Major Outer Membrane Protein Vaccine Research Articles

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.

Lack of neutralization of Chlamydia trachomatis infection by high avidity monoclonal antibodies to surface-exposed major outer membrane protein variable domain IV

Chlamydia trachomatis is the leading cause of sexually transmitted diseases causing frequent, long-lasting, and often asymptomatic recurrent infections resulting in severe reproductive complications. C. trachomatis is an intracellular Gram-negative bacterium with a biphasic developmental cycle in which extracellular, infectious elementary bodies (EB) alternate with the intracellular replicating reticulate bodies (RB). The outer membrane of EB consists of a tight disulfide cross-linking protein network. The most essential protein is the 42 kDa major outer membrane protein (MOMP) that contributes to the rigid structural integrity of the outer membrane. MOMP is a transmembrane protein with a β-barrel structure consisting of four variable domains (VD) separated by five constant domains. VDIV possesses surface-exposed species-specific epitopes recognized by the immune system and, therefore, functions as a candidate for vaccine development. To analyze the protective contribution of antibodies for a MOMP vaccine, we investigated the specificity and binding characteristics of two monoclonal antibodies (MAb)224.2 and MAb244.4 directed against C. trachomatis serovar D MOMP. By immunoelectron microscopy, we found that both MAb bind to the surface of C. trachomatis EB. By epitope mapping, we characterized the MOMP epitope as linear consisting of 6 amino acids: 322TIAGAGD328. By ELISA it was shown that both antibodies bind with a higher avidity to the chlamydial surface compared to binding to monomeric MOMP, indicating that the antibodies bind divalently to the surface of C. trachomatis EB. Despite strong binding to the chlamydial surface, the antibodies only partially reduced the infectivity. This may be explained by the observation that even though both MAb covered the EB surface, antibodies could not be regularly detected on EB after the uptake into the host cell.

A Novel Intranasal Vaccine With PmpGs + MOMP Induces Robust Protections Both in Respiratory Tract and Genital System Post Chlamydia psittaci Infection

Chlamydia psittaci (C. psittaci) is a crucial zoonotic pathogen that causes severe respiratory and reproductive system disease in humans and animals. In our pioneer study, polymorphic membrane protein G (PmpG) mediated attachment to host cells as the adhesions and induced immunity against C. psittaci infection. We hypothesize that multiple PmpG antigens adjuvanted with Vibrio cholerae ghost (VCG) and chitosan gel might trigger full protection via the intranasal route (i.n). In the present study, 40 SPF chickens were randomly divided into four groups, including the PmpGs + MOMP group (i.n), major outer membrane protein (MOMP) group (i.n), PmpGs (Pmp17G + Pmp20G + Pmp21G) group (i.n), and control groups (VCG + chitosan gel) (i.n). Post twice immunizations, the PmpGs + MOMP group yielded highly level-specific IgG, IgA antibodies, and lymphocyte proliferation. As for cytokines, IFN-γ expression was upregulated significantly, while IL-10 concentration was downregulated in the PmpGs + MOMP group compared with other groups. Post challenge, exudate inflammations in air sacs, bacterial loads in lungs, and bacterial shedding in throat swabs were reduced significantly in the PmpGs + MOMP group. In the second experiment, 100 breeder ducks were divided into the PmpGs + MOMP group (i.n), the commercial MOMP group (via intramuscular injection, i.m), the inactivated EBs group (i.n), and the control group (i.n), 25 ducks per group. Post challenge, the reduced egg production recovered soon in the inactivated EBs group and the PmpGs + MOMP group. Moreover, the aforementioned two groups induced higher robust IgG antibodies, lymphocyte proliferation, and IFN-γ secretions than the commercial MOMP vaccine did. Postmortem, lower bacterial loads of spleens were determined in the PmpGs + MOMP group and the inactivated EBs group. However, bacterial clearance of follicular membranes and shedding from the vaginal tract were not significant differences among the three tested groups. Furthermore, the PmpGs + MOMP group induced lower inflammations in the follicles and oviducts. Based on the above evidence, the combination of PmpGs and MOMP adjuvanted with chitosan gel and VCG via intranasal route could induce full protection both in the respiratory system and genital tract post C. psittaci infection. More importantly, the combination antigens are superior to the inactivated EBs antigen due to no contamination to the environment and less genital inflammation. The combination of PmpGs + MOMP adjuvanted with VCG and chitosan gel might be a promising novel vaccine by blocking C. psittaci infection from animals to human beings.

Capturing Complex Vaccine-Immune-Disease Relationships for Free-Ranging Koalas: Higher Chlamydial Loads Are Associated With Less IL17 Expression and More Chlamydial Disease.

