Intranasal vaccination with a Chimeric Chlamydial Antigen BD584 confers protection against Chlamydia trachomatis genital tract infection

Chlamydia is the most common sexually transmitted infection of bacterial origin, and a broadly protective vaccine is urgently needed given the largely asymptomatic nature of the infection and the severe reproductive sequelae in women with untreated infections. The primary aim of this study was to characterize the immune response to vaccination with BD584, a novel recombinant antigen consisting of three type III secretion (T3S) proteins, and to evaluate its effi cacy against a Chlamydia trachomatis challenge in three strains of mice. C57BL/6, BALB/c, and C3H/HeN mice were vaccinated three times intranasally with BD584 and CpG adjuvant, then challenged intravaginally with C. trachomatis. BD584/CpG vaccination induced strong cell-mediated and humoral immune responses in all three strains of mice, suggesting that this vaccine should be immunogenic in a genetically heterogenous population. BD584/CpG vaccination reduced vaginal shedding of C. trachomatis in C57BL/6 and C3H/HeN mice. Together, these results strengthen the rationale for further investigating the use of T3S proteins in a C. trachomatis vaccine. Research Article Intranasal vaccination with a Chimeric Chlamydial Antigen BD584 confers protection against Chlamydia trachomatis genital tract infection Steven Liang1 and James B Mahony1,2* 1The Research Institute of St. Joe’s Hamilton, St. Joseph’s Healthcare Hamilton, Hamilton, Ontario, Canada 2Department of Pathology and Molecular Medicine, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada Received: 06 July, 2020 Accepted: 13 July, 2020 Published: 14 July, 2020 *Corresponding author: James B Mahony, PhD, FCCM, FAAM, Professor Emeritus, Department of Pathology & Molecular Medicine, McMaster University, St. Joseph’s Healthcare Hamilton, 50 Charlton Ave. E., Hamilton, ON L8N 4A6, Canada, Tel: (905) 522-1155 ext 35013; Email: ORCID: https://orcid.org/0000-0002-8916-9819 https://www.peertechz.com


Introduction
Chlamydia trachomatis infections are the most prevalent sexually transmitted bacterial infections in the world. The World Health Organization estimates that 131 million new cases of C. trachomatis infection occur each year [1]. Up to 90% of women and 50% of men with these infections are asymptomatic [2] and consequently do not seek testing and treatment. Untreated infections in women can lead to many complications including Pelvic Infl ammatory Disease (PID), tubal factor infertility, and ectopic pregnancy [3]. Public health programs, including screening, partner identifi cation, and treatment, have contributed to improve infection control, but to curb the infection rates, there is a need for the development of an effective C. trachomatis vaccine [4] as a necessary next step to control this public health problem.
Almost immediately following the isolation of the causative agent of trachoma, human trials were initiated using vaccines based on whole Chlamydiae [5]. While these studies demonstrated that some protection can be achieved using whole-cell vaccines, protective effi cacy appeared to be serovar-specifi c, which, together with concerns about potentially detrimental antigens in intact bacteria, highlighted the need for a broadly protective subunit vaccine. Although fi ve decades of vaccine research efforts have led to the discovery of a large number of potential vaccine antigens [6,7], only one such vaccine has reached phase I clinical trials [8]. Therefore, sustained efforts are needed to identify and evaluate promising antigens for testing in humans.

BLASTp
Non-redundant protein sequences were searched with BLASTp using CopB, CopD, CT584, or MOMP from C. trachomatis serovar D (UW-3/Cx) as query sequences. Using the queries as references, the number of non-identical residues in each identifi ed C. trachomatis sequence was calculated, tabulated, and represented as a frequency of non-identical amino acids.
Cloning of BD584, which consists of the fi rst 100 amino acids of CT578 (CopB) and CT579 (CopD) and full length CT584, has been described previously [14]. Briefl y, PCR products containing the fi rst 300 nucleotides of copB, copD, and full length ct584 were generated from genomic DNA from C. trachomatis (UW-3/Cx) using primers with appropriate restriction sites and cloned into the multiple cloning site 1 of pETDuet-1 to generate pETDuet-1::BD584. All constructs included a His-tag for protein purifi cation via affi nity chromatography.

