A computational approach to developing a multi-epitope vaccine for combating Pseudomonas aeruginosa-induced pneumonia and sepsis.

Pseudomonas aeruginosa is a complex nosocomial infectious agent responsible for numerous illnesses, with its growing resistance variations complicating treatment development. Studies have emphasized the importance of virulence factors OprE and OprF in pathogenesis, highlighting their potential as vaccine candidates. In this study, B-cell, MHC-I, and MHC-II epitopes were identified, and molecular linkers were active to join these epitopes with an appropriate adjuvant to construct a vaccine. Computational tools were employed to forecast the tertiary framework, characteristics, and also to confirm the vaccine's composition. The potency was weighed through population coverage analysis and immune simulation. This project aims to create a multi-epitope vaccine to reduce P. aeruginosa-related illness and mortality using immunoinformatics resources. The ultimate complex has been determined to be stable, soluble, antigenic, and non-allergenic upon inspection of its physicochemical and immunological properties. Additionally, the protein exhibited acidic and hydrophilic characteristics. The Ramachandran plot, ProSA-web, ERRAT, and Verify3D were employed to ensure the final model's authenticity once the protein's three-dimensional structure had been established and refined. The vaccine model showed a significant binding score and stability when interacting with MHC receptors. Population coverage analysis indicated a global coverage rate of 83.40%, with the USA having the highest coverage rate, exceeding 90%. Moreover, the vaccine sequence underwent codon optimization before being cloned into the Escherichia coli plasmid vector pET-28a (+) at the EcoRI and EcoRV restriction sites. Our research has developed a vaccine against P. aeruginosa that has strong binding affinity and worldwide coverage, offering an acceptable way to mitigate nosocomial infections.

Selection of vaccine candidates against Pseudomonas koreensis using reverse vaccinology and a preliminary efficacy trial in Empurau (Tor tambroides).

This study marks the first utilization of reverse vaccinology to develop recombinant subunit vaccines against Pseudomonas koreensis infection in Empurau (Tor tambroides). The proteome (5538 proteins) was screened against various filters to prioritize proteins based on features that are associated with virulence, subcellular localization, transmembrane helical structure, antigenicity, essentiality, non-homology with the host proteome, molecular weight, and stability, which led to the identification of eight potential vaccine candidates. These potential vaccine candidates were cloned and expressed, with six achieving successful expression and purification. The antigens were formulated into two distinct vaccine mixtures, Vac A and Vac B, and their protective efficacy was assessed through in vivo challenge experiments. Vac A and Vac B demonstrated high protective efficacies of 100 % and 81.2 %, respectively. Histological analyses revealed reduced tissue damage in vaccinated fish after experimental infection, with Vac A showing no adverse effects, whereas Vac B exhibited mild degenerative changes. Quantitative real-time PCR results showed a significant upregulation of TNF-α and downregulation of IL-1ß in the kidneys, spleen, gills, and intestine in both Vac A- and Vac B-immunized fish after challenged with P. koreensis. Additionally, IL-8 exhibits tissue-specific differential expression, with significant upregulation in the kidney, gills, and intestine, and downregulation in the spleen, particularly notable in Vac A-immunized fish. The research underscores the effectiveness of the reverse vaccinology approach in fish and demonstrates the promising potential of Vac A and Vac B as recombinant subunit vaccines.

Defining the Mechanistic Correlates of Protection Conferred by Whole-Cell Vaccination against Pseudomonas aeruginosa Acute Murine Pneumonia.

