Recombinant Outer Membrane Protein Research Articles

A candidate glycoconjugate vaccine induces protective antibodies in the serum and intestinal secretions, antibody recall response and memory T cells and protects against both typhoidal and non-typhoidal Salmonella serovars.

Human Salmonella infections pose significant public health challenges globally, primarily due to low diagnostic yield of systemic infections, emerging and expanding antibiotic resistance of both the typhoidal and non-typhoidal Salmonella strains and the development of asymptomatic carrier state that functions as a reservoir of infection in the community. The limited long-term efficacy of the currently licensed typhoid vaccines, especially in smaller children and non-availability of vaccines against other Salmonella serovars necessitate active research towards developing a multivalent vaccine with wider coverage of protection against pathogenic Salmonella serovars. We had earlier reported immunogenicity and protective efficacy of a subunit vaccine containing a recombinant outer membrane protein (T2544) of Salmonella Typhi in a mouse model. This was achieved through the robust induction of serum IgG, mucosal secretory IgA and Salmonella-specific cytotoxic T cells as well as memory B and T cell response. Here, we report the development of a glycoconjugate vaccine, containing high molecular weight complexes of Salmonella Typhimurium O-specific polysaccharide (OSP) and recombinant T2544 that conferred simultaneous protection against S. Typhi, S. Paratyphi, S. Typhimurium and cross-protection against S. enteritidis in mice. Our findings corroborate with the published studies that suggested the potential of Salmonella OSP as a vaccine antigen. The role of serum antibodies in vaccine-mediated protection is suggested by rapid seroconversion with high titers of serum IgG and IgA, persistently elevated titers after primary immunization along with a strong antibody recall response with higher avidity serum IgG against both OSP and T2544 and significantly raised SBA titers of both primary and secondary antibodies against different Salmonella serovars. Elevated intestinal secretory IgA and bacterial motility inhibition by the secretory antibodies supported their role as well in vaccine-induced protection. Finally, robust induction of T effector memory response indicates long term efficacy of the candidate vaccine. The above findings coupled with protection of vaccinated animals against multiple clinical isolates confirm the suitability of OSP-rT2544 as a broad-spectrum candidate subunit vaccine against human infection due to typhoidal and non-typhoidal Salmonella serovars.

Structural Modeling of T9SS Outer Membrane Proteins and Their Complexes.

The type 9 secretion system (T9SS) is a recently discovered machinery that both transports cargo proteins across the Gram-negative bacterial outer membrane and attaches them to lipopolysaccharides on the extracellular surface. Outer membrane proteins (OMPs) are key components of the T9SS and are involved in both steps. In this chapter, we describe a method for the in silico modeling of T9SS OMPs and their complexes, and model validation. This is useful when the production of recombinant OMPs is difficult, and these protocols can also be applied to OMP complexes outside of the T9SS.

Characterization of Three New Outer Membrane Adhesion Proteins in Fusobacterium necrophorum.

Fusobacterium necrophorum, an anaerobic Gram-negative pathogen, causes necrotic cattle infections, impacting livestock health and the US feedlot industry. Antibiotic administration is the mainstay for treating F. necrophorum infections, although resistance hampers their effectiveness. Vaccination, especially targeting outer membrane proteins (OMPs) due to their antigenic properties and host specificity, offers an alternative to antibiotics. This study identified high-binding-affinity adhesion proteins from F. necrophorum using binding and pull-down assays with bovine adrenal gland endothelial cells (EJG). Four OMP candidates (17.5 kDa/OmpH, 22.7 kDa/OmpA, 66.3 kDa/cell surface protein (CSP), and a previously characterized 43 kDa OMP) were expressed as recombinant proteins and purified. Rabbit polyclonal antibodies to recombinant OMPs were generated, and their ability to inhibit bacterial binding in vitro was assessed. The results show that treatment with individual polyclonal antibodies against 43 kDa significantly inhibited bacterial adhesion, while other antibodies were less potent. However, combinations of two or more antibodies showed a more prominent inhibitory effect on host-cell adhesion. Thus, our findings suggest that the identified OMPs are involved in fusobacterial attachment to host cells and may have the potential to be leveraged in combination for vaccine development. Future in vivo studies are needed to validate their roles and test the feasibility of an OMP-based subunit vaccine against fusobacterial infections.

