The goal of this study was to determine if exosomes, isolated from Chlamydia muridarum infected H... more The goal of this study was to determine if exosomes, isolated from Chlamydia muridarum infected HeLa cells (C. muridarum-exosomes), induce protective immune responses in mice following vaccination using CpG plus Montanide as adjuvants. Exosomes, collected from uninfected HeLa cells and PBS, formulated with the same adjuvants, were used as negative controls. Mass spectrometry analyses identified 113 C. muridarum proteins in the C. muridarum-exosome preparation including the major outer membrane protein and the polymorphic membrane proteins. Vaccination with C. muridarum-exosomes elicited robust humoral and cell-mediated immune responses to C. muridarum elementary bodies. Following vaccination, mice were challenged intranasally with C. muridarum. Compared to the negative controls, mice immunized with C. muridarum-exosomes were significantly protected as measured by changes in body weight, lungs’ weight, and number of inclusion forming units recovered from lungs. This is the first repo...
Chlamydia trachomatis is the most frequently detected sexually transmitted bacterial pathogen in ... more Chlamydia trachomatis is the most frequently detected sexually transmitted bacterial pathogen in the world. Attempts to control these infections with screening programs and antibiotics have failed and, therefore, a vaccine is the best approach to control this epidemic. The Chlamydia major outer membrane protein (MOMP) is the most protective subunit vaccine so far tested. Protection induced by MOMP is, in part, dependent on its tertiary structure. We have previously described new recombinant antigens composed of the Neisseria lactamica PorB engineered to express the variable domains (VD) from Chlamydia muridarum MOMP. Here we tested antigens containing each individual MOMP VD and different VD combinations. Following immunization, mice were challenged intranasally with C. muridarum. Our results show that three constructs, PorB/VD1–3, PorB/VD1–4, and PorB/VD1–2–4, elicited high serum IgG titers in vivo, significant IFN-γ levels upon T cells re-stimulation in vitro, and evidence of prot...
Chlamydia trachomatis is the leading cause of infection-induced infertility in women. Attempts to... more 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 × 105 C. 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 con...
Two cationic liposomal adjuvants CAF01 and CAF09 were formulated with the native or the recombina... more Two cationic liposomal adjuvants CAF01 and CAF09 were formulated with the native or the recombinant Chlamydia muridarum major outer membrane protein (nMOMP and rMOMP). BALB/c mice were immunized with the four vaccine formulations using the subcutaneous followed by the intranasal (i.n.) routes. As positive controls mice were inoculated i.n. with live C. muridarum and negative controls received i.n. minimal essential medium (MEM). Four weeks after the last immunization mice were challenged i.n. with 10(4) inclusion forming units (IFU) of C. muridarum. Following the challenge the mice were weighed daily. At 10days post-challenge the mice were euthanized, their lungs weighed and the number of C. muridarum IFU determined. Serum collected the day before the challenge showed that all four groups of mice immunized with CAF01, or CAF09 and MOMP had significant C. muridarum-specific antibody titers. As determined by a T-cell lymphoproliferative assay, these four groups of mice also mounted ro...
Monoclonal antibodies (MAbs) E-4, E-21, and DIII A3, which recognize the same or similar overlapp... more Monoclonal antibodies (MAbs) E-4, E-21, and DIII A3, which recognize the same or similar overlapping peptides in the variable domain IV of the major outer membrane protein of Chlamydia trachomatis but differ in isotype, were used in a complement-independent (CI) in vitro neutralization assay. These MAbs had previously been shown to neutralize chlamydial infectivity in HeLa 229 cells in a complement-dependent assay. In this report, all three MAbs neutralized chlamydial infectivity in HaK cells in a CI assay. However, when HeLa cells were used as the host cell, MAb E-4 (immunoglobulin G2b [IgG2b]) and MAb DIII A3 (IgG2b) failed to neutralize infectivity, while MAb E-21 (IgG1) neutralized chlamydial infectivity. These findings are consistent with the proposal that because of the presence of Fc gamma RIII receptors, HeLa cells facilitate infectivity and thus block neutralization through the uptake of an IgG2b-chlamydia complex. Since Fc gamma RIII receptors do not bind or bind poorly to...
