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.

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.

Evaluation of the safety, immunogenicity and protective effect of an attenuated Pseudomonas plecoglossicida strain ΔgacS as the live vaccine for the large yellow croaker (Larimichthys crocea).

Pseudomonas plecoglossicida is one of most important pathogenic bacterial species in large yellow croaker and several other commercially valuable fish species. In our previous study, a GacS deficient mutant (ΔgacS) was constructed and its virulence showed substantially attenuated. In present study, the safety, immunogenicity and protective effect of the ΔgacS were evaluated in large yellow croaker as a live-attenuated vaccine candidate. It was shown that the ΔgacS strain exhibited good safety to large yellow croaker and there was no mortality or clinical symptoms observed in all fish that infected by ΔgacS strain with the doses range from 2 × 105～107 CFU per fish via intraperitoneal injection (IP) or immersion (IM), and almost all bacteria were cleaned up in the spleen of the fish at 14-day post infection. Specific antibodies could be detected at 7-day and 14-day post infection by direct agglutination method, and the valences of antibodies and bactericidal activities of the serum were significant increased with vaccination doses and vaccination time. Moreover, the expressions of some molecules and cytokines involved in specific immune responses were detected in the ΔgacS strain immunization group and control group. After challenged by the wild-type (WT) strain XSDHY-P, the relative percentage survival (RPS) showed highly correlated with the immunized dosage regardless of vaccination methods. It showed that the RPS of the IP groups were 39.47 %, 57.89 %, 71.05 % with the immune dosage in a descending order, respectively, and the RPS of the IM groups were 26.31 %, 36.84 %, 76.31 % with the immune dosage in a descending order, respectively. In summary, the ΔgacS strain exhibited safety and good protective effect to large yellow croaker and was a potential live vaccine candidate.

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.

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.

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.

Structural and biological insights into outer membrane protein lipotoxin F of Pseudomonas aeruginosa: Implications for vaccine application.

Due to the increasing antibiotic resistance of Pseudomonas aeruginosa (PA), an effective vaccine is urgently needed. However, no PA vaccine has been approved to date, and new protective antigens are needed to improve their efficacy. In this study, Luminex beads were used to identify new candidate antigens, after which their crystal structure was determined, and their potential contribution to bacterial pathogenesis was assessed in vitro and in vivo. Notably, a significant antibody response against the outer membrane protein LptF (lipotoxin F) was detected in sera from 409 volunteers. Moreover, vaccination with recombinant LptF conferred effective protection in an acute PA pneumonia model. The crystal structure showed that LptF comprises a 3-stranded ß-sheet (ß1-ß3) and three α-helices (α1-α3) that are organized in an α/ß/α/ß/α/ß pattern, which is structurally homologous to OmpA and related outer membrane proteins. In addition, LptF binds to peptidoglycan in an atypical manner, contributing to the pathogenesis and survival of PA under stress. Our data indicate that LptF is an important virulence factor and thus a promising candidate antigen for PA vaccines.

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.

Multicomponent Pseudomonas aeruginosa Vaccines Eliciting Th17 Cells and Functional Antibody Responses Confer Enhanced Protection against Experimental Acute Pneumonia in Mice.

The Gram-negative pathogen Pseudomonas aeruginosa is a common cause of pneumonia in hospitalized patients. Its increasing antibiotic resistance and widespread occurrence present a pressing need for vaccines. We previously showed that a P. aeruginosa type III secretion system protein, PopB, elicits a strong Th17 response in mice after intranasal (IN) immunization and confers antibody-independent protection against pneumonia in mice. In the current study, we evaluated the immunogenicity and protective efficacy in mice of the combination of PopB (purified with its chaperone protein PcrH) and OprF/I, an outer membrane hybrid fusion protein, compared with immunization with the proteins individually either by the intranasal (IN) or subcutaneous (SC) routes. Our results show that after vaccination, a Th17 recall response from splenocytes was detected only in mice vaccinated with PopB/PcrH, either alone or in combination with OprF/I. Mice immunized with the combination of PopB/PcrH and OprF/I had enhanced protection in an acute lethal P. aeruginosa pneumonia model, regardless of vaccine route, compared with mice vaccinated with either alone or adjuvant control. Immunization generated IgG titers against the vaccine proteins and whole P. aeruginosa cells. Interestingly, none of these antisera had opsonophagocytic killing activity, but antisera from mice immunized with vaccines containing OprF/I, had the ability to block IFN-γ binding to OprF/I, a known virulence mechanism. Hence, vaccines combining PopB/PcrH with OprF/I that elicit functional antibodies lead to a broadly and potently protective vaccine against P. aeruginosa pulmonary infections.

