Native Major Outer Membrane Protein Research Articles

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.

Improved protection against Chlamydia muridarum using the native major outer membrane protein trapped in Resiquimod-carrying amphipols and effects in protection with addition of a Th1 (CpG-1826) and a Th2 (Montanide ISA 720) adjuvant

A new vaccine formulated with the Chlamydia muridarum native major outer membrane protein (nMOMP) and amphipols was assessed in an intranasal (i.n.) challenge mouse model. nMOMP was trapped either in amphipol A8-35 (nMOMP/A8-35) or in A8-35 conjugated with Resiquimod (nMOMP/Resiq-A8-35), a TLR7/8 agonist added as adjuvant. The effects of free Resiquimod and/or additional adjuvants, Montanide ISA 720 (TLR independent) and CpG-1826 (TLR9 agonist), were also evaluated. Immunization with nMOMP/A8-35 alone administered i.n. was used as negative adjuvant-control group, whereas immunizations with C. muridarum elementary bodies (EBs) and MEM buffer, administered i.n., were used as positive and negative controls, respectively. Vaccinated mice were challenged i.n. with C. muridarum and changes in body weight, lungs weight and recovery of Chlamydia from the lungs were evaluated. All the experimental groups showed protection when compared with the negative control group. Resiquimod alone produced weak humoral and cellular immune responses, but both Montanide and CpG-1826 showed significant increases in both responses. The addition of CpG-1826 alone switched immune responses to be Th1-biased. The most robust protection was elicited in mice immunized with the three adjuvants and conjugated Resiquimod. Increased protection induced by the Resiquimod covalently linked to A8-35, in the presence of Montanide and CpG-1826 was established based on a set of parameters: (1) the ability of the antibodies to neutralize C. muridarum; (2) the increased proliferation of T-cells in vitro accompanied by higher production of IFN-γ, IL-6 and IL-17; (3) the decreased body weight loss over the 10 days after challenge; and (4) the number of IFUs recovered from the lungs at day 10 post challenge. In conclusion, a vaccine formulated with the C. muridarum nMOMP bound to amphipols conjugated with Resiquimod enhances protective immune responses that can be further improved by the addition of Montanide and CpG-1826.

A Novel LNP-Based <i>Chlamydia</i> Subunit Vaccine Formulation That Induces Th1 Responses without Upregulating IL-17 Provides Equivalent Protection in Mice as Formulations That Induced IL-17 and Th1 Cytokines

We evaluated novel Chlamydial vaccines, consisting of major outer membrane protein (MOMP) alone or in combination with polymorphic membrane proteins D (PmpD) and G (PmpG) using a C57BL/6 mouse model. Native MOMP (nMOMP) isolated from C. muridarum elementary bodies (EBs) and recombinant PmpD and PmpG proteins were adjuvanted with Monophosphoryl lipid A (MPLA), with either lipid nanoparticles (LNPs) or the cationic lipid dimethyldioctadecylammonium bromide (DDA). Antibody titers to C. muridarum nMOMP, and EBs were evaluated by ELISA, and T-cell responses were analyzed by intracellular cytokine staining (ICS). Protection from challenge was determined by qPCR. Vaccine immunized mice showed significantly higher antibody titers to nMOMP (P C. muridarum EBs (P C. muridarum EBs and PmpG. ICS analysis showed more robust CD4 + T-cell responses (IFN-γ/IL-2/TNF-a) in the DDA and LNP groups compared to the adjuvant alone group. The DDA + MPLA gave robust Th17 responses in comparison to MPLA and LNP group. Mice immunized with Chlamydia antigens also showed protection from C. muridarum challenge, by reduction in bacterial shedding for all groups (P < 0.003) compared to shedding from the adjuvant control. Both vaccine formulations generated robust immunological responses, and both were protective by reducing bacterial shedding after challenge. This data indicates equal protection can be achieved without the induction of Th17 responses.

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation.

Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. Chlamydia trachomatis Serovars D–K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available. MOMP accounts for ~ 60% of the chlamydial protein mass and is considered to be one of the lead vaccine candidates against C. trachomatis. We report on the spectroscopic analysis of C. trachomatis native MOMP Serovars D, E, F, and J as well as C. muridarum MOMP by size exclusion chromatography multi angle light scattering (SEC MALS), circular dichroism (CD) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR‐FTIR). MOMP was purified from the native bacterium grown in either adherent HeLa cells or in different suspension cell lines. Our results confirm that MOMP forms homo‐trimers in detergent micelles. The secondary structure composition of C. trachomatis MOMP was conserved across serovars, but different from composition of C. muridarum MOMP with a 13% (CD) to 18% (ATR‐FTIR) reduction in β‐sheet conformation for C. trachomatis MOMP. When Serovar E MOMP was isolated from suspension cell lines the α‐helix content increased by 7% (CD) to 13% (ATIR‐FTIR). Maintenance of a native‐like tertiary and quaternary structure in subunit vaccines is important for the generation of protective antibodies. This biophysical characterization of MOMP presented here serves, in the absence of functional assays, as a method for monitoring the structural integrity of MOMP.

Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, and protein misfolding. Here, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP-tNLP). The cell-free expressed mMOMP-tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP-tNLP complex in a 1-ml cell-free reaction. The mMOMP-tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP-tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.

Recombinant expression of Chlamydia trachomatis major outer membrane protein in E. Coli outer membrane as a substrate for vaccine research.

BackgroundChlamydia trachomatis is a human pathogen which causes a number of pathologies, including genital tract infections in women that can result in tubal infertility. Prevention of infection and disease control might be achieved through vaccination; however, a safe, efficacious and cost-effective vaccine against C. trachomatis infection remains an unmet medical need. C. trachomatis major outer membrane protein (MOMP), a β-barrel integral outer membrane protein, is the most abundant antigen in the outer membrane of the bacterium and has been evaluated as a subunit vaccine candidate. Recombinant MOMP (rMOMP) expressed in E. coli cytoplasm forms inclusion bodies and rMOMP extracted from inclusion bodies results in a reduced level of protection compared to the native MOMP in a mouse challenge model.ResultsWe sought to target the recombinant expression of MOMP to the E. coli outer membrane (OM). Successful surface expression was achieved with codon harmonization, utilization of low copy number vectors and promoters with moderate strength, suitable leader sequences and optimization of cell culture conditions. rMOMP was extracted from E. coli outer membrane, purified, and characterized biophysically. The OM expressed and purified rMOMP is immunogenic in mice and elicits antibodies that react to the native antigen, Chlamydia elementary body (EB).ConclusionsC. trachomatis MOMP was functionally expressed on the surface of E. coli outer membrane. The OM expressed and purified rMOMP elicits antibodies that react to the native antigen, Chlamydia EB, in a mouse immunogenicity model. Surface expression of MOMP could provide useful reagents for vaccine research, and the methodology could serve as a platform to produce other outer membrane proteins recombinantly.

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.

Vaccination with major outer membrane protein proteosomes elicits protection in mice against a Chlamydia respiratory challenge

Vaccines formulated with the Chlamydia muridarum native major outer membrane protein (nMOMP) have so far been shown to elicit the most robust protection against this pathogen. nMOMP is a membrane protein and therefore, detergents are used to keep it in solution. Detergents however, have toxic effects. To address this limitation, we tested a nMOMP proteosome vaccine and compared its ability to elicit protection against nMOMP solubilized in the detergent Z3-14. The two preparations were formulated with or without CpG + Montanide (C/M). As a control antigen we used ovalbumin. Mice vaccinated with nMOMP developed strong humoral and cell mediated Chlamydia-specific immune responses. Based on the IgG2a/IgG1 levels in serum and amounts of IFN-γ in splenocytes supernatants the immune responses were predominantly Th1-biased. The animals were subsequently challenged intranasally with 2 × 103Chlamydia inclusion forming units (IFU) and the course of the infection was followed for 10 days when the mice were euthanized. Based on changes in body weight, weight of the lungs and number of IFU recovered from the lungs, mice immunized with nMOMP-Ps and nMOMP + Z3-14 adjuvanted with C/M showed the most robust protection. In summary, nMOMP-Ps should be considered as Chlamydia vaccine candidates.

