Outer Membrane Vesicle of Neisseria Meningitidis Serogroup B As An

Annals of Microbiology, 59 (1) 145-149 (2009)

Outer membrane vesicle of Neisseria meningitidis serogroup B as an adjuvant to induce specific antibody response against the lipopolysaccharide of Brucella abortus S99

Ali SHARIFAT SALMANI1,2, Seyed Davar SIADAT1,3*, Dariush NOROUZIAN1, Jalal IZADI MOBARAKEH4, Maryam KHEIRANDISH5, Mehrangiz ZANGENEH6, Mohammad Reza AGHASADEGHI3, Mehdi NEJATI1, Mohammad Hossein HEDAYATI7, Arfa MOSHIRI8, Seyed Mehdi SADAT3

1Department of Bacterial Vaccines and Antigens Production, Pasteur Institute of Iran, 13169-43551, Tehran; 2Department of Microbiology, Islamic Azad University, Research and Science Branch, Tehran; 3Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran; 4Department of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran; 5Department of Immunology, Research Center of Iranian Blood Transfusion Organization; 6Department of Infectious Disease, Faculty of Medicine, Islamic Azad University, Tehran; 7Department of Quality Control, Pasteur Institute of Iran, Tehran; 8Department of Medical Laboratory Sciences, Iran University of Medical Sciences, Tehran, Iran

Received 3 June 2008 / Accepted 3 December 2008

Abstract - Brucellosis is a worldwide zoonosis and represents one of the most important public health problems in many countries, especially around the Mediterranean basin, Middle East, India and Central and South America. Currently subunit vaccines are being considered to develop effective vaccines for human which has been evidenced by vaccines currently available against the infections such as meningococcal diseases and influenza .The application of new adjuvants of microbial origins is also underway to design subunit vaccines promoting the immune responses to the antigenic determinant(s) of the vaccine. In order to explore the efficacy of Brucella abortus lipopolysaccharide (LPS) combined with different adjuvants and proteins (as a vaccine candidate) in the induction of immune response as an effective and long-lasting immunity against Brucella, we evaluated the outer membrane vesicle of Neisseria meningitidis serogroup B (GBMOMV) as a subcutaneous adjuvant and a part of a brucellosis candidate vaccine to induce high titres of specific anti-Brucella abortus S99 LPS in animal model (mice). The obtained results were compared with complete and incomplete Freund’s adjuvants (CFA and IFA). LPS+GBMOMV was the most immunogenic compound that stimulated following the first injection an increase in IgG titre of about 3.90, 3.18 and 1.58 fold higher than that produced against LPS, LPS+IFA and LPS+CFA, respectively. The highest anti-LPS IgG titre was detected two weeks after the third injection (the day 42) of LPS+GBMOMV. Purified GBMOMV can be considered as a safe subcutaneous adjuvant and a part of a candidate vaccine when combined with lipopolysaccharide of Brucella abortus S99.

Key words: lipopolysaccharide (LPS); Brucella abortus S99; outer membrane vesicle; adjuvant.

INTRODUCTION LPS also acts as one of the primary targets of the innate immunity. Brucella abortus strains may appear as either smooth (S) or Brucellosis is a worldwide zoonosis and represents one of the rough (R) strains, expressing smooth LPS (S-LPS) or rough LPS most important public health problems in many countries, espe- (R-LPS), as the major surface antigens, respectively (Carmichael, cially around the Mediterranean basin, Middle East, India and 1990). Most wild-type Brucellae have smooth LPS (S-LPS) con- Central and South America (Corbel, 1997). More than 500,000 sisting of three domains: Lipid A, Core oligosaccharide and O new cases are reported each year and, according to the World specific polysaccharide (O-antigen) (Jacques et al., 1991). Health Organization (1997) and this figure underestimates At present, there is no licensed vaccine against human the magnitude of the problem (Adlimoghadam et al., 2008). brucellosis. Live attenuated vaccines such as a derivative of Brucellosis leads to acute or chronic inflammation related to the Brucella abortus S19 (19-BA) have been tested in human trials. interaction of Brucella-derived products such as lipopolysaccha- For example it was used in the former Soviet Union but shown ride with the host cell. In fact Brucella LPS acts as a virulence to be insufficiently attenuated when tested in the USA (Spink et factor of the infection (Jimenez de Bagues et al., 2005; Lapaque al., 1962; Elberg et al., 1973). Currently, subunit vaccines are et al., 2005). Since naturally occurring strains lacking LPS show hot topics in the development and design of human brucellosis reduced survival, LPS is considered to be a major virulence factor. vaccine. Jacques et al. (1991) showed the efficacy of Brucella O-polysaccharide-BSA conjugate in protection against Brucella * Corresponding Author. E-mail: [email protected] melitensis H38. Other studies have been carried out to design 146 A. SHARIFAT SALMANI et al.