Immunoprophylactic potential of recombinant outer membrane protein of Salmonella enterica serovar Typhi against enteric fever in BALB/c mice

ObjectivesThis study was design to explore the immunogenic behavior of LamB and SpaO outer membrane protein of Salmonella serovar Typhi against enteric fever in BALB/c mice. MethodsSpaO or LamB genes of locally isolated strain of S.Typhi was amplified, cloned and expressed. Recombinant protein SpaO and LamB were purified and lyophilized to prepare aluminum hydroxide Gel precipitated vaccine. Clinical trials were performed in BALB/c mice. The Immunoprophylactic potential of recombinant SpaO or LamB alone and in combination at different concentrations were determined using Complement fixation test along with whole culture attenuated and commercial vaccine. ResultsThe immune response of rSpaO and rLamB proteins in combination at 500 µg/ml was significantly high at 48th day and maintained until 60thday. Protective immune potential of recombinant monovalent and multivalent vaccine assessed upon challenge in mice with 109 cells of S.Typhi virulent culture. Out of five, no mice could survive in control group although rSpaO and rLamB in combination at 500 µg/ml conferred 80 % protection. Whereas the monovalent rSpaO and rLamB vaccine elucidate protection 60 % or 40 % respectively. ConclusionFrom this study, it is conclude that combine vaccine rSpaO and rLamB effectively enhanced the immunological response and withstand the challenge of the causative agent more effectively as compared to the monovalent vaccines.

Serodiagnosis of human brucellosis by an indirect ELISA test using recombinant outer membrane protein 19 kDa (rOMP19) as an antigen

BackgroundBrucellosis remains one of the global health concerns that reemerges in recent years. Delayed or inaccurate diagnosis end to a long treatment duration and financial burden; therefore, finding a good antigen for detection of specific anti-Brucella antibodies is crucial. We intended to evaluate the serodiagnosis value of recombinant Brucella outer membrane protein 19 kDa (rOMP19) using indirect ELISA system compared with Rose Bengal test.ResultsThe OMP19 sequence was successfully cloned into pET-28a and produced in E. coli cells (DE3). After extraction and purification of rOMP19, this protein was used for designing indirect ELISA to detect anti-Brucella antibodies in 73 human sera, including 6 brucellosis-positive and 67 brucellosis-negative samples. The accuracy of rOMP19 ELISA was evaluated by receiver operating characteristic (ROC) curve and then compared with Rose Bengal plate test and a commercial anti-IgG Brucella ELISA kit. In comparison with Rose Bengal plate test, the area under the ROC curve was 0.985 (95% CI, 0.96–1.00). From coordinates of the curve, the optimal cut-off value was selected at 0.147, in which the diagnostic sensitivity was 100%, and the specificity was 94%. At this cut-off point, 10 samples were diagnosed as positive (6 true positives and 4 false positives), while negative samples were all correctly diagnosed. The results of our designed rOMP19 ELISA was the same as data obtained from commercial ELISA kit, which applied LPS as an antigen.ConclusionsWe concluded that OMP19 is an efficient antigen for the serodiagnosis of human brucellosis.

Recombinant Salmonella enterica OmpX protein expression and its potential for serologically diagnosing Salmonella abortion in mares.

Salmonella abortion in mares is caused by Salmonella enterica subspecies enterica serovar abortus equi infection and is characterized by premature (abortion) or non-viable fetus birth. Although all horses are susceptible to infection, the condition is more often clinically manifested in pregnant mares, with most abortions recorded in young females. In addition, nonspecific clinical disease signs and poorly sensitive and effective bacteriological diagnostic methods hinder rapid and reliable infection diagnoses. Immunochemical methods such as enzyme-linked immunosorbent assay (ELISA) and immunochromatography assays can facilitate effective and rapid diagnoses. However, they require highly specific and active antigens and antibodies. This study aimed to generate a recombinant S. enterica outer membrane protein X (OmpX) and evaluate its suitability for serological diagnosis of Salmonella abortion in mares. Outer membrane protein X from the S. enterica antigen was synthesized de novo and expressed in Escherichia coli using the pET28 vector. Transformed E. coli cells were cultured under different conditions to detect recombinant OmpX (rOmpX) expression, and rOmpX purification and refolding were both conducted using metal affinity chromatography. Refolded and purified rOmpX was characterized by western blotting, liquid chromatography with tandem mass spectrometry, and ELISA. After optimized rOmpX expression, a 23 kDa molecular weight protein was identified. Amino acid sequence analysis using Mascot program suggested that these peptides were the OmpX protein from S. enterica. High specificity and diagnostic efficiency were recorded when rOmpX was used in ELISA against 89 serum samples from aborted and contact mares. Recombinant outer membrane protein, in comparison to the O antigen, demonstrated better diagnostic characteristics against sera from mares who aborted and contact horses.