Pseudomonas aeruginosa is a Gram-negative pathogen that causes severe pulmonary infections associated with high morbidity and mortality in immunocompromised patients. The development of a vaccine against P. aeruginosa could help prevent infections caused by this highly antibiotic-resistant microorganism. We propose that identifying the vaccine-induced correlates of protection against P. aeruginosa will facilitate the development of a vaccine against this pathogen. In this study, we investigated the mechanistic correlates of protection of a curdlan-adjuvanted P. aeruginosa whole-cell vaccine (WCV) delivered intranasally. The WCV significantly decreased bacterial loads in the respiratory tract after intranasal P. aeruginosa challenge and raised antigen-specific antibody titers. To study the role of B and T cells during vaccination, anti-CD4, -CD8, and -CD20 depletions were performed prior to WCV vaccination and boosting. The depletion of CD4+, CD8+, or CD20+ cells had no impact on the bacterial burden in mock-vaccinated animals. However, depletion of CD20+ B cells, but not CD8+ or CD4+ T cells, led to the loss of vaccine-mediated bacterial clearance. Also, passive immunization with serum from WCV group mice alone protected naive mice against P. aeruginosa, supporting the role of antibodies in clearing P. aeruginosa We observed that in the absence of T cell-dependent antibody production, mice vaccinated with the WCV were still able to reduce bacterial loads. Our results collectively highlight the importance of the humoral immune response for protection against P. aeruginosa and suggest that the production of T cell-independent antibodies may be sufficient for bacterial clearance induced by whole-cell P. aeruginosa vaccination.

Construction of Genomic Library and High-Throughput Screening of Pseudomonas aeruginosa Novel Antigens for Potential Vaccines.

Hospital-acquired infections with Pseudomonas aeruginosa have become a great challenge in caring for critically ill and immunocompromised patients. The cause of high mortality is the presence of multi-drug resistant (MDR) strains, which confers a pressing need for vaccines. Although vaccines against P. aeruginosa have been in development for more than several decades, there is no vaccine for patients at present. In this study, we purified genomic DNA of P. aeruginosa from sera of patients affected, constructed genome-wide library with random recombinants, and screened candidate protein antigens by evaluating their protective effects in vivo. After 13-round of screening, 115 reactive recombinants were obtained, among which 13 antigens showed strong immunoreactivity (more than 10% reaction to PcrV, a well-characterized V-antigen of P. aeruginosa). These 13 antigens were: PpiA, PtsP, OprP, CAZ10_34235, HmuU_2, PcaK, CarAd, RecG, YjiR_5, LigD, KinB, RtcA, and PscF. In vivo studies showed that vaccination with PscF protected against lethal P. aeruginosa challenge, and decreased lung inflammation and injury. A genomic library of P. aeruginosa could be constructed in this way for the first time, which could not only screen candidate antigens but also in a high-throughput way. PscF was considered as an ideal promising vaccine candidate for combating P. aeruginosa infection and was supported for further evaluation of its safety and efficacy.

Exogenous remodeling of lung resident macrophages protects against infectious consequences of bone marrow-suppressive chemotherapy.

Infection is the single greatest threat to survival during cancer chemotherapy because of depletion of bone marrow-derived immune cells. Phagocytes, especially neutrophils, are key effectors in immunity to extracellular pathogens, which has limited the development of new approaches to protect patients with cancer and chemotherapy-induced neutropenia. Using a model of vaccine-induced protection against lethal Pseudomonas aeruginosa pneumonia in the setting of chemotherapy-induced neutropenia, we found a population of resident lung macrophages in the immunized lung that mediated protection in the absence of neutrophils, bone marrow-derived monocytes, or antibodies. These vaccine-induced macrophages (ViMs) expanded after immunization, locally proliferated, and were closely related to alveolar macrophages (AMs) by surface phenotype and gene expression profiles. By contrast to AMs, numbers of ViMs were stable through chemotherapy, showed enhanced phagocytic activity, and prolonged survival of neutropenic mice from lethal P. aeruginosa pneumonia upon intratracheal adoptive transfer. Thus, induction of ViMs by tissue macrophage remodeling may become a framework for new strategies to activate immune-mediated reserves against infection in immunocompromised hosts.

The protective effects of nasal PcrV-CpG oligonucleotide vaccination against Pseudomonas aeruginosa pneumonia.

