Recombinant Major Outer Membrane Protein Research Articles

CRISPR/Cas9-edited duck enteritis virus expressing Pmp17G of Chlamydia psittaci induced protective immunity in ducklings.

Chlamydia psittaci is threatening to the animal industry and human beings. Live attenuated duck enteritis virus (DEV) is considered a good vaccine vector. In the present study, the Pmp17G antigen of C. psittaci was expressed in DEV to construct a recombinant DEV-Pmp17G vaccine. The growing curve of the rDEV-Pmp17G vaccine was comparable to the parental DEV strain, and Pmp17G protein expression was detected in the cytosol and membrane of the infected host cells. A total of 30 ducklings assigned to 5 groups were used to evaluate the vaccine efficacy. The birds in the vaccine groups received 15 000 plaque forming units of the rDEV-Pmp17G vaccine via hypodermic injection. In contrast, the control groups received intramuscular inoculation with 1×103 embryo lethal dose of DEV vector or 50µg of commercial recombinant major outer membrane protein (MOMP) vaccine. The rDEV-Pmp17G vaccine induced significantly higher levels of IgG antibodies than the commercial MOMP did on day 14, and the IgG antibodies persisted for 28 days. Moreover, the rDEV-Pmp17G vaccine also induced higher levels of lymphocyte proliferations compared to the DEV vector. The vaccinated animals significantly reduced lesions and enhanced bacterial clearance in the lungs and throats compared to the MOMP immunization. Thus, the rDEV-Pmp17G vaccine induced persistent IgG antibodies and lymphocyte proliferation against C. psittaci infection.

Effects of prime-boost strategies on the protective efficacy and immunogenicity of a PLGA (85:15)-encapsulated Chlamydia recombinant MOMP nanovaccine.

To begin to optimize the immunization routes for our reported PLGA-rMOMP nanovaccine [PLGA-encapsulated Chlamydia muridarum (Cm) recombinant major outer membrane protein (rMOMP)], we compared two prime-boost immunization strategies [subcutaneous (SC) and intramuscular (IM-p) prime routes followed by two SC-boosts)] to evaluate the nanovaccine-induced protective efficacy and immunogenicity in female BALB/c mice. Our results showed that mice immunized via the SC and IM-p routes were protected against a Cm genital challenge by a reduction in bacterial burden and with fewer bacteria in the SC mice. Protection of mice correlated with rMOMP-specific Th1 (IL-2 and IFN-γ) and not Th2 (IL-4, IL-9, and IL-13) cytokines, and CD4+memory (CD44highCD62Lhigh) T-cells, especially in the SC mice. We also observed higher levels of IL-1α, IL-6, IL-17, CCL-2, and G-CSF in SC-immunized mice. Notably, an increase of cytokines/chemokines was seen after the challenge in the SC, IM-p, and control mice (rMOMP and PBS), suggesting a Cm stimulation. In parallel, rMOMP-specific Th1 (IgG2a and IgG2b) and Th2 (IgG1) serum, mucosal, serum avidity, and neutralizing antibodies were more elevated in SC than in IM-p mice. Overall, the homologous SC prime-boost immunization of mice induced enhanced cellular and antibody responses with better protection against a genital challenge compared to the heterologous IM-p.

Comparison of the immune effects of the Chlamydia abortus MOMP antigen displayed in different parts of bacterial ghosts.

Bacterial ghosts (BGs) are promising vaccine platforms owing to their high adjuvant properties and delivery efficiency. Heterologous antigens can be anchored to different parts of BGs using genetic engineering strategies to prepare vaccines. However, several key issues need to be resolved, including the efficient preparation of BGs and determining the optimal anchoring position of exogenous antigens in the BGs. Here, we prepared an efficient temperature-controlled lysis system using lysis gene E of phage PhiX174 and used the major outer membrane protein (MOMP) of Chlamydia abortus (C. abortus) as a model antigen to explore the optimal display location of exogenous antigens in BGs. We demonstrated that the constructed recombinant temperature-controlled lysis plasmid can still stably inhibit E gene expression at 37°C, and the lysis efficiency of E. coli can reach above 99.9%. Four recombinant MOMP Escherichia coli (E. coli) ghost vaccines were constructed using different anchor sequences. These vaccines all induced strong specific antibody responses and secrete high levels of IFN-γ in immunized mice and significantly increased the clearance of C. abortus in a mouse infection model. Notably, the strongest immune effect was observed when MOMP was displayed on the surface of E. coli ghosts (rECG-InpN-M), which resulted in the clearance of C. abortus in mice 6 days earlier than that with the recombinant MOMP vaccine. Altogether, we constructed an efficient BG temperature-controlled lysis system and provided a feasible strategy for developing a BG delivery platform with enhanced immune effects.