The ability to induce protection against a genital challenge was studied in BALB/c female mice wi... more The ability to induce protection against a genital challenge was studied in BALB/c female mice with three Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) preparations as well as an acellular vaccine consisting of the chlamydial outer membrane complex (COMC). The MOMP preparations were extracted with three different types of detergents, sodium dodecyl sulfate (SDS), n-octyl-beta-D-glucopyranoside (OGP), and Zwittergent 3-14 (Z3-14). A positive immunization control consisted of mice inoculated intranasally with 10(4) C. trachomatis MoPn inclusion-forming units (IFU). Mice inoculated with ovalbumin served as a negative control. Furthermore, a sham-immunized, nonchallenged group was included as a fertility control. Two weeks after the last immunization, the mice were challenged in the left ovarian bursa with 10(5) C. trachomatis MoPn IFU. Vaginal swabs were collected for culture, vaginal and serum samples were assayed for chlamydial-specific antibodies...
Due to its availability, nontoxicity, and biodegradability, chitosan has been heralded as a mater... more Due to its availability, nontoxicity, and biodegradability, chitosan has been heralded as a material with applications in antimicrobials, manufacturing, and certain biomedical fields. However, certain factors limiting the widespread use of chitosan include the lack of solubility in neutral solutions and limited response to biological triggers. Attempts to overcome these shortcomings have led to various modifications such as N-/O-carboxyalkyl modification or acylation. In this study, we attempted to increase chitosan’s aqueous solubility and simultaneoulsy add stimuli-responsiveness by conjugating an amino branch to the primary hydroxyl group via a temporary, acid-cleavable ketal linkage, resulting in ketalized chitosan (kC). We hypothesized that enginered chitosan with amine branches via ketal linkage would 1) increase the hydrophilicity, 2) promote cytosolic release in the mildly acidic endosome, and 3) regenerate chitosan back in its native antimicrobial form upon acid hydrolysis....
Chlamydia-specific antibody-secreting cells have been identified in conjunctiva and draining cerv... more Chlamydia-specific antibody-secreting cells have been identified in conjunctiva and draining cervical lymph nodes by an ELISPOT assay in a cynomolgus monkey model of trachoma. These local sites contained numbers of chlamydia-specific B cells that were higher than those in distant inguinal lymph nodes and peripheral blood. The numbers of chlamydia-specific immunoglobulin G-secreting B cells observed were 5 to 57 per 10(6) cells in conjunctiva and 24 to 996 per 10(6) cells in cervical lymph nodes during conjunctival infection or after challenge of immune monkeys with the chlamydial 57-kDa heat shock protein (hsp60). These studies demonstrate a large chlamydia-specific B-cell component in the conjunctiva during ocular chlamydial infection. These results are similar to our findings for chlamydia-specific T-cell responses.
The immunopathogenesis of ocular infection with Chlamydia trachomatis can be investigated in the ... more The immunopathogenesis of ocular infection with Chlamydia trachomatis can be investigated in the cynomolgus monkey model of trachoma. During ocular infection, both follicles (germinal centers) containing primarily B lymphocytes and a heavy perifollicular T cell infiltrate are observed. Limiting dilution analyses were performed on conjunctival and peripheral blood lymphocytes to quantify the proportion of antigen-specific cells elicited by ocular C. trachomatis infection. The frequencies of chlamydia-specific cells were 10-100 times higher in conjunctiva than in peripheral blood. Similar frequencies were observed during active infection and during the ocular delayed hypersensitivity response elicited in sensitized monkeys by topical challenge with a triton X-100 extract containing the 57 kD chlamydial stress protein. Given the antigen-specific proliferative response, these cells are most likely T lymphocytes. The finding of large numbers of antigen-specific conjunctival lymphocytes a...
In an attempt to confer long-term protective immunity, BALB/c female mice were immunized intranas... more In an attempt to confer long-term protective immunity, BALB/c female mice were immunized intranasally with 10(4) inclusion-forming units (IFU) of the Chlamydia trachomatis mouse pneumonitis biovar (MoPn). Animals were subsequently challenged in the ovarian bursa with 10(5) C. trachomatis MoPn IFU at 60, 120, or 180 days post-intranasal immunization. Two control groups were included in the study. One control was sham immunized and mock challenged, and another group was sham immunized and challenged with 10(5) C. trachomatis MoPn IFU. Vaginal cultures were collected at regular intervals following the intrabursal challenge. In comparison with the sham-immunized mice, the animals that were intranasally immunized with C. trachomatis had significant protection, as shown by a reduction in the number of animals that had positive vaginal cultures and by a decrease in the intensity and length of the shedding. Furthermore, histopathological characterization of the genital tract following chall...