An effective vaccine against Pseudomonas aeruginosa would be hugely beneficial to people who are susceptible to the serious infections it can cause. Vaccination against PcrV of the P. aeruginosa type III secretion system is a potential prophylactic strategy for improving the incidence and prognosis of P. aeruginosa pneumonia. Here, the effect of nasal PcrV adjuvanted with CpG oligodeoxynucleotide (CpG) was compared with a nasal PcrV/aluminum hydroxide gel (alum) vaccine. Seven groups of mice were vaccinated intranasally with one of the following: 1, PcrV-CpG; 2, PcrV-alum; 3, PcrV alone; 4, CpG alone; 5, alum alone; 6 and 7, saline control. Fifty days after the first immunization, anti-PcrV IgG, IgA and IgG isotype titers were measured; significant increases in these titers were detected only in the PcrV-CpG vaccinated mice. The vaccinated mice were then intratracheally infected with a lethal dose of P. aeruginosa and their body temperatures and survival monitored for 24 hr, edema, bacteria, myeloperoxidase activity and lung histology also being evaluated at 24 hr post-infection. It was found that 73% of the PcrV-CpG-vaccinated mice survived, whereas fewer than 30% of the mice vaccinated with PcrV-alum or adjuvant alone survived. Lung edema and other inflammation-related variables were less severe in the PcrV-CpG group. The significant increase in PcrV-specific IgA titers detected following PcrV-CpG vaccination is probably a component of the disease protection mechanism. Overall, our data show that intranasal PcrV-CpG vaccination has potential efficacy for clinical application against P. aeruginosa pneumonia.

[Study on Construction of Recombinant Bb-pGEX-OprI Vaccine of Pseudomonas aeruginosa and Its Protection Effect].

OBJECTIVE: To construct the recombinant Bb-pGEX-OprI vaccine of Pseudomonas aeruginosa (Pa) outer membrane protein I (OprI) and study its protection effect in mice against Pa. METHODS: The OprI gene was amplified by PCR,and cloned into pGEX-1λT to generate pGEX-OprI. The pGEX-OprI was transformed into Bifidobacterium bifidum(Bb) to construct recombinant Bb-pGEX-OprI vaccine by electroporation. After identification with double enzyme digestion,PCR and sequencing,the vaccine was then induced with IPTG,and its expression was analyzed and identified by SDS-PAGE and Western blot respectively. Twenty-one mice were randomly divided into 3 groups and vaccinated by intragastric administration with Bb-pGEX-OprI,Bb-pGEX-1λT and Bb respectively. All mice were challenged with PA01 strain at 4 weeks after the first vaccination. At 2 weeks after the challenge,mice were sacrificed to separate their lungs,and the numbers of bacterial colonies in lungs were counted. Venous blood was collected before vaccination,at 4 weeks after the first vaccination and 2 weeks after the challenge of PA01 strain. The serum IgG,IgG subclasses and IgE were detected by routine ELISA. RESULTS: The OprI gene of 194 bp was successfully amplified by PCR. Double enzyme digestion,PCR and sequencing confirmed that the OprI gene was successfully cloned into pGEX-1λT and pGEX-OprI was successfully transformed into Bb,constructing the Bb-pGEX-OprI vaccine. SDS-PAGE indicated that Bb-pGEX-OprI vaccine expressed an OprI-GST fusion protein with the relative molecular mass of approximately 32×103. Western blot verified that the fusion protein could be specifically identified by the sera of mice infected with Pa. The number of bacterial colonies in lung of Bb-pGEX-OprI vaccine group was lower than that of Bb-pGEX-1λT or Bb control ( P<0.01). The levels of serum IgG,IgG2b,IgG3 and IgE in Bb-pGEX-OprI vaccine group rose at 4 weeks after the first vaccination and 2 weeks after the challenge successively. The levels of serum antibodies in Bb-pGEX-OprI vaccine group were higher than those in Bb-pGEX-1λT or Bb control at the same time point ( P<0.01 or P<0.05). CONCLUSION: The recombinant Bb-pGEX-OprI vaccine was successfully constructed and produced an effective humoral immune response against the Pa infection.

