A Novel LNP-based Chlamydia subunit vaccine formulation that induces Th1 responses without upregulating IL-17 provides equivalent protection in mice as formulations that induced IL-17 and Th1 cytokines

Sexually transmitted Chlamydia infections can lead to the development of debilitating diseases such as chronic pelvic pain, ectopic pregnancy, pelvic inflammatory disease, and infertility. It has been proposed that immunity against Chlamydia infection and disease may be determined by a balance of IL-17 signaling. We sought to evaluate novel formulations for a candidate Chlamydia vaccine, consisting of Chlamydia major outer membrane protein (MOMP) alone or in combination with polymorphic membrane protein D (PmpD) and polymorphic membrane protein G (PmpG) as target immunogens. Native MOMP (nMOMP) isolated from C. muridarum elementary bodies (EBs) and recombinant PmpD and PmpG proteins were adjuvanted with Monophosphoryl lipid A (MPLA), in one of two formulations containing either lipid nanoparticles (LNPs) or the cationic lipid dimethyldioctadecylammonium bromide (DDA). Antibody titers to C. muridarum, nMOMP, and EBs were evaluated by ELISA, and T-cell responses by intracellular cytokine staining (ICS). Protection from challenge was determined by qPCR and gross pathology. All mice immunized with the new vaccine formulations showed significantly higher antibody titers to nMOMP (P<0.001) and C. muridarum EBs (P<0.001), when compared to the negative control group (adjuvant alone). Antibody titers in vaccine groups with Monophosphoryl lipid A (MPLA)+LNP were higher as compared to the MPLA+DDA group (P<0.001) except for groups 6 (Cm nMOMP+PmpG+PmpD p73+PmpD p82+MPLA+DDA) vs 7 (Cm nMOMP+PmpG+PmpD p73+PmpD p82+MPLA+LNP) for both C. muridarum EBs and PmpG; the groups were not statistically significant. ICS analysis showed more robust CD4+ T-cell responses (IFN-γ/IL-2/TNF-α) in the dimethyldioctadecylammonium digestion

control groups (9)(10)(11)(12). The Pmp family of proteins are good T-cell antigens and potential targets for a T-cell focused vaccine (9,11,13). Native MOMP has been shown to elicit a strong humoral immune response (14). A vaccine that includes antigens that provide both a T-cell and antibody response against Chlamydia may provide optimal protection against both initial and repeat infection than a vaccine profile that only elicits a Th1 or Th2 responses (11). IL-17 mediated immunity has been shown to play a role in the control of intracellular pathogens, including Chlamydia (10,15) However, there is also evidence for a pathogenic role through enhanced recruitment of polymorphonuclear neutrophils and inflammatory monocytes in the UGT. The Th17-mediated inflammation has been associated with tissue destruction, intra-abdominal adhesions and increases in splenic weights in the mouse model (15)(16)(17). We tested the role of Th1 and/or Th17 T-cells in affording protection from Chlamydia infection and their ability to cause pathology in the upper genital tract utilizing two vaccine formulations.
We have evaluated numerous conserved Chlamydia antigen targets (data not shown) with minimal protection or reduction in bacterial burden observed following immunization and intravaginal challenge from most targets. Interestingly, we have observed protection with vaccine formulations including nMOMP similar to what has been published in the literature (6)(7)(8)18). Since MOMP is a good humoral antigen, we also looked for antigens that would elicit cell-mediated immune responses. Immunoproteomic vaccine studies found that four polymorphic membrane proteins (PmpE, PmpF, PmpG and PmpH) are immunodominant, recognized by various MHC class II haplotypes and are protective in mouse models (9)(10)(11). PmpG/F have been shown to be the most immunodominant of the Pmps (11). Therefore, based on protection with nMOMP observed in our laboratory; and publications citing protective immunity for both MOMP and polymorphic membrane proteins, with PmpG/F being most immunodominate of the pmps, we focused on nMOMP, PmpG, PmpD passenger domain (PmpD-p73) and PmpD translocator domain (PmpD-p82) as candidate vaccine targets in the current study. Additionally, various adjuvants were included to drive the cell mediated immune response toward Th1, and/or Th17 and the role of T-helper population in conferring protection was evaluated. To determine the role of Th17 mediated immunity, vaccine formulations in some test groups were adjuvanted with the cationic lipid Immunized groups also showed protection from challenge with C. muridarum, as evidenced by a reduction in bacterial shedding from the vagina for all groups (P<0.003) compared to shedding from the adjuvant control, Group 1. Taken together, the data suggest that the vaccines formulated with either MPLA+LNP or MPLA+DDA elicited strong immunological responses and both were protective by reducing bacterial shedding after challenge.
Furthermore, equal protection is achieved with or without a Th17 response.