Vaccination and challenge
C57BL/6, BALB/c, and C3H/HeN female mice were manipulate host cell biology to ensure successful invasion.
Surface exposed T3S proteins are therefore attractive vaccine candidates as they are essential for infection and amenable to neutralization by antibodies. While they have been successfully used in the development of vaccines against other pathogenic bacteria [9][10][11], only within the last few years have they been tested as vaccine antigens against Chlamydia. Recently, Koroleva et al. reported that immunization with the T3S needle protein (TC_0037), an ortholog of C. trachomatis CdsF, protected mice against C. muridarum infection and the associated pathology [12]. A multistage vaccine containing proteins associated with the T3S system (CdsF and CT584), inclusion membrane (IncA and CT229), and outer membrane (MOMP and PmpG) conferred greater protection against infection and pathology than vaccines targeting outer membrane proteins alone [13]. Previously, our group has shown that C57BL/6 mice immunized with BD584, a fusion protein consisting of highly conserved C. trachomatis T3S components CopB, CopD, and CT584, were protected against infection and upper genital tract (UGT) pathology upon challenge with the mouse pathogen C. muridarum [14]. However, given that signifi cant differences exist between C. muridarum and C. trachomatis infections [15,16], vaccine-mediated protection against C. muridarum does not imply protection against C. trachomatis, and it is important to determine the effectiveness of BD584 vaccination against the human pathogen. Furthermore, a more detailed analysis of the cell-mediated and humoral immune response to BD584 vaccination will enhance our understanding of BD584mediated protective immunity.
Most Chlamydia vaccine studies utilizing the murine model have been conducted using a single inbred mouse strain, which is not ideal given the genetic heterogeneity of humans, suggesting the need for vaccine studies using animals with different genetic backgrounds. Moreover, use of animals with genetically determined differences in immune predispositions can also provide insight into host factors that contribute to effective anti-chlamydial immunity. In this study, we used C57BL/6 mice (Th1-polarized; Chlamydia-resistant), C3H/HeN mice (Th1-polarized; Chlamydia-susceptible), and BALB/c mice (Th2-polarized; Chlamydia-susceptible) to model human genetic heterogeneity and to identify potential correlates of BD584-mediated protection [17,18]. We show here that intranasal immunization with BD584 elicits robust cell-mediated and humoral immune responses in addition to strong mucosal immunity in all three strains of mice and that BD584 immunization confers protection against C. trachomatis infection in a strain dependent way.

Ethics statement
This study was carried out in accordance with the Canadian

Collection of vaginal washes and blood
Seven days after the fi nal immunization, vaginal washes were collected for antibody analysis for fi ve consecutive days by pipetting twice with 30 μl of PBS in and out of the vagina to give a total of 60 μl per mouse per day and stored at -70°C.
Blood (submandibular) was collected 14 days after the fi nal immunization, allowed to clot at room temperature for 30 minutes, centrifuged at 1,000 x g for 10 minutes at 4°C, and serum was removed and stored at -70°C.

Antibody ELISA
Antigen-specifi c antibodies were quantifi ed by Enzyme-

Splenocyte recall assay
To determine cell-mediated immune responses, another

Statistical analysis
GraphPad Prism 6 was used for statistical analysis.
Differences were assessed using Student's t test for parametric data and Mann-Whitney test for nonparametric data, one-way ANOVA when comparing more than two groups, or two-way ANOVA when comparing more than one independent variable.

CopB, CopD, and CT584 are highly conserved among C. trachomatis serovars
A C. trachomatis vaccine should be effective against all C.
trachomatis serovars, and one way to achieve this would be to include antigens that are highly conserved. Therefore, we began our investigation by comparing the degree of amino acid variability in CopB, CopD, and CT584, using MOMP as a reference antigen. As shown in Figure 1, alignment of known sequences of each protein showed that CopB, CopD, and CT584 have, on average, 0.46, 0.55, and 0.02 non-identical amino acids per 100 residues, respectively, which are signifi cantly fewer compared to MOMP, which averages 4.16 non-identical amino acids per 100 residues. These results show that CopB, CopD, and CT584 are signifi cantly more conserved than MOMP in C. trachomatis, which is an advantage for subunit vaccine for a pathogen with multiple serotypes.