Chlamydia muridarumrecombinant MOMP nanovaccine induces cross-reactive antibodies against Chlamydia trachomatishuman serovars

Abstract Chlamydia trachomatis (CT), caused by various serovars, remains the leading sexually transmitted bacterial infection worldwide. An efficient delivery system is critical to developing whole or subunit vaccines against CT, and some vaccine developmental efforts are targeting biodegradable nanoparticles-based vaccines with encapsulated antigens. We developed a nanovaccine employing the recombinant major outer membrane protein (rMOMP) of C. muridarum (Cm) encapsulated in poly(lactic acid-co-glycolic acid) 85/15 (PLGA-rMOMP). We previously compared the humoral responses of two priming routes, subcutaneous (SC) or intramuscular (IM-p), followed by two SC-boosts to evaluate rMOMP-specific serum antibodies [IgG2a, IgG2b and IgG1] produced by PLGA-rMOMP immunization of female BALB/c mice. Here we used an elementary body (EB)-specific ELISA to investigate the ability of systemic antibodies produced in immunized mice to recognize Cm and to cross-recognize human CT-serovars D, F and J. Serum from mice inoculated with PBS encapsulated PLGA served as negative control. We observed high recognition of Cm specific total IgG (8-fold) and isotypes IgG2a (8-fold), IgG2b (32-fold), IgG1 (8-fold) by SC in comparison to IM-p immunizations. We also observed IgG antibodies recognizing CT-serovars D, F and J from mice immunized via both routes. Evaluation of the Th1 (IgG2a, IgG2b)/Th2 (IgG1) antibody titer ratios revealed that immunization via the SC route induced predominantly Th1 antibodies recognizing both Cm and human CT-serovars. Our data show that mice immunized with PLGA-rMOMP produced high levels of systemic antibodies recognizing Cm, but more importantly, there was a robust serological cross-recognition of the human CT-serovars. This research was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number NIH-R21AI111159, NIH-NIGMS-RISE (1R25GM106995-01) and the National Science Foundation (NSF)-CREST (HRD-1241701) and NSF-HBCU-RISE (HRD-1646729) grants.

Expression of recombinant Omp18 and MOMP of Campylobacter jejuni and the determination of their suitability as antigens for serological diagnosis of campylobacteriosis in animals.

Campylobacteriosis causes gastrointestinal tract lesions in adults and children and may result in severe complications. The primary sources of infection are infected animals and animal products. Immunochemical methods effectively diagnose intestinal infections but require highly specific antigens to detect their antibodies. This study aimed to obtain two recombinant immunogenic antigens of Campylobacter jejuni, an outer membrane protein with a molecular weight of 18 kDa (Omp18) and the major outer membrane protein (MOMP) with a molecular weight of 45 kDa, and evaluate their suitability for the serological diagnosis of campylobacteriosis using immunochromatographic assay (ICA). The C. jejuni Omp18 and MOMP gene sequences were synthesized de novo (Macrogen, Korea) and cloned into the pET32 expression plasmid. Using these genetic constructs, electrocompetent cells of the Escherichia coli BL21 strain were transformed and cultured under various conditions. Antigens were purified and refolded using metal affinity chromatography. The properties of the purified proteins were studied by western blotting, liquid chromatography with tandem mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). We developed two recombinant E. coli BL21 cells producing rOmp18 and Recombinant MOMP (rMOMP) antigens with molecular weights of 36 and 64 kDa, respectively. Amino acid sequence analysis of the obtained antigens showed complete homology with the reference sequences in the PubMed NCBI database. Western blotting using positive-control sera demonstrated the specificity of the recombinant antigens. The results of ELISA with 94 bovine sera showed the interaction of recombinant antigens with specific antibodies. The obtained rOmp18 and rMOMP antigens can detect antibodies in the serum of infected or recovered animals and can be used to develop ICA.

