Immunological evaluation of OMP-F of native Iranian Pseudomonas aeruginosa as a protective vaccine

Introduction: This study involved 300 Pseudomonas aeruginosa strains isolated from patients admitted in four Tehran hospitals. Using standard O-specific typing sera, they were all grouped into 16 strains out of 17 known P. aeruginosa. The strains were lyophilized and each was given a code according to the Collection of Standard Bacteria, Pasteur Institute of Iran (CSBPI) for further investigations. Methodology: Among all clinical samples, CSBPI: 16-190 was the most prevalent P. aeruginosa serotype which showed a high agglutination titer (4+, 320) against homologous O-specific typing sera. This serotype was selected for extraction of P. aeruginosa major outer membrane vesicles (OMP-F). OMP-F vesicles were extracted and purified according to the Deoxycholate Ultracentrifuge Differentiation Technique. Purity and molecular weight of OMP-F were determined by SDS-PAGE and the ability of OMP-F vesicles to induce high titers of antibody in rabbit, which was shown as a sharp antibody-antigen precipitation line in the agarose gel immune-diffusion technique. Results: Passive immunization of mice with anti-rabbit OMP-F antisera induced a high level of protection when the mice were postchallenged with 2×LD50 of live P. aeruginosa CSBPI: 16-190. Furthermore, active immunization of mice with 50 μg of OMP-F could protect mice against 2×LD50 of live homologous (100% protection) and 15 heterologous native Iranian P. aeruginosa serotypes with 50100% level of protection. Conclusions: These investigations indicate that purified OMP-F of CSBPI: 16-190 can be regarded as a safe protective immunogen in vaccinothrapy against all P. aeruginosaimmunotype isolated in Iran.


Introduction
Pseudomonas aeruginosa, a ubiquitous environmental Gram-negative microorganism, is a major cause of hospital-acquired infections [1,2].It is one of the most causatives against of nosocomial infections worldwide and prophylaxis is a standard goal for its control and prevention [3].Being parasitic and saprophytic in nature, P. aeruginosa generally is responsible for acute and chronic lung infections in artificially ventilated [4] and in cystic fibrosis patients [5], and for septicemia in immunocompromised patients, including transplant and cancer patients, as well as patients with severe burn wounds [6].Using the International Antibody Typing Standard (IATS), P. aeruginosa can be classified into 17 serotypes [7], but about 16 serotypes are prevalent in Iran [8].Serotype CSBPI: 16/190 was one of the most cross-reactive of serotypes.
Among Iranian isolates, LPS or detoxified derivatives from P. aeruginosa provide good protective activities in animal models, but their pathophysiological adverse effects limit their application in vaccines.Therefore, major outer membrane proteins (OMP-F) from serotype CSBPI: 16/190 with a molecular weight of 37-39 KDa can be regarded as a safe and reliable vaccine candidate.Different antigens of P. aeruginosa, such as the outer membrane proteins (OMPs), LPS, toxins, pilli and flagella, have been investigated as possible targets for the development of vaccines.Vaccination with outer membrane protein antigens has been shown to be efficacious against P. aeruginosa infection in a number of studies using killed whole cells [9], isolated outer membrane proteins [10][11][12][13] and purified outer membrane preparations [14].The P. aeruginosa major constitutive porin protein, OMP-F, which has previously been shown to be antigenic [10,15] and has high homology among Pseudomonas strains [12,16], was also chosen as a vaccine target [17].This protein has been shown to provide protection in a mouse model of systemic infection [10], a mouse burn infection model, and in rodent models of acute [18] and chronic lung infection [12].In the present study, OMP-F was obtained from Iranian serotype CSBPI: 16/190 through a mild extraction procedure as described by Classen et al. [19], then the immunological characteristics of the OMP-F was evaluated through passive and active immunization challenges with homologous and heterologous P. aeruginosa serotypes in mouse models.

Bacterial strain
Strain CSBPI-16-190 was selected from 300 P. aeruginosa biochemically and serologically confirmed clinical isolates [8].The selection was based on the cross-reactivity between the antibody raised against the heat-inactivated CSBPI-16-190 and other P. aeruginosa isolates belonging to serotypes 1-15.Active protection was tested using 300 P. aeruginosa serotypes 1-15 isolated from clinical samples [8].This strain was further selected for extraction of OMP-F.

Culture conditions
P. aeruginosa CSBPI-16-190 was cultured into 10 liters of synthetic P. aeruginosa medium [20] using a Novo-paljas Bioreactor (Contact-flow B.V., Bilthoven, Netherlands) at optimum cultural conditions.At the end of logarithmic phase of growth, the cells were harvested by centrifuge at 3000 rpm for 45 minutes at 4°C.

Extraction of OMP-F
OMP-F was extracted from P. aeruginosa CSBPI-16-190; using the method described by Classen et al. [19] with mild modifications as follows: Cells were washed three times with phosphate buffered saline (PBS, pH = 7.2) and suspended in 7.5 times its wet-weight with 0.1 M Tris-HCl buffer containing 10 mM EDTA.After two hours of shaking at room temperature, 20 times (V/V) 0.1 M Tris-Buffer containing 10 mM EDTA and 100 grams of sodium deoxycholate were added to the cell suspension.After 10 minutes of shaking, the suspension was centrifuged at 8000 rpm for one hour at 4°C.The cells were then discarded and the  supernatant was centrifuged at 42000 rpm for two hours.