Ethics statement
All animal experiments were approved by the Merck & Co., Inc., Kenilworth, NJ, USA, Institutional Animal Care and Use Committee (IACUC). All procedures were performed in accordance with our institution's IACUC guidelines in strict accordance with the recommendations in the Guide for Care and Use of Laboratory Animals of the National Institutes of Health. Mice were housed in large mouse containers (n = 10 mice/box and n = 5 mice/box for ICS) with micro isolator lids, and the rooms were maintained with controlled humidity and temperature, and 12-hour light-dark cycles. All containers had nestlets and animals were provided standard chow (Purina 5001 rodent diet) and water ad libitum. The physical condition of the animals was monitored daily (7 days a week), and any health changes were noted. At the end of the study, animals were euthanized using CO2 inhalation (10% -30% CO2 in the air mixture inhaled per minute). No animals were found to be moribund or to have any adverse advents throughout the study.
Cell culture and propagation of Chlamydia muridarum C. muridarum was obtained from Bernard Arulanandam (UTSA San Antonio, Texas). HeLa 229 cells were used for propagation of C. muridarum. HeLa 229 cells were grown in Eagle's Minimal Essential Medium (EMEM; ATCC, Manassas, VA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT), 50 g/ml vancomycin (Sigma, St. Louis, MO), and 10 g/ml gentamicin (Gibco; Carlsbad, CA). Host cells were seeded into tissue culture flasks at a cell density of 5 x 10 5 cells/ml and incubated overnight at 37°C in 5% CO 2 to achieve a confluent monolayer. Cell monolayers were infected with a Chlamydia stock diluted in sucrose-phosphate-glutamate (SPG) buffer. The C. muridarum strain was cultured for 48 hours. The Chlamydiae were harvested from the infected cells and purified by centrifugation through 30% Renograffin (Bracco Diagnostics, Princeton, NJ) and stored frozen at -80°C.

Amino Acid Analysis
Vapor phase hydrolysis was performed with constant boiling 6 N HCl at 150 °C for 1 hour using an ELDEX (Napa, CA) H/D workstation. Protein hydrolysates and amino acid standards were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) using the Waters AccQ Tag Ultra derivatization kit according to the manufacturer's protocol. AQC reacts with primary and secondary amines to form stable fluorescent derivatives that can be separated by RP-HPLC. The fluorescent AQC derivatives were separated on an AccQ Tag Ultra C18, 1.7 µm, 2.1 x 100 mm column using a modified gradient program.

Native MOMP purification from infected cells (HeLa)
Native MOMP (nMOMP) was purified by extraction using sonication of intact Chlamydia-

Mice
Female C57BL/6 mice were purchased from Taconic Farms (Hudson, NY). At the time of study start, mice were 6-7 weeks of age, with food and water provided ad libitum. Animals were randomly assigned to cages upon receipt from the vendor. All animal experiments in these studies were approved by the Institutional Animal Care and Use Committee (IACUC), our institution's IACUC guidelines in strict accordance with the recommendations in the Guide for Care and Use of Laboratory Animals of the National Institutes of Health. Animals were treated and assessed in numerical order groups 1-8.

Murine model and specimen collection
Study design is shown in Figure 1. Fifteen animals per group (10 for challenge and 5 for ICS) were immunized by subcutaneous (s.c.) route with the following combinations of adjuvants and antigens as seen in Table 1  At spleen harvest or at the end of the study, animals were euthanized using CO2 inhalation (10% -30% CO2 in the air mixture inhaled per minute). No animals were found to be moribund throughout the study. Gross pathology was assessed on day 118. to the adjuvant only control group, significant T-cell responses (IFN-γ and TNF-) in EB exposed mice were only detected towards MOMP and PmpG ( Figure 2). Additionally, these responses were much lower in magnitude as compared with the subcutaneous protein vaccinated groups.
As expected, potent IL-17 responses were detected from mice in groups that were vaccinated with DDA containing formulations ( Figure 2D), and such responses were not induced by the LNP formulations or C. muridarum EB exposure. Additionally, IL-2 cytokine production trended higher in the groups which received DDA ( Figure 2C).
As polyfunctional T-cell responses have been shown to correlate with protection in many infectious disease models, we evaluated the percent of total responses in each group which produced all four cytokines (IFN-γ/IL-2/TNF-/IL-17), any combination of the three, two or any one cytokine ( Figure 2). Primarily due to significantly increased IL-17 and IL-2 production in the DDA adjuvanted vaccine groups ( Figure 2C and 2D), a higher percentage of polyfunctional T-cells were detected in the DDA adjuvanted vaccine groups as compared to the LNP or EB exposed groups ( Figure 2E).