A recombinant Chlamydia muridarum MOMP encapsulated PLGA 85/15 nanovaccine induced antibodies elicited cross-recognition of serovar-D elementary bodies

Abstract Chlamydia trachomatis (Ct), caused by various serovars, remains the leading sexually transmitted bacterial infection worldwide. An efficient delivery system is critical to developing whole or subunit vaccines against Ct, and vaccine developmental efforts are rapidly targeting biodegradable nanoparticles based vaccines with encapsulated antigens. We have developed a nanovaccine employing the recombinant major outer membrane protein (rMOMP) of C. muridarum (Cm) encapsulated in poly (lactic acid-co-glycolic acid) (PLGA) 85/15 (PLGA-rMOMP). We previously compared the humoral responses of priming routes; subcutaneous (SC) or intramuscular (IM-p) followed by two sc-boosts to evaluate rMOMP specific serum antibodies [Th1 (IgG2a, IgG2b), Th2 (IgG1)] produced by PLGA-rMOMP immunization of female BALB/c mice. Here we explored the ability of systemic antibodies produced in immunized mice to recognize UV-inactivated whole elementary bodies (EB) of Cm and one of the prevalent human serovar D (Ct-D) using antigen-specific ELISA. Serum from phosphate-buffered saline (PBS) or PBS encapsulated (PLGA-PBS) groups of mice served as a negative control to validate the EB recognition by immunized sera. We observed higher EB specific total IgG (4-fold) and isotypes IgG2b (16-fold), IgG1 (2-fold) antibodies recognizing Ct-D compared to Cm for the SC and IM-p immunization groups. Evaluation of the Th1 (IgG2b)/Th2 (IgG1) antibody titer ratio revealed a higher Th1 antibody recognition of Cm (4-fold) and Ct-D (16-fold), for the SC and IM-p immunization routes. Our data show that mice immunized with PLGA-rMOMP produced Th1 systemic antibodies against EB, but more importantly, there was robust serological cross recognition of the human serovar D. This research was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R21AI111159, NIH-NIGMS-RISE (1R25GM106995-01) and the National Science Foundation (NSF)-CREST (HRD-1241701) and NSF-HBCU-RISE (HRD-1646729) grants.

Cell-free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development.

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (<1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

Encapsulation of Recombinant MOMP in Extended-Releasing PLGA 85:15 Nanoparticles Confer Protective Immunity Against a Chlamydia muridarum Genital Challenge and Re-Challenge.

Recently we reported the immune-potentiating capacity of a Chlamydia nanovaccine (PLGA-rMOMP) comprising rMOMP (recombinant major outer membrane protein) encapsulated in extended-releasing PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. Here we hypothesized that PLGA-rMOMP would bolster immune-effector mechanisms to confer protective efficacy in mice against a Chlamydia muridarum genital challenge and re-challenge. Female BALB/c mice received three immunizations, either subcutaneously (SC) or intranasally (IN), before receiving an intravaginal challenge with C. muridarum on day 49 and a re-challenge on day 170. Both the SC and IN immunization routes protected mice against genital challenge with enhanced protection after a re-challenge, especially in the SC mice. The nanovaccine induced robust antigen-specific Th1 (IFN-γ, IL-2) and IL-17 cytokines plus CD4+ proliferating T-cells and memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) phenotypes in immunized mice. Parallel induction of antigen-specific systemic and mucosal Th1 (IgG2a, IgG2b), Th2 (IgG1), and IgA antibodies were also noted. Importantly, immunized mice produced highly functional Th1 avidity and serum antibodies that neutralized C. muridarum infectivity of McCoy fibroblasts in-vitro that correlated with their respective protection levels. The SC, rather than the IN immunization route, triggered higher cellular and humoral immune effectors that improved mice protection against genital C. muridarum. We report for the first time that the extended-releasing PLGA 85:15 encapsulated rMOMP nanovaccine confers protective immunity in mice against genital Chlamydia and advances the potential towards acquiring a nano-based Chlamydia vaccine.