An egg-based medium was found to be superior to the conventional Wang transport medium and the re... more An egg-based medium was found to be superior to the conventional Wang transport medium and the recently developed biphasic medium for the preservation of Campylobacter jejuni in the laboratory. Strains of C. jejuni preserved in egg-based medium maintained at 4 degrees C were viable for over 3 months. The survival of C. jejuni in egg-based medium held at room temperature (27 +/- 2 degrees C) was also relatively longer than in Wang transport medium and biphasic medium.
Pregnant BALB/c mice were inoculated intravaginally on day 5 of gestation with the Chlamydia trac... more Pregnant BALB/c mice were inoculated intravaginally on day 5 of gestation with the Chlamydia trachomatis mouse pneumonitis biovar. Animals that received 10(5), 10(6), or 10(7) inclusion-forming units (IFU) of C. trachomatis delivered prematurely on days 15 to 16 of gestation. A focal inflammatory infiltrate was observed in the wall of the uterus on the day 14 of gestation in animals inoculated with 10(5) IFU. In this group of mice, immunohistochemical analysis showed chlamydial inclusions in the endometrium and fetal membranes.
The ability to induce protection against a genital challenge was studied in BALB/c female mice wi... more The ability to induce protection against a genital challenge was studied in BALB/c female mice with three Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) preparations as well as an acellular vaccine consisting of the chlamydial outer membrane complex (COMC). The MOMP preparations were extracted with three different types of detergents, sodium dodecyl sulfate (SDS), n-octyl-beta-D-glucopyranoside (OGP), and Zwittergent 3-14 (Z3-14). A positive immunization control consisted of mice inoculated intranasally with 10(4) C. trachomatis MoPn inclusion-forming units (IFU). Mice inoculated with ovalbumin served as a negative control. Furthermore, a sham-immunized, nonchallenged group was included as a fertility control. Two weeks after the last immunization, the mice were challenged in the left ovarian bursa with 10(5) C. trachomatis MoPn IFU. Vaginal swabs were collected for culture, vaginal and serum samples were assayed for chlamydial-specific antibodies...
The goal of this study was to determine if exosomes, isolated from Chlamydia muridarum infected H... more The goal of this study was to determine if exosomes, isolated from Chlamydia muridarum infected HeLa cells (C. muridarum-exosomes), induce protective immune responses in mice following vaccination using CpG plus Montanide as adjuvants. Exosomes, collected from uninfected HeLa cells and PBS, formulated with the same adjuvants, were used as negative controls. Mass spectrometry analyses identified 113 C. muridarum proteins in the C. muridarum-exosome preparation including the major outer membrane protein and the polymorphic membrane proteins. Vaccination with C. muridarum-exosomes elicited robust humoral and cell-mediated immune responses to C. muridarum elementary bodies. Following vaccination, mice were challenged intranasally with C. muridarum. Compared to the negative controls, mice immunized with C. muridarum-exosomes were significantly protected as measured by changes in body weight, lungs’ weight, and number of inclusion forming units recovered from lungs. This is the first repo...
Chlamydia trachomatis is the most frequently detected sexually transmitted bacterial pathogen in ... more Chlamydia trachomatis is the most frequently detected sexually transmitted bacterial pathogen in the world. Attempts to control these infections with screening programs and antibiotics have failed and, therefore, a vaccine is the best approach to control this epidemic. The Chlamydia major outer membrane protein (MOMP) is the most protective subunit vaccine so far tested. Protection induced by MOMP is, in part, dependent on its tertiary structure. We have previously described new recombinant antigens composed of the Neisseria lactamica PorB engineered to express the variable domains (VD) from Chlamydia muridarum MOMP. Here we tested antigens containing each individual MOMP VD and different VD combinations. Following immunization, mice were challenged intranasally with C. muridarum. Our results show that three constructs, PorB/VD1–3, PorB/VD1–4, and PorB/VD1–2–4, elicited high serum IgG titers in vivo, significant IFN-γ levels upon T cells re-stimulation in vitro, and evidence of prot...
Chlamydia trachomatis is the leading cause of infection-induced infertility in women. Attempts to... more 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 × 105 C. 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 con...