Vaccine immunizations result in high ELISA titers to nMOMP, C. muridarum EBs, PmpG and
PmpD Animals were immunized with combinations of recombinant and native extracted proteins as per the scheme depicted in Table 1. An adjuvant control group with MPLA, DDA and LNP was used as a comparator against all test vaccine groups (10 animals per group). Each of the following combinations of antigens were formulated with MPLA+DDA or MPLA+LNP: nMOMP; nMOMP + PmpG; and nMOMP + PmpG + PmpD-p73 + PmpD-p82 (Table 1) High systemic antibody titers were seen overall in this study. The LNP+MPLA adjuvanted groups had higher antibody titers when compared to MPLA+DDA adjuvanted group.
Interestingly higher titers of antibodies in the LNP+MPLA groups did not translate to better clearance of Chlamydia following the intravaginal challenge. There can be several potential explanations for this observation, including higher amplitude of serum antibody titer may not reflect the antibody titers in the mucosal compartment thus not translating into improved protection from vaginal challenge. Furthermore, the functionality of the antibodies raised could be different for the two groups of adjuvanted formulations. We plan to evaluate functional mucosal responses in future studies.
In these studies, we observed a reduction in overall bacterial shedding in vaccine test groups that were statistically better than the adjuvant control group. We observed that an Future studies could include for academic purposes, intranasal immunization with live EBs to test whether this immunization route would be more resistant to genital tract pathology consistent with the literature.
Our nMOMP immunized groups conferred protection from challenge similar to previous publications (6)(7)(8)18). For those groups combining MOMP and either PmpD or PmpG or a combination of both PmpD and PmpG (PmpDG), we did not see an increased reduction of bacterial shedding compared to nMOMP alone after intravaginal challenge. This data was in contrast to previous reports that showed the combination of Pmp E, F, G, H + MOMP reduced shedding over MOMP alone, PmpG alone, or the combination of Pmps (11).
However, it should be noted that there are marked differences between the studies performed herein and those reported previously. Differences include the combination of Pmps, protein purification methods, study designs (antigen dose, adjuvants), assessment following challenge (sample collection, Chlamydia shedding assay), all of which may theoretically result in the lack of increased protection for the combination vaccine image.
The Pmps in our study were selected based on recombinant protein expression and availability; a future study would need to include additional recombinantly expressed Pmps such as E, F, and H. In addition to including more Pmps in a future study, a stricter alignment using the same protein purification methods, the use of same adjuvants, and assessment following challenge is warranted for a closer comparison between the data sets. Further, a direct comparison of the reagents and antigens generated from the previous studies with our reagents and vaccine combinations in a side by side comparison would be beneficial to elucidate why the results were not similar.

Conclusions
In conclusion, we found that Chlamydia vaccines based on nMOMP provided modest protection against intravaginal challenge with Chlamydia muridarum. The efficacy of these vaccines could not be improved either by the incorporation of additional Pmp antigens, or by formulating the nMOMP antigen with adjuvants that either increased circulating anti-nMOMP IgG, or by eliciting a Th17+ response to nMOMP. An efficacious vaccine will require finding the optimal combination of antigens with several targets included and an adjuvant/immunization route combination that results in multifunctional CD4+ T cells and humoral responses elicited systemically and in the mucosal compartments.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
All authors are employee of Merck & Co., Inc., West Point, PA, USA

Ethics Approval and Consent to Participate
All animal experiments were approved by the Merck & Co., Inc., Kenilworth, NJ, USA, Institutional Animal Care and Use Committee (IACUC). All procedures were performed in accordance with our institution's IACUC guidelines in strict accordance with the recommendations in the Guide for Care and Use of Laboratory Animals of the National Institutes of Health. Mice were housed in large mouse containers (n = 10 mice/box) with micro isolator lids, and the rooms were maintained with controlled humidity and temperature, and 12-hour light-dark cycles. All containers had nestlets and animals were provided standard chow (Purina 5001 rodent diet) and water ad libitum. The physical condition of the animals was monitored daily (7 days a week), and any health changes were noted.
At the end of the study, animals were euthanized using CO2 inhalation (10% -30% CO2 in the air mixture inhaled per minute). No animals were found to be moribund throughout the study.

Authors' Contribution
MB is the primary author, and performed recombinant protein cloning and expression, and  3C. Total IgG titers to C. muridarum EBs 3D. Total IgG titers to PmpD p73 3E.