Amphipols stabilize the Chlamydia major outer membrane protein and enhance its protective ability as a vaccine

The native major outer membrane protein (nMOMP) from Chlamydia was purified in its trimeric form using the zwitterionic detergent Z3-14. In aliquots from this preparation, Z3-14 was exchanged for amphipol (APol) A8-35. CD analysis showed that trapping with A8-35 improved the thermostability of nMOMP without affecting its secondary structure. Recombinant MOMP (rMOMP) was also formulated with Z3-14 or A8-35. Four groups of mice were vaccinated with nMOMP/Z3-14, nMOMP/A8-35, rMOMP/Z3-14 or rMOMP/A8-35 using CpG and Montanide as adjuvants. A positive control group was inoculated intranasally with live Chlamydia and a negative control group with culture medium. Mice were challenged intranasally with live Chlamydia and protection was assessed based on changes in body weight, the weight of the lungs and the number of chlamydial inclusion forming units recovered from the lungs 10 days after the challenge. Overall, vaccines formulated with nMOMP elicited better protection than those using rMOMP. Furthermore, the protection afforded by nMOMP/A8-35 was more robust than that achieved with nMOMP/Z3-14. In contrast, no differences in protection were observed between rMOMP/Z3-14 and rMOMP/A8-35 preparations. These findings suggest that the higher protection conferred by nMOMP/A8-35 complexes most likely results from a better preservation of the native structure of MOMP and/or from a more efficient presentation of the antigen to the immune system, rather than from an adjuvant effect of the amphipol. Thus, amphipols can be used in vaccine formulations to stabilize a membrane-protein component and enhance its immunogenicity.

Immunogenicity of a vaccine formulated with the Chlamydia trachomatis serovar F, native major outer membrane protein in a nonhuman primate model

To determine the ability of a vaccine formulated with the genital Chlamydia trachomatis, serovar F, native major outer membrane protein (Ct-F-nMOMP), to induce systemic and mucosal immune responses, rhesus macaques ( Macaca mulatta) were immunized three times by the intramuscular (i.m.) and subcutaneous (s.c.) routes using CpG-2395 and Montanide ISA 720 VG, as adjuvants. As controls, another group of M. mulatta was immunized with ovalbumin instead of Ct-F-nMOMP using the same formulation and routes. High levels of Chlamydia-specific IgG and IgA antibodies were detected in plasma, vaginal washes, tears, saliva, and stools from the Ct-F-nMOMP immunized animals. Also, high neutralizing antibody titers were detected in the plasma from these animals. Monkeys immunized with ovalbumin had no detectable Chlamydia-specific antibodies. Furthermore, as measured by a lymphoproliferative assay, significant Chlamydia-specific cell-mediated immune responses were detected in the peripheral blood mononuclear cells (PBMC) from the rhesus macaques vaccinated with Ct-F-nMOMP when compared with the animals immunized with ovalbumin. In addition, the levels of two Th1 cytokines, IFN-γ and TNF-α, were significantly higher in the animals immunized with Ct-F-nMOMP when compared with those from the monkeys immunized with ovalbumin. To our knowledge, this is the first time that mucosal and systemic immune responses have been investigated in a nonhuman primate model using a subunit vaccine from a human genital C. trachomatis serovar.

Induction of protection in mice against a respiratory challenge by a vaccine formulated with the Chlamydia major outer membrane protein adjuvanted with IC31 ®

IC31 ®, a novel adjuvant, has been shown to be effective by increasing the levels of IFN-γ in animal models when delivered with several antigens. Here, we tested the ability of IC31 ®, to enhance the protective ability of the Chlamydia trachomatis native major outer membrane protein (nMOMP). BALB/c mice were immunized by the intramuscular (i.m.) and subcutaneous (s.c.) routes with nMOMP + IC31 ®. Another group of animals was immunized with nMOMP + Alum and as a negative control mice were immunized with ovalbumin (Ova) + IC31 ®. Animals immunized with nMOMP + IC31 ® developed high Chlamydia-specific IgG titers. The serum levels of IgG1 were higher than those of the IgG2a. T cells, from the spleens of mice immunized with IC31 ®-adjuvanted nMOMP demonstrated a strong lymphoproliferative reaction to Chlamydia elementary bodies (EB) compared with the groups immunized with nMOMP + Alum or Ova + IC31 ®. A similar comparison between these groups of mice revealed that the levels of IFN-γ in the supernatants from stimulated T-cells were significantly higher in animals immunized with nMOMP + IC31 ®. Following an intranasal challenge with C. trachomatis, the mice immunized with IC31 ®-adjuvanted nMOMP showed significant protection. The change in body weight, an indication of the severity of the infection, was significantly less reduced in mice immunized with nMOMP + IC31 ®. Furthermore, the weight of the lungs, as well as the pulmonary Chlamydia burden, was significantly lower in the animals immunized with nMOMP + IC31 ® when compared with the groups immunized with nMOMP + Alum or with Ova + IC31 ®. In conclusion, these results provide the rationale for further preclinical testing of IC31 ® using other chlamydial antigens, and support the potential evaluation of this adjuvant in human vaccines against Chlamydia.