Two cationic liposomal adjuvants CAF01 and CAF09 were formulated with the native or the recombina... more Two cationic liposomal adjuvants CAF01 and CAF09 were formulated with the native or the recombinant Chlamydia muridarum major outer membrane protein (nMOMP and rMOMP). BALB/c mice were immunized with the four vaccine formulations using the subcutaneous followed by the intranasal (i.n.) routes. As positive controls mice were inoculated i.n. with live C. muridarum and negative controls received i.n. minimal essential medium (MEM). Four weeks after the last immunization mice were challenged i.n. with 10(4) inclusion forming units (IFU) of C. muridarum. Following the challenge the mice were weighed daily. At 10days post-challenge the mice were euthanized, their lungs weighed and the number of C. muridarum IFU determined. Serum collected the day before the challenge showed that all four groups of mice immunized with CAF01, or CAF09 and MOMP had significant C. muridarum-specific antibody titers. As determined by a T-cell lymphoproliferative assay, these four groups of mice also mounted ro...
Monoclonal antibodies (MAbs) E-4, E-21, and DIII A3, which recognize the same or similar overlapp... more Monoclonal antibodies (MAbs) E-4, E-21, and DIII A3, which recognize the same or similar overlapping peptides in the variable domain IV of the major outer membrane protein of Chlamydia trachomatis but differ in isotype, were used in a complement-independent (CI) in vitro neutralization assay. These MAbs had previously been shown to neutralize chlamydial infectivity in HeLa 229 cells in a complement-dependent assay. In this report, all three MAbs neutralized chlamydial infectivity in HaK cells in a CI assay. However, when HeLa cells were used as the host cell, MAb E-4 (immunoglobulin G2b [IgG2b]) and MAb DIII A3 (IgG2b) failed to neutralize infectivity, while MAb E-21 (IgG1) neutralized chlamydial infectivity. These findings are consistent with the proposal that because of the presence of Fc gamma RIII receptors, HeLa cells facilitate infectivity and thus block neutralization through the uptake of an IgG2b-chlamydia complex. Since Fc gamma RIII receptors do not bind or bind poorly to...
The ability to induce protection against a genital challenge was studied in BALB/c female mice wi... more The ability to induce protection against a genital challenge was studied in BALB/c female mice with three Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) preparations as well as an acellular vaccine consisting of the chlamydial outer membrane complex (COMC). The MOMP preparations were extracted with three different types of detergents, sodium dodecyl sulfate (SDS), n-octyl-beta-D-glucopyranoside (OGP), and Zwittergent 3-14 (Z3-14). A positive immunization control consisted of mice inoculated intranasally with 10(4) C. trachomatis MoPn inclusion-forming units (IFU). Mice inoculated with ovalbumin served as a negative control. Furthermore, a sham-immunized, nonchallenged group was included as a fertility control. Two weeks after the last immunization, the mice were challenged in the left ovarian bursa with 10(5) C. trachomatis MoPn IFU. Vaginal swabs were collected for culture, vaginal and serum samples were assayed for chlamydial-specific antibodies...
Due to its availability, nontoxicity, and biodegradability, chitosan has been heralded as a mater... more Due to its availability, nontoxicity, and biodegradability, chitosan has been heralded as a material with applications in antimicrobials, manufacturing, and certain biomedical fields. However, certain factors limiting the widespread use of chitosan include the lack of solubility in neutral solutions and limited response to biological triggers. Attempts to overcome these shortcomings have led to various modifications such as N-/O-carboxyalkyl modification or acylation. In this study, we attempted to increase chitosan’s aqueous solubility and simultaneoulsy add stimuli-responsiveness by conjugating an amino branch to the primary hydroxyl group via a temporary, acid-cleavable ketal linkage, resulting in ketalized chitosan (kC). We hypothesized that enginered chitosan with amine branches via ketal linkage would 1) increase the hydrophilicity, 2) promote cytosolic release in the mildly acidic endosome, and 3) regenerate chitosan back in its native antimicrobial form upon acid hydrolysis....
Chlamydia-specific antibody-secreting cells have been identified in conjunctiva and draining cerv... more Chlamydia-specific antibody-secreting cells have been identified in conjunctiva and draining cervical lymph nodes by an ELISPOT assay in a cynomolgus monkey model of trachoma. These local sites contained numbers of chlamydia-specific B cells that were higher than those in distant inguinal lymph nodes and peripheral blood. The numbers of chlamydia-specific immunoglobulin G-secreting B cells observed were 5 to 57 per 10(6) cells in conjunctiva and 24 to 996 per 10(6) cells in cervical lymph nodes during conjunctival infection or after challenge of immune monkeys with the chlamydial 57-kDa heat shock protein (hsp60). These studies demonstrate a large chlamydia-specific B-cell component in the conjunctiva during ocular chlamydial infection. These results are similar to our findings for chlamydia-specific T-cell responses.