Generation of a novel affibody molecule targeting Chlamydia trachomatis MOMP

Chlamydia trachomatis (C. trachomatis) is the leading cause of preventable blindness worldwide and the most prevalent cause of bacterial sexually transmitted diseases. At present, there is no available vaccine, and recurrences after antibiotics treatment are substantial problems. Major outer membrane protein (MOMP) accounts for 60% of the outer mass of C. trachomatis, functioning as trimeric porin, and it is highly antigenic. Therefore, MOMP is the most promising candidate for vaccine developing and target therapy of Chlamydia. Affibody, a new class of affinity ligands derived from the Z-domain in the binding region of Staphylococcus aureus protein A, has been the focus of researchers as a viable alternative to antibodies. In this study, the MOMP-targeted affibody molecule (ZMOMP:461) was screened by phage-displayed peptide library. Further, the affinity and specificity were characterized by surface plasmon resonance (SPR) and Western blot. Immunofluorescence assay (IFA) indicated that the MOMP-binding affibody could recognize native MOMP in HeLa229 cells infected C. trachomatis. Immunoprecipitation assay confirmed further that ZMOMP:461 molecule specifically recognizes the epitope on relaxed trimer MOMP. Our findings provide strong evidence that affibody molecule (ZMOMP:461) serves as substitute for MOMP antibody for biological applications and has a great potential for delivering drugs for target therapy.Key points• We screened a novel affibody molecule ZMOMP:461 targeting Chlamydia trachomatis MOMP.• ZMOMP:461 recognizes the recombinant and native MOMP with high affinity and specificity.• ZMOMP:461 could be internalized into live target cells.

Detection and identification of adhesins involved in adhesion of Campylobacter jejuni to chicken skin

Campylobacter jejuni is the leading cause of bacterial food poisoning worldwide. Chickens are considered to be one of the major reservoirs of Campylobacter infection in humans due to colonization of their intestinal tract. When the chickens are slaughtered and processed, the entire skin of the carcass becomes contaminated with campylobacters. We observed that the number of C. jejuni attached to chicken skin was reduced significantly after treatment of the skin with sodium hydroxide followed by washing with PBS, implying that adhesion factors involved in binding to C. jejuni may exist on skin. Such potential binding-related proteins present in alkaline extracts of the skin surface were detected by a two-dimensional overlay assay and identified by liquid chromatography mass spectrometry (LC-MS). Chicken serum albumin (CSA) was identified as a major protein in these alkaline extracts and confirmed by ELISA to bind specifically to C. jejuni. Moreover, using the same approach, flagellar hook protein E (FlgE) and major outer membrane protein (MOMP) in C. jejuni were identified as bacterial adhesins that bound to the CSA. The ability to bind CSA was also confirmed using recombinant FlgE and MOMP of C. jejuni expressed in Escherichia coli. The present findings suggest that adhesins expressed on C. jejuni cells may bind specifically via proteins present on the skin, as well as by physical attachment.

Vaccination with the recombinant major outer membrane protein elicits long-term protection in mice against vaginal shedding and infertility following a Chlamydia muridarum genital challenge

Implementation of a vaccine is likely the best approach to curtail Chlamydia trachomatis infections. The aim of this study was to determine the ability of a vaccine formulated with the recombinant major outer membrane protein (MOMP) and Th1 and Th2 adjuvants, delivered by combinations of systemic and mucosal routes, to elicit long-term protection in mice against a genital challenge with Chlamydia muridarum. As a negative control, mice were vaccinated with the recombinant Neisseria gonorrhoeae porinB, and the positive control group was immunized with C. muridarum live elementary bodies (EB). The four vaccines formulated with MOMP, as determined by the titers of IgG and neutralizing antibodies in serum, proliferative responses of T-cells stimulated with EB and levels of IFN-γ in the supernatants, elicited robust humoral and cellular immune responses over a 6-month period. Groups of mice were challenged genitally at 60, 120, or 180 days postimmunization. Based on the number of mice with positive vaginal cultures, number of positive cultures, length of time of shedding, and number of inclusion forming units recovered, MOMP vaccinated groups were significantly protected. To assess fertility, when the vaginal cultures became negative, female mice were caged with male mice and the outcome of the pregnancy evaluated. As determined by the number of pregnant mice and the number of embryos, two of the vaccine formulations protected mice up to 180 days postimmunization. To our knowledge this is the first subunit of Chlamydia vaccine that has elicited in mice significant long-term protection against a genital challenge.

