Production of recombinant chimeric protein of Pseudomonas aeruginosa virulence factors and assessment of its immunogenicity in mice

doi:10.21203/rs.3.rs-3572584/v1

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Production of recombinant chimeric protein of Pseudomonas aeruginosa virulence factors and assessment of its immunogenicity in mice

https://doi.org/10.21203/rs.3.rs-3572584/v1

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Escalating mortality rates attributed to drug-resistant Pseudomonas aeruginosa and the substantial complications arising from its infections present a global health crisis. Consequently, the need for innovative therapeutic approaches, notably immunogenic vaccines, has become imperative. Despite extensive research, it is still uncertain whether Pseudomonas aeruginosa immunization can effectively protect against acute and chronic infections. Additionally, the immune response triggered by this immunization is not strong enough against these bacteria. This study aims to devise a chimeric vaccine encompassing key virulence factors of P. aeruginosa that play roles in distinct infection phases. Moreover, these factors possess the capacity to interact with immune receptors, thereby enhancing innate and acquired immune reactions against this bacterium. In this investigation, an in-depth analysis of the P. aeruginosa profile informs the design of the antigenic chimeric structure named PAV3. This construct incorporates lectin, HIV TAT peptide, the N-terminal fragment of exotoxin A, and the Epi8 outer membrane protein F (OprF). Employing an immunoinformatic approach, B cell epitopes, HTL epitopes, CTL epitopes, and IFN-γ are predicted. The PAV3 construct is then expressed in E. coli strain BL21 and purified using affinity chromatography. Immunogenicity assessment of PAV3 entails measuring total IgG, IgG2a, and IgG1, along with specific cytokines (IFN-γ and IL-4) associated with Th1 and Th2 responses, respectively, in vaccinated BALB/c mice. Vaccination with the chimeric vaccine in BALB/c mice triggers a robust Th1 response characterized by elevated production of IgG2a and IFN-γ. This bodes well for inducing potent humoral and cellular immune responses, affording heightened protection.

However, the introduction of alum adjuvant alongside PAV3 redirects the immune response toward Th2, leading to reduced protection against internal P. aeruginosa challenges compared to PAV3 alone. In summation, the designed vaccine elicits innate, humoral, and cellular immune responses, offering a promising strategy to manage host-P. aeruginosa interactions and surmount challenges associated with P. aeruginosa vaccination.

Biological sciences/Immunology

Biological sciences/Microbiology

Chimeric structure

Immunoinformatic

Pseudomonas aeruginosa

Vaccine

Pseudomonas aeruginosa, a rod-shaped member of the Gamma proteobacteria class, is a Gram-negative bacterium. It is a ubiquitous opportunistic pathogen that can infect individuals with conditions like cancer, cystic fibrosis (CF), severe burns, or weakened immune systems¹. Pseudomonas aeruginosa plays a substantial role in hospital-acquired infections, accounting for 17% of cases of nosocomial pneumonia, 11% of urinary tract infections, and 8% of bloodstream infections. Furthermore, it is a major contributor to mortality resulting from respiratory infections². Pseudomonas aeruginosa's antibiotic resistance, particularly in the case of multidrug-resistant (MDR) strains, poses a significant and alarming challenge. This has prompted the Centres for Disease Control and Prevention (CDC) to categorize it as a major hospital-acquired pathogen, as the efficacy of antibiotics against it continues to decline.³. Therefore, understanding the pathogenesis of P. aeruginosa and developing new therapeutic approaches that can strengthen the eradication of bacteria without selective pressure, whether through natural or to induce and reduce the complications and mortality associated with infections caused by this bacterium can be a breakthrough. Pathogenic bacteria face the challenging task of interacting with host cells and orchestrating complex molecular and cellular networks to counter host defense strategies, such as colonization and dissemination. Therefore, understanding the pathogen-host relationship is essential for combating these bacteria⁴. Many pathogenic bacteria possess a wide array of virulence factors that engage essential host components, leading to cellular damage. This enables bacterial survival during intracellular life stages, infection of neighboring cells, and extracellular expansion^5,6. An alternative to antibiotics is immunoprophylaxis and immunotherapy, utilizing vaccines to empower the immune system. Vaccines for P. aeruginosa trigger targeted immune responses to prevent infections, reduce complications, and limit bacterial spread in healthcare. In cystic fibrosis and other vulnerable groups, these vaccines are crucial, improving long-term outcomes, decreasing exacerbations, and enhancing quality of life⁷. Considerable research has been dedicated to designing vaccines against P. aeruginosa initially; surface antigens and cell wall components were targeted, to induce opsonizing antibodies that could clear P. aeruginosa infections. Later studies focused on various antigenic elements and virulence factors aiming to target and neutralize these factors, thereby reducing the bacterium's ability to cause harm to the bacterium these factors including lipopolysaccharides, ETA, flagella, pili, outer membrane proteins, type III secretion system proteins, high molecular weight polysaccharides, alginate, and DNA^8,9. However, these studies have not yielded an optimal vaccine design. Developing a successful vaccine against P. aeruginosa presents numerous hurdles owing to the bacterium's distinct attributes and its capacity to elude the immune response^10,11. The primary obstacle in designing a vaccine against P. aeruginosa lies in differentiating between the acute and chronic phases of infection, as the bacterium adeptly adjusts to its infection site. The acute stage of P. aeruginosa infection is characterized by rapid onset and often occurs in individuals with compromised immune systems or in healthcare settings. Acute infections can lead to severe symptoms, including systemic inflammation, tissue damage, and organ dysfunction. Prompt recognition and treatment of acute P. aeruginosa infections are essential to prevent the progression of chronic infection and associated complications. Pathogens strategically select virulence factors throughout acute and chronic stages based on environmental conditions and exhibit significant antigenic diversity. These variations make it difficult to select a single target antigen or combination of antigens that can provide broad protection against P. aeruginosa infection^5,12,13. Most of the existing vaccines are designed based on some limited factors of acute or chronic stages and cannot cover all stages of Pseudomonas infection. The second challenge for an evolved effective vaccine is the immune escape strategies of P. aeruginosa has developed various strategies to elude the host immune system, showcasing an array of virulence factors including toxins, outer membrane proteins, and secretion systems. That can alter the effective immune response to type 2 immunity as an inefficient and harmful immune response, impairing the function of immune cells and other important mechanisms in immunity such as autophagy^7,14,15. In addition, the ability of this bacterium to live intracellularly and extracellularly and the difference in the effective immune response to each of these steps highlights the need for a balanced immune response that includes both humoral and cellular immunity. In most of the designed vaccines against P. aeruginosa, this bacterium is considered an extracellular pathogen, and naturally, the basis of vaccine design is to stimulate a Type 2 immune response (Th2), accompanied by predominant humoral immunity and the production of a high level of non-opsonizing non-functional antibodies, without considering the importance of cellular immunity. This approach will certainly not succeed in eradicating both extracellular and intracellular bacteria¹⁶. However, recent findings indicate that a balanced combination of humoral and cellular arms is necessary to create effective immunity against this bacterium. This requires the induction of Type 1 immunity (Th1), which can induce an acceptable level of functional and opsonizing antibody subclasses, and balanced cellular immunity^7,17,18. This enhanced strategy could play a crucial role in eradicating both extracellular and intracellular bacteria, effectively preventing the bacteria's sustained survival within host cells and the progression to a chronic infection stage ^19,20. These evasion strategies complicate the design of a vaccine that can effectively stimulate a robust and long-lasting immune response against P. aeruginosa. To overcome the first challenge and limitation in the size of synthetic genes and the limited capacity of heterologous carriers in expressing complex and multi-valent proteins, recent studies on vaccine design have focused on the strategies of protein-based vaccines, including polytopes and chimeric vaccines. Polytopic or chimeric vaccines represent a new category of non-composite vaccines, composed of epitopes or portions of multiple antigens from one or several proteins, linked together by a linker. This enables the utilization of multiple pathogenic factors from both the acute and chronic infection stages, offering the potential for an effective defense against various disease-causing agents^21,22. Overcoming to second challenge of the P. aeruginosa vaccine we need to shift the immune response to an effective response which requires a comprehensive and multi-faceted approach. It involves enhancing both innate and adaptive immunity through different strategies. In our study, we enhanced innate through stimulation special immunoreceptors such as TLR4 and CD14 which can help initiate a robust and effective response against P. aeruginosa. This may involve using immunomodulatory agents such as several peptides as autoadjuvants to boost their activity and effectiveness in combating P. aeruginosa infections^14,23–25. To enhance and stimulate the adaptive immune response, we utilized a successful chimeric vaccine containing both B cell and T cell epitopes. This vaccine can activate both T cells and B cells, resulting in the production of targeted antibodies and cellular responses against P. aeruginosa antigens^14,26. Antibodies play a crucial role in neutralizing the bacterium and aiding in its removal, whereas T cells orchestrate immune responses and directly eliminate infected cells. Another advantage of these vaccines is the potential to integrate adjuvants into the vaccine composition, offering vaccine designers this valuable opportunity. Additionally, the utilization of immunomodulatory therapies can assist in fine-tuning the immune response to a more efficacious state^14,26–29. These therapies aim to balance the immune system's response, reducing excessive inflammation while maintaining an adequate defense against P. aeruginosa. This may involve the use of autoadjuvant for shifting imunoresponse to type 1 immunity for induced strong humoral and cellular furthermore activation of autophagy for clearness of intracellular P. aeruginosa.

