Chlamydia trachomatis (C.t.) is the leading cause of bacterial sexually transmitted diseases worldwide. Globally, the total number of Chlamydia cases was estimated to be around 100 million adults in 2008 (1). The largest burden of disease from C.t. is in women, where untreated genital infections may lead to severe complications such as pelvic inflammatory disease, ectopic pregnancy, and infertility.

The choice of immunization routes is critical when defining future vaccine strategies against a genital infection like Chlamydia. A strategic tool can be to combine several delivery routes to influence both the immune profile and localization of vaccine responses. Regarding the immune profile, it is widely believed that an optimal Chlamydia vaccine will need to elicit mucosal immunity comprising both neutralizing antibodies (Abs) and cell-mediated immunity (2–8). Several Chlamydia studies have determined that interferon gamma (IFNγ)-producing CD4 T cells play a direct protective role during infection, as bactericidal IFNγ targets C.t. while it is intracellular (6, 9–14). In addition, CD4 T cells also play a role through cognate interactions with antigen (Ag)-specific B cells leading to differentiation of high-affinity long-lived memory and plasma cells (15–17). Although Abs are not essential during primary infection, evidence suggests that they can play a significant role by decreasing initial infectious load through neutralization and possible complement activation (3, 4, 6, 7, 18–21). In the vagino-cervix of humans, IgG is the predominant secreted isotype relative to secretory IgA (SIgA) (22). However, SIgA has several advantages over IgG, e.g., it is more resistant to protease cleavage and is up to 10 times more effective than monomeric Ig’s in neutralizing pathogens (23). Importantly, it has been shown that the concentration of IgA in the human endocervix inversely correlates with C.t. load (24, 25) and in accordance with that we recently found that the presence of vaginal SIgA correlated with accelerated clearance of C.t. in infected minipigs (26). Therefore, vaccination protocols and delivery routes stimulating mucosal IgA are a subject of intense research (3). Interestingly, Th17 cells have been recognized as a key accelerator of mucosal immunity and IgA secretion (27, 28). Th17 cells display a great degree of plasticity, capable of acquiring functional characteristics of follicular helper T cells, which can induce IgA-isotype switching (29–31).

When defining vaccine strategies against genital Chlamydia infections, the selection of any specific immunization route or combination of routes is highly relevant. Systemic T cells induced by parenteral immunization routes can migrate freely through organs such as the spleen and liver, whereas mucosal organs like the airways and genital tract (GT) are restrictive for entry of circulating T cells (32). Thus, mucosal immunization is required to generate or permit entry of circulating activated T cells to establish a local tissue-resident memory T cell (TRM) pool, which composes a separate compartment from the circulating memory pool (33–37). TRMs provide early in situ responses upon mucosal Ag reexposure and their significance to C.t. vaccine strategies was evident in a recent study of Stary et al., suggesting that optimal C.t. clearance required both a first wave of GT-seeded CD4 TRMs followed by a second wave of infection-induced recruitment of circulating memory T cells (36). Another advantage of mucosal immunization routes is that, unlike parenteral immunization, it favors the generation of SIgA Abs (26, 31, 38–40). Mucosal compartments such as the respiratory tract or intestine have organized immune inductive sites, where immune responses are initiated (36, 41). However, the GT lacks these sites (42–47) and alternative mucosal vaccine strategies should, therefore, be considered. It has been reported that intranasal (IN) immunization can induce humoral and cellular memory responses in both the respiratory mucosa and the GT (3, 26, 31, 38, 39, 48, 49). However, IN immunization does not always induce strong immune responses by itself, largely depending on the adjuvant/Ag combination (31, 50). Recent work has shown that sequential combinations of parenteral and nasal immunization routes represent a strategic tool to modulate immunity, magnitude, and localization of the vaccine responses (31, 40, 50, 51). Recently, we showed that parenterally primed Th17 cells acquired airways tropism following a nasal vaccine boost, which facilitated IgA⁺ B cells in the lung (31). In addition, we showed that intramuscular prime followed by nasal boost caused accelerated C.t. clearance in minipigs correlating with a SIgA response in the GT (26).

