Cost of postoperative complications after general surgery at a major Canadian academic centre.

BACKGROUND: In a fiscally constrained health care environment, the need to reduce unnecessary spending is paramount. Postoperative complications contribute to hospital costs and utilization of health care resources. OBJECTIVE: The purpose of this observational study was to identify the cost associated with complications of common general surgery procedures performed at a major academic hospital in Toronto, Ontario. METHODS: The institutional National Surgical Quality Improvement Program database was used to identify complications in patients who underwent general surgical procedures at our institution from April 2015 to February 2018. A mix of elective and emergent cases was included: bariatric surgery, laparoscopic appendectomy, laparoscopic cholecystectomy, thyroidectomy, right hemicolectomy and ventral incisional hernia repair. The total cost for each visit was calculated by adding all the aggregate costs of inpatient care. Median total costs and the breakdown of cost components were compared in cases with and without complications. RESULTS: A total of 2713 patients were included. Nearly 6% of patients experienced at least one complication, with an incidence ranging from 1.1% after bariatric surgery to 23.8% after right hemicolectomy. The most common type of complication varied by procedure. Median total costs were significantly higher in cases with complications, with a net increase ranging from $2989 CAD (35% increase) after bariatric surgery to $10 459 CAD (161% increase) after ventral incisional hernia repair. CONCLUSION: Postoperative complications after both elective and emergent general surgery procedures add substantially to hospital costs. Quality improvement initiatives targeted at decreasing postoperative complications could significantly reduce costs in addition to improving patient outcomes.

A Recombinant Chlamydia trachomatis MOMP Vaccine Elicits Cross-serogroup Protection in Mice Against Vaginal Shedding and Infertility.

BACKGROUND: Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Here, we determined the ability of a C. trachomatis recombinant major outer membrane protein (rMOMP) vaccine to elicit cross-serogroup protection. METHODS: Female C3H/HeN mice were vaccinated by mucosal and systemic routes with C. trachomatis serovar D (UW-3/Cx) rMOMP and challenged in the ovarian bursa with serovars D (UW-3/Cx), D (UCI-96/Cx), E (IOL-43), or F (N.I.1). CpG-1826 and Montanide ISA 720 were used as adjuvants. RESULTS: Immune responses following vaccination were more robust against the most closely related serovars. Following a genital challenge (as determined by number of mice with positive vaginal cultures, number of positive cultures, number of inclusion forming units recovered, and number of days with positive cultures) mice challenged with C. trachomatis serovars of the same complex were protected but not those challenged with serovar F (N.I.1) from a different subcomplex. Females were caged with male mice. Based on fertility rates, number of embryos, and hydrosalpinx formation, vaccinated mice were protected against challenges with serovars D (UW-3/Cx), D (UCI-96/Cx), and E (IOL-43) but not F (N.I.1). CONCLUSIONS: This is the first subunit vaccine shown to protect mice against infection, pathology, and infertility caused by different C. trachomatis serovars.

Characterization of the Horizontal and Vertical Sexual Transmission of Chlamydia Genital Infections in a New Mouse Model.

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide, and there is a need to control this epidemic. So far there is no established animal model in which both the horizontal and the vertical transmission of Chlamydia can be studied. To implement a horizontal sexual transmission model, male mice were inoculated in the meatus urethra with Chlamydia muridarum and they were caged with naive female mice. Urine and vaginal swab specimens were collected for culture. To study vertical transmission, newborns were euthanized and specimens were cultured. As controls, females were mated with sham-infected male mice. All C. muridarum-inoculated male mice had positive urine cultures. As determined by serology, all females caged with C. muridarum-inoculated males became infected, and 93% of them had positive vaginal swab specimen cultures. More females mated with C. muridarum-infected male mice (35%) than females mated with sham-infected male mice (0%) were infertile (P < 0.05). Also, C. muridarum-infected females delivered significantly fewer pups (3.8 ± 3.2/mouse) than control females (6.3 ± 1.6/mouse) (P < 0.05). Of the newborn mice, 32% were C. muridarum positive either in the lungs or in the intestines. Female mice housed with sham-infected males had no positive vaginal swab specimen cultures or C. muridarum-positive pups. This new mouse model of horizontal and vertical sexual transmission of Chlamydia closely parallels C. trachomatis sexual transmission in humans and may be a good model system to better understand the pathogenesis of these infections.