Induction of protective immunity by vaccination against Chlamydia trachomatis using the major outer membrane protein adjuvanted with CpG oligodeoxynucleotide coupled to the nontoxic B subunit of cholera toxin

The present study was undertaken to test the efficacy of immunization with the native major outer membrane protein (nMOMP) of Chlamydia trachomatis mouse pneumonitis (MoPn) serovar in combination with a novel immunostimulatory adjuvant consisting of CpG oligodeoxynucleotide (ODN) linked to the nontoxic B subunit of cholera toxin (CTB-CpG) to elicit a protective immune response to C. trachomatis. High levels of Chlamydia-specific IgG antibodies were detected in the sera from BALB/c mice immunized intramuscularly and subcutaneously (i.m. + s.c.) with the nMOMP/CTB-CpG vaccine or with nMOMP adjuvanted with a mixture of CT and CpG ODN (CT + CpG). Further, these immunization schemes gave rise to significant T-cell-mediated Chlamydia-specific immune responses. No Chlamydia-specific humoral or cell-mediated immune responses were detected in the control mice vaccinated with ovalbumin together with either CTB-CpG or CT + CpG. Following an intranasal challenge with C. trachomatis the groups of mice immunized with nMOMP plus CTB-CpG, CT + CpG or live C. trachomatis were found to be protected based on their change in body weight and lung weight as well as number of inclusion forming unit recovered from the lungs, as compared with control groups immunized with ovalbumin plus either adjuvants. Interestingly, IFN-γ-producing CD4 +, but not CD8 +, T-cells showed a significant correlation with the outcomes of the challenge. In conclusion, nMOMP in combination with the novel adjuvant CTB-CpG elicited a significant antigen-specific antibody and cell-mediated immune responses as well as protection against a pulmonary challenge with C. trachomatis.

Infection-induced antibodies against the major outer membrane protein ofCampylobacter jejunimainly recognize conformational epitopes

The major outer membrane protein (MOMP) of Campylobacter jejuni is an abundant surface protein with a pore-forming function and may be a potential candidate for vaccine development. Despite the fact that MOMP is immunogenic and the recombinant MOMP (rMOMP) can be readily produced in Escherichia coli, the nature of the antibody response to MOMP during in vivo infection is not well understood. In this study, various methods involving detergent replacement and liposome reconstitution were used to refold rMOMP, and antibody responses to MOMP elicited in Campylobacter-colonized chickens were evaluated using sera from chickens either naturally or experimentally infected by C. jejuni. The results demonstrated that proteoliposomes restored the reactivity of rMOMP to rabbit antibodies elicited by native MOMP, indicating the recovery of native MOMP conformation by this refolding method. Importantly, sera from naturally or experimentally infected chickens reacted weakly with denatured rMOMP, but strongly with rMOMP reconstituted in proteoliposome, suggesting that the chicken antibody response to MOMP is predominantly directed against conformational epitopes. These observations provide direct evidence for conformation-dependent humoral responses to MOMP induced by Campylobacter infection, demonstrate that C. jejuni MOMP is immunogenic in its natural host and suggest that proteoliposomes may be potentially used for the evaluation of rMOMP-based vaccines.

Codon usage bias in Chlamydia trachomatis and the effect of codon modification in the MOMP gene on immune responses to vaccinationThis paper is one of a selection of papers in this Special Issue, entitled International Symposium on Recent Advances in Molecular, Clinical, and Social Medicine, and has undergone the Journal's usual peer-review process.