Intranasal Delivery of Antigen-Coated Polymer Particles Protects against Pseudomonas aeruginosa Infection.

Pseudomonas aeruginosa is an opportunistic human pathogen that is intrinsically resistant to multiple antibiotics, causing severe and persistent infections in immunocompromised individuals. This bacterium has been listed as a priority pathogen by the WHO in 2017, and there is no vaccine available for human use. In this study, 10 vaccine candidate antigens were selected for particulate vaccine design. We engineered Escherichia coli to assemble biopolymer particles (BPs) that were either coated with epitopes (Ag) derived from OprF/I-AlgE proteins or PopB or PopB-Ag or coated with single or double copies of epitopes (10Ag and 10Ag(2x)) derived from OprF, OprI, AlgE, OprL, PopB, PilA, PilO, FliC, Hcp1, and CdrA. Antigen-coated BPs showed a diameter of 0.93-1.16 µm with negative surface charge. Antigens attached to BPs were identified by mass spectrometry. Vaccination with BP-Ag, BP-PopB, BP-PopBAg, PB-10Ag, and BP-10Ag(2x) with and without Alhydrogel adjuvant induced significant antigen-specific humoral and cell-mediated immune responses in mice. All particulate vaccines with Alhydrogel induced protection in an acute pneumonia murine model of P. aeruginosa infection, contributing to up to 80% survival when administered intramuscularly, and the addition of Alhydrogel boosted immunity. The BP-10Ag(2x) vaccine candidate showed the best performance and even induced protective immunity in the absence of Alhydrogel. Intramuscular administration of the BP-10Ag(2x) without Alhydrogel vaccine resulted in 60% survival. Intranasal vaccination induced immunity, contributing to about 90% survival. Overall, our data suggest that vaccination with BPs coated with P. aeruginosa antigens induce protective immunity against P. aeruginosa infections. The possibility of intranasal delivery will strongly facilitate administration and use of BP vaccines.

A novel inactivated whole-cell Pseudomonas aeruginosa vaccine that acts through the cGAS-STING pathway.

Pseudomonas aeruginosa infection continues to be a major threat to global public health, and new safe and efficacious vaccines are needed for prevention of infections caused by P. aeruginosa. X-ray irradiation has been used to prepare whole-cell inactivated vaccines against P. aeruginosa infection. However, the immunological mechanisms of X-ray-inactivated vaccines are still unclear and require further investigation. Our previous study found that an X-ray-inactivated whole-cell vaccine could provide protection against P. aeruginosa by boosting T cells. The aim of the present study was to further explore the immunological mechanisms of the vaccine. Herein, P. aeruginosa PAO1, a widely used laboratory strain, was utilized to prepare the vaccine, and we found nucleic acids and 8-hydroxyguanosine in the supernatant of X-ray-inactivated PAO1 (XPa). By detecting CD86, CD80, and MHCII expression, we found that XPa fostered dentritic cell (DC) maturation by detecting. XPa stimulated the cGAS-STING pathway as well as Toll-like receptors in DCs in vitro, and DC finally underwent apoptosis and pyroptosis after XPa stimulation. In addition, DC stimulated by XPa induced CD8+ T-cell proliferation in vitro and generated immunologic memory in vivo. Moreover, XPa vaccination induced both Th1 and Th2 cytokine responses in mice and reduced the level of inflammatory factors during infection. XPa protected mice in pneumonia models from infection with PAO1 or multidrug-resistant clinical isolate W9. Chronic obstructive pulmonary disease (COPD) mice immunized with XPa could resist PAO1 infection. Therefore, a new mechanism of an X-ray-inactivated whole-cell vaccine against P. aeruginosa infection was discovered in this study.

Outbreak of Pseudomonas aeruginosa mastitis in a dairy cow herd in northern Greece and its control with an autogenous vaccine.