Therefore, in this study, a chimeric vaccine strategy based on epitopes was employed, as we believed that this approach has the necessary potential for controlling P. aeruginosa infection. In our present study, we have created a chimeric vaccine named PAV3 by integrating the entire LecB protein (115-1) at the N-terminus with the second transmembrane domain of HIV TAT protein (57 − 47), a 28 kDa fragment of the N-terminal of Exotoxin A (ETA) (277 − 26), and the Epi8 epitope from OprF (341 − 311) at the C-terminal region of the chimeric vaccine. The immunogenic properties of this construct were evaluated both alone and in conjunction with alum adjuvant.

Selection and Retrieval of P. aeruginosa Proteins for PAV3 Chimeric Vaccine Development

In the development of the PAV3 chimeric vaccine, it is crucial to integrate complete virulence factors or specific fragments of these factors. This integration plays a pivotal role in effectively tackling both the acute and chronic phases of infection. One important feature in selecting a virulence factor is the surface accessibility of these molecules for immune recognition. This step of virulence factor screening was performed by examining the proteome of this bacterium and using CELLO and PSORTb tools to determine their extracellular location or presence in the outer membrane. In the subsequent stage, the antigenicity of the screened proteins was analyzed using the VaxiJen web server, and proteins with suitable antigenicity were selected. Then, the proteins were further investigated based on their functional roles in bacterial pathogenesis and their presence in different stages of acute and chronic infections. Finally, proteins with different epitopes were examined, and proteins with high epitope density for B cells, HTL cells, CTL cells and IFN-γ were selected which included Fucose binding lectin (UniProt ID: Q9HYN5), Exotoxin A (UniProt ID: P11439), OprF (UniProt ID: P13794), and HIV TAT protein (UniProt ID: A6MI22). To build the vaccine, these chosen proteins, whether in their entirety or as fragments, are melded together using flexible and hydrophobic GS linkers. Subsequent steps involve using bioinformatics techniques²¹,²⁶. Ultimately, to enhance the chimeric vaccine's construction for expression in the E. coli system, codon optimization and necessary modifications are executed and the sequence is submitted to NCBI by submission ID: 2769062. Specifically, NcoI and NotI restriction sites are strategically introduced at the N- and C-terminal ends of the DNA sequence, respectively. For the successful expression of the vaccine, the SnapGene tool is employed to insert the sequence containing the restriction sites into the pET-22b vector, following the methodology outlined by Adames et al. in 2015³⁰.

Ethics statement

This study strictly adhered to animal care and use protocols by the Regulations for the Administration of Experimental Animals, which received approval from The Animal Ethical and Experimental Committee of Shahed University under the ethical code IR.SHAHED.REC.1399.152 (available at https://ethics.research.ac.ir/IR.SHAHED.REC.1399.152). During surgical procedures, a combination of Ketamine/Xylazine anesthesia was administered, and utmost efforts were made to mitigate any potential distress.

Bacterial strains and culture method

The Pseudomonas aeruginosa reference strain PAO1 was used. The strain was cultivated in Luria Bertani broth and then subjected to washing and subsequent dilution with sterile PBS to attain an appropriate cellular concentration. The concentration was determined through spectrophotometric measurements at 600 nm (OD600)³¹.

Cloning, expression, and purification of recombinant proteins

The chimeric antigen structure (PAV3) comprising lectin (1-115), HIV TAT peptide (47–57), N-terminal fragment of exotoxin A (26–277), and Epi8 outer membrane protein F (311–341) was amplified from the genome of P. aeruginosa strain PAO1. The amplified structure was then integrated into an expression vector based on the pET22b plasmid, positioned between the NotI and NcoI restriction sites. To facilitate expression and purification, a His-tag was affixed to the N-terminus of each protein. The synthesis of the DNA sequence encoding the recombinant PAV3 was outsourced to Sinaclon Company (Tehran, Iran), and the sequence was incorporated into the pET22b vector using the aforementioned restriction sites. In the chimeric structure, two linkers, "GGGGS" and "GSGGSG," were introduced between the three proteins (Fig. 2). Subsequently, the expression vectors were introduced into E. coli BL21 strain and the bacterial culture were grown in LB media supplemented with ampicillin (100 µg/ml) until the optical density at 600 nm (OD600) reached 0.6. Induction of recombinant protein expression occurred by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to achieve a final concentration of 0.2 mM, followed by overnight incubation at 16°C. The expressed proteins were found in inclusion bodies and were purified using a Ni-NTA column (Qiagen) as per the manufacturer's guidelines. Additionally, the approach outlined by Reichelt et al.²⁶ was utilized to eliminate endotoxins simultaneously using Triton X-114. The identity and purity of the eluted proteins were verified through SDS-PAGE and Western blot analysis using a His-tag antibody conjugate (Invitrogen). The quantification of the purified proteins was carried out employing the Bradford assay³⁰.

Toxicity test in mice

Before starting the immunization, the recombinant protein was examined for toxicity. For this purpose, 100 micrograms (equivalent to 10 doses) of recombinant protein were injected intraperitoneally into 5 mice. For one week, the injection site was examined for wound formation, allergic reactions, inflammation, and mortality of animals³².