Transcervical Inoculation with Chlamydia trachomatis Induces Infertility in HLA-DR4 Transgenic and Wild-Type Mice.

Chlamydia trachomatis is the leading cause of infection-induced infertility in women. Attempts to control this epidemic with screening programs and antibiotic therapy have failed. Currently, a vaccine to prevent C. trachomatis infections is not available. In order to develop an animal model for evaluating vaccine antigens that can be applied to humans, we used C. trachomatis serovar D (strain UW-3/Cx) to induce infertility in mice whose major histocompatibility complex class II antigen was replaced with the human leukocyte antigen DR4 (HLA-DR4). Transcervical inoculation of medroxyprogesterone-treated HLA-DR4 transgenic mice with 5 × 105C. trachomatis D inclusion forming units (IFU) induced a significant reduction in fertility, with a mean number of embryos/mouse of 4.4 ± 1.3 compared to 7.8 ± 0.5 for the uninfected control mice (P < 0.05). A similar fertility reduction was elicited in the wild-type (WT) C57BL/6 mice (4.3 ± 1.4 embryos/mouse) compared to the levels of the WT controls (9.1 ± 0.4 embryos/mouse) (P < 0.05). Following infection, WT mice mounted more robust humoral and cellular immune responses than HLA-DR4 mice. As determined by vaginal shedding, HLA-DR4 mice were more susceptible to a transcervical C. trachomatis D infection than WT mice. To assess if HLA-DR4 transgenic and WT mice could be protected by vaccination, 104 IFU of C. trachomatis D was delivered intranasally, and mice were challenged transcervically 6 weeks later with 5 × 105 IFU of C. trachomatis D. As determined by severity and length of vaginal shedding, WT C57BL/6 and HLA-DR4 mice were significantly protected by vaccination. The advantages and limitations of the HLA-DR4 transgenic mouse model for evaluating human C. trachomatis vaccine antigens are discussed.

Imaging of effector memory T cells during a delayed-type hypersensitivity reaction and suppression by Kv1.3 channel block.

Effector memory T (Tem) cells are essential mediators of autoimmune disease and delayed-type hypersensitivity (DTH), a convenient model for two-photon imaging of Tem cell participation in an inflammatory response. Shortly (3 hr) after entry into antigen-primed ear tissue, Tem cells stably attached to antigen-bearing antigen-presenting cells (APCs). After 24 hr, enlarged Tem cells were highly motile along collagen fibers and continued to migrate rapidly for 18 hr. Tem cells rely on voltage-gated Kv1.3 potassium channels to regulate calcium signaling. ShK-186, a specific Kv1.3 blocker, inhibited DTH and suppressed Tem cell enlargement and motility in inflamed tissue but had no effect on homing to or motility in lymph nodes of naive and central memory T (Tcm) cells. ShK-186 effectively treated disease in a rat model of multiple sclerosis. These results demonstrate a requirement for Kv1.3 channels in Tem cells during an inflammatory immune response in peripheral tissues. Targeting Kv1.3 allows for effector memory responses to be suppressed while central memory responses remain intact.

Increased immunoaccessibility of MOMP epitopes in a vaccine formulated with amphipols may account for the very robust protection elicited against a vaginal challenge with Chlamydia muridarum.