subunit vaccines using other components and conjugated com- 37 ± 1 °C. After 72 h of incubation, the bacteria were inactivated pounds such as porins and smooth lipopolysaccharide, recom- with 0.5% phenol, harvested and washed as previously described binant ribosomal proteins and anti-OPS specific monoclonal (Shapouri et al., 2008). antibodies (Cloeckaert et al., 1992, 1993; Oliveira and Splitter, 1996; Winter et al., 1988). LPS extraction and purification. S-LPS was extracted by hot Nowadays adjuvants are extensively used as immuno-stimu- phenol-water method. In brief, 50 g wet weight of cells was sus- lator and immuno-modulator compounds to design subunit vac- pended in 170 ml of distilled water, followed by the addition of cines .The adjuvants of microbial origins are more considerable 190 ml of 90% (v/v) phenol at 66 °C. After 30 min, the mixture among the other currently used adjuvants. The outer membrane was centrifuged and phenol layer was removed. The LPS was vesicle (OMV) of Neisseria meningitidis is among the newly stud- precipitated by cold methanol and dissolved in 0.1 M Tris buffer. ied components with microbial origin, which could be applied as For further enzymatic digestion Proteinase K, DNase and RNase an adjuvant. The potency of OMV as a carrier (conjugated to a were added to reduce protein and nucleic acid contamination of hapten) is now proved (Siadat et al., 2007a, 2007b). the extracted LPS samples and then samples were freeze dried. Among the described adjuvant properties of OMV is that the Alternatively LPS could be extracted through Butanol method mucosal and systemic antibody responses to an influenza virus since the industrial use of phenol is currently forbidden (Sharifat vaccine are greatly augmented when co-administered with OMV Salmani et al., 2008). (Haneberg et al., 1998). In addition, the adjuvant properties of OMV-derived particles have been demonstrated for potential can- Chemical analysis. Concentration of the extracted LPS samples cer vaccines (Estevez et al., 1999). Overall, the previous studies was measured by chromogenic LAL (Limulus Amebocyte Lysate) have shown that the predominant outer membrane proteins assay (Cambrex, USA). Ketodeoxyoctanate content (KDO), which (OMPs) (PorA, PorB and RmpM) from N. meningitidis present is a unique component of LPS, was determined colorimetrically by in the Meningococci B Cuban vaccine had differing capacities to thiobarbituric acid methods using pure KDO as the standard with prime the immune system. some modifications as previously described (Sharifat Salmani et Oliver et al. (2002) have demonstrated that the application al., 2008). Protein content was measured by the modified Lowry of OMV from Neisseria lactamica is an alternative approach for a method and nucleic acid contamination was determined through meningococcal vaccine. Immunization with N. lactamica OMV has measuring A-260 (Shapouri et al., 2008). provided protection against the lethal challenges with a range of meningococcal strains in a mouse model of meningococcal Outer membrane vesicle preparation. OMVs were prepared disease. It has also been shown that immunization of mice with as previously described (Claassen et al., 1996). In brief, N. men- N. lactamica OMV induces cross-reactive antibodies with N. meningitidis serogroup B strain (CSBPI, G-245) was grown under ingitidis and primes for an enhanced response to N. meningitidis controlled submerge cultural condition in a fermentor containing immunization (Sanchez et al., 2002). In addition, OMV isolated modified Frantz medium (Siadat and Nourozian) at 36 ± 1 °C from commensal Neisseria can be a delivery vehicle for heterolo- for 24 h up to the early stationary phase. The outer membrane gous vaccine antigens. Sardinas et al. (2006) have employed a vesicles (OMVs) were extracted in 0.1 M Tris-HCl buffer, pH 8.6, hepatitis B surface antigen (HBsAg) vaccine as a model antigen containing 10 mM EDTA and 0.5% w/v deoxycholate. Purification to assess the adjuvant properties of N. lactamica OMV. of the OMVs was done by sequential centrifugation at 20,000 x The effect of Brucella abortus LPS combined with different g for 30 min followed by ultracentrifugation at 125,000 x g for 2 adjuvants and proteins (as a subunit vaccine candidate) to induce h; the pelleted OMVs were homogenized in phosphate buffered the immune response as an effective and long-lasting immunity saline (PBS), pH 7.2. Throughout the process, thiomersal (100 against Brucella could be studied as a proposed method (Van de mg/l) was added as preservative. verg et al., 1996; Bhattacharjee et al., 2002, 2006). In paral- lel with adjuvant properties of OMV Bhattacharjee et al. (2002) Immunization schedule. Brucella abortus extracted S-LPS (10 have developed a vaccine composed of purified LPS of Brucella μg) was injected subcutaneously into the four different groups of melitensis 16M noncovalent complex with the OMV of N. menin- animal models (each group consisted of 10 mice): gitidis serogroup B and studied the immunological properties of S-LPS: without any adjuvant; this vaccine candidate. In a similar study, they have reported that LPS+CFA: 10 μg of S-LPS dissolved in 100 μl of complete the purified B.melitensis LPS+GBMOMV noncovalent complex Freund’s adjuvant (CFA) (Sigma Chemical Co, USA); vaccine could be a safe candidate as a vaccine against human LPS+IFA: 10 μg of S-LPS dissolved in 100 μl of incom plete brucellosis (Bhattacharjee et al., 2006). Freund’s adjuvant (IFA) (Sigma Chemical Co); In the present study, we designed a subunit brucellosis LPS+GBMOMV: 10 μg of S-LPS plus 50 μg of OMV as an adju- vaccine composed of B. abortus S99 LPS with N. meningitidis vant. serogroup B OMV as a noncovalent complex and then evaluated The booster injections were carried out in 14th and 28th days specific antibody response against the LPS of B. abortus S99. after the first injection. The immunized animals were bled on the days 0, 14, 28 and 42 and the immune sera were separated, pooled and kept in -20 °C. MATERIALS AND METHODS Enzyme Linked Immunosorbent Assay (ELISA). Specific Bacterial strain and culture conditions. Brucella abortus antibody molecules produced against the extracted LPS of B. S-99 (biovar 1) was obtained from the type bacteria collection of abortus were demonstrated through ELISA. Total specific IgG Pasteur institute of Iran (25th Km of Tehran-Karaj Highway, Iran). and IgM molecules, elicited against the S-LPS of B. abortus The bacteria were routinely grown on Brucella agar medium were detected. The ELISA was performed in 96-well flat-bottom (Merck, Darmstadt, Germany) at 37 ± 1 °C for 72 h. Then, the polystyrene microtitre plates. The wells were coated with the bacteria were propagated into a 60 L fermentor (Nova-Paljas, extracted S-LPS of B. abortus S99 at a concentration of 10 μg/ Contact-flow B.V, Netherlands) with a working volume of 40 L at ml in PBS azide (0.014 M sodium chloride, 0.02% sodium azide, Ann. Microbiol., 59 (1), 145-149 (2009) 147