Immunization of mice

Female BALB/c mice aged 6–7 weeks were procured from the Pasteur Institute of Iran. All procedures involving animal subjects followed the guidelines set forth by the European Communities Council Directive of 24 November 1986 (86/609/EEC). In the context of active immunization, the purified proteins dissolved in PBS buffer were blended in a 1:1 volume ratio with Al (OH)3 (Pierce). Intramuscular injections of 200 µl of the resulting emulsion, comprising 30 µg of protein, along with PBS buffer plus adjuvant, or PBS buffer alone (control), were administered to the mice on days 0, 14, and 21. Following this regimen, the immunized mice were employed in cytokine and challenge experiments. On day 28, the mice were subjected to infection^33,34.

P. aeruginosa infection

One week after the final immunization on day 28, the mice were infected to establish the acute pneumonia model by with intubation-mediated intratracheal inoculation (IMIT) method (Fig. 1). Within each group, mice were anesthetized using Ketamine/Xylazine and then subjected to intratracheal injection with a lethal dose of P. aeruginosa isolates to assess survival rates. The occurrences of mortality within each group were documented every 12 hours over a 7-day observation period following the challenge. The established lethal dose for P. aeruginosa strain PAO1 was 1.0 × 10⁷ colony-forming units (CFUs). For evaluations associated with bacterial burdens, histopathology, inflammatory cells, and cytokine analysis, mice were intratracheally infected with 5.0 × 10⁶ CFUs of PAO1. In the sub-lethal challenge group, the general health condition of the mice was assessed based on criteria including piloerection, posture, locomotion, breathing rate, and nasal secretion. A composite disease score was assigned as follows: unaffected (0–1), slightly affected (2–4), moderately affected (5–7), and severely affected (8–10)^31,35.

After 24 hours post-infection, the bacterial burdens were quantified, and the survival rate of the experimental mice was monitored for 7 days.

(Fig. 1)

Bacterial Burden Assessment

For the assessment of bacterial burden in the pneumonia-affected lung tissues, tissue samples were collected at 8 and 24 hours following the challenge. The gathered lung tissues were weighed and subsequently homogenized in 1 ml of sterile PBS buffer. To investigate the potential systemic dissemination of bacteria, samples from the liver, spleen, and blood were obtained from each animal. These samples were also weighed and homogenized in PBS. To quantify the bacterial load, all of the homogenized samples were plated onto LB plates using a technique of serial dilution by a factor of 10. Subsequently, the plates were incubated at 37°C for 20 hours to promote bacterial growth. The quantification of Colony-Forming Units (CFUs) per gram of tissue (CFUs/g) was accomplished by enumerating the bacterial colonies on each plate³⁶.

Isolation and cultivation of spleen cells

After two weeks of the final immunization, mice spleens were removed and placed in RPMI medium with antibiotics. Spleen cells were released and separated using centrifugation and Ficoll. Viable cells were counted and 200,000 were placed in each well of a 24-well microplate. After 24 hours, cells were primed with 10 µg of pure protein and control wells were set with PBS and PHA. After another 24 hours, supernatant was collected and stored at -70°C. Fresh medium was added, and after 24 hours, the microplates were centrifuged, and the supernatant was collected again for testing and frozen at -70°C³⁷.

MTT cell proliferation Assay

The MTT cell proliferation assay is a colorimetric method used to quantify cell proliferation. In this assay, 100 µl of MTT solution (composed of 10 µl of 5 mg/ml MTT in 90 µl of RPMI) was added to spleen cell-containing wells. Microplates were then incubated for 4 hours at 37°C with 5% CO2. After centrifugation, supernatants were removed, and 200 µl of DMSO was added to each well. Following an overnight incubation at room temperature, absorbance was measured at 570 nm using a spectrophotometer. This assay was employed to assess lymphocyte proliferation and proliferation rate under various conditions, including cell-to-protein interaction (in vivo), restimulation with PHA (in vitro), and non-stimulated conditions (ex vivo). Comparison was made to control groups, including PBS and negative controls. Mitochondrial activity was quantified as a percentage absorbance variation relative to the negative control, calculated using the formula: [(absorbance value in stimulated cells / average absorbance value in unstimulated cells) − 1] × 100. Positive and negative percentage values indicated increased or decreased mitochondrial activity compared to unstimulated cells. This assay offers insights into cellular proliferation dynamics and responses under different conditions^38,39.

Cytokine assays using the ELISA method

The cells were isolated from immunized mice and plated with 2 million splenic cells in RPMI-1640 culture medium in each well of the plate. The cells were stimulated by adding 10 milliliter (µg/ mL) of a peptide mixture. Then, the cells were incubated at 37°C with 5% CO2 for 72 hours. After that, centrifugation was performed at 300 rpm for 10 minutes, and the supernatant from each well was collected and stored at -70°C until the measurement of cytokines at 70°C. Cytokines were determined using a mouse eBioscience commercial ELISA kit for Interferon-gamma and Interleukin-4, respectively, according to the manufacturer’s instructions^40,41.

Determination of mouse-specific antibody titers and antibody typing via ELISA

For ELISA, eBioscience kits were used for IgG Total and IgG2a, and Bioassay Technology Laboratory kits were used for IgG1 and IgA To cover the plate, 100 µl of the first antibody or capture antibody, prepared at a dilution of 1/100, was added and coated onto the plate surface, which was then sealed with a plate sealer and incubated at 37 ° C for one hour. The wells were first filled with 300 µl of washing solution and then emptied. Washing of the wells was performed three times. 200 µl of blocking solution was added to each well, and the plate was sealed and incubated at room temperature for half an hour. The washing steps (same as step two) were repeated. According to the kit instructions, the standard was prepared by diluting it in a dilution buffer to a concentration of 1000 ng/ml. 100 µl of each sample directly from the cell culture plate wells was transferred to the microplate wells for ELISA. Assuming a concentration of 1000 ng/ml, serial dilutions of 1/4, 1/8, 1/16, 1/32, and 1/64 of the first standard were added to two rows of microplate wells. The last two wells were used as blanks and filled with the dilution buffer. The plate was sealed with a plate sealer and incubated at room temperature for one hour. Plate washing was performed as in step two. To detect antibodies, a dilution of 1/20000 was used for IgG2a, and a dilution of 1/100000 was used for IgG (the second antibody was conjugated with HRP). The plate was sealed with a specific plate sealer and incubated at room temperature for one hour. Washing was performed as in step two.1000 µl of TMB (Tetramethylbenzidine) substrate solution and H2O2 were added to each well, and the plate was incubated in darkness at room temperature for twenty minutes. To stop the colour reaction, 50 µl of 2 M sulfuric acid solution was added to all wells, and the optical density of each well was measured at a wavelength of 450 nm using an ELISA reader. A reference filter was used to correct the absorbance at 620 nm^40,41.

Bactericidal activity of serum

This test examines the effectiveness of antibodies generated through complement factors in conditions outside the body. Bactericidal antibodies, by activating the complement cascade, initiate the formation of the membrane attack complex, ultimately leading to bacterial lysis. To perform this test, 25 µl of heat-inactivated mouse serum (at 56°C for 30 minutes) was mixed with a ratio of 40:1 and 12.5 µl of 10% rabbit baby serum as the complement source. Additionally, 12.5 µl of P. aeruginosa PAO1 bacterial suspension, approximately 10^6 × 1 colony-forming units, were mixed in sterile microtubes. Control tubes were missing one of the serum components, complement, or bacteria. After adding all components to the wells, 10 µl from each well were transferred to Muller-Hinton agar plates after dilution and incubated at 37°C for 18 hours (T1). Following incubation, the T0 and T1 plates were colony-counted, and the bacterial counts were compared⁴².