P-Value
The sediment was dissolved in 15 ml DW containing 3% sucrose, and then filter-sterilized and lyophilized.
SDS-PAGE electrophoresis was performed in 10% gel using standard-size markers (Figure 1) and the protein content was measured using the Lowry method with BSA as a reference.

Production of hyper immune antibody
Hyper immune antibody was prepared by intramuscular injection of 500 μg of the P. aeruginosa OMP-F vesicle along with Fronds incomplete adjuvant into a group of three New Zealand white rabbits weighing 1.5 -2 kg, on days 0, 7, 14, 21 and 28.Seven days after the last injection, serum was collected from each immunized rabbit and pooled.The precipitation reaction of hyper immune antiserum against the purified OMP-F was evaluated by double agarose gel immunodiffusion technique.

Passive immunization
Passive immunization was performed as described by Gilleland et al. [12] (Table 1).A group of five young adult mice weighing 18-20 grams received 0.1ml of hyper immune antiserum against OMP-F by I/V injection.PBS (pH = 7.2) was used as the control [12].

Active immunization
Active immunization against homologous and heterologous serotypes was performed using animal models (mice) by subcutaneous injection of 50 µg of OMP-F according to the methods of Pier et al. [21].Active protection of OMP-F vesicle of CSBPI: 16-190 P. aeruginosa was evaluated against homologous and heterologous isolates (Table 2).

Pyrogenic test
Pyrogenic tests were performed according to the British Pharmacopeia (2007) annex 2.6.8, through intravenous injection of purified OMP-F into the group of three rabbits weighing 2 to 2.5 kg.

Abnormal toxicity test
The purified OMP-F was tested for abnormal toxicity by intra peritoneal injection of 100 µg purified OMP-F into five mice weighing 17-22 g and 500 µg purified OMP-F into two guinea-pigs weighing 250-350g.The test was considered satisfactory if the animals survived for at least seven days without weight loss.

Results
OMP-F was extracted from CSBPI: 16/190 as a result of the high cross-reactivity between this serotype and all the 300 P. aeruginosa isolates.Furthermore, this serotype was the most common pathogenic isolate from Iranian hospitals [8].
The molecular weight of OMP-F was found to be 37-39 KDa by SDS-PAGE electrophoresis (Figure 1) and the high precipitation line of reaction in agarose gel immuno diffusion was observed between purified OMP-F and its hyper-immune rabbit antibodies (Figure 2).
The results of the toxicity and pyrogenicity tests showed that OMP-F was safe and did not cause any adverse effects after injection into the animals.
As shown in Table 1, passive immunization of mice with hyper immune OMP-F antiserum protects mice against 2×LD50 of the live homologous serotype.Moreover, good protective activity of the OMP-F was observed by active immunization of mice against 2×LD50 of live homologous and heterologous serotypes (Table 2).It seems that there is a close immunological cross-reactivity among OMP-F isolated from different P. aeruginosa isolates.

Discussion
Today, the protective activities of subunit vaccines-mainly major outer membrane proteins of many Gram-negative bacteria-are well-known [12,22,23].There is no reliable P. aeruginosa vaccine produced so far, although many attempts have been made [10,14,24,25].In 1984, Gilleland et al. used the porin of P. aeruginosa as a protective vaccine in mice [10].Pier et al. induced good protective activity by immunizing mice with capsular polysaccharide of P. aeruginosa [21].However, this activity was not observed in man and in 2000 Lee et al. [26] reported that OMPs elicit antibodies with protective efficacy against P. aeruginosa infection in burn patients.In 2006, Cripps et al. used heat-killed P. aeruginosa as a vaccine against the infection, but this immunogen caused serious adverse effects.
In the present investigation, out of 300 P. aeruginosa strains isolated from clinical samples of hospitalized patients in Iran, serotype CSBPI: 16/190 was selected to extract OMP-F because this serotype was the most pathogenic isolate prevalent in Iran and also showed O-antigenic similarities with all 300 isolates [8].Our results also indicate that purified OMP-F induced not only good protective efficacy against homologous serotypes of P. aeruginosa infection, but also protected the mice against all 15 heterologous isolates, suggesting that there is good immunological cross-reactivity among purified OMP-F of different serotypes.Therefore, OMP-F extracted from serotype CSBPI: 16/190 was considered to be a reliable and safe immunogen with broad spectrum activity in regard to its application in therapeutic vaccines or prophylaxis.Thus purified OMP-F isolated from serotype CSBPI: 16/190 can be a safe and effective candidate subunit vaccine against infection caused by different P. aeruginosa serotypes.

Table 1 .
Passive immunization in mice with hyper immune serum against 2×LD50 of P. aeruginosa

Table 2 .
Active immunization of OMP-F vesicle of P. aeruginosa CSBPI: 16/190 in mice challenged with 2×LD50 of live cell homologous and heterologous isolates