Immunoassay-based evaluation of rOmp28 protein as a candidate for the identification of Brucella species.

Introduction. Brucellosis is an important bacterial zoonosis, re-emerging as a serious public health concern in developing countries. Two major species, Brucella melitensis and Brucella abortus, cause recurrent facile infection in human. Therefore, rapid and accurate diagnosis for early disease control and prevention is needed in areas with low disease burden.Hypothesis. This study evaluated the sandwich enzyme-linked immunosorbent assay (ELISA) (S-ELISA) immunoassay for potential use of whole-cell (WC) and recombinant outer-membrane protein (rOmp28)-derived IgG polyclonals in sensitive detection of Brucella.Aim. Immunoassay-based WC detection of Brucella species in important sub-clinical matrices at lower limits of detection.Methodology. We purified recombinant rOmp28 with Ni-NTA gel affinity chromatography and produced IgG polyclonal antibodies (pAbs) using BALB/c mice and New Zealand white female rabbits against different antigens (Ags) of Brucella. Checkerboard sandwich ELISA and P/N ratio (optical density of 'P' positive test sample to 'N' negative control) were used for evaluation and optimization of the study. The pAbs were characterized using Western blot analysis and different matrices were spiked with WC Ag of Brucella.Results. Double-antibody S-ELISA was developed using WC Ag-derived rabbit IgG (capture antibody at 10 µg ml-1) and rOmp28-derived mice IgG (detection antibody at 100 µg ml-1) with a detection range of 102 to 108 cells ml-1 and a limit of detection at 102 cells ml-1. A P/N ratio of 1.1 was obtained with WC pAbs as compared to 0.6 and 0.9 ratios with rOmp28-derived pAbs for detecting B. melitensis 16M and B. abortus S99, respectively. An increased P/N ratio of 4.4 was obtained with WC Ag-derived rabbit IgG as compared to 4.2>4.1>2.4 ratios obtained with rabbit IgGs derived against cell envelope (CE), rOmp28 and sonicated antigen (SA) of Brucella with high affinity for rOmp28 Ag analysed on immunoblots. The rOmp28-derived mice IgG revealed two Brucella species at P/N ratios of 11.8 and 6.3, respectively. Upon validation, S-ELISA detected Brucella WCs in human whole blood and sera samples with no cross-reactivity to other related bacteria.Conclusion. The developed S-ELISA is specific and sensitive in early detection of Brucella from different matrices of clinical and non-clinical disease presentation.

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

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

In Silico Analysis and Immune Response of YaeT Protein Against Riemerella anatipestifer in Ducks.

Riemerella anatipestifer (RA) is one of the most harmful bacterial pathogens in waterfowl and causes enormous economic loss worldwide. Due to weak cross-immunity protection against different serotypes of RA, inactivated and attenuated vaccines are only effective for RA of specific serotypes. In this paper, outer membrane protein YaeT in RA was analyzed through bioinformatics, in vivo, and in vitro assays. Homology, physicochemical and structural properties, transmembrane domains, and B-cell binding epitopes were investigated. The recombinant outer membrane protein YaeT was then inoculated into Cherry Valley ducks to analyze its immune protection against RA. Results showed that the protein was conservative in different RA strains and had sufficient B-cell binding epitopes. The immunized duck serum contains high-affinity antibodies that could activate complement and promote the opsonophagocytosis of RA by phagocytes. After RA challenge, the survival rate of the YaeT protein-immunized ducks was 80%.

Immunogenicity and protective efficacy of OmpA subunit vaccine against Aeromonas hydrophila infection in Megalobrama amblycephala: An effective alternative to the inactivated vaccine.