Efficacy comparison of adjuvants in PcrV vaccine against Pseudomonas aeruginosa pneumonia.

Vaccination against the type III secretion system of P. aeruginosa is a potential prophylactic strategy for reducing the incidence and improving the poor prognosis of P. aeruginosa pneumonia. In this study, the efficacies of three different adjuvants, Freund's adjuvant (FA), aluminum hydroxide (alum) and CpG oligodeoxynucleotide (ODN), were examined from the viewpoint of inducing PcrV-specific immunity against virulent P. aeruginosa. Mice that had been immunized intraperitoneally with recombinant PcrV formulated with one of the above adjuvants were challenged intratracheally with a lethal dose of P. aeruginosa. The PcrV-FA immunized group attained a survival rate of 91%, whereas the survival rates of the PcrV-alum and PcrV-CpG groups were 73% and 64%, respectively. In terms of hypothermia recovery after bacterial instillation, PcrV-alum was the most protective, followed by PcrV-FA and PcrV-CpG. The lung edema index was lower in the PcrV-CpG vaccination group than in the other groups. PcrV-alum immunization was associated with the greatest decrease in myeloperoxidase in infected lungs, and also decreased the number of lung bacteria to a similar number as in the PcrV-FA group. There was less neutrophil recruitment in the lungs of mice vaccinated with PcrV-alum or PcrV-CpG than in those of mice vaccinated with PcrV-FA or PcrV alone. Overall, in terms of mouse survival the PcrV-CpG vaccine, which could be a relatively safe next-generation vaccine, showed a comparable effect to the PcrV-alum vaccine.

Preparation of Pseudomonas aeruginosa alginate-flagellin immunoconjugate.

Mucoid strains of Pseudomonas aeruginosa are closely associated with chronic pulmonary infections. In this report we describe a straightforward approach to conjugate high molecular weight alginate to type b-flagellin (FLB) and investigation of its bioactivity. The conjugation process was performed by using ADH and EDAC. The endotoxin was eliminated from the candidate vaccine by LPS removal resin followed by LAL test. The bioconjugate molecules were verified by simultaneously determination of polysaccharide/protein content followed by gel filtration chromatography and FTIR spectroscopy. Groups of eight BALB/c mice were injected intranasally with 5 µg (per each nostril) of purified alginate, FLB and conjugated alginate-FLB with two week intervals. The functional activity of the vaccine was evaluated by ELISA and opsonophagocytosis tests. Vaccination with the alginate-FLB conjugate induced a significant (P = 0.0033) rise in alginate specific IgG in mice. At all dilution ranges, the opsonic activity of the conjugate vaccine antisera was significantly higher than alginate alone (61.9% vs. 17.3% at 1:4 dilution; P = 0.0067). The alginate-FLB conjugate could elicit high specific antibodies titer against alginate by improving its immunogenicity. In addition, the antisera raised against conjugate vaccine act as a suitable opsonin for phagocytosis of the mucoid strains of P. aeruginosa.

Salmonella Typhimurium strain expressing OprF-OprI protects mice against fatal infection by Pseudomonas aeruginosa.

Pseudomonas aeruginosa poses a major threat to human health and to the mink industry. Thus, development of vaccines that elicit robust humoral and cellular immunity against P. aeruginosa is greatly needed. In this study, a recombinant attenuated Salmonella vaccine (RASV) that expresses the outer membrane proteins fusion OprF190-342 -OprI21-83 (F1I2) from P. aeruginosa was constructed and the potency of this vaccine candidate assessed by measuring F1I2-specific humoral immune responses upon vaccination through s.c. or oral routes. S.C. administration achieved higher serum IgG titers and IgA titers in the intestine and induced stronger F1I2-specific IgG and IgA titers in lung homogenate than did oral administration, which resulted in low IgG titers and no local IgA production. High titers of IFN-γ, IL-4, and T-lymphocyte subsets induced a mixed Th1/Th2 response in mice immunized s.c., indicating elicitation of cellular immunity. Importantly, when immunized mice were challenged with P. aeruginosa by the intranasal route 30 days after the initial immunization, s.c. vaccination achieved 77.78% protection, in contrast to 41.18% via oral administration and 66.67% via Escherichia coli-expressed F1I2 (His-F1I2) vaccination. These results indicate that s.c. vaccination provides a better protective response against P. aeruginosa infection than do oral administration and the His-F1I2 vaccine.