PHI (Pathogen-Host Interactions) network construction and Enrichment analysis for P. aeruginosa

To construct the P. aeruginosa PHI network, we combined pathogen-host interaction data from various sources, including a STRING server ⁴³ and IntAct database⁴⁴. Using the Cytoscape StringApp and the human proteome, we generated a human protein-protein interaction network that was influenced by P. aeruginosa Furthermore for conducting Functional Enrichment Analysis and to calculate the significant annotations related to the biological processes and Reactome signaling pathways for our analysis, we utilized the DAVID server with a Benjamini-Hochberg false discovery rate (FDR) threshold of < 0.001. The resulting enrichment analysis was visualized using the Cytoscape software, and significant terms related to the pathogenesis of P.aeruginosa were manually curated. By performing this functional enrichment analysis, We were able to identify key biological processes and signaling pathways associated with P.aeruginosa pathogenesis and in next step selected key proteins for docking analysis^45,46.

Molecular Docking Analysis

The process of antigenic agents binding to target immunoreceptors is essential for generating an effective immune system response. To analyze how the PAV3 vaccine binds to TLR 4 (PDB ID: 5IJB) CD14 (DB ID: 4GLP) and TLR2 (PDB ID: 6NIG), researchers used molecular docking analysis through the HDOCK server. The HDOCK server is a comprehensive tool that combines homology search, template-based modeling, structure prediction, macromolecular docking, and biological information integration for efficient protein-protein docking. It uses individual protein structures and ambiguous information to guide the process. Unlike other docking servers, HDOCK can take amino acid sequences as input and incorporates an intrinsic scoring function to aid macromolecule docking. It also stands out for its hybrid docking approach, which incorporates experimental binding site data and small-angle X-ray scattering for more accurate docking and post-docking processes⁴⁷.

Investigation of immunomodulatory activity of PAV3 vaccine by influence on signaling pathways

The Immunomodulatory activity of the PAV3 vaccine was analyzed through various approaches. Firstly, based on the PHI analysis, the PAV3 vaccine was assessed for its interaction with proinflammatory host target proteins. Additionally, enrichment analysis of biological processes and signaling pathways provided further evidence. Furthermore, docking and MD simulations were performed to investigate the interactions between the PAV3 vaccine and the target proteins with pro-inflammatory activity in P. aeruginosa infection^48–50.

Sequence retrieval and construct design

We utilized various bioinformatics approaches to screen key proteins based on their important function in the pathogenicity of P. aeruginosa PAO1 during chronic and acute infection stages. These proteins were selected for having a high epitope density, including B-cell, HTL, and CTL epitopes, along with the potential to induce IFN-γ via MHC class II. Sequentially, a recombinant chimeric vaccine was meticulously constructed through the fusion of distinct protein fragments. This chimeric vaccine is composed of four distinct constituents: the full LecB protein (1–115) occupying the N-terminal, the HIV TAT-protein transduction domain (PTD) (47–57), a segment from ETA (26–277) localized in the N-terminal 28 kDa region, and the C-terminal epitope (Epi8) of OprF (311–341) situated at the protein's C-terminal extremity. To ensure effective partitioning and adaptability between the various domains, hydrophobic linkers (GGGGS) were integrated between the fragments. The schematic representation of the PAV3 chimeric vaccine's arrangement is visually depicted in Fig. 2A and the final protein sequence of the PAV3 chimeric vaccine is presented in Fig. 2B. For the proficient cloning and expression of the chimeric protein, E. coli BL21 was selected as the host organism. Codon optimization catering to E. coli expression requisites was meticulously executed, relying upon bioinformatics methodologies. Subsequently, the cloning procedure was facilitated by employing the SnapGene tool. The target sequence was seamlessly integrated into the pET22b (+) vector, positioned between the NcoI and NotI restriction sites. This vector was endowed with six-histidine residues at the C-terminal terminus, strategically positioned to facilitate affinity-based purification (Fig. 3).

(Fig. 2)

(Fig. 3)

Protein purification and analysis

In this section (3–15 to 3–18) of the study, the expression and characterization of a non-tagged chimeric protein were investigated. After culturing and inducing transformed bacteria, the chimeric protein's expression level was assessed using SDS-PAGE gel under different conditions. An induced protein of approximately 46 kDa was observed, matching the predicted weight of the non-tagged protein. Optimal expression conditions were determined: 1 mM IPTG induction at 28°C for 6 hours, resulting in higher expression compared to other conditions. The expression vector initially contained a His-Tag for purification and detection. To confirm protein identity, bacterial lysates, and purified protein underwent SDS-PAGE and Western blotting. Only non-tagged protein bands displayed histidine labeling after induction and purification. For purification, a Nickel Agarose column was employed, followed by dialysis. SDS-PAGE analysis before and after purification confirmed the successful purification of desired proteins, with a molecular weight of around 45 kDa. Protein concentration was determined using the Bradford method after protein precipitation and concentration using Millipore filters. Spectrophotometry at 595 nm indicated a protein concentration of 100 mg/ml (Fig. 4).

(Fig. 4)

Sterility and toxicity tests in mice

To assess the sterility of the protein solution, it was cultured on a TSB medium, and no growth was observed. For toxicity evaluation, the protein was injected intraperitoneally into Syrian mice. No mortality or significant weight loss was observed compared to the control group over one week. Furthermore, histopathological examination and Hematoxylin-Eosin (H&E) staining of the liver in these mice did not reveal any toxic effects (Fig. 5).

(Fig. 5)

PAV3 vaccination, P. aeruginosa lung infection protection and severity assessment, bacterial burden determination

Timing immunization of BALB/c mice was carried out by intraperitoneal injection of the recombinant protein. The injections were administered on days 0, 14, and 21 in three doses. Immunological challenges and assays were performed on days 28 and 35 after the initial vaccination. PAV3 protection efficacy against P. aeruginosa acute lung infection for bacterial burden and severity. Considering that the LD50 value was determined to be 1.0 × 10^{^8} CFUs, the mice were challenged with doses of LD50, 2×LD50, and 4×LD50 intratracheally. The survival percentages of each group after the challenge with these doses were plotted as survival percentage graphs. Based on these results, significant protection was observed in the group of mice immunized with the recombinant protein compared to the control group with a P-value < 0.0001. Additionally, the challenge with the LD50 dose showed a significant reduction in disease severity scores in vaccinated mice compared to the control (PBS) and alum groups (Fig. 6). The results of the assessment of P. aeruginosa bacterial load in the lungs, blood, and spleen of mice in the vaccinated and non-vaccinated groups, 18 hours after the challenge, are shown in Fig. 7. The immunized mice exhibited a significant reduction (P < 0.001) in bacterial counts in the lungs, blood, and spleen compared to the control group. The outcomes of this study indicated that POH demonstrated a capacity to partially impede the growth and colonization of P. aeruginosa in vivo. Subsequent histological evaluation revealed that the lungs of mice immunized with the recombinant vaccine exhibited diminished alveolar disruption, decreased vascular permeability, and lessened deposition of bacterial microcolonies within the alveoli post-infection, in contrast to those in the Al (OH) 3 group. Conversely, mice subjected to Al (OH) 3 immunization displayed typical pathological alterations. In contrast, mice immunized with POH displayed a reduction in inflammatory cell infiltration, bleeding, and tissue damage when compared to the Al (OH) 3-immunized group.