The overall aim of this study was to evaluate whether an optimized multivisit parenteral prime and nasal boost could be turned into a two-visit strategy by combining the subcutaneous (SC) and IN immunization route simultaneously (SIM) and thereby develop a robust immunization protocol favoring Th1/Th17 and IgA responses in the GT. An obvious advantage of this strategy is a reduction in immunization visits, which would reduce the risk of missed doses in the immunization schedule. We used a vaccine consisting of recombinant C.t. proteins known to elicit Chlamydia-specific T cell responses and neutralizing Abs (3, 52). The Ags were coadministered with CAF01, an adjuvant known to shape the immune response toward Th1 and Th17 cells independent of the choice of Ag (53–56). We show that the SIM immunization strategy generates strong peripheral Th1/Th17 responses with increased Th1/Th17 responses in the airways, which disseminate to the GT although in low numbers. SIM also generates systemic Ab responses and, in contrast to parenteral vaccination alone, promotes a significant IgA response in the GT following vaginal C.t. challenge.

A combined mucosal and systemic immunization strategy induces both Chlamydia-specific T cells and mucosal IgA responses.

The aim was to investigate whether it was possible to elicit Chlamydia-specific T cell and Ab (IgA) responses in the GT by administering a Chlamydia vaccine simultaneously, by the SC and IN routes. To start with, it was evaluated how this immunization strategy would affect both the peripheral Th1/Th17 and mucosal IgA responses compared to separate SC followed by IN immunization. Recently, we showed that the latter immunization strategy resulted in high peripheral Th17 responses, which following nasal boost were pulled to the lungs and facilitated induction of mucosal IgA (31). Mice were vaccinated three times with 2 weeks intervals using both routes simultaneously or three SC immunizations followed by two IN boosts, referred to as the PB protocol. A SC-only immunized group was included as control. Five weeks post last immunization, splenic T cells were restimulated in vitro with vaccine Ags to measure Ag-specific IFNγ or IL-17 responses using FACS (CD4⁺CD44^high) (Figures 1A,B). Overall, we observed similar peripheral T cell responses in SIM-, PB-, and SC-immunized mice. There was a tendency toward higher responses in the PB group, although not statistically significant. Equal levels of Ag-specific IgG1 were measured in serum and lungs of all the immunized groups (Figures 1C,D), while significantly higher Ag-specific IgA responses were induced in the SIM- and PB-immunized mice compared to the SC immunized (Figures 1E,F), confirming that mucosal immunization favors the generation of IgA Abs. Thus, SIM immunization mediated comparable T cell and IgA responses to the PB immunization strategy.

It is becoming increasingly clear that combinations of different delivery routes for priming and boosting represent vaccination strategies that can modulate not only localization and magnitude but also the type of immune responses elicited including long lasting memory responses (31, 40, 50, 51). In the current study, we demonstrate that similar to the PB immunization protocol, simultaneous use of the SC and IN immunization routes elicits high T cell and IgA/IgG1 responses in serum and the lung, which disseminates further to the GT. This was in contrast to SC immunization, which results in strong IgG1 but not IgA responses. For IN vaccination, we only found minor mucosal and peripheral IgG1 and IgA responses, which is consistent with previous findings using CAF01 as adjuvant (31, 50), suggesting that CAF01 is not an optimal mucosal adjuvant. Consequently, the high IgA response found in SIM-immunized mice most likely is a result of the strong peripheral T cell response being pulled into the airways. Here, these cognate T cells can participate in linked interactions with Ag-specific B and induce IgA responses (15, 17). The presence of Chlamydia-specific Abs in the GT following SIM immunization is highly relevant with regard to the ongoing debate on the impact of Abs toward C.t. infection (3, 21, 62). We previously demonstrated that Hirep1 adjuvanted with CAF01 resulted in neutralizing Abs (3, 52), which play a critical role in controlling infection. Thus, adoptive transfer of serum Abs facilitated significant early control of the infection (3). In support of this, we found that parenteral priming of minipigs followed by IN boosting using CAF01-adjuvanted Hirep1/CTH93 elicited vaginal SIgA, which accelerated C.t. clearance compared to parenteral immunization (26). However, the relative role of vaginal IgA involved in protection against C.t. is unclear and must be balanced against the contribution of neutralizing IgG. This is exemplified by a parallel study in minipigs that were primed intramuscularly without mucosal boost, which suggested protection even without IgA, if the IgG titers are sufficiently high (33). Because of that, characterizing the vaginal IgA responses generated by the SIM immunization strategy was of particular interest. We found that following vaccination, B220⁺ B cells and IgA⁺ memory B cells localized in the lung parenchyma and importantly also spread to the GT. It should be noted that these data are limited in power since we had to pool samples from multiple mice to obtain enough cells for analysis. However, this finding is in line with several previous studies describing a close link between GT and the upper airways (3, 26, 31, 38, 39, 48, 49). Following infection we measured vaccine-specific IgA-secreting cells in the GT 7 days p.i. in the SIM-vaccinated group. Whether these local IgA plasma cells originate from lung-derived memory B cells that migrate immediately after nasal immunization or from systemic memory pool is subject to further work. The SIM vaccination strategy induces slightly stronger or similar peripheral Th17 and Th1 responses compared to SC immunization. However, important differences are found in the selective homing of T cells to the lungs tissue in the SIM-vaccinated animals, which is in agreement with a recent study of Ciabattini et al. They found that parenteral priming elicits high peripheral T cell responses, which respond strongly to nasal CAF01 boosting both in the periphery and mucosa (50). They also found that nasal T cell priming is poorly responsive to parenteral boosting (50). In parallel, they showed that the route used for boosting has great impact on the local effector responses, i.e., nasal priming followed by nasal boosting, but not parenteral boosting, enhances the IgA response (40, 50). It was, therefore, of most interest to find that simultaneous use of the parenteral and mucosal routes directed strong systemic and mucosal T cell and IgA responses.

