Recombinant Outer Membrane Protein Research Articles

The Role of Extracellular Loops in the Folding of Outer Membrane Protein X (OmpX) of Escherichia coli.

The outer membrane of Gram-negative bacteria acts as an additional diffusion barrier for solutes and nutrients. It is perforated by outer membrane proteins (OMPs) that function most often as diffusion pores, but sometimes also as parts of larger cellular transport complexes, structural components of the cell wall, or even as enzymes. These OMPs often have large loops that protrude into the extracellular environment, which have promise for biotechnological applications and as therapeutic targets. Thus, understanding how modifications to these loops affect OMP stability and folding is critical for their efficient application. In this work, the small outer membrane protein OmpX was used as a model system to quantify the effects of loop insertions on OMP folding and stability. The insertions were varied according to both hydrophobicity and size, and their effects were determined by assaying folding into detergent micelles in vitro by SDS-PAGE and in vivo by isolating the outer membrane of cells expressing the constructs. The different insertions were also examined in molecular dynamics simulations to resolve how they affect OmpX dynamics in its native outer membrane. The results indicate that folding of OMPs is affected by both the insert length and by its hydrophobic character. Small insertions sometimes even improved the folding efficiency of OmpX, while large hydrophilic inserts reduced it. All the constructs that were found to fold in vitro could also do so in their native environment. One construct that could not fold in vitro was transported to the OM in vivo, but remained unfolded. Our results will help to improve the design and efficiency of recombinant OMPs used for surface display.

Efficacy of recombinant subunit OMP and hly vaccines against Aeromonas hydrophila in Rohu (Labeo rohita).

The main aim of the current study was to clone and express a new outer membrane protein (OMP) and haemolysin (hly) from a pathogenic Aeromonas hydrophila and to investigate their potential as a vaccine candidate against A. hydrophila infection in Rohu (Labeo rohita). The OMP and hly genes were cloned in pET-30b vector and recombinant plasmids pET-30b-OMP and pET-30b-hly were constructed, which were then transferred into Escherichia coli BL21 (DE3). The recombinant E.coli BL21 (DE3) was induced by IPTG, and the OMP and hly proteins were expressed highly. The proteins OMP and hly were estimated in 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Their molecular weights were found to be 40 kD and 68 kD. The expressed proteins OMP and hly were purified by Ni-NTA His-Bind Resin column, and the immunogenicity was confirmed by Western blotting. The fishes (L.rohita) were divided into IV groups, and the group I fishes were treated with phosphate saline, the II and III group were immunized with the purified OMP and hly recombinant proteins, and the fishes were treated IV group with combined OMP and hly for 10 days. After 10 days of treatment, the fishes of all the four groups were challenged with virulent A.hydrophila. The results revealed that vaccinated fish showed significantly improved haematological profile, phagocytic activity, myeloperoxidase activity and total immunoglobulin levels on the 5th and 10th days. The non-vaccinated group (Group I) showed 100% mortality, whereas the mixture of recombinant OMP (r-OMP) and hly (r-hly) protein-treated groups (Group IV) exhibited higher survival rate (80%). Relatively, expression of pro- and anti-inflammatory cytokines (IL-1β, IL-10 and TGF-β), c-type and g-type lysozymes were significantly up-regulated in heart and kidney of vaccinated groups compared with the non-vaccinated group. Our results revealed that OMP and hly genes were effective vaccine candidates in the aquaculture system and could be used as recombinant subunit vaccine for diseases caused by pathogenic A.hydrophila.

Overexpression of the Bam Complex Improves the Production of Chlamydia trachomatis MOMP in the E. coli Outer Membrane.

A licensed Chlamydia trachomatis (Ct) vaccine is not yet available. Recombinant Chlamydia trachomatis major outer membrane protein (Ct-MOMP), the most abundant constituent of the chlamydial outer membrane complex, is considered the most attractive candidate for subunit-based vaccine formulations. Unfortunately, Ct-MOMP is difficult to express in its native structure in the E. coli outer membrane (OM). Here, by co-expression of the Bam complex, we improved the expression and localization of recombinant Ct-MOMP in the E. coli OM. Under these conditions, recombinant Ct-MOMP appeared to assemble into a β-barrel conformation and express domains at the cell surface indicative of correct folding. The data indicate that limited availability of the Bam complex can be a bottleneck for the production of heterologous OM vaccine antigens, information that is also relevant for strategies aimed at producing recombinant OMV-based vaccines.