Overall, the findings emphasize that the degree of lung damage was markedly lower in the POH-immunized group compared to the control groups (Fig. 8).

(Fig. 6).

(Fig. 7)

(Fig. 8)

Splenocytes pproliferation assay

The memory immune response was examined after culturing splenocytes in the RPMI medium and adding the chimeric protein to the culture medium. In each group of lymphocytes, PHA was used as a mitogen to induce proliferation under in vitro conditions. Additionally, a group of lymphocytes remained unstimulated under ex vivo conditions. The result of splenocyte proliferation in response to stimulation with the chimeric protein as the main antigen is represented as the stimulation index (SI) in Fig. 9A, indicating the continued proliferation of lymphocytes. After vaccination, a remarkable difference in spleen size is observed between the protein-chimeric vaccinated mice and the non-immunized mice (Fig. 8).

Cytokine response Patterns

In this study, different mouse groups were individually investigated. According to this method, the spleens of mice from various groups were isolated and homogenized, and the splenocytes were cultured in the presence of the antigen for 12 hours. The culture supernatants from these splenocytes were then examined for cytokine production (Fig. 9B).

Interferon-gamma (IFN-γ) production

Based on the results presented in highest level of IFN-γ production was observed in splenocytes activated with the antigen in the group of mice immunized with the PAV3 structure. The IFN-γ level in this group was measured at 497 pg/ml, showing a significant difference compared to the other groups.

Interleukin-4 (IL-4) production

In the group of mice vaccinated with the PAV3 structure, the level of IL-4 significantly decreased compared to the group of mice immunized with alum along with PAV3. There was no significant difference in IL-4 production between the control groups (PBS and alum).

Determination of IgG Total, IgG2a, IgG1, and IgA levels in serum using ELISA

Fourteen days after the last antigen injection, the overall levels of IgG Total, IgG2a, IgG1, and IgA antibodies were examined in the serum of both test and control mouse groups using ELISA. A comparison of their levels was performed and shown in Fig. 9C. The PAV3 structure alone (Group 3) induced higher levels of the IgG2a subclass and IgA class antibodies, whereas PAV3 along with alum demonstrated a higher ability to induce IgG Total and IgG1 antibody responses. The production levels of all types of antibodies in both vaccinated groups significantly increased compared to the control and alum groups (> P-Value < 0.0001/0). Additionally, the IgG1/IgG2a ratios in mice vaccinated with PAV3 decreased when compared to the PAV3-alum formulations.

Serum bactericidal assay

The bactericidal activity associated with serum antibodies produced against the PAV3 structure was examined using an external complement source. The results obtained demonstrated a significant difference in bactericidal activity between the immunized mouse groups and the control groups (> P-Value < 0.0001) (Fig. 9D).

PAV3 vaccination triggers both Th1 and Th2 responses.

To assess cytokine levels, splenocytes from vaccinated mice were stimulated with recombinant proteins. Notably, the splenocytes from vaccinated mice displayed a significantly elevated IFN-γ/IL-4 ratio upon stimulation with the corresponding recombinant proteins, in comparison to both the Al (OH)3 control group and the PAV3-alum group (P < 0.0001, Fig. 9B). It's important to note that IFN-γ and IL-4 serve as markers for Th1 and Th2 responses, respectively. Furthermore, the PAV3 group exhibited the highest cytokine production. This suggests that mice immunized with PAV3 showcased a more pronounced Th1-biased response upon encountering the antigen.

An intriguing observation emerged where IL-4 levels in mice immunized with PAV3-alum were notably higher than those in mice immunized with PAV3 alone. Moreover, the antigen-specific IgG2a level for PAV3 alone was approximately 160 times greater than IgG1 (Fig. 9C). It's noteworthy that IgG1 and IgG2a are indicative of Th2 and Th1 responses, respectively. These findings robustly suggest the induction of a Th1-biased response, in alignment with the observed cytokine production trend in the supernatant. This underscores the capability of PAV3 alone to act as an autoadjuvant, effectively stimulating Th1 responses against P. aeruginosa.

(Fig. 9)

PHI network construction and Enrichment analysis for P. aeruginosa:

Analysing the PHI interactome may unveil new vaccine targets against P. aeruginosa. The generated PPI network consisted of 38 important proteins influenced by P. aeruginosa Lung Infection.

Functional enrichment analysis of these genes included annotations related to GO biological processes, KEGG, and Reactome signaling pathways, with a focus on the most important and influential biological processes and signaling pathways in the P. aeruginosa Lung Infection. Indicating significant annotations (p-value < 0.001). Furthermore, enrichment analysis of PHI genes revealed their involvement in LPS signalling, NF-κB signaling, TLR4-TRIF Signaling and Autophagy, Inflammation response, Apoptotic process, and Apoptotic process with p-value < 0.001. These findings highlight the influence of PHI genes in these specific pathways. Selection of signaling and key proteins as candidates for immunomodulatory and vaccine interventions were selected as candidates for the in-docking analysis (Fig. 10)

(Fig. 10)

Molecular docking analysis

We evaluated the binding energies of PAV3 vaccine with TLR4 and CD14 by examining their molecular docking complexes which are − 325.22 and − 342.45respectively. The recorded binding energies provide insights into the stability of the interaction, as lower binding energies indicate higher binding affinity (Fig. 11). Additionally, we performed further analysis of the docked PAV3- TLR2 to identify the binding site residues involved in the interaction of vaccine-TLR2 (Fig. 12).

(Fig. 11)

(Fig. 12)

Investigation of immunomodulatory activity of PAV3 vaccine by influence on signaling pathways

To determine of Immunomodulatory activity of the PAV3 vaccine, we analyzed biological processes, signaling pathways, and function predictions for PHI genes (Fig. 13). Based on the results, we identified the TLR4 signaling, TNFα/AP-1 signaling and necroptosis and autophagy pathway as a target for important proteins and their related signaling in P. aeruginosa infection. This pathway plays a crucial role in a cytokine storm, a severe inflammatory response, and tissue damage.

(Fig. 13)

P. aeruginosa is a dangerous bacterium, especially for immunocompromised individuals, causing hospital-acquired infections and antibiotic resistance. To address this threat, there's a need for a better understanding of its pathogenesis and developing innovative treatments to reduce complications and mortality, as recognized by the CDC ³¹,⁵¹,⁵². P. aeruginosa, a bacterial pathogen, has complex mechanisms for interacting with host cells and causing infections. Understanding this interaction is vital for effective treatment. P. aeruginosa possesses virulence factors that harm host cells⁵²,⁵³.