In a dairy cow herd consisted of herd of 200 lactating Holstein-Friesian cows and heifers, clinical signs of mastitis in 40 out of 170 animals were observed. Treatments with antibiotics were proved ineffective. Milk bacterial cultures from 15 affected animals revealed Pseudomonas aeruginosa. An autogenous vaccine was administered subcutaneously, twice in a month period, to all adults. Cases of clinical mastitis declined significantly (p⟨0.0001) during next 3 months.

Designing multi-epitope vaccine candidates against functional amyloids in Pseudomonas aeruginosa through immunoinformatic and structural bioinformatics approach.

Pseudomonas aeruginosa (P. aeruginosa) displays high drug resistance and biofilm-mediated adaptability, which makes its infections difficult to treat. Alternative intervention methods and targets have made such infections treatment manageable. One of the biofilm components, functional amyloids of Pseudomonas (Fap) is correlated positively with virulence and mucoidy phenotype found in infection in cystic fibrosis (CF) patients. Extracellular accessibility, conservation across P. aeruginosa isolates and linkage with lung infections phenotype in CF patients, makes Fap a promising intervention target. Furthermore, the reported effect of bacterial amyloid on neuronal function and immune response makes it a targetable candidate. In the current study, Fap C protein and its immediate interactions were explored to extract antigenic T-cell and B-cell epitopes. A combination of epitopes and peptide adjuvants has been linked to derive vaccine candidate structures. The vaccine candidates were validated for antigenicity, allergenicity, physiochemical properties, stability and interactions with TLRs and MHC alleles. Immunosimulation studies have demonstrated that vaccines elicit Th1 dominated response, which can assist in good prognosis of infection in CF patients.

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.

Understanding Pseudomonas aeruginosa-Host Interactions: The Ongoing Quest for an Efficacious Vaccine.

Pseudomonas aeruginosa is a leading cause of chronic respiratory infections in people with cystic fibrosis (CF), bronchiectasis or chronic obstructive pulmonary disease (COPD), and acute infections in immunocompromised individuals. The adaptability of this opportunistic pathogen has hampered the development of antimicrobial therapies, and consequently, it remains a major threat to public health. Due to its antimicrobial resistance, vaccines represent an alternative strategy to tackle the pathogen, yet despite over 50 years of research on anti-Pseudomonas vaccines, no vaccine has been licensed. Nevertheless, there have been many advances in this field, including a better understanding of the host immune response and the biology of P. aeruginosa. Multiple antigens and adjuvants have been investigated with varying results. Although the most effective protective response remains to be established, it is clear that a polarised Th2 response is sub-optimal, and a mixed Th1/Th2 or Th1/Th17 response appears beneficial. This comprehensive review collates the current understanding of the complexities of P. aeruginosa-host interactions and its implication in vaccine design, with a view to understanding the current state of Pseudomonal vaccine development and the direction of future efforts. It highlights the importance of the incorporation of appropriate adjuvants to the protective antigen to yield optimal protection.

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.

Immunopotentiation of the engineered low-molecular-weight pilin targeting Pseudomonas aeruginosa: A combination of immunoinformatics investigation and active immunization.

Several vaccine candidates have been introduced for immunization against Pseudomonas aeruginosa strains. Despite extensive efforts in recent decades, there is no accurate immunogenic candidate against this pathogen in the market yet. Due to the rapid increase in several drug-resistant strains, P. aeruginosa has caused various health concerns worldwide. It encodes many specific virulence features, which can be used as an appropriate vaccine candidate. The primary stage of the pathogenesis of P. aeruginosa is the expression of many dynamic adhesive molecules, such as type IV pili (T4P), which acts as a principal colonization factor. It has been confirmed that three different subtypes of T4P, including type IVa (T4aP), type IVb (T4bP) and tight adherence (Tad) pili are expressed by P. aeruginosa. The IVa fimbriae type is almost the main cause of challenges to design an effective pili based-immunotherapy method. Nevertheless, in terms of heterogeneity, variability and hidden conserved binding site of T4aP, this attitude has been remained controversial and there is no permitted human study based on IVa pilin commercially. The engineered synthetic peptide-based vaccines are highly talented to mimic the target. In this research, for the first time, some dominant immunogenic features of the Flp protein, such as both B- and T-cell-associated epitopes, presence of IgE-associated epitopes, solvent-accessible surface area were evaluated by analytical immunoinformatics methods. In addition, we designed the engineered Flp pilin as an effective immunogenic substance against several clinically important P. aeruginosa strains. Moreover, by practical active immunization approaches, the humoral and cellular immune response against the extremely conserved region of the engineered synthetic Flp (EFlp) formulated in Montanide ISA 266 compared to the control group. The results of active immunization against EFlp significantly signified that EFlp-Montanide ISA 266 (EFLP-M) strongly could induce both humoral and cellular immune responses. We concluded that Flp pilin has therapeutic potential against numerous clinically significant P. aeruginosa strains and can be served as a novel immunogen for further investigations for development of effective immunotherapy methods against P. aeruginosa as a dexterous pathogen.