There is a need to implement a vaccine to protect against Chlamydia trachomatis infections. To test a new vaccine, mice were immunized with the Chlamydia muridarum native major outer membrane protein (nMOMP) solubilized with either amphipol A8-35 or the detergent Z3-14. OVA was used as a negative control, and mice were inoculated intranasally with C. muridarum as positive controls. Animals vaccinated with nMOMP mounted strong Chlamydia-specific humoral and cell-mediated immune responses. Mice vaccinated with nMOMP/A8-35 had a higher ratio of Abs to denatured elementary bodies (EB) over live EB, recognized more synthetic MOMP peptides and had higher neutralizing titers than sera from mice immunized with nMOMP/Z3-14. T cell lymphoproliferative responses and levels of IFN-γ were also higher in mice vaccinated with nMOMP/A8-35 than with nMOMP/Z3-14. Following immunization, animals were challenged intravaginally with C. muridarum. On the basis of the number of mice with positive vaginal cultures, length of vaginal shedding, total number of positive vaginal cultures, and number of Chlamydia inclusion forming units recovered, nMOMP/A8-35 elicited a more robust protection than nMOMP/Z3-14. By depleting T cells with Abs, we determined that CD4(+) and not CD8(+) T cells mediated the protection elicited by nMOMP/A8-35. Mice were subsequently mated, and based on the number of pregnant mice and number of embryos, animals that were vaccinated with nMOMP/A8-35 or nMOMP/Z3-14 had fertility rates equivalent to the positive control group immunized with live EB and the fertility controls. In conclusion, increased accessibility of epitopes in the nMOMP/A8-35 preparation may account for the very robust protection against infection and disease elicited by this vaccine.

A vaccine formulated with the major outer membrane protein can protect C3H/HeN, a highly susceptible strain of mice, from a Chlamydia muridarum genital challenge.

C3H/HeN female mice were vaccinated with native Chlamydia muridarum major outer membrane protein (MOMP), using Montanide+CpG or Alum+CpG as adjuvants. Negative control groups were immunized with ovalbumin (OVA) and the same adjuvants. As positive control, mice were inoculated intranasally with live Chlamydia. Mice were challenged in the ovarian bursa with 10(5) C. muridarum inclusion forming units. Six weeks after the genital challenge the animals were caged with male mice and monitored for pregnancy. Mice vaccinated with MOMP+Montanide+CpG developed high levels of C. muridarum-specific antibodies, with a high IgG2a/IgG1 ratio and neutralizing titres. Animals immunized using Alum+CpG had low antibody levels. Cellular immune responses were significantly higher in mice vaccinated with MOMP and Montanide+CpG, but not with Alum+CpG, when compared with negative controls. Following the genital challenge, only 20% (4/20) of mice vaccinated with MOMP+CpG+Montanide had positive vaginal cultures whereas 100% (9/9) of mice immunized with MOMP+CpG+Alum had positive cultures. Of the positive control animals inoculated with live Chlamydia only 15% (3/20) had positive vaginal cultures. In contrast, 100% (20/20) of mice immunized with OVA+CpG+Montanide, or minimal essential medium, had positive cultures. Following mating, 80% (16/20) of mice vaccinated with MOMP+CpG+Montanide, and 85% (17/20) of animals inoculated intranasally with live C. muridarum carried embryos in both uterine horns. No protection against infertility was observed in mice immunized with MOMP and CpG+Alum or OVA. In conclusion, this is the first time that a subunit vaccine has been shown to elicit a protective immune response in the highly susceptible C3H/HeN strain of mice against an upper genital challenge.

Activation of CD1d-restricted natural killer T cells can inhibit cancer cell proliferation during chemotherapy by promoting the immune responses in murine mesothelioma.

We studied the impact of natural killer T (NKT) cell activation by alpha-galactocysylceramide (α-GalCer, α-GC) on cancer cell repopulation during chemotherapy in murine mesothelioma. The number of NKT cells was found to be increased during the development of murine mesothelioma. NKT cells specifically recognize α-GC through CD1d resulting in their activation and expansion. Tumor-bearing mice were treated with chemotherapy once weekly, and α-GC was followed after each cycle of chemotherapy. Anti-tumor effect was evaluated on wild-type (WT) and CD1d knockout (CD1dKO) mice. Cancer cell proliferation and apoptosis were evaluated by Ki67 and TUNEL immunohistochemistry. CD4(+) and CD8(+) T cell proportion and activation in tumor, spleen, draining lymph node and peripheral blood were determined by flow cytometry, and gene expression of activated T cell-related cytokines was quantified by reverse transcription PCR. NKT cells were identified by CD1d-α-GC-tetramer staining. In WT mice, tumor growth delay was achieved by cisplatin (Cis), and this effect was improved in combination with α-GC, but α-GC alone had little effect. Cancer cell proliferation during chemotherapy was significantly inhibited by α-GC, while cancer cell death was significantly upregulated. α-GC following chemotherapy resulted in NKT cell expansion and an increase of interferon-γ production in the draining lymph node, blood and spleen. Gene expression of immune-associated cytokines was upregulated. Strikingly, the percentage of inducible T cell co-stimulator(+)CD4 T cells, Th17/Tc17 cells increased in splenocytes. In CD1d KO mice, however, Cis alone was less effective and Cis + α-GC provided no additional benefit over Cis alone. α-GC alone had minimal effect in both mice. NKT activation between cycles of chemotherapy could improve the outcome of mesothelioma treatment.