While vaccines have been considered, designing an effective one remains a challenge. Extensive research on various antigens has not yet yielded a successful vaccine. More studies are required to develop an effective P. aeruginosa vaccine. Immunization against P. aeruginosa poses challenges due to its ability to adapt to different stages of infection and intelligently select virulence factors. The design of an effective vaccine requires consideration of both acute and chronic stages of infection. P. aeruginosa possesses various mechanisms to counteract the immune system, including inhibiting phagocytosis and intracellular survival, which leads to resistance to antibiotics and inefficient immune responses. Most vaccine designs focus on extracellular bacteria, emphasizing a Th2-biased immune response and non-opsonizing antibodies while neglecting the importance of cellular immunity⁵⁴,⁵⁵,⁵⁶. However, recent findings suggest that a balanced immune response, including Th1-biased cellular immunity and effective opsonizing antibodies, is necessary for effective immunity against P. aeruginosa. A successful vaccine should utilize multiple virulence factors from both acute and chronic stages, as well as modulate the host immune system toward effective immunity⁷. The constraints of gene size and vector capacity impede the creation of multivalent vaccines. To address these limitations, Epitope-based and polytop/chimeric vaccines, combining epitopes for B lymphocytes and MHC receptors, from various proteins, aim to optimize immune responses, often using adjuvants for improved effectiveness²¹. In vaccine design, understanding the pathogen-host interaction is crucial and Pathogens manipulate the immune response, so it's vital to determine the effective immune response type. Balancing Th1 and Th2 immune responses is key. Type 1 responses involve both cellular and humoral components and could be effective against both intracellular and extracellular pathogens, while type 2 responses primarily focus on humoral immunity and aid in parasite elimination and inflammation resolution. P. aeruginosa, with both intracellular and extracellular aspects, requires a combination of humoral and cellular immunity⁵⁷, ⁵⁸. Th1 stimulation is vital for intracellular clearance, Antibodies induced by type 1 immunity enhance cellular immunity through opsonization.. Assessing the Th1/Th2 balance is essential for tailoring vaccine strategies. Cytokine patterns IFN-γ / IL-4 and antibody subclasses guide this process, helping design vaccines that target specific pathogens effectively⁵⁷. The designed vaccine in the study successfully Balanced Th1/Th2 responses and induced a type 1 immune response against P. aeruginosa, promoting effective immune responses for clearance. The vaccine cleared the bacterium, induced long-term immunity, and facilitated tissue repair. The results demonstrated the effectiveness of vaccine design strategies in inducing type 1 immunity. our study takes a unique approach by developing the PAV3 chimeric vaccine.

in this study with several bioinformatics approaches selected important virulence factors and designed a PAV3 vaccine that can overcome to P. aeruginosa vaccine challenge and limitation in the size and production of the subunit vaccine ²⁶. This study aimed to design a protein structure that can simultaneously possess antigenic and adjuvant properties and provide protective immunity against P. aeruginosa in BALB/c mice. The antigenic component of this structure is formed by the fusion of whole or partial segments of important bacterial pathogen factors, both in the acute and chronic infection stages. These segments were selected based on their epitopic characteristics for B and T cells, aiming to stimulate both humoral and cellular arms of the immune system according to the Th2/Th1 balance theory. In this study, the non-complex polytop vaccine PAV3 was constructed by fusing the complete LecB protein (115-1) at the N-terminal with the second transmembrane domain protein TAT from HIV (57 − 47), the 28 kDa N-terminal fragment of exotoxin A (ETA) (277 − 26), and the Epi8 epitope from OprF (341 − 311) at the C-terminal region. The immunogenic properties of this construct, both alone and in conjunction with alum adjuvant, were evaluated.

Exotoxin A is a bacterial toxin that disrupts protein synthesis in cells as an ADP-ribosyl transferase, dampening the host's immune response and causing tissue damage⁵⁹. It hinders macrophages, weakens phagocytosis, and impairs bacterial clearance⁶⁰,⁶¹. Anti-ETA antibodies neutralize ETA, reducing tissue invasion and enhancing survival in infected mice. Produced by around 90% of clinical bacterial strains, ETA is a vital virulence factor with antigenic properties. Neutralizing antibodies against ETA are developed successfully⁶²,⁶³. The CD91 receptor governs ETA entry and immune function, activating antigen-presenting cells, and plays a role in the production of cytokines, priming of T cells, and maturation of immune cells. Understanding the Ia-CD91 interaction informs vaccine design and immune activation⁶⁴,⁶⁵. The design of a vaccine against ETA involves utilizing a specific fragment of the toxin that has good accessibility, B cell epitopes, and stable characteristics without enzymatic or toxic activities. It contains potent epitopes for cellular immunity and antibody production, enabling the immune system to target ETA-infected cells and prevent tissue damage²⁶, ⁶⁶. OprF, a pivotal protein in P. aeruginosa, holds a multifaceted role in infection and immune interactions ⁶⁷. It also participates in associations with LecB and Psl, affecting the stability of biofilms. Antibodies against OprF and LecB disrupt these interactions, thwarting biofilm formation. OprF's interactions with the immune system encompass C3b, IFN-γ, and neutrophil elastase. OprF acts as a C3b receptor, instigating membrane attack complex formation. However, polysaccharide Psl can impede this interaction, diminishing phagocytosis. Furthermore, OprF can adjust gene expression in response to IFN-γ, intensifying virulence factors. By targeting OprF in vaccination strategies, we can enhance antigen presentation, and antibody production, and disrupt biofilm formation, promising advancements in combatting P. aeruginosa infections⁶⁸,⁶⁹,⁷⁰. Our study discusses the immunogenicity and importance of the C-terminal region, particularly epitope 8 (epi8), of the OprF protein in P. aeruginosa. As previous studies have shown fusion vaccine design with Epi8, OprF, OprI, and flagellin improved antibody production, complement activation, and bacterial clearance, especially in preventing mucosal colonization through intranasal immunization⁷¹. other studies demonstrate that epi8 induces a potent immune response, including high levels of IFN-γ and IgA/IgG2a antibodies. The epi8 epitope shows good binding affinity to MHC-II and has cross-reactivity with homologous proteins. It plays a crucial role in pathogenesis, adhesion, biofilm formation, and immune modulation. Several studies confirm the immunogenicity and opsonizing antibody production of OprF, primarily through epitopes in the C-terminal region⁷²,⁷³. Our bioinformatics study on PAV3²⁶, ⁷⁴ demonstrated that the epi8 epitope of OprF has high immunogenic potential and exposed the presence of high score conformational and Linear B-cell, HTL, and IFN- γ epitope. Furthermore, Molecular docking and dynamics with TLR4-MD2 and epi8 demonstrated that this peptide can be considered a candidate as a TLR4 agonist for facilitating immune response and vaccine could be capable of eradicating P. aeruginosa via inducing a Th1dominant response; although triggering strong cellular and humoral immune responses which is Aligned and matched with experimental results in this study. Lectins are glycan-binding proteins that, although lacking catalytic activity, are capable of generating a series of cellular signalling events. Findings indicate that LecB and OprF are two virulence factors that interact with each other and impact key functions related to P. aeruginosa pathogenesis⁷⁵,⁷⁶. These proteins mutually modulate their functions and affect host interactions, cytotoxicity, tissue damage, lung severity, colonization, biofilm formation, IFN-γ binding, virulence factor expression, and immune modulation. Fucose disrupts their interaction via the lectin-binding site⁷⁷, ⁷⁸,⁷⁹. LecB links OprF and Psl for biofilm formation. Mutations affect biofilm quality. LecB is relevant in acute and chronic infection, a potential immune system target⁸⁰, ⁸¹. LecB contains epitopes that activate B cells, HTLs, and CTLs. Therefore, understanding LecB and its binding site is vital for drug and vaccine development. In our study, we harnessed B cell epitopes within the lectin binding site to produce antibodies that can hinder lectin function, prevent bacterial adherence to host cells, disrupt pathogenic processes, interfere with interactions with other virulence factors, and ultimately, protect against chronic P. aeruginosa infection⁸².