Mannose-modified chitosan microspheres enhance OprF-OprI-mediated protection of mice against Pseudomonas aeruginosa infection via induction of mucosal immunity.

Pseudomonas aeruginosa is an opportunistic pathogen that localizes to and colonizes mucosal tissue. Thus, vaccines that elicit a strong mucosal response against P. aeruginosa should be superior to other vaccination strategies. In this study, to stimulate rapid and enhanced mucosal immune responses, mannose-modified chitosan microspheres loaded with the recombinant outer membrane protein OprF190-342-OprI21-83 (FI) (FI-MCS-MPs) of P. aeruginosa were developed as a potent subunit vaccine for mucosal delivery. FI-MCS-MPs were successfully obtained via the tripolyphosphate ionic crosslinking method. Confocal and immunohistochemical analyses indicated that FI-MCS-MPs exhibited the ability to bind the macrophage mannose receptor (MMR, CD206) in vitro and in vivo. After intranasal immunization of mice with FI-MCS-MPs, FI-specific humoral immune responses were detected, measured as local IgM antibody titers in lung tissue slurry; IgA antibody titers in nasal washes, bronchoalveolar lavage (BAL), and intestinal lavage; and systemic IgA and IgG antibody titers in serum. FI-MCS-MPs induced early and high mucosal and systemic humoral antibody responses comparable to those in the group vaccinated with unmodified mannose. High levels of IFN-γ and IL-4 in addition to T lymphocyte subsets induced a mixed Th1/Th2 response in mice immunized with FI-MCS-MPs, resulting in the establishment of cellular immunity. Additionally, when immunized mice were challenged with P. aeruginosa via the nasal cavity, FI-MCS-MPs demonstrated 75 % protective efficacy. Together, these data indicate that mannose-modified chitosan microspheres are a promising subunit delivery system for vaccines against P. aeruginosa infection.

Development of a nano-emulsion based multivalent protein subunit vaccine against Pseudomonas aeruginosa.

Pseudomonas aeruginosa (Pa) is an opportunistic bacterial pathogen responsible for severe hospital acquired infections in immunocompromised and elderly individuals. Emergence of increasingly drug resistant strains and the absence of a broad-spectrum prophylactic vaccine against both T3SA+ (type III secretion apparatus) and ExlA+/T3SA- Pa strains worsen the situation in a post-pandemic world. Thus, we formulated a candidate subunit vaccine (called ExlA/L-PaF/BECC/ME) against both Pa types. This bivalent vaccine was generated by combining the C-terminal active moiety of exolysin A (ExlA) produced by non-T3SA Pa strains with our T3SA-based vaccine platform, L-PaF, in an oil-in-water emulsion. The ExlA/L-PaF in ME (MedImmune emulsion) was then mixed with BECC438b, an engineered lipid A analogue and a TLR4 agonist. This formulation was administered intranasally (IN) to young and elderly mice to determine its potency across a diverse age-range. The elderly mice were used to mimic the infection seen in elderly humans, who are more susceptible to serious Pa disease compared to their young adult counterparts. After Pa infection, mice immunized with ExlA/L-PaF/BECC/ME displayed a T cell-mediated adaptive response while PBS-vaccinated mice experienced a rapid onset inflammatory response. Important genes and pathways were observed, which give rise to an anti-Pa immune response. Thus, this vaccine has the potential to protect aged individuals in our population from serious Pa infection.

Phylogenetic Tracing of Evolutionarily Conserved Zonula Occludens Toxin Reveals a "High Value" Vaccine Candidate Specific for Treating Multi-Strain Pseudomonas aeruginosa Infections.