The immunopathogenesis of ocular infection with Chlamydia trachomatis can be investigated in the ... more The immunopathogenesis of ocular infection with Chlamydia trachomatis can be investigated in the cynomolgus monkey model of trachoma. During ocular infection, both follicles (germinal centers) containing primarily B lymphocytes and a heavy perifollicular T cell infiltrate are observed. Limiting dilution analyses were performed on conjunctival and peripheral blood lymphocytes to quantify the proportion of antigen-specific cells elicited by ocular C. trachomatis infection. The frequencies of chlamydia-specific cells were 10-100 times higher in conjunctiva than in peripheral blood. Similar frequencies were observed during active infection and during the ocular delayed hypersensitivity response elicited in sensitized monkeys by topical challenge with a triton X-100 extract containing the 57 kD chlamydial stress protein. Given the antigen-specific proliferative response, these cells are most likely T lymphocytes. The finding of large numbers of antigen-specific conjunctival lymphocytes a...
In an attempt to confer long-term protective immunity, BALB/c female mice were immunized intranas... more In an attempt to confer long-term protective immunity, BALB/c female mice were immunized intranasally with 10(4) inclusion-forming units (IFU) of the Chlamydia trachomatis mouse pneumonitis biovar (MoPn). Animals were subsequently challenged in the ovarian bursa with 10(5) C. trachomatis MoPn IFU at 60, 120, or 180 days post-intranasal immunization. Two control groups were included in the study. One control was sham immunized and mock challenged, and another group was sham immunized and challenged with 10(5) C. trachomatis MoPn IFU. Vaginal cultures were collected at regular intervals following the intrabursal challenge. In comparison with the sham-immunized mice, the animals that were intranasally immunized with C. trachomatis had significant protection, as shown by a reduction in the number of animals that had positive vaginal cultures and by a decrease in the intensity and length of the shedding. Furthermore, histopathological characterization of the genital tract following chall...
An egg-based medium was found to be superior to the conventional Wang transport medium and the re... more An egg-based medium was found to be superior to the conventional Wang transport medium and the recently developed biphasic medium for the preservation of Campylobacter jejuni in the laboratory. Strains of C. jejuni preserved in egg-based medium maintained at 4 degrees C were viable for over 3 months. The survival of C. jejuni in egg-based medium held at room temperature (27 +/- 2 degrees C) was also relatively longer than in Wang transport medium and biphasic medium.
Pregnant BALB/c mice were inoculated intravaginally on day 5 of gestation with the Chlamydia trac... more Pregnant BALB/c mice were inoculated intravaginally on day 5 of gestation with the Chlamydia trachomatis mouse pneumonitis biovar. Animals that received 10(5), 10(6), or 10(7) inclusion-forming units (IFU) of C. trachomatis delivered prematurely on days 15 to 16 of gestation. A focal inflammatory infiltrate was observed in the wall of the uterus on the day 14 of gestation in animals inoculated with 10(5) IFU. In this group of mice, immunohistochemical analysis showed chlamydial inclusions in the endometrium and fetal membranes.
The ability to induce protection against a genital challenge was studied in BALB/c female mice wi... more The ability to induce protection against a genital challenge was studied in BALB/c female mice with three Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) preparations as well as an acellular vaccine consisting of the chlamydial outer membrane complex (COMC). The MOMP preparations were extracted with three different types of detergents, sodium dodecyl sulfate (SDS), n-octyl-beta-D-glucopyranoside (OGP), and Zwittergent 3-14 (Z3-14). A positive immunization control consisted of mice inoculated intranasally with 10(4) C. trachomatis MoPn inclusion-forming units (IFU). Mice inoculated with ovalbumin served as a negative control. Furthermore, a sham-immunized, nonchallenged group was included as a fertility control. Two weeks after the last immunization, the mice were challenged in the left ovarian bursa with 10(5) C. trachomatis MoPn IFU. Vaginal swabs were collected for culture, vaginal and serum samples were assayed for chlamydial-specific antibodies...
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