To our surprise, we found that SC- and SIM-vaccinated mice were equally well protected. There can be several reasons for that. As outlined above, the impact of IgA in the GT might be masked by the contribution of high tittered neutralizing IgG responses. In support of this, we found similar levels of in vitro neutralizing capacity in sera from SIM- and SC-immunized mice (3). The very modest numbers of tissues resident T cells following immunization in SIM vs. SC groups are probably outnumbered by the strong peripheral T cell responses induced by both SC and SIM immunization, which is rapidly recruited (day 2–3 p.i.) into the GT following C.t. infection. Nonetheless, the SIM protocol might have a more pronounced impact if you wait longer after vaccination, when peripheral Ab responses have diminished. By that time, the presence of local plasma and memory B cells may play a critical role in controlling C.t. infections. Furthermore, in circumstances where C.t. would ascend to the upper GT with the risk of causing pathology, IgA might be particularly relevant as IgA was found in higher amount in the human cervical mucus (upper GT) compared to the cervicovaginal mucus (lower GT), and thus might be essential in preventing bacterial spreading to the fallopian tubes (22). Likewise, when peripheral T cell responses has diminished longer after immunization, even low TRM responses might be relevant as a first wave of Chlamydia-specific T cells, as demonstrated by Stary et al. (36). In conclusion, we identify a novel vaccination strategy for Chlamydia vaccines. Simultaneous parenteral and nasal immunizations protect mice from C.t. infection. SIM induces strong circulating and mucosal Chlamydia-specific Th1/Th17 cell and Ab responses and, in contrast to systemic PB, includes a significant IgA response in the GT following vaginal challenge. This strategy is important for future vaccine schemes as heterologous immunizations given simultaneously will reduce the number of immunization visits, which is critical for vaccine compliance and, therefore, overall efficacy at the population level.

Animal experiments were conducted in accordance with national Danish guidelines (Amendment #1306 of November 23, 2007) for animal experiments as approved by the Danish Animal Experiments Inspectorate, Ministry of Justice and in accordance with EU Directive 2010/63EU for animal experiments. All techniques/procedures were refined to provide maximum comfort and minimal stress to the animals. Experiments performed at Statens Serum Institute have been approved by the governmental Animal Experiments Inspectorate under licenses 2013-15-2934-00978.

JW planned the study, performed the experiments, and made the laboratory analysis, statistics, interpreted data, and drafted the figures and manuscript. FF and PA planned the study, interpreted data, and revised figures and the manuscript. MS planned the study and performed some experiments and laboratory analysis. AO revised the figures and manuscript. All the authors approved the final manuscript.

PA, AO, and FF are coinventors on a patent application relating to Chlamydia trachomatis vaccines. All rights have been assigned to Statens Serum Institute, a Danish not-for-profit governmental institute. The other 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.