Extensively drug-resistant Pseudomonas aeruginosa infections are emerging as a significant threat associated with adverse patient outcomes. Due to this organism's inherent properties of developing antibiotic resistance, we sought to investigate alternative strategies such as identifying "high value" antigens for immunotherapy-based purposes. Through extensive database mining, we discovered that numerous Gram-negative bacterial (GNB) genomes, many of which are known multidrug-resistant (MDR) pathogens, including P. aeruginosa, horizontally acquired the evolutionarily conserved gene encoding Zonula occludens toxin (Zot) with a substantial degree of homology. The toxin's genomic footprint among so many different GNB stresses its evolutionary importance. By employing in silico techniques such as proteomic-based phylogenetic tracing, in conjunction with comparative structural modeling, we discovered a highly conserved intermembrane associated stretch of 70 amino acids shared among all the GNB strains analyzed. The characterization of our newly identified antigen reveals it to be a "high value" vaccine candidate specific for P. aeruginosa. This newly identified antigen harbors multiple non-overlapping B- and T-cell epitopes exhibiting very high binding affinities and can adopt identical tertiary structures among the least genetically homologous P. aeruginosa strains. Taken together, using proteomic-driven reverse vaccinology techniques, we identified multiple "high value" vaccine candidates capable of eliciting a polarized immune response against all the P. aeruginosa genetic variants tested.

An unnatural amino acid dependent, conditional Pseudomonas vaccine prevents bacterial infection.

Live vaccines are ideal for inducing immunity but suffer from the need to attenuate their pathogenicity or replication to preclude the possibility of escape. Unnatural amino acids (UAAs) provide a strategy to engineer stringent auxotrophies, yielding conditionally replication incompetent live bacteria with excellent safety profiles. Here, we engineer Pseudomonas aeruginosa to maintain auxotrophy for the UAA p-benzoyl-L-phenylalanine (BzF) through its incorporation into the essential protein DnaN. In vivo evolution using an Escherichia coli-based two-hybrid selection system enabled engineering of a mutant DnaN homodimeric interface completely dependent on a BzF-specific interaction. This engineered strain, Pa Vaccine, exhibits undetectable escape frequency (<10-11) and shows excellent safety in naïve mice. Animals vaccinated via intranasal or intraperitoneal routes are protected from lethal challenge with pathogenic P. aeruginosa PA14. These results establish UAA-auxotrophic bacteria as promising candidates for bacterial vaccine therapy and outline a platform for expanding this technology to diverse bacterial pathogens.

Pseudomonas aeruginosa outer membrane vesicles modulate host immune responses by targeting the Toll-like receptor 4 signaling pathway.

Bacteria can naturally secrete outer membrane vesicles (OMVs) as pathogenic factors, while these vesicles may also serve as immunologic regulators if appropriately prepared. However, it is largely unknown whether Pseudomonas aeruginosa OMVs can activate inflammatory responses and whether immunization with OMVs can provide immune protection against subsequent infection. We purified and identified OMVs, which were then used to infect lung epithelial cells in vitro as well as C57BL/6J mice to investigate the immune response and the underlying signaling pathway. The results showed that OMVs generated from P. aeruginosa wild-type strain PAO1 were more cytotoxic to alveolar epithelial cells than those from quorum-sensing (QS)-deficient strain PAO1-ΔlasR. The levels of Toll-like receptor 4 (TLR4) and proinflammatory cytokines, including interleukin-1ß (IL-1ß) and IL-6, increased following OMV infection. Compared with lipopolysaccharide (LPS), lysed OMVs in which the membrane structures were broken induced a weak immune response. Furthermore, expression levels of TLR4-mediated responders (i.e., cytokines) were markedly downregulated by the TLR4 inhibitor E5564. Active immunization with OMVs or passive transfer of sera with a high cytokine quantity acquired from OMV-immunized mice could protect healthy mice against subsequent lethal PAO1 challenges (1.5 × 10(11) CFU). Collectively, these findings indicate that naturally secreted P. aeruginosa OMVs may trigger significant inflammatory responses via the TLR4 signaling pathway and protect mice against pseudomonal lung infection.