A nanovaccine formulation of Chlamydia recombinant MOMP encapsulated in PLGA 85:15 nanoparticles augments CD4+ effector (CD44high CD62Llow) and memory (CD44high CD62Lhigh) T-cells in immunized mice

Vaccine developmental strategies are utilizing antigens encapsulated in biodegradable polymeric nanoparticles. Here, we developed a Chlamydia nanovaccine (PLGA-rMOMP) by encapsulating its recombinant major outer membrane protein (rMOMP) in the extended-releasing and self-adjuvanting PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. PLGA-rMOMP was small (nanometer size), round and smooth, thermally stable, and exhibited a sustained release of rMOMP. Stimulation of mouse primary dendritic cells (DCs) with PLGA-rMOMP augmented endosome processing, induced Th1 cytokines (IL-6 and IL-12p40), and expression of MHC-II and co-stimulatory (CD40, CD80, and CD86) molecules. BALB/c mice immunized with PLGA-rMOMP produced enhanced CD4+ T-cells-derived memory (CD44high CD62Lhigh), and effector (CD44high CD62Llow) phenotypes and functional antigen-specific serum IgG antibodies. In vivo biodistribution of PLGA-rMOMP revealed its localization within lymph nodes, suggesting migration from the injection site via DCs. Our data provide evidence that the PLGA (85:15) nanovaccine activates DCs and augments Chlamydia-specific rMOMP adaptive immune responses that are worthy of efficacy testing.

Chlamydia recombinant MOMP encapsulated in PLGA 85/15 nanoparticles admixed with Escherichia coli dmLT mucosal adjuvant enhanced protection against a genital challenge in subcutaneously immunized BALB/c mice

Abstract There is an urgent need to develop a vaccine against Chlamydia trachomatis, the most reported bacterial-sexually transmitted infection globally. We developed a Chlamydia nanovaccine by employing PLGA [poly (D, L-lactic-co-glycolic acid) 85/15 nanoparticles encapsulated with recombinant MOMP (major outer membrane protein) of Chlamydia](named PLGA-rMOMP) that partially protected mice against a genital challenge. Here we hypothesized that admixing PLGA-rMOMP with the Escherichia coli dmLT (double-mutant heat-labile enterotoxin (LT)-R192G/L211A) mucosal adjuvant will improve the protective efficacy. Six groups of BALB/c mice were subcutaneously immunized three-times at two-week intervals with various immunogens and then challenged intravaginally with 105 inclusions forming units (IFUs) of C. muridarum. Vaginal swabs were collected every three days up to three weeks from all mice and analyzed by indirect immunofluorescence to compare the IFUs between groups of mice. Our results showed maximal protection provided in mice immunized with PLGA-rMOMP admixed with dmLT evidently by their lower IFUs as compared with the other mice groups. Moreover, the PLGA-rMOMP adjuvanted mice produced elevated MOMP-specific serum IgG2b (Th1) as compared to IgG1 (Th2) antibodies, which is indicative of an effective Th1 immune response. Interestingly splenic T-cells from these mice also produced enhanced IL-17 and IL-2 MOMP-specific cytokines after the genital challenge in comparison to the other groups of mice. Our results indicate that the E. coli dmLT mucosal adjuvant improves the immunogenicity and the protective efficacy of the PLGA-rMOMP nanovaccine against a genital challenge in mice.

Protection of outbred mice against a vaginal challenge by a Chlamydia trachomatis serovar E recombinant major outer membrane protein vaccine is dependent on phosphate substitution in the adjuvant

ABSTRACT Chlamydia trachomatis is the most common bacterial sexually-transmitted pathogen for which there is no vaccine. We previously demonstrated that the degree of phosphate substitution in an aluminum hydroxide adjuvant in a TLR-4-based C. trachomatis serovar E (Ser E) recombinant major outer membrane protein (rMOMP) formulation had an impact on the induced antibody titers and IFN-γ levels. Here, we have extended these observations using outbreed CD-1 mice immunized with C. trachomatis Ser E rMOMP formulations to evaluate the impact on bacterial challenge. The results confirmed that the rMOMP vaccine containing the adjuvant with the highest phosphate substitution induced the highest neutralizing antibody titers while the formulation with the lowest phosphate substitution induced the highest IFN-γ production. The most robust protection was observed in mice vaccinated with the formulation containing the adjuvant with the lowest phosphate substitution, as shown by the number of mice with positive vaginal cultures, number of positive cultures and number of C. trachomatis inclusion forming units recovered. This is the first report showing that vaccination of an outbred strain of mice with rMOMP induces protection against a vaginal challenge with C. trachomatis.