The Outer Membrane Proteins and Their Synergy Triggered the Protective Effects against Pathogenic Escherichia coli.

Colibacillosis caused by pathogenic Escherichia coli (E. coli) is one of the most serious infectious diseases, causing an extensive burden on animal husbandry and the human healthcare system. Vaccination is one of the ideal ways to prevent E. coli infection. In this work, recombinant outer membrane protein A (rOmpA), outer membrane protein C (rOmpC) and BamA (rBamA) from E. coli O78 (CVCC CAU0768) were expressed in a prokaryotic expression system with the concentration of 1–2 mg/mL after purification. Considerable immune responses could be triggered in mice that were immunized with these recombinant proteins, high antibody titers, high total IgG level and various antibody isotypes were detected in antisera after booster immunizations. Moreover, mice immunized with several recombinant proteins in combination showed a higher survival rate with the challenge of homologous strain E. coli O78 and a more significant cross-protection effect against heterologous strain E. coli O157:H7 (CICC 21530) in vivo than those of immunized alone. The antisera from immunized mice showed high affinity to multiple strains of Escherichia, Shigella and Salmonella in vitro, indicating that recombinant outer membrane proteins from E. coli O78 had the potential to be developed into universal antigenic substances against not only E. coli but also a variety of Gram-negative bacteria. rOmpA was considered as the most immunogenic protein in this work and the combination of different proteins could further enhance the immune response of immunized mice, which provided the reference for the construction of novel antigens with higher efficiency.

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.

Australian Aboriginal Otitis-Prone Children Produce High-Quality Serum IgG to Putative Nontypeable Haemophilus influenzae Vaccine Antigens at Lower Titres Compared to Non-Aboriginal Children.

BackgroundNontypeable Haemophilus influenzae (NTHi) is the most common bacterial otopathogen associated with otitis media (OM). NTHi persists in biofilms within the middle ears of children with chronic and recurrent OM. Australian Aboriginal children suffer exceptionally high rates of chronic and recurrent OM compared to non-Aboriginal children. NTHi protein vaccines comprised of antigens associated with both adhesion and persistence in a biofilm are under development and could be beneficial for children with chronic and recurrent OM. Understanding the ontogeny of natural antibody development to these antigens provides insight into the value of vaccinating with particular antigens.MethodsAn in-house multiplex fluorescent bead immunoassay was used to measure serum IgG titres and avidity for three putative vaccine antigens: recombinant soluble PilA (rsPilA), ChimV4, and outer membrane protein 26 (OMP26) in sera from Australian Aboriginal otitis-prone children (n=77), non-Aboriginal otitis-prone children (n=70) and non-otitis-prone children (n=36). Serum IgG titres were adjusted for age, and geometric mean concentrations (GMCs) were compared between groups using a univariate analysis model. Antibody avidity was calculated as a relative avidity index and compared between groups using ANOVA.ResultsAustralian Aboriginal otitis-prone children had lower serum IgG titres to rsPilA and ChimV4 than non-Aboriginal otitis-prone children (p<0.001), and non-otitis-prone children (p<0.020). No differences were observed between serum IgG titres from non-Aboriginal otitis-prone children and non-otitis-prone children. There were also no differences in the proportion of high avidity IgG specific for these antigens between these groups. Serum IgG titres to OMP26 were similar between all groups (p>0.670) although otitis-prone children had a higher proportion of high avidity antibodies to this antigen.ConclusionsAustralian Aboriginal otitis-prone children had lower serum IgG titres to 2/3 major NTHi vaccine candidate antigens, suggesting these children are unable to develop persistent IgG responses due to repeated NTHi exposure. These reduced IgG titres may relate to earlier and more frequent exposure to diverse NTHi strains in Aboriginal children through carriage or infection. These data suggest that Aboriginal children may benefit from immunisation with vaccines containing these antigens to increase titres of protective antibodies.

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.