Background: Chlamydial disease is a major factor negatively affecting koala populations. Vaccination is a promising management option that would result in immune-mediated protection against disease. Measuring and assessing vaccine efficacy can be challenging owing to both direct and indirect interactions caused by vaccination. In this study, we investigate vaccine-immune-chlamydial load-disease relationships from MOMP (major outer membrane protein) vaccine trials to protect healthy free-ranging koalas against Chlamydia-related diseases.Methods: We created a priori hypotheses based on data sources and perceived direct and indirect interactions from koalas vaccinated 6 months prior. Each hypothesis was tested as a structural equation model separately for either the urogenital or the ocular site to evaluate possible causality among measured variables. Model averaging was used as multiple models fit the data, and the strength of relationships was examined through averaged coefficients and the raw data.Results: We found more relationships in urogenital models as compared to ocular models, particularly those with interleukin 17 (IL17) mRNA expression compared to models with interferon gamma (IFNγ) expression. In the averaged model with IL17, urogenital chlamydial load was positively associated with disease and negatively associated with IL17 expression. MOMP vaccination had a trending effect for reducing urogenital chlamydial load and also had a strong effect on increasing IL17 expression. Not surprisingly, urogenital chlamydial load was a positive predictor for the development of urogenital disease at 6 months post-vaccination.Conclusions: Despite multiple potential sources of variation owing to the koalas in this study being free-ranging, our analyses provide unique insights into the effects of vaccinating against Chlamydia. Using structural equation modeling, this study has helped illuminate that the expression of the immune cytokine IL17 is linked to MOMP vaccination, and animals with a high urogenital chlamydial load expressed less IL17 and were more likely to develop disease, enhancing previous investigations. Going beyond univariate statistics, the methods used in this study can be applied to other preclinical vaccination experiments to identify important direct and indirect factors underpinning the effects of a vaccine.

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.

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.

Immunization of a wild koala population with a recombinant Chlamydia pecorum Major Outer Membrane Protein (MOMP) or Polymorphic Membrane Protein (PMP) based vaccine: New insights into immune response, protection and clearance.

We assessed the effects of two different single-dose anti-Chlamydia pecorum (C. pecorum) vaccines (containing either Major Outer Membrane Protein (3MOMP) or Polymorphic Membrane Protein (Pmp) as antigens) on the immune response of a group of wild koalas. Both vaccines elicited a systemic humoral response as seen by the production of anti-chlamydial IgG antibodies in more than 90% of vaccinated koalas. A mucosal immune response was also observed, with an increase in Chlamydia-specific mucosal IgG and/or IgA antibodies in some koalas post-vaccination. Both vaccines elicited a cell-mediated immune response as measured by the production of the cytokines IFN-γ and IL-17 post-vaccination. To determine the level of protection provided by the vaccines under natural conditions we assessed C. pecorum infection loads and chlamydial disease status of all vaccinated koalas pre- and post-vaccination, compared to a non-vaccinated cohort from the same habitat. The MOMP vaccinated koalas that were infected on the day of vaccination showed significant clearance of their infection at 6 months post-vaccination. In contrast, the number of new infections in the PMP vaccine was similar to the control group, with some koalas progressing to disease. Genotyping of the ompA gene from the C. pecorum strains infecting the vaccinated animals, identified genetic variants of ompA-F genotype and a new genotype ompA-O. We found that those animals that were the least well protected became infected with strains of C. pecorum not covered by the vaccine. In conclusion, a single dose vaccine formulated with either recombinant PmpG or MOMP can elicit both cell-mediated and humoral (systemic and mucosal) immune responses, with the MOMP vaccine showing clearance of infection in all infected koalas. Although the capability of our vaccines to stimulate an adaptive response and be protective needs to be fully evaluated, this work illustrates the necessity to combine epitopes most relevant to a large panel of variable strains with an efficient adjuvant.

Humoral immune responses in koalas (Phascolarctos cinereus) either naturally infected with Chlamydia pecorum or following administration of a recombinant chlamydial major outer membrane protein vaccine

The development of a vaccine is a key strategy to combat the widespread and debilitating effects of chlamydial infection in koalas. One such vaccine in development uses recombinant chlamydial major outer membrane protein (rMOMP) as an antigen and has shown promising results in several koala trials. Previous chlamydial vaccine studies, primarily in the mouse model, suggest that both cell-mediated and antibody responses will be required for adequate protection. Recently, the important protective role of antibodies has been highlighted. In our current study, we conducted a detailed analysis of the antibody-mediated immune response in koalas that are either (a) naturally-infected, and/or (b) had received an rMOMP vaccine. Firstly, we observed that naturally-infected koalas had very low levels of Chlamydia pecorum-specific neutralising antibodies. A strong correlation between low IgG total titers/neutralising antibody levels, and higher C. pecorum infection load was also observed in these naturally-infected animals. In vaccinated koalas, we showed that the vaccine was able to boost the humoral immune response by inducing strong levels of C. pecorum-specific neutralising antibodies. A detailed characterisation of the MOMP epitope response was also performed in naturally-infected and vaccinated koalas using a PepScan epitope approach. This analysis identified unique sets of MOMP epitope antibodies between naturally-infected non-protected and diseased koalas, versus vaccinated koalas, with the latter group of animals producing a unique set of specific epitope-directed antibodies that we demonstrated were responsible for the in vitro neutralisation activity. Together, these results show the importance of antibodies in chlamydial infection and immunity following vaccination in the koala.

