydroxide adjuvant (AlOH). AlOH is traditionally known to generate high titers of systemic IgG combined with Th2 immunity17 and has also been shown to be immunogenic when formulated with CTH522 in humans8. This vaccine would also provide information as to the degree to which AlOH-induced immunity correlates with protection. In our post-exposure model, CTH522/AlOH generated systemic IgG antibodies that were neutralizing (Fig. 6a–c), and a GT IgG recall response after reinfection (Fig. 6d). However, no IgA recall response in the GT post reinfection was observed (Fig. 6d). We also did not observe any protection against reinfection with this vaccine (Fig. 6f and Supplementary Fig. 4). Post reinfection, we did not observe increased numbers of CD4 T cells in the GT of CTH522/AlOH vaccinated animals (Fig. 6g). In vitro stimulation with the vaccine antigen of splenocytes led to increased secretion of IL-5 and IL-13, but stimulation of GT cells did not (Fig. 6h, i). Furthermore, IFNγ, TNFα, IL-2 or IL-17 was not expressed by neither spleen or GT cells following stimulation with CTH522 (data not shown).Fig. 6: Testing CTH522/AlOH as a post-exposure vaccine.After the first infection and antibiotics treatment, groups of female B6C3F1 mice were vaccinated three times with two weeks intervals. 13 weeks after the first infection, the mice were given a second infection with 1.5 × 103 IFU of C.t. SvD. At day 7 the animals were analyzed. a, b total CTH522-specific IgG in serum (n = 8) as titration curve with calculated AUC. c Serum neutralization titers (n = 8). Points indicate medians with 25th and 75th percentiles and dotted lines show reciprocal 50% neutralization titers. For the AlOH group the 50% neutralization titer was >12,800. d, e IgG and IgA titers in GT measured in a 1:25 dilution of vaginal wash (n = 8). f Bacterial levels were measured at day 7 post infection no 2 (n = 12–16). g % CD4 T cells out of all cells in the GT (gating strategy in Supplementary Fig. 1). h, i Genital tract or spleen cells were stimulated with CTH522 in vitro for 72 h and secreted IL-13 and IL-5 were measured by MSD (n = 12 pooled in pairs). Cytokine values are raw data with background subtraction. Negative values reflect noise. Bars indicate means ± SD. Statistical significance was evaluated by an unpaired t test. **p < 0.01, ****p < 0.0001, ns: not significant.Full size imageIn another experiment we focused on the later stages post reinfection. In this experiment, at day 42 post reinfection, 3 out of 8 animals showed bacterial levels below the detection limit in the CTH522/AlOH vaccinated group. In contrast, 7 out of 8 CTH522/CAF®01 vaccinated mice did not show any bacteria, whereas 5 out of 8 animals among the non-vaccinated animals were below the detection limit (Supplementary Fig. 5). Increased numbers of viable bacteria found in GT of the CTH522/AlOH animals correlated with increased numbers of CT603-specific T cells in the GT. CT603 was used as a marker for infection-induced T cells as it is strongly recognized in previously infected humans18 and in infected mice (Dietrich et al., non-published observations). Reduced bacterial counts in CTH522/CAF®01 animals correlated with an increase in CTH522-specific T cells.In summary, despite generating systemic Th2 immunity, neutralizing antibodies, and an IgG recall response, CTH522/AlOH failed to recruit CTH522-specific T cells to the GT at day 7 post reinfection, did not induce an IgA recall response, and did not protect against reinfection in the early or late phases post reinfection. In fact, there was even a trend to an increased bacterial load in the later stages of infection compared to non-vaccinated mice.Gross pathology examination in CTH522/CAF®01 vaccinated animalsTo examine whether the protection against bacterial numbers of CTH522/CAF®01 vaccine had an effect on development of pathology, we performed gross pathology analysis of genital tracts at day 36–42 post reinfection on four independent experiments. Experiment 3 was also analyzed at day 78 post reinfection. All samples were blinded and scored according to the examples shown in Fig. 7a. The data showed that in all four experiment the CTH522/CAF®01 exibited a lower score compared to the non-vaccinated animals (Fig. 7b and Supplementary Figs. 6–10). This was also observed at day 78 in experiment 3 (Fig. 7b and Supplementary Fig. 9). In this experiment we also analyzed the number