Type I IFN signalling is required for cationic adjuvant formulation (CAF)01-induced cellular immunity and mucosal priming

Despite being in the midst of a global pandemic of infections caused by the pathogen Chlamydia trachomatis, a vaccine capable of inducing protective immunity remains elusive. Given the C. trachomatis mucosal port of entry, a formulation compatible with mucosal administration and capable of eliciting potent genital tract immunity is highly desirable. While subunit vaccines are considered safer and better tolerated, these are typically poorly immunogenic and require co-formulation with immune-potentiating adjuvants. However, of the adjuvants licensed for use in humans, very few drive robust cellular responses, a pre-requisite for protection against C. trachomatis infection. Recently, the cationic adjuvant formulations (CAF) have been shown to induce robust humoral and cellular immunity in pre-clinical models of chlamydia, malaria and tuberculosis (TB). Here, we demonstrate that CAF01 induces potent immune responses when combined with the major outer membrane protein (MOMP) of C. trachomatis following parenteral immunisation and also as part of a heterologous prime/boost regime. We show that a subcutaneous prime with CAF01-adjuvanted recombinant MOMP licenses antigen-specific immunity at distant mucosal sites which can be activated following oral antigen re-encounter in the absence of concomitant adjuvant stimulation. Finally, we shed light on the mechanism(s) through which CAF01 elicits robust antigen-specific immunity to co-formulated MOMP via type I interferon (IFN) signalling.

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

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. 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. 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). This is the first subunit vaccine shown to protect mice against infection, pathology, and infertility caused by different C. trachomatis serovars.

PLGA-encapsulated Chlamydia recombinant MOMP enhances serum and mucosal Th1 antibodies including memory and effector T-cells via subcutaneous prime-boost as compared to the intramuscular priming route immunization of mice

Abstract Vaccine developmental efforts are rapidly targeting biodegradable nanoparticles-based vaccines with encapsulated antigen. We have developed a PLGA 85/15 (poly (lactic-co-glycolic acid) Chlamydia nanovaccine by encapsulating its recombinant MOMP (major outer membrane protein) that induced robust adaptive immune responses and provided enhanced protection against C. muridarum (Cm) challenge in subcutaneously immunized mice. In the current study, we compared the immunogenicity and efficacy of priming routes; subcutaneous (sc) or intramuscular (im) followed by two sc-boosts to evaluate serum and mucosal antibodies [Th1 (IgG2a, IgG2b), Th2 (IgG1)] as well as memory and effector T-cells activation after immunization and Cm challenge of mice. We observed significantly high levels of MOMP-specific serum and mucosal antibodies with high avidity especially for Th1 antibodies. A significant increase in Th1 serum and mucosal antibodies with even higher avidity was observed after genital tract Cm challenge in the sc-prime-boost immunized mice. Evaluation of the Th1/Th2 antibody titer ratio revealed that the nanovaccine evoked a higher Th1 and lower Th2 response, which skewed to a dominated Th1 antibody response after bacterial exposure to sc-prime mice. In addition, CD4+ memory (CD44highCD62Lhigh) and effector (CD44highCD62Llow) T-cells proliferation was about two-fold higher in the sc-prime mice. Herein, we reveal that the subcutaneous prime-boost immunization with the nanovaccine was more effective in enhancing the Th1 antibody response and memory and effector T-cell responses against a chlamydial genital tract challenge of mice.

Obtaining an ELISA test based on a recombinant protein of Chlamydia trachomatis.

Chlamydia trachomatis is considered as a public health problem due to its high prevalence and increased rates of gynecological disorders. The major outer membrane protein (MOMP) of this bacterium is the most abundant protein in its membrane and has been evaluated not only as a vaccine development candidate but also is used in many diagnostic tests. The MOMP weighs 69kDa and contains four variable segments (VS 1-4) separated by constant regions. Several research groups have developed recombinant single-variable segments of MOMP expressed in Escherichia coli cytoplasm. But, all variable segments have been used minimally for the diagnosis of a chlamydial infection. In this experiment, the authors obtained the recombinant MOMP of C. trachomatis (rMOMP) in E. coli rMOMP and extracted, purified, and partially characterized it. This was later used to identify anti-Chlamydia trachomatis antibodies in sera of infertile patients by immunodetection assays, enzyme-linked immunosorbent assay (ELISA), and indirect immunofluorescence tests. The ELISA test showed high sensitivity and low specificity of 100 and 58.3%, respectively. The above results obtained were linked to the cross-reactivity of antibodies against C. pneumoniae or C. psittaci. Hence, an evaluation was performed to obtain an optimized test for the diagnosis of C. trachomatis infection.