Long-term stability of a vaccine formulated with the amphipol-trapped major outer membrane protein from Chlamydia trachomatis.

Chlamydia trachomatis is a major bacterial pathogen throughout the world. Although antibiotic therapy can be implemented in the case of early detection, a majority of the infections are asymptomatic, requiring the development of preventive measures. Efforts have focused on the production of a vaccine using the C. trachomatis major outer membrane protein (MOMP). MOMP is purified in its native (n) trimeric form using the zwitterionic detergent Z3-14, but its stability in detergent solutions is limited. Amphipols (APols) are synthetic polymers that can stabilize membrane proteins (MPs) in detergent-free aqueous solutions. Preservation of protein structure and optimization of exposure of the most effective antigenic regions can avoid vaccination with misfolded, poorly protective protein. Previously, we showed that APols maintain nMOMP secondary structure and that nMOMP/APol vaccine formulations elicit better protection than formulations using either recombinant or nMOMP solubilized in Z3-14. To achieve a greater understanding of the structural behavior and stability of nMOMP in APols, we have used several spectroscopic techniques to characterize its secondary structure (circular dichroism), tertiary and quaternary structures (immunochemistry and gel electrophoresis) and aggregation state (light scattering) as a function of temperature and time. We have also recorded NMR spectra of (15)N-labeled nMOMP and find that the exposed loops are detectable in APols but not in detergent. Our analyses show that APols protect nMOMP much better than Z3-14 against denaturation due to continuous heating, repeated freeze/thaw cycles, or extended storage at room temperature. These results indicate that APols can help improve MP-based vaccine formulations.

Safety and efficacy of C. muridarum vaccines adjuvanted with CpG-1826 and four concentrations of Montanide-ISA-720-VG.

It is recommended that the adjuvant Montanide ISA 720 VG be used at a concentration of 70% v/v. At this concentration, Montanide causes at the site of immunization a local granuloma that can last for several weeks. To determine the safety and protective efficacy of a Chlamydia muridarum MOMP vaccine, formulated with CpG-1826 and four different concentrations of Montanide (70%, 50%, 30% and 10%), BALB/c (H-2d) female mice were immunized twice intramuscularly. Local reactogenicity was significant for vaccines formulated with 70% or 50% Montanide but not for those inoculated with 30% or 10% Montanide. Robust humoral and cell mediated memory immune responses were elicited by the 70%, 50% and 30% Montanide formulations. Mice were challenged intranasally with 104 C. muridarum inclusion forming units (IFU). Based on changes in body weight, lungs's weight and number of IFU recovered, mice vaccinated with the 70%, 50% and 30% Montanide formulations were significantly protected, but not mice receiving 10% Montanide. To conclude, we recommend the 30% Montanide concentration to be tested in humans and animal models to determine its safety and efficacy, in comparison to the 70% Montanide concentration currently used. The 30% Montanide formulation could significantly facilitate licensing of this adjuvant for human use.

Structural Assessment of Chlamydia trachomatis Major Outer Membrane Protein (MOMP)-Derived Vaccine Antigens and Immunological Profiling in Mice with Different Genetic Backgrounds.