SOLUBILIZATION AND REFOLDING TECHNIQUES TO ATTAIN Candidatus liberibacter ASIATICUS OMP PROTEIN FROM INCLUSION BODIES

The OMP is a conserved and major outer membrane protein presented on the surface of Candidatus Liberibacter asiaticus (CLas). High expression level of recombinant outer membrane protein (OMP) in Escherichia coli led to form inclusion bodies (IBs) in cytoplasmic space which is an inactive form of protein and needs a solubilizing and refolding process. The aim of this study was to compare different methods for solubilization and refolding of recombinant OMP expressed in E. coli. For this aim, preliminary washing process was performed by Triton X-100 and sodium deoxycholate. To solubilize IBs, reducing agents; guanidine-hydrochloride, urea, β-mercaptoethanol and dithiothritol were applied. The results of washing process showed that applying of 0.5% TritonX-100 (v/v) is led to highest protein recovery. The results obtained from solubilization step revealed that applying guanidine-hydrochloride denaturing agent is led to obtain more solubilized protein. Furthermore, adding supplementary β-mercaptoethanol in denaturing buffer resulted to improve protein recovery during solubilization process. Presence of arginine in refolding buffer containing high urea concentration was led to significant decreases in aggregation. As a result, the best recovery of OMP was obtained at a concentration 0.5 M of Triton X-100, the high efficiency of solubility was found in pH 8 in the presence of guanidine-hydrochloride and the best efficiency of refolding was attained by final buffer containing 2M urea and 0.5 M L-Arginine. These results suggested a suitable procedure to produce appropriate recombinant OMP for preparation of poly and monoclonal antibodies.

Identification and Evaluation of Recombinant Outer Membrane Proteins as Vaccine Candidates Against Klebsiella pneumoniae.

Klebsiella pneumoniae found in the normal flora of the human oral and intestinal tract mainly causes hospital-acquired infections but can also cause community-acquired infections. To date, most clinical trials of vaccines against K. pneumoniae have ended in failure. Furthermore, no single conserved protein has been identified as an antigen candidate to accelerate vaccine development. In this study, we identified five outer membrane proteins of K. pneumoniae, namely, Kpn_Omp001, Kpn_Omp002, Kpn_Omp003, Kpn_Omp004, and Kpn_Omp005, by using reliable second-generation proteomics and bioinformatics. Mice vaccinated with these five KOMPs elicited significantly higher antigen-specific IgG, IgG1, and IgG2a. However, only Kpn_Omp001, Kpn_Omp002, and Kpn_Omp005 were able to induce a protective immune response with two K. pneumoniae infection models. These protective effects were accompanied by the involvement of different immune responses induced by KOMPs, which included KOMPs-specific IFN-γ-, IL4-, and IL17A-mediated immune responses. These findings indicate that Kpn_Omp001, Kpn_Omp002, and Kpn_Omp005 are three potential Th1, Th2, and Th17 candidate antigens, which could be developed into multivalent and serotype-independent vaccines against K. pneumoniae infection.

Evaluation of chimeric proteins for serological diagnosis of brucellosis in cattle.

Background and Aim:An accurate diagnosis of Brucella-infected animals is one of the critical measures in eradication programs. Conventional serological tests based on whole-cell (WC) antigens and detecting antibodies against pathogen-associated lipopolysaccharide might give false-positive results due to the cross-reactivity with other closely related bacteria. This study evaluated the serological potential of Brucella spp. chimeric outer membrane proteins (Omps) as antigens in an indirect enzyme-linked immunosorbent assay (i-ELISA).Materials and Methods:The chimeric gene constructs of the most immunodominant regions of Brucella Omps 25+31, 25+19, and 19+31 were cloned into the pET28a expression vectors and transformed into Escherichia coli BL21 (DE3). The serological potential of chimeric proteins compared with single recombinant Omps (rOmps)19, 25, and/or 31 were studied on blood serum samples of (i) a rabbit immunized with killed Brucella abortus 19WC, (ii) mice immunized with single rOmps, (iii) cows seropositive for brucellosis by rose Bengal test, and (iv) cattle naturally and/or experimentally infected with brucellosis.Results:E. coli BL21 actively produced Brucella chimeric rOmps, the concentration of which reached a maximum level at 6 h after isopropyl-b-D-1-thiogalactopyranoside stimulation. Target proteins were antigenic and expressed in an active state, as recognized by rabbit anti-B. abortus antibodies in an i-ELISA and western blotting. Murine antibodies against the single rOmps reacted with chimeric antigens, and conversely, antichimeric antibodies found their epitopes in single proteins. Brucella chimeric rOmps showed higher antigenicity in blood sera of seropositive cattle kept in the hotbed of the infection and/or experimentally challenged with brucellosis than single proteins.Conclusion:Brucella chimeric recombinant outer membrane proteins could be a potential antigen candidate for developing an ELISA test for accurate diagnosis of bovine brucellosis.