Flagellin stimulates protective lung mucosal immunity: role of cathelicidin-related antimicrobial peptide.

TLRs are required for generation of protective lung mucosal immune responses against microbial pathogens. In this study, we evaluated the effect of the TLR5 ligand flagellin on stimulation of antibacterial mucosal immunity in a lethal murine Pseudomonas aeruginosa pneumonia model. The intranasal pretreatment of mice with purified P. aeruginosa flagellin induced strong protection against intratracheal P. aeruginosa-induced lethality, which was attributable to markedly improved bacterial clearance, reduced dissemination, and decreased alveolar permeability. The protective effects of flagellin on survival required TLR5 and were observed even in the absence of neutrophils. Flagellin induced strong induction of innate genes, most notably the antimicrobial peptide cathelicidin-related antimicrobial peptide. Finally, flagellin-induced protection was partially abrogated in cathelicidin-related antimicrobial peptide-deficient mice. Our findings illustrate the profound stimulatory effect of flagellin on lung mucosal innate immunity, a response that might be exploited therapeutically to prevent the development of gram-negative bacterial infection of the respiratory tract.

[Production of hybrid protein OprF-OprL of Pseudomonas aeruginosa].

AIM: Production of preparation consisting of amino acid sequences of 2 proteins of outer membrane--OprF and OprI--of P. aeruginosa and study of its protective properties from experimental P. aeruginosa infection. MATERIALS AND METHODS: Nucleotide sequences coding OprF protein (1 kb) as well as its C-terminal region (0.6 kb) and OprI protein (0.25 kb) were integrated into pQE-30 plasmid (QIAGEN). And oprF gene (C-terminal region of oprF in variant 2) and oprI gene were combined and cloned sequentially into a single vector. E. coli M15 strain cells (QIAGEN) were used for the production of producent strains of recombinant proteins. Protein products were analyzed by electrophoresis in polyacrylamide gel by Lammle. Purification of recombinant proteins was performed by affinity chromatography in Ni-sepharose columns. Live virulent culture P. aeruginosa PA-170015 strain was used for the analysis of protective properties of recombinant proteins. RESULTS: 2 hybrid recombinant proteins were produced including amino acid sequences of F and I proteins of outer membrane (OprF and OprI) of P. aeruginosa. Recombinant protein 1 included whole size sequences of OprF and OprI and protein 2--C-terminal region (including amino acid residues 192-342) of OprF and whole size sequence of OprI. These recombinant proteins after 2 immunizations protected mice from the experimental intraperitoneal infection with P. aeruginosa. Hybrid protein consisting of whole size sequences had the best protective effect. CONCLUSION: The results obtained open a perspective for further immunobiological testing of hybrid recombinant protein OprF-OprI with the aim of creating immunopreparations for prophylaxis of P. aeruginosa infection.

Pseudomonas aeruginosa PAO1 outer membrane vesicles-diphtheria toxoid conjugate as a vaccine candidate in a murine burn model.

Pseudomonas aeruginosa is an opportunistic pathogen considered a common cause of nosocomial infection with high morbidity and mortality in burn patients. Immunoprophylaxis techniques may lower the mortality rate of patients with burn wounds infected by P. aeruginosa; consequently, this may be an efficient strategy to manage infections caused by this bacterium. Several pathogenic Gram-negative bacteria like P. aeruginosa release outer membrane vesicles (OMVs), and structurally OMV consists of several antigenic components capable of generating a wide range of immune responses. Here, we evaluated the immunogenicity and efficacy of P. aeruginosa PA-OMVs (PA-OMVs) conjugated with the diphtheria toxoid (DT) formulated with alum adjuvant (PA-OMVs-DT + adj) in a mice model of burn wound infection. ELISA results showed that in the group of mice immunized with PA-OMVs-DT + adj conjugated, there was a significant increase in specific antibodies titer compared to non-conjugated PA-OMVs or control groups. In addition, the vaccination of mice with PA-OMVs-DT + adj conjugated generated greater protective effectiveness, as seen by lower bacterial loads, and eightfold decreased inflammatory cell infiltration with less tissue damage in the mice burn model compared to the control group. The opsonophagocytic killing results confirmed that humoral immune response might be critical for PA-OMVs mediated protection. These findings suggest that PA-OMV-DT conjugated might be used as a new vaccine against P. aeruginosa in burn wound infection.