Chlamydia trachomatis (Ct) is the most common cause of bacterial sexually transmitted infections (STIs) worldwide. Ct infections are often asymptomatic in women, leading to severe reproductive tract sequelae. Development of a vaccine against Chlamydia is crucial. The Chlamydia major outer membrane protein (MOMP) is a prime vaccine antigen candidate, and it can elicit both neutralizing antibodies and protective CD4+ T cell responses. We have previously designed chimeric antigens composed of immunogenic variable regions (VDs) and conserved regions (CDs) of MOMP from Chlamydia muridarum (Cm) expressed into a carrier protein (PorB), and we have shown that these were protective in a mouse model of Cm respiratory infection. Here, we generated corresponding constructs based on MOMP from Ct serovar F. Preliminary structure analysis of the three antigens, PorB/VD1-3, PorB/VD1-4 and PorB/VD1-2-4, showed that they retained structure features consistent with those of PorB. The antigens induced robust humoral and cellular responses in mice with different genetic backgrounds. The antibodies were cross-reactive against Ct, but only anti-PorB/VD1-4 and anti-PorB/VD1-2-4 IgG antibodies were neutralizing, likely due to the antigen specificity. The cellular responses included proliferation in vitro and production of IFN-γ by splenocytes following Ct re-stimulation. Our results support further investigation of the PorB/VD antigens as potential protective candidates for a Chlamydia subunit vaccine.

CT584 Is Not a Protective Vaccine Antigen against Respiratory Chlamydial Challenge in Mice.

Background:Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen in humans worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and decentralized production of recombinant protein vaccine antigens. Methods: Here, we use CFPS to produce the full-length putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for evaluation as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) (RIBM, Tsukuba, Japan) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four weeks later by an i.m. boost before respiratory challenge with 104 inclusion forming units (IFU) of Chlamydia muridarum. Results: Immunization with CT584 generated robust antibody responses but weak cell-mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lung weights, and the presence of high numbers of IFUs in the lungs. Conclusion: While CT584 was not a protective vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens make it an attractive technique for antigen production.

Evaluation in mice of cell-free produced CT584 as a Chlamydia vaccine antigen.

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and de-centralized production of recombinant protein vaccine antigens. Here, we use CFPS to produce the putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for use as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four-weeks later by an i.m. boost before respiratory challenge with 104 inclusion forming units (IFU) of Chlamydia muridarum. Immunization with CT584 generated robust antibody responses but weak cell mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lungs' weights and the presence of high numbers of IFUs in the lungs. While CT584 alone may not be the ideal vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens makes it an attractive technique for antigen production.

Sequence, structure prediction, and epitope analysis of the polymorphic membrane protein family in Chlamydia trachomatis.

The polymorphic membrane proteins (Pmps) are a family of autotransporters that play an important role in infection, adhesion and immunity in Chlamydia trachomatis. Here we show that the characteristic GGA(I,L,V) and FxxN tetrapeptide repeats fit into a larger repeat sequence, which correspond to the coils of a large beta-helical domain in high quality structure predictions. Analysis of the protein using structure prediction algorithms provided novel insight to the chlamydial Pmp family of proteins. While the tetrapeptide motifs themselves are predicted to play a structural role in folding and close stacking of the beta-helical backbone of the passenger domain, we found many of the interesting features of Pmps are localized to the side loops jutting out from the beta helix including protease cleavage, host cell adhesion, and B-cell epitopes; while T-cell epitopes are predominantly found in the beta-helix itself. This analysis more accurately defines the Pmp family of Chlamydia and may better inform rational vaccine design and functional studies.

Vaccination with the recombinant major outer membrane protein elicits antibodies to the constant domains and induces cross-serovar protection against intranasal challenge with Chlamydia trachomatis.