In this study, we investigate immune-boosting compounds and adjuvants to enhance vaccines, with a specific focus on the adjuvant capabilities of lectins. Plant lectins like ArtinM activate immune receptors, such as TLR2, promoting macrophage polarization. Similarly, bacterial lectins, such as Toxoplasma gondii TgMIC1 and Paracoccidioides brasiliensis TgMIC1, interact with TLR2 and TLR4, leading to cellular signaling, NF-kB activation, IL-12 cytokine production, and a Th1-directed immune response. These findings offer insights for improving vaccine efficacy ⁸³. In the present study, the bacterial lectin LecB from P. aeruginosa was used in the vaccine's structure. This lectin bears significant structural and functional similarities to plant and bacterial lectins, suggesting an expectation of its interaction with immune system receptors and its potential modulatory effects. In this study, docking analyses were performed on the PAV3 with TLR2 which demonstrated vaccine in the location of LecB attaches to this receptor with high affinity and a low energy level (Fig. 11). This could substantiate LecB's adjuvant and modulatory role and provide a rationale for the similar effects observed in the PAV3 vaccine and ArtinM-based vaccines in terms of immune system stimulation, as supported by immunoinformatics and laboratory results. These effects include an increase in IFN-γ induction, a decrease in IL-4, and consequently a redirection of the immune system towards a type 1 immune response, along with the induction of humoral immunity marked by an appropriate level of antibodies, predominantly IgG2a subtypes. Additionally, these effects can be activated macrophages and DCs, characterized by enhanced phagocytic and cytotoxic capabilities of innate immune cells, a reduction in their inflammatory properties, and thereby a decrease in tissue damage and pathogenesis (Fig. 8, 9). It suggests that this approach could be highly effective in combating the immune system against P. aeruginosa, which exhibits intracellular and extracellular life forms. The study also demonstrated potential high-affinity binding between the designed vaccine and CD14, in addition to TLR2 and TLR4 receptors. This interaction promotes optimal signaling through TLR2, Th1 immune response, macrophage activation, TLR4 endocytosis, and TRIF (TIR domain-containing adaptor-inducing interferon-β) pathway activation, contributing to enhanced immune responses. Simultaneous stimulation of TLR2 and TLR4 has synergistic effects on immune signaling and modulation, resulting in improved immune system direction and Th1-biased responses. These findings have important implications for vaccines²³, ⁸⁰, ³⁹,⁸⁴,⁸⁵.

HIV Tat protein's role in regulating the immune system involves attracting monocytes, facilitating activation and infection. Protein vaccines face challenges in stimulating immune responses, but incorporating the HIV Tat protein transduction domain (PTD) enhances antigen delivery into cells, promotes Th1 responses, and improves antibody production, cytokine release, and immune cell activation. This results in a stronger cellular and humoral immune response⁸⁶,⁸⁷, ⁸⁸. This study explored the use of aluminum salts (alum) as vaccine adjuvants for P. aeruginosa vaccines. Alum was initially thought to enhance immune responses by antigen accumulation and presentation but was found to interact with innate immune receptors and impact adaptive immunity. Aluminum crystal vaccines interact with dendritic cell membranes, promoting antigen uptake and activating T cells. However, alum induces an innate type 2 immune response, which may not be suitable for P. aeruginosa vaccines requiring Th1 and cytotoxic T lymphocyte responses. Using alum with the PAV3 vaccine may counteract disease-promoting factors but may also cause inflammation and impact immune response balance⁸⁹,⁹⁰,⁹¹,⁹². As our findings also demonstrated alum adjuvant to the vaccine reduces its protective effect by manipulating the immune response towards Th2 and significantly decreasing the IFN-γ/IL-4 and IgG2a/IgG1 response patterns compared to the vaccine alone.

Studying the immunomodulatory effects of the PAV3 vaccine on signaling pathways is vital for comprehending the interplay between P. aeruginosa and the immune system. While the host's innate defense mechanisms, such as cell death, autophagy, and inflammation, are pivotal in countering intracellular bacteria, their significance has often been underestimated in the context of P. aeruginosa infections¹⁵. For determination of Immunomodulatory activity of PAV3 vaccine, we analyzed biological processes, signaling pathways, and function predictions for PHI genes (Fig. 10). Based on the results, we identified the TLR4 signaling, TNFα/AP-1 signaling and necroptosis and autophagy pathway as a target for important proteins and their related signaling in P. aeruginosa infection. This pathway plays a crucial role in cytokine storm, a severe inflammatory response and tissue damage. On of important mechanisms in host defense against P. aeruginosa is autophagy, which is a conserved cellular process vital for both innate and adaptive immunity. It contributes to antigen presentation (including cross-presentation), intracellular and extracellular pathogens elimination, antimicrobial peptide production, inflammation regulation, immune cell development, and cytokine modulation. The bacterium can counteract autophagy with specific virulence factors. The factors involved in this process include ETA, the T3SS secretion system, OprF, and lectins⁹³,⁹⁴,⁹⁵. P. aeruginosa induces autophagy via TLR4, involving LPS and the TRIF-dependent pathway. CD14 aids TLR4 activation and internalization, leading to increased recognition by the TLR2 complex. This process enhances autophagy, type I interferon production, mitophagy, reactive oxygen species, macrophage phagocytosis, and bacterial clearance⁹⁶, ⁹⁷. The PAV3 vaccine effectively activates the TLR4 and CD14 pathway, mimicking immunoreceptor agonists, and promotes autophagy to combat P. aeruginosa infection.

The bacterium evades autophagy intracellularly through the type III secretion system (T3SS) and by degrading phagosomes. However, genes MgtC and OprF can regulate and stimulate the T3SS and inhibite autophagy, it enhances invasion, inflammation, and assists in bacterial dissemination and escape⁹⁸,⁹⁹. As demonstrated in Pre-treatment with anti-OprF mAb significantly reduces bacterial cell invasion of host cells by affecting T3SS¹⁰⁰. This PAV3 vaccine by utilizing epi8 epitopes produces anti-OprF antibodies, which reduce the invasive capacity of T3SS, stimulate autophagy decrease tissue damage, and improve bacterial clearance. P. aeruginosa employs various tactics and virulence factors to bolster external invasion, impede phagocytosis for intracellular survival, and manipulate the immune response in its favour. In the extracellular phase, ETA secretion disrupts EF2a signaling, hindering autophagy¹⁰¹. ETA also triggers caspase 1, which cleaves TRIF and inhibits autophagy promote non-inflammatory cell death while inducing inflammatory cell deaths like necrosis and pyroptosis, intensifying inflammation and tissue damage ¹⁰². Hence, using domain Ia of ETA in a chimeric vaccine aims to generate antibodies that block toxin binding, preventing its entry into host cells and preserving autophagy. P. aeruginosa uses various strategies to steer the host immune response toward a Th2 phenotype, promoting chronic inflammation and tissue damage. This Th2-biased response, resulting from both intracellular and extracellular P. aeruginosa infections, leads to reduced bacterial clearance, worsening lung function, and more severe clinical outcomes. Notably, autophagy and Th2 responses inhibit each other, with Th2 cytokines opposing autophagy¹⁰³,⁹³. By promoting a Th2 response during P. aeruginosa infection, autophagy is suppressed, allowing intracellular bacteria to survive, reducing clearance, and increasing the risk of transitioning from acute to chronic infection. In cystic fibrosis, autophagy is further impaired due to gene mutations. Therapies like rapamycin that enhance autophagy can improve bacterial clearance and reduce inflammation in epithelial cells with CFTR mutations²⁴. Previous vaccines against P. aeruginosa have failed due to the bacterium's complex behaviours and interactions with the immune system, including autophagy inhibition, type 2 immune responses, chronic inflammation, and phagocytosis blockage. To tackle this bacterium successfully, innovative vaccine strategies are needed. Modulating autophagy is a promising therapeutic approach that can strengthen the host's immune system and mitigate inflammation, offering potential benefits in fighting various pathogens, including P. aeruginosa⁷, ¹⁰, ⁶.