Oligomerization of IC43 resulted in improved immunogenicity and protective efficacy against Pseudomonas aeruginosa lung infection.

IC43, a truncate form of outer membrane proteins OprF190-342 and OprI21-83 from Pseudomonas aeruginosa, is a promising candidate antigen and exists as monomer in solution. In this study, we generated the heptamer of IC43 by carrier protein aided oligomerization, which was confirmed by gel-filtration and chemical cross-linking analysis. The carrier protein naturally exists as a homo-heptamer, and IC43 was displayed on the surface of the carrier protein in the fusion protein. Immunization with this fusion protein resulted in increased level of antigen specific IgG antibodies and higher survival rate after infection. The improved efficacy was correlated with lower bacteria burden, inflammation and tissue damage in the lungs of immunized mice. Further studies revealed that immunization with this fusion protein resulted in increased levels of IL-4 and antigen specific IgG1, suggesting a stronger Th2 immune response was induced. The improved immunogenicity may be attributed to the exposure of more epitopes on the antigen, which was confirmed by results from immune-dominant peptide mapping and passive immunization. These results demonstrated a possible strategy to improve the immunogenicity of an antigen by carrier protein aided oligomerization.

Protective Potential of Conjugated P. aeruginosa LPS -PLGA Nanoparticles in Mice as a Nanovaccine.

BACKGROUND: Pseudomonas aeruginosa has an important role in nosocomial infections. OBJECTIVE: To evaluate biological activity of the detoxified LPS (D-LPS) entrapped into Poly lactic-co-glycolic acid (PLGA) nanoparticles. MATERIALS: LPS was extracted and detoxified from the P. aeruginosa strain PAO1. The D-LPS, conjugated to the PLGA nanoparticles with 1-ethyl-3-dimethyl aminopropyl carbodiimide (EDAC) and N-hydroxy-succinimide (NHS). The connection was evaluated by FTIR (Fourier transform infrared), Zetasizer, and Atomic Force Microscope (AFM). The BALB/c mice injected intramuscularly with the D-LPS-PLGA with two-week intervals and then challenged two weeks after the last immunization. The bioactivity of the induced specific antisera and cytokines responses against D-LPS-PLGA antigen was assessed by ELISA. RESULTS: D-LPS-PLGA conjugation was confirmed by FTIR, Zetasizer, and AFM. The ELISA results showed that D-LPS was successful in the stimulation of the humoral immune response. The immune responses raised against the D-LPS-PLGA, significantly decreased bacterial titer in the spleen of the immunized mice after challenge with PAO1 strain in comparison with the control groups. CONCLUSION: The conjugation of the bacterial LPS to the PLGA nanoparticle increased their functional activity by decrease in bacterial dissemination and increase the killing of opsonized bacteria.

Immunological considerations in the development of Pseudomonas aeruginosa vaccines.

Pseudomonas aeruginosa is an opportunistic human pathogen capable of causing a wide range of potentially life-threatening infections. With multidrug-resistant P. aeruginosa infections on the rise, the need for a rationally-designed vaccine against this pathogen is critical. A number of vaccine platforms have shown promising results in pre-clinical studies, but no vaccine has successfully advanced to licensure. Growing evidence suggests that an effective P. aeruginosa vaccine may require Th17-type CD4+ T cells to prevent infection. In this review, we summarize recent pre-clinical studies of P. aeruginosa vaccines, specifically focusing on those that induce Th17-type cellular immunity. We also highlight the importance of adjuvant selection and immunization route in vaccine design in order to target vaccine-induced immunity to infected tissues. Advances in cellular immunology and adjuvant biology may ultimately influence better P. aeruginosa vaccine platforms that can protect targeted human populations.