of CD8 T cells in the GT as previous reports have implicated these cells in the development of pathology19,20,21. The results showed that whereas the CTH522/CAF®01 group showed approximately the same number of CD8 T cells from day 3 to 78 post infection 2, non-vaccinated animals showed an increase in CD8 T cell numbers between day 35 and 78 (Supplementary Fig. 11).Fig. 7: Gross pathology scores of vaccinated and non-vaccinated animals.a Example of how the genital tracts were scored. b Gross-pathology analysis of the genital tract from four independent experiments (group sizes were from 5 to 8 mice) that all received a post exposure vaccine with CTH522/ CAF®01, a reinfection, and was analyzed at day 36–42 post reinfection. Exp. 3 was also analyzed at day 78 post reinfection. Genital tract was photographed and scored: “−“ no/mild pathology, “+” moderate pathology, “++” severe pathology. Table show all the scores from the four experiments.Full size imageThus, based on gross pathology analyses, post exposure vaccination with CTH522/CAF®01 did not cause vaccine-enhanced pathology, and in addition did not show an enhanced recruitment of CD8 T cells.DiscussionPrevious studies have shown that an infection with Chlamydia trachomatis provide very efficient protection against a reinfection11. Our data confirmed this, excluding this model as a post-exposure vaccine-testing model. However, animals that received antibiotics one week after the first infection only developed a partially protective natural immunity against a reinfection and thus provided an adequate model in which to test post-exposure vaccines (Fig. 2). In addition, it could be speculated that as the mice receive the second infection at a later age, that also contribute to their suceptibility to reinfection. In this study our focus was on developing the model, and to demonstrate that the model could be used to measure post exposure vaccine-induced protection against reinfection (measured by reduction in bacterial numbers, early and late after reinfection). Moreover, our aim was to characterize the correlates of protection against infection by testing several clinically relevant chlamydia vaccines (inducing different immune responses).Both UV-SvD/CAF®01 and CTH522/CAF®01 post-exposure vaccines generated a systemic CMI response consisting of multifunctional Th1 and Th17 T cells as well as an increased IgG titer. This immunological profile correspond to what has previously been shown for CAF®01 in preventive vaccines6,7, demonstrating that the pre-existing infection-induced Th1 T cell immunity did not affect the ability of CAF®01 to exert its immunological Th1/Th17 profile (Figs. 2 and 3). CTH522/CAF®01 could even increase the quality of existing Th1 responses, since the majory of Th1 T cells in non-vaccinated animals only produced IFNγ in contrast to CTH522/CAF®01 vaccinated animals where the Th1 T cells produced multiple cytokines (Fig. 4). This is in agreement with previous studies using a CAF®01 adjuvanted post-exposure tuberculosis vaccine22 and it shows that despite the existence of natural-induced T cells, specific for a given antigen, CAF®01 can still prime new T cells with increased quality against the same antigen, instead of merely boosting existing antigen-specific T cells.Following reinfection, vaccinated animals showed fast recruitment of circulating Th1/Th17 cells, which correlated with an increase in the percentage of neutrophils, macrophages and dendritic cells (Supplementary Fig. 2). This is in agreement with other studies showing that the cytokines IFNγ and IL-17 lead to recruitment and/or activation of innate cells6,23. Following a reinfection, CTH522/CAF®01 vaccinated animals showed a significant, CD4 dependent, reduction in bacterial numbers. Importantly, the increased recruitment of vaccine-specific Th1/Th17 T cells to the GT did not lead to increased influx of CD8 T cells, and did not cause enhanced immuno-pathology (Fig. 7 and supplementary Fig. 11). In contrast to CAF®01, immunity induced by the Th2 adjuvant AlOH did not protect against reinfection. This is in agreement with a previous study where CTH522/AlOH showed less protection against a primary infection24. This suggests that AlOH may not be the ideal adjuvant for a Chlamydia vaccine. Lack of protection was observed at day 7 post reinfection. In the later stages of infection, we measured slightly increased bacterial numbers in