Transcriptome analysis of immune response in recombinant cell vaccine expressing OmpK vaccinated juvenile seabass (lates calcarifer) head kidney against vibrio harveyi infection

Seabass (Lates calcarifer) are high value fish occupying a market niche, and is susceptible to infection by pathogenic bacteria such as Vibriosis. In this study the effect of intraperitoneal administration of recombinant outer membrane protein (OMP) of inactivated cells vaccine; r-OmpK on hematological parameters for monitoring fish health and histopathological examination of seabass were investigated. The results revealed that r-OmpK affect some hematological parameters positively in juvenile bass. The r-OmpK vaccinated seabass showed mild histological alterations in the vital organs (spleen, kidney and intestine) when compared to PBS control groups. Further we sought to understand the mechanisms of molecular immunity of L. calcarifer against V. harveyi infection. We carried out an extensive analysis of the transcriptome of L. calcarifer head kidney upon vaccination trial with r-OmpK recombinant cell vaccine and challenged with V. harveyi pathogenic strain VH1. Average reads of 92.74 million and 116.17 million were obtained from head kidney of juvenile L. calcarifer immunized with phosphate buffered saline (PBS) and r-OmpK (10 9 cfu/mL) recombinant cell vaccine, respectively. Also, the analysis revealed that a higher number of genes and pathways were regulated in response to infection with V. harveyi at 48 h post infection compared to the PBS controls. Based on the differential expressed genes (DEGs) around 0.31 % of total unigenes were differentially expressed between the two vaccinated groups. Protein-protein interaction analysis predicts glycolysis pathway, alanine, aspartate and glutamate metabolism pathway, pyruvate metabolism pathway, MAPK signaling pathway, metabolic pathways, insulin signaling pathway and focal adhesion pathway in head kidney of infected fish, which might be useful in understanding biological processes and further studies in the direction of pathways. We reported the novel immune-related signalling pathways, namely complement and coagulation cascades, chemokine signalling pathway, and toll-like receptor signalling pathway having great importance in immunity and anti-inflammation.

Development of IMBs-qPCR detection method for Yersinia enterocolitica based on the foxA gene.

Yersinia enterocolitica is an important zoonotic pathogen, which seriously endangers food-safety risk. In this study, the recombinant outer membrane protein OmpF and its antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Y. enterocolitica in food samples, combining the quantitative PCR detection with primers of virulence factor gene foxA for Yersinia enterocolitica contamination. The results showed that the capture efficiency of approximately 80% using anti-OmpF antibody-immunomagnetic beads and linearly dependent capture under 101-105CFU/mL Y. enterocolitica compared with less than 10% capture of other bacteria. The detection limit of 64CFU/mL was obtained based on foxA gene PCR detection combined with capture of the anti-OmpF antibody-immunomagnetic beads to detect Yersinia enterocolitica in artificially contaminated milk and pork samples. Compared to the culture method, the developed IMBs-qPCR method has higher consistency, was less time consuming, which taken together provides an effective alternative method for rapid detection of Y. enterocolitica in food.

Comparative analysis of the main outer membrane proteins of Brucella in the diagnosis of brucellosis

Brucellosis has placed a heavy economic burden on numerous countries and has consumed considerable medical resources worldwide. To improve the specificity and sensitivity of serological methods for diagnosing brucellosis, it is important to develop new diagnostic antigens. Brucella outer membrane proteins(omps) possess good immunogenicity, but there is a scarcity of comparative studies of these proteins in the clinical diagnosis of brucellosis. In this study, six recombinant Brucella outer membrane proteins, omp10, omp16, omp19, omp25, omp31 and BP26, were expressed in prokaryotic cells and utilized as diagnostic antigens. The clinical sera of humans, bovines and goats with brucellosis were analyzed by indirect ELISA using these proteins, lipopolysaccharide(LPS) and Rose Bengale Ag, served as positive-control antigens. In diagnosing human and goat serum, BP26 exhibited the highest diagnostic accuracy of 96.45% and 95.00%, respectively, while omp31 exhibited the strongest ability to detect Brucella in bovine serum with an accuracy of 84.03%. Cross-reaction experiments also confirmed that the diagnostic specificities of omp31 and BP26 were higher than those of the LPS and Rose Bengale Ag antigens. The results of this study indicate that omp31 and BP26 are candidate antigens with high potential application value in the clinical diagnosis of brucellosis.