BD584/CpG vaccination induces robust cell-mediated immune responses
Cell-mediated immunity has been reported to be essential for protection against Chlamydia [15,20]. Therefore, we examined whether vaccination with BD584 and CpG induces antigen-specifi c cell-mediated immune responses. C57BL/6, BALB/c, and C3H/HeN mice were intranasally vaccinated with BD584/CpG or PBS, and BD584-induced production of cytokines from splenoctyes was analyzed 7 days later.
Antigen-specifi c splenocyte production of IFN- and IL-17 in all three strains of mice indicates that a mixed Th1 and

BD584/CpG vaccination elicits robust systemic and mucosal humoral immune responses
While cell-mediated immunity is essential for protection against C. trachomatis, accumulating evidence suggests that a C. trachomatis vaccine should also generate strong humoral immunity. Therefore, C57BL/6, BALB/c, and C3H/HeN mice were intranasally vaccinated three times, three weeks apart, and antibody responses were assessed two weeks after the fi nal Non-redundant protein sequences were searched with BLASTp using CopB, CopD, CT584, or MOMP from C. trachomatis urogenital isolates serovar D (UW-3/Cx) as query sequences. The degree of amino acid variability of identifi ed C. trachomatis sequences were tabulated and presented as the mean frequency of non-identical residues (per 100 amino acids) with SD depicted. Amino acid variability in CopB, CopD, and CT584 was compared to that in MOMP (reference antigen) using oneway ANOVA followed by Dunn's multiple comparison test. ( Figure 4). Taken together, these results show that BD584/CpG is highly immunogenic in all three strains of mice, and that the subtype of antibodies associated with antigen-specifi c Th1 and Th2 responses is at least partially dependent on the animal's genetic background.

BD584/CpG immunization reduces bacterial shedding upon intravaginal C. trachomatis challenge
Next, we evaluated the ability of BD584/CpG immunization to protect against C. trachomatis infection as measured by reduction in bacterial shedding following intravaginal challenge. As shown in Figures 5A and 5B, C57BL/6 mice immunized with BD584/CpG showed a nearly 100-fold reduction of C. trachomatis shedding on days 7 and 10 post challenge compared to the PBS group. Shedding in the unvaccinated group had ceased on day 10 and no difference was seen in bacterial shedding between the two groups after day 14. Reduction in shedding was seen as early as day 3 post challenge, although the difference at this time point was not statistically signifi cant. Compared to C57BL/6 mice, C3H/HeN mice immunized with BD584/CpG exhibited less reduction in shedding. BD584/CpG immunized C3H/HeN mice shed 4.7-and 1.9-fold less C. trachomatis on day 3 and day 7, respectively, compared to the PBS group. An area under the curve (AUC) analysis, which allows for a simultaneous comparison of both the magnitude and duration of infection, showed that BD584/CpG immunization reduced the overall bacterial load by 96% in C57BL/6 mice and 75% in C3H/HeN mice ( Figure 5C). Interestingly, no statistically signifi cant reduction in shedding was observed at any point for BALB/c mice. Overall, these results demonstrate that BD584/ CpG vaccination reduces C. trachomatis shedding upon challenge in C57BL/6 and C3H/HeN mice.