Vaccination of Koalas with a Recombinant Chlamydia pecorum Major Outer Membrane Protein Induces Antibodies of Different Specificity Compared to Those Following a Natural Live Infection

Chlamydial infection in koalas is common across the east coast of Australia and causes significant morbidity, infertility and mortality. An effective vaccine to prevent the adverse consequences of chlamydial infections in koalas (particularly blindness and infertility in females) would provide an important management tool to prevent further population decline of this species. An important step towards developing a vaccine in koalas is to understand the host immune response to chlamydial infection. In this study, we used the Pepscan methodology to identify B cell epitopes across the Major Outer Membrane Protein (MOMP) of four C . pecorum strains/genotypes that are recognized, either following (a) natural live infection or (b) administration of a recombinant MOMP vaccine. Plasma antibodies from the koalas naturally infected with a C . pecorum G genotype strain recognised the epitopes located in the variable domain (VD) four of MOMP G and also VD4 of MOMP H. By comparison, plasma antibodies from an animal infected with a C . pecorum F genotype strain recognised epitopes in VD1, 2 and 4 of MOMP F, but not from other genotype MOMPs. When Chlamydia-free koalas were immunised with recombinant MOMP protein they produced antibodies not only against epitopes in the VDs but also in conserved domains of MOMP. Naturally infected koalas immunised with recombinant MOMP protein also produced antibodies against epitopes in the conserved domains. This work paves the way for further refinement of a MOMP-based Chlamydia vaccine that will offer wide cross-protection against the variety of chlamydial infections circulating in wild koala populations.

Antigenic specificity of a monovalent versus polyvalent MOMP based Chlamydia pecorum vaccine in koalas (Phascolarctos cinereus)

Chlamydia continues to be a major pathogen of koalas. The bacterium is associated with ocular, respiratory and urogenital tract infections and a vaccine is considered the best option to limit the decline of mainland koala populations. Over the last 20 years, efforts to develop a chlamydial vaccine in humans have focussed on the use of the chlamydial major outer membrane protein (MOMP). Potential problems with the use of MOMP-based vaccines relate to the wide range of genetic diversity in its four variable domains. In the present study, we evaluated the immune response of koalas vaccinated with a MOMP-based C. pecorum vaccine formulated with genetically and serologically diverse MOMPs. Animals immunised with individual MOMPs developed strong antibody and lymphocyte proliferation responses to both homologous as well as heterologous MOMP proteins. Importantly, we also showed that vaccine induced antibodies which effectively neutralised various heterologous strains of koala C. pecorum in an in vitro assay. Finally, we also demonstrated that the immune responses in monovalent as well as polyvalent MOMP vaccine groups were able to recognise whole chlamydial elementary bodies, illustrating the feasibility of developing an effective MOMP based C. pecorum vaccine that could protect against a range of strains.

Immune responses induced by the recombinant major outer membrane protein vaccine against Chlamydia trachomatis E serotype in rhesus monkeys