Therapeutic effect of a Chlamydia pecorum recombinant major outer membrane protein vaccine on ocular disease in koalas (Phascolarctos cinereus).

Chlamydia pecorum is responsible for causing ocular infection and disease which can lead to blindness in koalas (Phascolarctos cinereus). Antibiotics are the current treatment for chlamydial infection and disease in koalas, however, they can be detrimental for the koala’s gastrointestinal tract microbiota and in severe cases, can lead to dysbiosis and death. In this study, we evaluated the therapeutic effects provided by a recombinant chlamydial major outer membrane protein (MOMP) vaccine on ocular disease in koalas. Koalas with ocular disease (unilateral or bilateral) were vaccinated and assessed for six weeks, evaluating any changes to the conjunctival tissue and discharge. Samples were collected pre- and post-vaccination to evaluate both humoral and cell-mediated immune responses. We further assessed the infecting C. pecorum genotype, host MHC class II alleles and presence of koala retrovirus type (KoRV-B). Our results clearly showed an improvement in the clinical ocular disease state of all seven koalas, post-vaccination. We observed increases in ocular mucosal IgA antibodies to whole C. pecorum elementary bodies, post-vaccination. We found that systemic cell-mediated immune responses to interferon-γ, interleukin-6 and interleukin-17A were not significantly predictive of ocular disease in koalas. Interestingly, one koala did not have as positive a clinical response (in one eye primarily) and this koala was infected with a C. pecorum genotype (E’) that was not used as part of the vaccine formula (MOMP genotypes A, F and G). The predominant MHC class II alleles identified were DAb*19, DAb*21 and DBb*05, with no two koalas identified with the same genetic sequence. Additionally, KoRV-B, which is associated with chlamydial disease outcome, was identified in two (29%) ocular diseased koalas, which still produced vaccine-induced immune responses and clinical ocular improvements post-vaccination. Our findings show promise for the use of a recombinant chlamydial MOMP vaccine for the therapeutic treatment of ocular disease in koalas.

Vaccination of koalas (Phascolarctos cinereus) against Chlamydia pecorum using synthetic peptides derived from the major outer membrane protein.

Chlamydia pecorum is a mucosal infection, which causes debilitating disease of the urinary tract, reproductive tract and ocular sites of koalas (Phascolarctos cinereus). While antibiotics are available for treatment, they are detrimental to the koalas’ gastrointestinal tract microflora leaving the implementation of a vaccine as an ideal option for the long-term management of koala populations. We have previously reported on the successes of an anti-chlamydial recombinant major outer membrane protein (rMOMP) vaccine however, recombinant protein based vaccines are not ideal candidates for scale up from the research level to small-medium production level for wider usage. Peptide based vaccines are a promising area for vaccine development, because peptides are stable, cost effective and easily produced. In this current study, we assessed, for the first time, the immune responses to a synthetic peptide based anti-chlamydial vaccine in koalas. Five healthy male koalas were vaccinated with two synthetic peptides derived from C. pecorum MOMP and another five healthy male koalas were vaccinated with full length recombinant C. pecorum MOMP (genotype G). Systemic (IgG) and mucosal (IgA) antibodies were quantified and pre-vaccination levels compared to post-vaccination levels (12 and 26 weeks). MOMP-peptide vaccinated koalas produced Chlamydia-specific IgG and IgA antibodies, which were able to recognise not only the genotype used in the vaccination, but also MOMPs from several other koala C. pecorum genotypes. In addition, IgA antibodies induced at the ocular site not only recognised recombinant MOMP protein but also, whole native chlamydial elementary bodies. Interestingly, some MOMP-peptide vaccinated koalas showed a stronger and more sustained vaccine-induced mucosal IgA antibody response than observed in MOMP-protein vaccinated koalas. These results demonstrate that a synthetic MOMP peptide based vaccine is capable of inducing a Chlamydia-specific antibody response in koalas and is a promising candidate for future vaccine development.