Discussion
In this study, we show that vaccination with the conserved Previously, we have shown that intranasal immunization with C. trachomatis BD584/CpG reduces shedding and incidence of hydrosalpinx upon intravaginal challenge with C. muridarum [14]. Given that BD584 was cloned from the C. trachomatis genome, its ability to afford cross-species protection speaks to its conserved nature and its potential as a broadly protective immunogen for all 18 C. trachomatis serovars. The amino acid identity of BD584 between CT and Cmu is 57% while the identify between the ocular, genital and LGV strains of C. trachomatis is 92%. We have shown recently that antibodies raised against Thus, while BD584/CpG vaccination appears to have no effect on the duration of infection, it can reduce the magnitude of infection, which has the potential to reduce the incidence of disease by limiting transmission of infections. Ideally, however, a vaccine would confer sterilizing immunity, but this is diffi cult to evaluate in the C. trachomatis mouse model since reliable infection requires the use of a high inoculum that could overwhelm an otherwise sterilizing immune response [16]. In a previous study, we showed that BD584 vaccination reduced shedding of C. muridarum assessed by quantitative PCR, which does not necessarily imply a reduction in the shedding of live organisms [14]. In the current study, we show that BD584 vaccination reduced shedding of infectious C. trachomatis, which was quantifi ed by culture, thus providing stronger evidence for vaccine effi cacy.
BD584 vaccination induced robust cell-mediated and humoral immune responses in all three strains of mice, suggesting that the antigen should be immunogenic in a genetically heterogenous population. Splenocytes from BD584/CpG immunized mice exhibited increased IFN- and IL-17 production with no detectable IL-4 production following in vitro BD584 stimulation, suggesting that a mixed Th1 and Th17 response was generated by vaccination. This was expected given that BD584 was delivered intranasally, a route that preferentially generates Th17 responses [21], with a Th1 polarizing adjuvant, CpG [22]. Vaccinated C57BL/6 mice had signifi cantly lower levels of BD584-specifi c IgG and IgA compared to vaccinated BALB/c and C3H/HeN mice, but exhibited the highest level of protection against shedding, suggesting that an increase in the magnitude of BD584specifi c humoral responses, at least above a certain level, may not necessarily improve protection against infection. Instead, genetically determined differences in innate and adaptive immunity likely accounted for the difference in vaccine effi cacy between the three strains. Increased susceptibility to Chlamydia infection in C3H/HeN mice, compared to C57BL/6 mice, may have masked the protection afforded by BD584/ CpG vaccination [18]. However, it is unclear why BALB/c mice were not signifi cantly protected by vaccination. Compared to vaccinated BALB/c mice, vaccinated C57BL/6 and C3H/HeN mice had a signifi cantly higher BD584-specifi c IgG2a/c to IgG1 ratio, suggesting a more Th1 polarized response. However, whether higher IgG2a/c directly enhances protection via Fcdependent mechanisms [23,24], or is merely a surrogate marker for a protective Th1 response [25], or is associated with an unknown mechanism of protection, remains unknown. A high IgG2a/Ig1 ratio may also be functionally similar to a high IgG/IgA ratio; in both cases, the higher ratio may refl ect a greater engagement with Fc-dependent effector functions. We did not evaluate pathology in this study. In a previous study using C. muridarum we showed that UGT pathology viz. hydrosalpinx was reduced by 85% after vaccination with BD584. Importantly, an immune-correlates analysis of an HIV vaccine clinical trial found that higher antigen-specifi c IgG and lower antigen-specifi c IgA correlated with protection [26]. The authors suggested that IgA interfered with protective IgG effector functions. Interestingly, in the current study, we also found that the two strains of mice protected by BD584 vaccination exhibited higher BD584-specifi c IgG/IgA ratios than the unprotected strain. However, conclusive evidence for the role of specifi c antibody isotypes and subtypes will need to come from mechanistic studies, which are beyond the scope of the current study. Experiments are underway to better understand the mechanisms of BD584-mediated protection. Given t hat different strains of Chlamydia infect different animals (mice and humans) and different immune mechanisms may be required to clear chlamydial infections in different animals, the protection provided by BD584 should be evaluated additional animal models. Although cell-mediated responses obtained in this study appeared to be good, and this is important in clearing an intracellular pathogen infection, the duration of immune protection could not be evaluated in this study, because C. trachomatis infection doesn't persist in non-vaccinated mice. The use of progesterone treated animals may help elucidate the question of duration.