Chlamydia trachomatis is a kind of obligate intracellular bacterial pathogen that causes ocular and sexually transmitted diseases. In this study, we analyzed the codon usage patterns of the C. trachomatis mouse pneumonitis biovar (MoPn) and Homo sapiens. We found large differences between MoPn and human codon usages. To enhance the expression of Chlamydia protein in mammalian cells, the DNA sequence encoding the major outer-membrane protein (MOMP) of MoPn was modified to substitute the human-preferred codons for rarely used codons. The huma-optimized MOMP gene was synthesized and cloned into the pcDNA3 vector, as was the wild-type MOMP gene. The protein expression levels of the human-optimized MOMP and wild-type MOMP genes were compared. The experiments showed that the human-optimized MOMP gene produced significantly higher levels of MOMP protein than the wild-type MOMP, both in vitro and in vivo, but no obvious difference was observed in the levels of modified and native MOMP mRNA expression. The immunogenicity of the 2 constructs was examined using BALB/c mice following intramuscular immunization. The results showed that the mice immunized with the human-optimized MOMP produced higher levels of antigen-specific IgG antibody and showed stronger delayed-type hypersensitivity reactions and proliferative T cell responses than those immunized with the wild-type MOMP. Antigen-specific stimulation of spleen cells obtained from human MOMP DNA immunized mice produced higher levels of interferon-gamma than those obtained from wild-type MOMP DNA immunized mice. Taken together, the data show that human-optimized codon optimization can significantly enhance the gene expression and immunogenicity of the C. trachomatis MOMP DNA vaccine.

Immunization with the Chlamydia trachomatis major outer membrane protein, using adjuvants developed for human vaccines, can induce partial protection in a mouse model against a genital challenge

To test several vaccines for Chlamydia trachomatis we vaccinated BALB/c and C3H/HeN female mice with a purified preparation of the native mouse pneumonitis (MoPn) major outer membrane protein (MOMP). The MOMP was formulated with anyone of three different adjuvants MF59, LT-K63 or LT-R72. As a negative control the animals were immunized with ovalbumin. Positive controls were inoculated intranasally (i.n.) with 10 4 inclusion-forming units (IFU) of C. trachomatis MoPn. High levels of Chlamydia-specific antibodies were detected in the serum and vaginal washes of the mice immunized with MOMP. Using a lymphoproliferative assay (LPA) a significant response was obtained in splenocytes from most of the groups of mice vaccinated with MOMP. Two weeks after the last immunization the mice were challenged in the left ovarian bursa with 10 5 IFU of C. trachomatis MoPn and vaginal cultures were collected for a period of 6 weeks. Overall, BALB/c and C3H/HeN mice immunized with MOMP showed a decrease in the severity and length of the infection but the difference with the controls was not statistically significant. Following mating the percentage of mice with bilateral fertility was not significantly different between mice vaccinated with MOMP and their respective ovalbumin-immunized controls. However, the C3H/HeN mice immunized with MOMP using MF59 or LTR72 as adjuvants had significantly more embryos per mouse than the control groups. In conclusion, mice immunized with native MOMP and adjuvants developed for human vaccines showed significant Chlamydia-specific immune response and a limited protection against infection and long-term sequelae.

Molecular characterization of the major outer membrane protein of Haemophilus somnus

The major outer membrane protein (MOMP) of Haemophilus somnus shows antigenic and molecular mass diversity that forms the basis of a preliminary grouping system for H. somnus strains. In this study, the gene encoding MOMP of H. somnus strain 8025 was cloned in three overlapping fragments by PCR techniques, and then sequenced. The gene consists of a 1164-bp open reading frame encoding a deduced 380-amino acid protein with a 19-amino acid signal sequence, giving a mature protein with a calculated molecular mass of 39,913 Da. Significant homology was found between MOMP and porin protein sequences of bacteria in Pasteurellaceae species. When expressed in Escherichia coli, the protein from the MOMP gene directed by the T7 promoter was identical in size (approximately 40 kDa) to native MOMP and reacted with MOMP-specific antibodies. Comparisons of the MOMP gene sequences from six unrelated strains of H. somnus to that of strain 8025 revealed that the genes of three MOMP type 1 strains were highly conserved with that of strain 8025 in length and sequence. However, two MOMP type 3c strains and one MOMP type 3a strain differed markedly from the MOMP of strain 8025 in their 3'-terminal halves. Their deduced MOMP amino acid sequences differed in sequence (3c, 80.5 and 82.7% identity; 3a, 62.4% identity) and in length (3c, 384 and 376; 3a, 316), indicating that the molecular differences are the basis of antigenicity and molecular mass differences of H. somnus MOMP. In the predicted MOMP secondary structure, the variable sequences primarily mapped to putative surface-exposed loops, and a variable and surface-exposed epitope of MOMP-specific antibody was identified in the seventh-largest loop. These findings are useful for understanding the structural and immunological characteristics of H. somnus.