CTH522/AlOH vaccinated animals compared to CTH522/CAF®01 vaccinated animals, which correlated with an increased infection-induced CMI response (Supplementary Fig. 5). Lack of protection with the CTH522/AlOH vaccine could not be explained by lack of neutralizing antibodies in serum. What this means for the role of IgG is not clear. IgG can neutralize the bacteria and activate phagocytes, but it can also correlate with increased GT immunopathology or even with reduced protection in humans19,25,26. However, a recent publication showed that neutralization rates were significantly higher in sera from women with spontaneous resolution versus those with a persisting infection27. In mice, adoptive transfer of IgG from CTH522/CAF®01 vaccinated animals to naïve animals led to protection against infection, but primarily in the early phases of the infection, and it was suggested that this might be due to lack of protective CD4 T cells9. Thus, we favor the view that IgG do play a protective role, but in particularly in combination with Th1/Th17 T cell immunity, and it could be suggested that the further the bacteria ascend into the upper genital tract tissue, the more important a CMI response becomes9, and it should be noted that we used a TC animal model that bypass the vagina where it could be speculated that IgG is particularly important. Concerning the CMI response, lack of protection despite Th2 immunity indicate that Th1 and Th17 T cells are the most important T cells in terms of protection.Compared to AlOH, the CAF®01-adjuvanted vaccine facilitated an IgA recall response post reinfection, suggesting that CAF®01 immunity may support an IgA response. In support of this, in humans vaccinated with a CAF®01-containing adjuvant, we observed mucosal IgA at nasal, vaginal and ocular mucosa10,28. There are several reasons why local IgA in the GT is interesting. IgA is considered to be an anti-inflammatory isotype and is a poor activator of the complement system29. Following an infection, in both females and males, IgA is observed in the reproductive tract mucosa (vaginal fluid or prostatic fluid), and in male prostatic fluid IgA dominate over IgG30,31,32. In women, secretory IgA in cervical secretion, specific for C.t., demonstrated an inverse correlation to C.t. load33. In female mice, IgA protected against infection34 and in male mice IgA in prostatic fluid was also found to be protective35,36. Thus, a vaccine that can increase the IgA titer may be advantageous. CAF®01 is known to generate Th17 T cells. Th17 T cells have previously been suggested to be implicated in an IgA response in the intestines due to their ability to convert into a follicular helper T cell (TFH cell) phenotype37 and to upregulate Poly-Ig receptor38,39.Protective immunity against a primary infection have been shown to involve Th1/Th17 T cell immunity6,7,14,24,40,41,42,43,44,45,46. Although several studies have shown that Th1 CD4 T cells play a protective role during a C.t. infection in mice14,47, other recent studies have questioned the dominant protective role of Th1 T cells48,49, and showed that protective immunity could develop in a Tbet KO mouse49, where Th17 T cells, but not bonafied Th1 T cells, were present50,51. Our study also show recruitment of both Th1 and Th17 T cells following reinfection. Although CD4 depletion abrogated protection, future experiments are required to determine the protective role of these individual T cell subsets.Previously, the CTH522/CAF®01 vaccine, as a preventive vaccine, has been shown to protect against development of pathology6. In the present study, the same vaccine as a post exposure vaccine reduced bacterial levels both early and late after reinfection (supplementary Fig. 5), which was associated with an increased recruitment of CD4 T cells, but without concurrent recruitment of CD8 T cells (supplementary Fig. 11). Overall these vaccinated animals had a lower gross pathology score compared to the non-vaccinated animals at day 35–42, and we saw no sign of vaccine-enhanced pathology, suggesting that CTH522/CAF®01 is a safe post-exposure vaccine. However, it should be noted that, to which degree these data, obtained with a murine model, can be translated to humans is not known.In summary, our results demonstrate that our reinfection animal model can be used to measure post-exposure vaccine protection, and that protection required CD4 T cells and correlated with