This study holds great significance in the current research context because the designed chimeric vaccine PAV3has the ability to simultaneously bind to TLR4 via N-terminal 28 kDa region of ETA and attach to TLR2 through LecB in its N-terminal end, with a highly suitable binding affinity (Fig. 11, 12). Due to the importance of the CD14 molecule in TLR2 and TLR4 activation the interaction of the designed vaccine with these immune system receptors could play a vital role in enhancing the vaccine's immunogenicity. Thus, the attachment of the designed vaccine in our study to CD14, in addition to the main TLR2 and TLR4 receptors, optimally activates the TLR2 signaling pathway, guides the immune response towards Th1, activates M1 macrophages, induces TLR4 internalization, and activates the TRIF pathway and autophagy, all of which play a crucial role in promoting autophagy and inducing type I interferon and Th1 immune response. The simultaneous stimulation of these two types of TLRs has a synergistic effect on the activation of signaling pathways and immune system modulation toward Th1 direction. This aligns with the simulation results of the immune response through bioinformatics methods and laboratory experiments, demonstrating the vaccine's potential to steer towards the Th1 response by assessing IFN-γ/IL-4 levels and IgG2a/IgG1 ratios, as well as activating CTLs and macrophages. These immunogenic features of the vaccine, in addition to the importance of humoral immunity and antibody production in the extracellular life of P. aeruginosa, as extensively discussed in the preceding sections, are highly relevant in immune system modulation and direction. This could have a significant impact, as it can clear intracellular pathogens through autophagy, recognize and lyse bacterial reservoir cells through CTL activation, and expose bacteria to the immune system or extracellular drugs, preventing bacterial resistance and chronic infection.

In this study, the PAV3 chimera protein was designed based on the profiling of P. aeruginosa. It consists of LecB protein, Tat peptide from HIV, N-terminal fragment of ETA, and Epi8 protein from OprF. The PAV3 structure was successfully replicated and expressed in E. coli. The effective immunogenicity of the PAV3 structure against P. aeruginosa infection is attributed to its appropriate design, which incorporates key pathogenic factors of the bacterium, resulting in high epitope density for B and T cell responses. Surprisingly, the adjuvant property of the PAV3 structure was unexpected, as it contains Epi8 from OPRF and LecB in its structure. These components can bind to TLR4, TLR2, and CD14 receptors, suggesting that the PAV3 structure may induce its own adjuvant and immunomodulatory properties by stimulating these receptors. The PAV3 protein alone exhibits high immunogenicity and directs the immune response towards effective Th1 immunity, as indicated by the increased IFN-γ/IL-4 cytokine ratio and IgG2a/IgG1 immunoglobulin ratio. It provides strong protection against P. aeruginosa infection. However, the addition of alum adjuvant to the vaccine reduces its protective effect by manipulating the immune response towards Th2 and significantly decreasing the IFN-γ/IL-4 and IgG2a/IgG1 response patterns compared to the vaccine alone. The results of this study demonstrate that the PAV3 chimera protein is a suitable structure that can be used and further investigated in the design of vaccines against P. aeruginosa. Additionally, the bioinformatics calculations performed to predict epitopes and simulate immune system responses and docking were consistent with the laboratory findings, emphasizing the importance of this field in future vaccine design.

OprF: Protein F

CF: Cystic fibrosis

MDR: Multidrug-resistant

CDC: Centres for Disease Control and Prevention

ETA: Exotoxin A

CFU: Colony-forming units

TMB : Tetramethylbenzidine

PHI : Pathogen-host interaction

SI: Stimulation index

IFN-γ: Interferon-gamma

IL-4: Interleukin-4

PTD: protein transduction domain

Alum: Aluminum salts

T3SS: Type III secretion system

CD14: Cluster of differentiation 14

TLR2: TLR2 - Toll-like receptor 2

TLR4: TLR2 - Toll-like receptor 2

TRIF: TIR-domain-containing adapter-inducing interferon-β

AP-1: Activator protein 1

Acknowledgments:

The authors would like to express our sincere gratitude to all those who contributed to the successful completion of this manuscript, which reflects the culmination of research carried out in the shahed and shahid beheshti Universitys Laboratory.

Funding resources

This study was financially supported by alzahra university and shahed university in Tehran, Iran.

Authors' contributions

Study concept and design: S. Amin nezhad, Ahya Abdi-Alia, Tooba Ghazanfari, Mojgan Bandehpour. Methodology: S. Amin nezhad, Ahya Abdi-Alia, Tooba Ghazanfari, Mojgan Bandehpour. Data management and analysis: S. Aminnezhad, Yousef Azarakhsh. Research and review: S. Amin nezhad, Ahya Abdi-Alia, Tooba Ghazanfari, Mojgan Bandehpour. Writing, review, and editing: S. Amin nezhad, Yousef Azarakhsh. This version has been read and approved by all authors.

Conflicts of Interest

The authors declare no other relevant competing interests.

Arrive Statement

We want to confirm that our study is indeed reported in accordance with the ARRIVE guidelines as outlined in (https://arriveguidelines.org). Furthermore, we can affirm that our study has received ethical approval as required. Ethical considerations and approval processes are fundamental to the responsible conduct of our research, and we have taken all necessary steps to comply with these standards.

Data availability

We have submitted gene sequences to NCBI repository and accession numbers [BankIt2774033] and protein sequence to NCBI repository and accession number [BankIt2774033] and waiting for release of gene and protein sequence data.

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Production of recombinant chimeric protein of Pseudomonas aeruginosa virulence factors and assessment of its immunogenicity in mice

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Abstract

Figures

Introduction

Material and method

Ethics statement

Bacterial strains and culture method

Cloning, expression, and purification of recombinant proteins

Toxicity test in mice

Immunization of mice

Bacterial Burden Assessment

Isolation and cultivation of spleen cells

MTT cell proliferation Assay

Cytokine assays using the ELISA method

Determination of mouse-specific antibody titers and antibody typing via ELISA

Bactericidal activity of serum

Molecular Docking Analysis

Investigation of immunomodulatory activity of PAV3 vaccine by influence on signaling pathways

Results

Sequence retrieval and construct design

Protein purification and analysis

Sterility and toxicity tests in mice

Splenocytes pproliferation assay

Cytokine response Patterns

Interferon-gamma (IFN-γ) production

Interleukin-4 (IL-4) production

Determination of IgG Total, IgG2a, IgG1, and IgA levels in serum using ELISA

Serum bactericidal assay

Molecular docking analysis

Discussion

CONCLUSION

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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