[Objective] To observe the specific immune responses induced by the recombinant major outer membrane protein (rMOMP) vaccine against Chlamydia trachomatis E serotype in rhesus monkeys.[Methods] Six rhesus monkeys were equally divided into three groups:adjuvant and protein group vaccinated with purified rMOMP and Freund's adjuvants,adjuvant group immunized with Freund's adjuvants only,and control group immunized with phosphate buffer.All the rhesus monkeys were intramuscularly immunized in the triceps brachii for 3 times at a 2-week interval.Two weeks after the last vaccination,serum,vaginal wash and venous blood samples were collected from the rhesus monkeys,and lymphocytes were isolated from the blood samples.Enzyme linked immunosorbent assay (ELISA) was performed to determine the specific IgG antibody and interferon level in sera and secretory IgA (sIgA) level in wash samples,and methyl thiazolyl tetrazolium (MTT) assay to evaluate the proliferation of lymphocytes after stimulation with Chlaraydia trachomatis serotype E elementary bodies.Delayed hypersensitivity was observed in rhesus monkeys challenged by inactivated Chlamydia trachomatis serotype E elementary bodies.In vitro antibody neutralization assay was conducted with the serum from rhesus monkeys.Indirect immunofluorescenee was used to detect Chlamydia trachomatis in exfoliative vaginal cells from rhesus monkeys from week 1 to 10 after challenge with Chlamydia trachomatis.Data were statistically analyzed by using one-way analysis of variance and least significant difference (LSD) test with the SPSS 14.0 software.[Results] The adjuvant and protein group differed statistically from the adjuvant group and control group in the serum level of specific IgG antibody (1.718 ± 0.213 vs.0.841 ± 0.315 and 0.791 ±0.437,both P＜ 0.05),interferon ((1086 ± 121.730) ng/L vs.(409 + 53.440) ng/L and (162 ± 48.046) ng/L,both P＜ 0.05),lymphocyte proliferation index (7.012 ± 1.026 vs.4.473 ± 1.850 and 1A26 ± 1.104,both P＜0.01 ) and the diameter of nodus in delayed hypersensitivity assay ( ( 1 1 ± 2.134) mm vs.(3 ± 0.914) mm and 0,both P ＜ 0.01 ).After attack,the exfoliative cells kept positive for Chlamydia trachomatis in the adjuvant and protein group from week 1 to 5,and in the other 2 groups from week 1 to 10,but were negative in the adjuvant and protein group from week 6 to 10.[Conclusion] The rMOMP vaccine can induce a specific,protective,humoral and cellular immune response against Chlamydia tracbomatis in rhesus monkeys. Key words: Chlamydia trachomatis Bacterial outer membrane proteins; Vaccines; Animal experimentation

A TLR2 agonist is a more effective adjuvant for a Chlamydia major outer membrane protein vaccine than ligands to other TLR and NOD receptors

Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial pathogen in the World and there is an urgent need for a vaccine to prevent these infections. To determine what type of adjuvant can better enhance the immunogenicity of a Chlamydia vaccine, we formulated the recombinant major outer membrane protein (Ct-rMOMP) with several ligands for Toll-like receptors (TLR) and the nucleotide-binding oligomerization domain (NOD) including Pam2CSK4 (TLR2/TLR6), Poly (I:C) (TLR3), monophosphoryl lipid A (TLR4), flagellin (TLR5), imiquimod R837 (TLR7), imidazoquinoline R848 (TRL7/8), CpG-1826 (TLR9), M-Tri-DAP (NOD1/NOD2) and muramyldipeptide (NOD2). Groups of female BALB/c mice were immunized intramuscularly (i.m.) three times with the Ct-rMOMP and each one of those adjuvants. Four weeks after the last immunization the mice were challenged intranasally (i.n.) with 104C. trachomatis mouse pneumonitis (MoPn) inclusion forming units (IFU). As negative antigen control, mice were immunized with the Neisseria gonorrhoeae recombinant porin B (Ng-rPorB) and the same adjuvants. As a positive vaccine control, mice were inoculated i.n. with 104IFU of MoPn. The humoral and cell mediated immune responses were determined the day before the challenge. Following the challenge the mice were weighed daily and, at 10 days post-challenge (p.c.), they were euthanized, their lungs weighted and the number of IFU in the lungs counted. As determined by the IgG2a/IgG1 ratio in the sera, mice immunized with Ct-rMOMP+Pam2CSK4 showed a strong Th2 biased humoral immune response. Furthermore, these mice developed a robust cellular immune response with high Chlamydia-specific T cell proliferation and levels of IFN-γ production. In addition, based on changes in body weight, weight of the lungs and number of IFU recovered from the lungs, the mice immunized with Ct-rMOMP+Pam2CSK4, were better protected against the i.n. challenge than any group of mice immunized with Ct-rMOMP and the other adjuvants. In conclusion, Pam2CSK4 should be evaluated as a candidate adjuvant for a C. trachomatis vaccine.

Detection of antibodies to the major outer membrane protein of Chlamydia trachomatis using an in vitro transcription-translation radioimmunoprecipitation assay

A radioimmunoprecipitation assay (RIPA) using in vitro translated protein was developed to measure serum antibodies to the major outer membrane protein (MOMP) of C. trachomatis. Polyclonal animal antisera to C. trachomatis serovars C, E and F reacted to serogroup-specific determinants by RIPA, as compared to species-specific epitopes by Western blot. Antibody responses in monkeys immunized systemically or mucosally with a candidate MOMP vaccine were assessed by RIPA and ELISA with elementary bodies (EB-ELISA). Unlike EB-ELISA, RIPA showed a significant difference in pre-challenge antibody levels between systemically and mucosally immunized animals. Additionally, only RIPA showed a significant inverse correlation between antibody level at time of challenge and microbiologic response measured as median inclusion forming units (IFU) in culture (r = −0.89; P < 0.001). RIPA using in vitro translated proteins is a useful method to measure antibody responses to specific C. trachomatis antigens and may be more informative than EB-ELISA and Western blot for assessing the immunogenicity of MOMP vaccines.