Aeromonas hydrophila is a kind of zoonotic pathogen, which can cause bacterial septicemia in fish and bring huge economic losses to global aquaculture. Outer membrane proteins (Omps) are conserved antigens of Aeromonas hydrophila, which can be developed as subunit vaccines. To evaluate the protective efficacy of inactivated vaccine and recombinant outer membrane protein A (OmpA) subunit vaccine against A. hydrophila in juvenile Megalobrama amblycephala, the present study investigated the immunogenicity and protective effects of both vaccines, as well as the non-specific and specific immune response of M. amblycephala. Compared with the non-vaccinated group, both inactivated and OmpA subunit vaccines improved the survival rate of M. amblycephala upon infection. The protective effects of OmpA vaccine groups were better than that of the inactivated vaccine groups, which should be attributed to the reduced bacterial load and enhanced host immunity in the vaccinated fish. ELISA assay showed that the titer of serum immunoglobulin M (IgM) specific to A. hydrophila up-regulated significantly in the OmpA subunit vaccine groups at 14 d post infection (dpi), which should contribute to better immune protective effects. In addition, vaccination enhanced host bactericidal abilities might also attribute to the regulation of the activities of hepatic and serum antimicrobial enzymes. Moreover, the expression of immune-related genes (SAA, iNOS, IL-1 β, IL-6, IL-10, TNF α, C3, MHC I, MHC II, CD4, CD8, TCR α, IgM, IgD and IgZ) increased in all groups post infection, which was more significant in the vaccinated groups. Furthermore, the number of immunopositive cells exhibiting different epitopes (CD8, IgM, IgD and IgZ) that were detected by immunohistochemical assay had increased in the vaccinated groups post infection. These results show that vaccination effectively stimulated host immune response (especially OmpA vaccine groups). In conclusion, these results indicated that both the inactivated vaccine and OmpA subunit vaccine could protect juvenile M. amblycephala against A. hydrophila infection, of which OmpA subunit vaccine provided more effective immune protection and can be used as an ideal candidate for the A. hydrophila vaccine.

Effects of different cytoplasmic chaperones on outer membrane solubility in Escherichia coli; rOMP25 modeling

In this study, for the first time in the literature, is investigated the effects of different chaperones on outer transmembrane membrane protein solubility by modeling the expression of the recombinant Omp25 outer membrane protein, which is one of the main antigens against brucellosis and known to form inclusion bodies in the cytoplasm.

Immune Protective Efficacy of Recombinant Outer Membrane Protein H of Pasteurella multocida B:2 Associated with Hemorrhagic Septicemia in Pakistan

Immune Protective Efficacy of Recombinant Outer Membrane Protein H of Pasteurella multocida B:2 Associated with Hemorrhagic Septicemia in Pakistan

Development of a diagnostic indirect ELISA test for detection of Brucella antibody using recombinant outer membrane protein 16 kDa (rOMP16).

Brucellosis is considered as one of the important global zoonotic diseases that causes medical as well as economic problems especially in tropical countries. The illness has no specific pathognomonic signs; therefore, the rapid and accurate diagnosis of the disease has a very important role in preventing the Brucella spillover and treatment. The purpose of this study was to design a new indirect ELISA test for detection of human brucellosis based on using recombinant Brucella abortus outer membrane protein 16 kDa (rOMP16) as an antigen. OMP16 gene of B. abortus was initially synthesized and cloned in pET-21d vector and then expressed in Escherichia coli cells. The expression was confirmed by the SDS-PAGE, western blotting and dot blotting. The purified protein was coated in ELISA plates and an indirect ELISA was performed on 70 human serum samples. The results were evaluated with a commercial IgG ELISA kit and Rose Bengal plate agglutination tests as reference tests. Diagnostic performance of designed OMP16 ELISA test in comparison with Rose Bengal plate test revealed 100% of sensitivity, 95.00% of specificity and good Fleiss kappa agreement, whereas, where it was compared to commercial ELISA kit, it revealed very good kappa agreement with 100% of sensitivity and 100% of specificity in cut-off value of 0.13. It was concluded that OMP 16 kDa could be acceptable alternative antigen for detecting Brucella IgG antibody with high accuracy.

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.