0.01 M sodium phosphate; pH 7.5) and incubated overnight at most immunogenic compound, promoting produced IgG titre 37 °C. The wells were washed three times with PBS azide. The following the first injection, 3.9, 3.18 and 1.58-fold higher wells were blocked by adding a blocking buffer (1% casein in PBS- than IgG titre elicited against LPS, LPS+IFA and LPS+CFA, azide) and then incubated again at 37 °C for 1 h. Serial two-fold respectively. The application of LPS+CFA led to the production dilutions of the sera were made on the plates. Then, the plates of 1.98 and 2.5-fold higher anti-LPS IgG titre as compared to kept at 25 °C for 16 h. Next, HRP-labelled goat anti-mouse IgG LPS+IFA and LPS, respectively. The anti-LPS IgG titre follow- was added to the wells at a concentration of 1 μg/ml and the ing the injection of LPS+IFA did not significantly increase in plates were incubated at 25 °C for 16 h. Afterwards, the sub- comparison with the induced titre following LPS injection. The strate of HRP (TMB) was added to the wells at a concentration of booster injections (especially the first booster) were effective to 1 mg/ml and the plates were incubated at room temperature for significantly increase the titre of anti-B. abortus S99 LPS IgG in 30 min. Finally stop solution (50 μl) was added to the wells and all the groups (P < 0.05). The first and second booster injections absorbance was measured at 405 nm using an automatic ELISA of LPS+GBMOMV caused 6 and 12.4-fold higher titres compared plate reader. The antibody titres are expressed in OD units and to the anti-LPS IgG titre induced after the first injection. The calculated by multiplying the reciprocal dilution of the serum by highest anti-LPS IgG titre was detected two weeks after the third the OD at that dilution. injection (the day 42) of LPS+GBOMV (Table 1). Anti-B. abortus S99 LPS IgM titre showed a significant Statistical methods. Antibody titres of groups of mice were increase (P < 0.05) after the injection of LPS+GBMOMV in expressed as means ± standard deviations. The significance of comparison to the immunization with LPS and LPS+IFA. The differences in ELISA titres was determined by Student’s t test. application of LPS+CFA significantly increased the anti-B. abortus S99 LPS IgM titre in comparison with the application of LPS and LPS+IFA (P < 0.05), but the difference of the elicited titres RESULTS following the application of LPS+CFA and LPS+GBOMV was not significant (P > 0.05) (Table 2). LPS characterization The LPS content of the extracted sample (measured by the chromogenic LAL assay) was 108 endotoxin units (EU)/ml. The KDO DISCUSSION content came out to be 1.3%. Also the protein content of the sample (measured by Lowry method) was < 2.0 and the nucleic As mentioned in the Results section, anti-B. abortus S99 LPS IgG acid content was < 1%. titres exponentially and significantly increased in all the groups comparing to the control group. The high titres of anti-B.abortus Enzyme linked immunosorbent assay S99 LPS IgG in comparison with the control group indicate the Subcutaneous immunization with B. abortus S99 LPS induced immunogenicity of B. abortus S99 LPS and the fidelity of our LPS the increase of specific anti-LPS IgG titre in all the groups. extraction procedure which dose not interfere with the natural Among the four injected compounds, LPS+GBMOMV was the and immunogenic structure of LPS.