Mucosal vaccination with a multivalent, live-attenuated vaccine induces multifactorial immunity against Pseudomonas aeruginosa acute lung infection.

Many animal studies investigating adaptive immune effectors important for protection against Pseudomonas aeruginosa have implicated opsonic antibody to the antigenically variable lipopolysaccharide (LPS) O antigens as a primary effector. However, active and passive vaccination of humans against these antigens has not shown clinical efficacy. We hypothesized that optimal immunity would require inducing multiple immune effectors targeting multiple bacterial antigens. Therefore, we evaluated a multivalent live-attenuated mucosal vaccination strategy in a murine model of acute P. aeruginosa pneumonia to assess the contributions to protective efficacy of various bacterial antigens and host immune effectors. Vaccines combining 3 or 4 attenuated strains having different LPS serogroups were associated with the highest protective efficacy compared to vaccines with fewer components. Levels of opsonophagocytic antibodies, which were directed not only to the LPS O antigens but also to the LPS core and surface proteins, correlated with protective immunity. The multivalent live-attenuated vaccines overcame prior problems involving immunologic interference in the development of O-antigen-specific antibody responses when closely related O antigens were combined in multivalent vaccines. Antibodies to the LPS core were associated with in vitro killing and in vivo protection against strains with O antigens not expressed by the vaccine strains, whereas antibodies to the LPS core and surface proteins augmented the contribution of O-antigen-specific antibodies elicited by vaccine strains containing a homologous O antigen. Local CD4 T cells in the lung also contributed to vaccine-based protection when opsonophagocytic antibodies to the challenge strain were absent. Thus, multivalent live-attenuated vaccines elicit multifactorial protective immunity to P. aeruginosa lung infections.

Efficacy of a conjugate vaccine containing polymannuronic acid and flagellin against experimental Pseudomonas aeruginosa lung infection in mice.

Vaccines that could effectively prevent Pseudomonas aeruginosa pulmonary infections in the settings of cystic fibrosis (CF) and nosocomial pneumonia could be exceedingly useful, but to date no effective immunotherapy targeting this pathogen has been successfully developed for routine use in humans. Evaluations using animals and limited human trials of vaccines and their associated immune effectors against different P. aeruginosa antigens have suggested that antibody to the conserved surface polysaccharide alginate, as well as the flagellar proteins, often give high levels of protection. However, alginate itself does not elicit protective antibody in humans, and flagellar vaccines containing the two predominant serotypes of this antigen may not provide sufficient coverage against variant flagellar types. To evaluate if combining these antigens in a conjugate vaccine would be potentially efficacious, we conjugated polymannuronic acid (PMA), containing the blocks of mannuronic acid conserved in all P. aeruginosa alginates, to type a flagellin (FLA) and evaluated immunogenicity, opsonic killing activity, and passive protective efficacy in mice. The PMA-FLA conjugate was highly immunogenic in mice and rabbits and elicited opsonic antibodies against mucoid but not nonmucoid P. aeruginosa, but nonetheless rabbit antibody to PMA-FLA showed evidence of protective efficacy against both types of this organism in a mouse lung infection model. Importantly, the PMA-FLA conjugate vaccine did not elicit antibodies that neutralized the Toll-like receptor 5 (TLR5)-activating activity of flagellin, an important part of innate immunity to flagellated microbial pathogens. Conjugation of PMA to FLA appears to be a promising path for developing a broadly protective vaccine against P. aeruginosa.