increased recruitment of multifunctional Th1/Th17 T cells and increased IgA titer in the GT. The fact that CTH522/CAF®01 can protect against infection, both as a pre- or post-exposure vaccine, and that it did not lead to vaccine-enhanced genital pathology is encouraging concerning a future Phase-II trial with this vaccine, where risk groups, such as pre-exposed individuals, are being considered.Materials and methodsEthics statementExperiments 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.AnimalsStudies 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 infectionChlamydia 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 treatmentAzithromycin 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 modelMice 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 immunizationMice 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 burdenQuantification 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 37o 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 preparationPrior 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-ELISANunc 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 assayNeutralization 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 analysisMSD 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 cytometryFor 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 analysisCell 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.Gross pathologyGenital tract was photographed, blinded and scored: “−” no/mild pathology, “+” moderate pathology, “++” severe pathology.IllustrationsAll illustrations were created in BioRender.com. Follmann, F. (2025), https://BioRender.com/b56l742.
Data availability
Data supporting the findings of this study are available from the corresponding author on reasonable request. This includes analysis files and raw data related to Chlamydial IFU calculations, flow cytometry assays, neutralization assays, MSD assays and ELISA.
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Download referencesAcknowledgementsWe are grateful for Annika de Caprétz and the rest of the staff at the experimental animal facility at Statens Serum Institut for handling the animal experiments. The project was funded by the Danish Research Council, Project ID: DFF – 2032-00012B. Illustrations were created in BioRender. Follmann, F. (2025) https://BioRender.com/b56l742.Author informationAuthors and AffiliationsStatens Serum Institut, Department of Infectious Disease Immunology, Copenhagen, DenmarkNina Dieu Nhien Tran Nguyen, Sharmila Subratheepam, Safia Guleed, Kristoffer Mazanti Melchiors, Anja Weinreich Olsen, Katharina Wørzner, Frank Follmann & Jes DietrichAuthorsNina Dieu Nhien Tran NguyenView author publicationsYou can also search for this author inPubMed Google ScholarSharmila SubratheepamView author publicationsYou can also search for this author inPubMed Google ScholarSafia GuleedView author publicationsYou can also search for this author inPubMed Google ScholarKristoffer Mazanti MelchiorsView author publicationsYou can also search for this author inPubMed Google ScholarAnja Weinreich OlsenView author publicationsYou can also search for this author inPubMed Google ScholarKatharina WørznerView author publicationsYou can also search for this author inPubMed Google ScholarFrank FollmannView author publicationsYou can also search for this author inPubMed Google ScholarJes DietrichView author publicationsYou can also search for this author inPubMed Google ScholarContributionsConceived and designed experiments: J.D., F.F. and N.N. Performed experiments: N.N., S.G. and S.S. Analyzed the data: J.D., F.F., N.N., S.G., K.W., A.O.L. and K.M. Drafted and edited the paper: J.D., F.F. and N.N. 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.Corresponding authorCorrespondence to
Jes Dietrich.Ethics declarations
Competing interests
A.W.O. and F.F. are co-inventors of a patent (WO2014146663A1) relating to C.t. vaccines. All rights have been assigned to Statens Serum Institut, a Danish not-for-profit governmental institute. The remaining authors declare no competing interests.
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Reprints and permissionsAbout this articleCite this articleNguyen, N.D.N.T., Subratheepam, S., Guleed, S. et al. Post-exposure vaccine protection of CTH522/CAF®01 against reinfection with Chlamydia trachomatis requires Th1/Th17 but not Th2-immunity.
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