Over-expression of gene encoding heat shock protein 70 from Mycobacterium tuberculosis and its evaluation as vaccine adjuvant

Background: Heat shock proteins (Hsps) are evolutionary ancient and highly conserved molecular chaperons found in prokaryotes as well as eukaryotes. Hsp70 is a predominant member of Hsp family. Microbial Hsp70s (mHsp70s) have acquired special significance in immunity since they have been shown to be potent activators of the innate immune system and generate specific immune responses against tumours and infectious agents. Objectives: The present study was aimed to clone express and purify recombinant Hsp70 from the Mycobacterium tuberculosis and characterise it immunologically. The study also aimed at determining the potential of recombinant M. tuberculosis heat shock protein (rMTB-Hsp70) as adjuvant or antigen carrier. Materials and Methods: Cloning of M. tuberculosis heat shock protein (MTB-Hsp70) amplicon was carried out using the pGEMT-Easy vector although for expression, pProExHTb prokaryotic expression vector was used. Purification of recombinant Hsp70 was carried out by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. For immunological characterization and determining the adjuvant effect of MTB-Hsp70, BALB/c mice were used. The data obtained was statistically analysed. Results: Hsp70 gene was cloned, sequenced and the sequence data were submitted to National Center for Biotechnology Information (NCBI). Recombinant MTB-Hsp70 was successfully over-expressed using the prokaryotic expression system and purified to homogeneity. The protein was found to be immunodominant. Significant adjuvant effect was produced by the rMTB-Hsp70 when inoculated with recombinant outer membrane protein 31; however, effect was less than the conventionally used the Freund’s adjuvant. Conclusion: Protocol standardised can be followed for bulk production of rHsp70 in a cost-effective manner. Significant adjuvant effect was produced by rMTB-Hsp70; however, the effect was than Freund’s adjuvant. Further, studies need to be carried out to explore its applicability as carrier of antigen.

Immune response in dairy cattle against combined foot and mouth disease and haemorrhagic septicemia vaccine under field conditions

BackgroundFoot-and-mouth disease (FMD) and Haemorrhagic septicemia (HS) are two important diseases that are known to have caused significant economic losses to the cattle industry. Accordingly, vaccinations have been recognized as an efficient method to control and prevent both of the above-mentioned diseases. This study aimed to determine the immune response to FMD virus antigens and the recombinant outer membrane protein of HS (rOmpH) of Pasteurella multocida in cattle administered as a combination vaccine and compare antibody titers with the two vaccines given independently, under field conditions. Dairy cattle were divided into three groups. Each group was immunized with different vaccine types according to the vaccination program employed in this study. Antibody responses were determined by indirect ELISA, liquid phase blocking ELISA (LPB-ELISA) and viral neutralization test (VNT). Furthermore, the cellular immune responses were measured by lymphocyte proliferation assay (LPA).ResultsThe overall antibody titers to HS and FMDV were above cut-off values for the combined FMD-HS vaccine in this study.The mean antibody titer against HS after the first immunization in the combined FMD-HS vaccine groups was higher than in the HS vaccine groups. However, no statistically significant differences (p > 0.05) were observed between groups. Likewise, the antibody titer to the FMDV serotypes O/TAI/189/87 and Asia 1/TAI/85 determined by LPB-ELISA in the combined vaccine were not statistically significantly different when compared to the FMD vaccine groups. However, the mean VNT antibody titer of combined vaccine against serotype O was significantly higher than the VN titer of FMD vaccine groups (p < 0.05). Moreover, the LPA results showed that all vaccinated groups displayed significantly higher than the negative control (p < 0.05). Nevertheless, no differences in the lymphocyte responses were observed in comparisons between the groups (p > 0.05).ConclusionsThe combined FMD-HS vaccine formulated in this study could result in high both antibody and cellular immune responses without antigenic competition. Therefore, the combined FMD-HS vaccine can serve as an alternative vaccine against both HS and FMD in dairy cattle under field conditions.

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.

Evaluation of the diagnostic potential of recombinant leptospiral OMP A-like protein (Loa22) and transmembrane (OmpL37) protein in latex agglutination test for serodiagnosis of leptospirosis in animals.