Design, expression and functional characterization of a synthetic gene encoding the Chlamydia trachomatis major outer membrane protein

A synthetic gene coding for the Chlamydia trachomatis serovar L2 major outer membrane protein (MOMP) was designed, constructed and expressed in Escherichia coli. The native amino acid sequence was reverse translated and the resulting nucleotide combinations manipulated in order to evenly distribute 25 unique restriction sites along the length of the gene while retaining the native amino acid sequence. The synthetic gene was cloned into a T7 promoter-controlled plasmid (pET-3a) and the expressed product was analyzed to assess antigenicity, cellular localization and function. Monoclonal antibodies specific for native MOMP reacted to the expressed product by immunoblot. Outer membrane fractionation confirmed that the processed protein was located in the outer membrane. MOMP expressed in E. coli and present in the outer membrane was shown to function as a general diffusion porin. This system provides the means to produce readily modifiable MOMP either in purified form or as a membrane-associated protein, and so facilitate the investigation of its functional, structural and antigenic properties.

Identification and characterization of T helper cell epitopes of the major outer membrane protein of Chlamydia trachomatis.

Chlamydia trachomatis serovars A, B, and C are the causative agents of trachoma, the world's leading cause of preventable blindness. Immunoprophylaxis is a possible approach to control trachoma. The chlamydial major outer membrane protein (MOMP) is thought to play an important role in the development of protective immunity against chlamydial infection, and is therefore considered to be a promising candidate antigen in the development of a trachoma vaccine. Much effort has been focused on the molecular characterization of B cell sites of the MOMP that elicit neutralizing antibodies. Neutralizing sites have been identified as linear epitopes that reside within variable domains (VDs) of the protein whose primary sequences vary among different serovars. No information exists on MOMP T helper (Th) cell antigenic determinants, which are likely critical components for the development of a successful chlamydial vaccine. We used overlapping synthetic peptides (25 mers) representing the entire primary sequence of serovar A MOMP in T cell proliferation assays to identify T cell antigenic determinants of this molecule. Eight synthetic peptides (A-2, A-3, A-7, A-8, A-11, A-22, A-23, and A-24) stimulated proliferative responses of splenic T cells isolated from MOMP-immunized A/J mice. To ascertain if these peptides functioned as Th cell antigens, we determined their ability to prime A/J mice in vivo to produce an anamnestic IgG response specific to the MOMP. Mice primed with synthetic peptides A-8 (106-130) or A-23 (331-355) produced IgG antibodies reactive with the native MOMP and with the synthetic peptides corresponding to surface-accessible serovar-specific epitopes located in VD I and serogroup-specific epitopes located in VD IV of the protein. We synthesized the A-8 and A-23 peptides with the VD I sequence as colinear chimeric peptides. Immunization of mice with the T/B cell peptides produced high titered antibodies against the VD I sequence, and these antibodies reacted with the native MOMP and intact chlamydiae. The MOMP sequences containing these Th cell epitopes are conserved among the MOMP genes of different C. trachomatis serovars, indicating that they are common Th cell antigenic sites. Thus, the Th cell epitopes contained within these peptides, in combination with different trachoma serovar-specific B cell neutralizing determinants, may be useful in the development of a synthetic or recombinant trivalent trachoma vaccine.

Surface-exposed Epitopes on the Major Outer-membrane Protein of Chlamydia trachomatis Defined with Peptide Antisera

The surface exposure of computer-predicted, linear B-cell epitopes on the major outer-membrane protein (MOMP) of Chlamydia trachomatis serovar B was assessed using antibodies raised against synthetic peptides in conjunction with immunogold transmission electron microscopy. Several of the chosen peptides elicited antibodies which reacted with both denatured and native MOMP. The majority of the exposed epitopes were found within the variable segments of MOMP. For each of the epitopes identified, the extent of their surface accessibility varied both among individual organisms and different developmental forms. Evidence for two distinct subspecies-specific epitopes within VS4 is presented.