To determine the ability of the major outer membrane protein (MOMP) to elicit cross-serovar protection, groups of mice were immunized by the intramuscular (i.m.) and subcutaneous (s.c.) routes with recombinant MOMP (rMOMP) from Chlamydia trachomatis serovars D (UW-3/Cx), E (Bour), or F (IC-Cal-3) or Chlamydia muridarum strain Nigg II using CpG-1826 and Montanide ISA 720 VG as adjuvants. Negative-control groups were immunized i.m. and s.c. with Neisseria gonorrhoeae recombinant porin B (Ng-rPorB) or i.n. with Eagle's minimal essential medium (MEM-0). Following vaccination, the mice developed antibodies not only against the homologous serovar but also against heterologous serovars. The rMOMP-vaccinated animals also mounted cell-mediated immune responses as assessed by a lymphoproliferative assay. Four weeks after the last immunization, mice were challenged i.n. with 10(4) inclusion-forming units (IFU) of C. muridarum. The mice were weighed for 10 days and euthanized, and the number of IFU in their lungs was determined. At 10 days postinfection (p.i.), mice immunized with the rMOMP of C. muridarum or C. trachomatis D, E, or F had lost 4%, 6%, 8%, and 8% of their initial body weight, respectively, significantly different from the negative-control groups (Ng-rPorB, 13%; MEM-0, 19%; P < 0.05). The median number of IFU recovered from the lungs of mice immunized with C. muridarum rMOMP was 0.13 × 10(6). The median number of IFU recovered from mice immunized with rMOMP from serovars D, E, and F were 0.38 × 10(6), 7.56 × 10(6), and 11.94 × 10(6) IFU, respectively. All the rMOMP-immunized animals had significantly less IFU than the Ng-rPorB (40 × 10(6))- or MEM-0 (70 × 10(6))-immunized mice (P < 0.05). In conclusion, vaccination with rMOMP can elicit protection against homologous and heterologous Chlamydia serovars.

Toll-like receptor 2-dependent activity of native major outer membrane protein proteosomes of Chlamydia trachomatis.

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen and the etiologic agent of blinding trachoma. Intracellular signaling pathways leading to host cell inflammation and innate immunity to Chlamydia include those mediated by Toll-like receptors (TLRs) and nucleotide binding oligomerization domain 1 (Nod1) protein. In epithelial cells, TLR-dependent signaling contributes to local immune responses via induction of inflammatory mediators. There is evidence that TLR3, TLR4, and, particularly, TLR2 are critical for Chlamydia-mediated host cell activation and pathology. Despite the importance of TLR2, major chlamydial TLR2 antigens have not been identified so far. Numerous bacterial porins are known TLR2 agonists, i.e., porins from Neisseriae, Shigella, Salmonella, Haemophilus influenzae, and Fusobacterium nucleatum, which share structural and functional similarities with the chlamydial major outer membrane protein (MOMP), a strong antigen candidate for a potential vaccine against C. trachomatis. We describe the ability of purified, detergent-free MOMP to signal via TLR2 in vitro in TLR-overexpressing cells and TLR2-competent human reproductive tract epithelial cell lines. Using MOMP formed in pure protein micelles (proteosomes), we show the induction of TLR2-dependent interleukin-8 (IL-8) and IL-6 secretion in vitro, the involvement of TLR1 as a TLR2 coreceptor, and the activation of both NF-κB and mitogen-activated protein (MAP) kinase intracellular pathways. Interestingly, MOMP proteosomes induce cytokine secretion in endocervical epithelial cells (End/E6E7) but not in urethral epithelial cells (THUECs). A detailed understanding of the TLR2-dependent molecular mechanisms that characterize the effect of MOMP proteosomes on host cells may provide new insights for its successful development as an immunotherapeutic target against Chlamydia.

Determination of the safety and efficacy of recombinant Chlamydia muridarum MOMP vaccines, formulated with CpG-1826 and 70%, 50%, 30% or 10% concentrations of Montanide ISA-720 VG, to elicit protective immune responses against a C. muridarum respiratory challenge.

To determine the safety and protective efficacy of a C. muridarum MOMP vaccine, formulated with CpG-1826 and four different concentrations of Montanide ISA 720 VG (70%, 50%, 30% and 10%), BALB/c mice were immunized twice intramuscularly. Local reactogenicity was significant for vaccines formulated with 70% and 50% Montanide but not in mice receiving 30% and 10% Montanide. Robust humoral and cell mediated memory immune responses were elicited by the 70%, 50% and 30% Montanide formulations. Mice were challenged intranasally with C. muridarum and, at day 10 post-challenge, mice were euthanized. Based on changes in body weight, lung's weight and number of IFU recovered, mice vaccinated with the 70%, 50% and 30% Montanide formulations were significantly protected, but not mice receiving 10% Montanide. To conclude, we recommend the 30% Montanide concentration to be tested in humans and animal models to determine its safety and efficacy, in comparison to the 70% Montanide concentration currently used. The 30% Montanide formulation will significantly facilitate licensing for human use.