Leptospirosis is a re-emerging zoonotic disease of animals and humans caused by pathogenic Leptospira, which has major public health concerns. The study is aimed to express the recombinant outer membrane protein (OMP) A-like protein (rLoa22) and transmembrane (rOmpL37) protein of Leptospira interrogans serovar Hardjo in the Escherichia coli and their evaluation as a diagnostic antigen in the latex agglutination test (LAT) to detect anti-leptospiral antibodies in the sera of animals. The Loa22 and OmpL37 genes lacking signal peptide coding sequences were individually amplified (522 and 963bp), by polymerase chain reaction, and directionally cloned into a pETite N-His Kan vector for expression. The expressed purified proteins were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblot, which confirmed leptospiral specific reactive protein with a molecular weight of ~19 and 36kDa, respectively. The sensitized latex beads coated with these OM proteins separately were evaluated in LAT using cattle sera of microscopic agglutination test (MAT) confirmed positive (n=53) and negative (n=52) cases of leptospirosis. The rLoa22 LAT and rOmpL37 LAT revealed the relative diagnostic sensitivity of 94·34 and 96·23%, diagnostic specificity of 92·31 and 96·15% and accuracy of 93·33 and 96·19%, with the excellent agreement of Cohen's kappa value of 0·87 and 0·92, respectively. After extensive evaluation, this rapid recombinant protein-based field diagnostic test can be applied as a screening test for the detection of anti-leptospiral antibodies in the sera of animals in the field conditions.

Recombinant outer membrane protein OmpC induces protective immunity against Aeromonashydrophila infection in Labeorohita

Aeromonashydrophila is an opportunistic pathogen that causes enormous loss to aquaculture industry. The outer membrane proteins of Aeromonas help in bacterium-host interaction, and are considered to be potential vaccine candidates. In the present study, we evaluated immunogenicity and protective efficacy of recombinant OmpC (rOmpC) of A. hydrophila in Indian major carp, Labeorohita. The rOmpC-vaccinated fish produced specific anti-rOmpC antibodies with a significant antibody titer, and the antisera could specifically detect the rOmpC in the cell lysates of Escherichia coli expressing rOmpC and cross-react with different Aeromonas lysates, indicating the suitability of the anti-rOmpC antisera to detect Aeromonas infection. A significant increase was noted in ceruloplasmin level, myeloperoxidase and anti-protease activities in transient and temporal manner the sera of the rOmpC-immunized fish as compared to PBS-control fish. Higher agglutination- and hemolytic activity titers in the anti-rOmpC antisera indicate stimulation of innate immunity. Expression of immune-related genes comprising various acute phase proteins, cytokines and inflammatory response molecules were modulated in the head kidney of rOmpC-immunized L. rohita. While IgM, IL1β, and TLR-22 were significantly up-regulated at early time points (3 h–72 h), the others showed a transient augmentation at both early and later time points (SOD, lysozymes C and G, NKEF-B, C3, CXCa and TNF-α) in the rOmpC-immunized L. rohita in comparison to PBS-injected controls. These data suggest that the rOmpC-induced immune response is temporally regulated to confer immunity. In vivo challenge of the rOmpC-immunized fish with A. hydrophila showed significantly greater survival when compared to PBS-injected control fish. Thus, our results highlight the immunomodulatory role of rOmpC and demonstrate its protective efficacy in L. rohita, along with the use of anti-rOmpC antisera in detecting Aeromonas infections.

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.

ROmpF and OMVs as efficient subunit vaccines against Salmonella enterica serovar Enteritidis infections in poultry farms

Salmonella enterica serovar Enteritidis remains the most prevalent serotype causing human salmonellosis through the consumption of contaminated foods, especially poultry products. The development of a subunit vaccine against S. Enteritidis can not only protect chickens against Salmonella infection in the poultry industry but also cut the transmission sources. In this study, both the expressed recombinant outer membrane protein F (rOmpF) and extracted outer membrane vesicles (OMVs) were developed as subunit vaccines against S. Enteritidis challenge in chickens. Immunization with the subunit vaccine could induce not only antibody production but also strong cell-mediated immune response. Both rOmpF plus QuilA adjuvant and OMVs alone had highly protective efficacy against S. Enteritidis challenge and rapidly decreased the colonization of bacteria in chicken. These findings revealed the potential application of rOmpF and OMVs as subunit vaccines in the poultry industry.