Evaluation of Four Adjuvant Combinations, IVAX-1, IVAX-2, CpG-1826+Montanide ISA 720 VG and CpG-1018+Montanide ISA 720 VG, for Safety and for Their Ability to Elicit Protective Immune Responses in Mice against a Respiratory Challenge with Chlamydia muridarum.

There is an urgent need to produce a vaccine for Chlamydia trachomatis infections. Here, using the Chlamydia muridarum major outer membrane protein (MOMP) as an antigen, four adjuvant combinations IVAX-1 (MPLA+CpG-1018+AddaVax), IVAX-2 (MPLA+CpG-1018+AS03), CpG-1826+Montanide ISA 720 VG (CpG-1826+Mont) and CpG-1018+Montanide ISA 720 VG (CpG-1018+Mont), were tested for their local reactogenicity and ability to elicit protection in BALB/c mice against a respiratory challenge with C. muridarum. Immunization with IVAX-1 or IVAX-2 induced no significant local reactogenicity following intramuscular immunization. In contrast, vaccines containing Montanide resulted in the formation of a local granuloma. Based on the IgG2a/IgG1 ratio in serum, the four adjuvant combinations elicited Th1-biased responses. IVAX-1 induced the highest in vitro neutralization titers while CpG-1018+Mont stimulated the lowest. As determined by the levels of IFN-γ produced by T-cells, the most robust cellular immune responses were elicited in mice immunized with CpG-1018+Mont, while the weakest responses were mounted by mice receiving IVAX-1. Following the respiratory challenge, mice immunized with CpG-1018+Mont lost the least amount of body weight and had the lowest number of C. muridarum inclusion-forming units (IFUs) in the lungs, while those receiving IVAX-2 had lost the most weight and had the highest number of IFUs in their lungs. Animals vaccinated with CpG-1826+Mont had the lightest lungs while those immunized using IVAX-2 had the heaviest. To conclude, due to their safety and adjuvanticity, IVAX formulations should be considered for inclusion in human vaccines against Chlamydia.

The Polymorphic Membrane Protein G Has a Neutral Effect and the Plasmid Glycoprotein 3 an Antagonistic Effect on the Ability of the Major Outer Membrane Protein to Elicit Protective Immune Responses against a Chlamydia muridarum Respiratory Challenge.

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen. The number of chlamydial infections continuous to increase and there is an urgent need for a safe and efficacious vaccine. To assess the ability of the Chlamydia muridarum polymorphic membrane protein G (PmpG) and the plasmid glycoprotein 3 (Pgp3) as single antigens, and in combination with the major outer-membrane protein (MOMP) to induce protection, BALB/c mice were immunized utilizing CpG-1826 and Montanide ISA 720 VG as adjuvants. Following vaccination with MOMP, significant humoral and cell-mediated immune responses were observed, while immunization with PmpG, or Pgp3, elicited weaker immune responses. Weaker immune responses were induced with MOMP+Pgp3 compared with MOMP alone. Following the intranasal challenge with C. muridarum, mice vaccinated with MOMP showed robust protection against body-weight loss, inflammatory responses in the lungs and number of Chlamydia recovered from the lungs. PmpG and Pgp3 elicited weaker protective responses. Mice immunized with MOMP+PmpG, were no better protected than animals vaccinated with MOMP only, while Pgp3 antagonized the protection elicited by MOMP. In conclusion, PmpG and Pgp3 elicited limited protective immune responses in mice against a respiratory challenge with C. muridarum and failed to enhance the protection induced by MOMP alone. The virulence of Pgp3 may result from its antagonistic effect on the immune protection induced by MOMP.

A modular vaccine platform enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens.

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. However, antigen display on OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. Here, we describe a universal approach for avidin-based vaccine antigen crosslinking (AvidVax) whereby biotinylated antigens are linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen-binding protein (SNAP) comprised of an outer membrane scaffold protein fused to a biotin-binding protein. We show that SNAP-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations are injected in mice, strong antigen-specific antibody responses are observed that depend on the physical coupling between the antigen and SNAP-OMV delivery vehicle. Overall, these results demonstrate AvidVax as a modular platform that enables rapid and simplified assembly of antigen-studded OMVs for application as vaccines against pathogenic threats.