TABLE 1 - Anti-Brucella abortus S99 LPS total IgG titre ± SD, 2 weeks after the first, second and third injections Compound Anti-Brucella abortus S99 LPS IgG titre, 2 weeks after first injection second (first booster) injection third (second booster) injection LPS 1868 ± 106 6828 ± 577 16809 ± 1276 LPS+IFA 2362 ± 118 10799 ± 840 24706 ± 2574 LPS+CFA 4678 ± 365 21828 ± 1828 52203 ± 3926 LPS+GBMOMV 7398 ± 390 45057 ± 2779 91993 ± 5665 Control group (normal saline) < 3 < 3 < 3 Titres are expressed in OD units. IFA: Incomplete Freund’s adjuvant, CFA: Complete Freund’s adjuvant, GBMOMV: Group B Meningococci Outer Membrane Vesicle.

TABLE 2 - Anti-Brucella abortus S99 LPS IgM titre ± SD, 2 weeks after the first, second and third injections Compound Anti-Brucella abortus S99 LPS IgM titre, 2 weeks after first injection second injection third injection LPS 446 ± 89 463 ± 112 422 ± 99 LPS+IFA 465 ± 125 479 ± 131 441 ± 107 LPS+CFA 576 ± 111 581 ± 98 539 ± 141 LPS+GBMOMV 604 ± 128 615 ± 118 568 ± 134 Control group (normal saline) < 2 < 2 < 2 Titres are expressed in OD units. IFA: Incomplete Freund’s adjuvant, CFA: Complete Freund’s adjuvant, GBMOMV: Group B Meningococci Outer Membrane Vesicle. 148 A. SHARIFAT SALMANI et al.

Two weeks after the first, second and third injections, the that our purified GBMOMV may be applied as a safe and potent highest titres of anti-B. abortus S99 LPS IgG were detected in the subcutaneous adjuvant to induce high titres of specific anti-B. group immunized with LPS+GBMOMV compound (7398 ± 390, abortus S99 IgG and may be used in candidate vaccines for 45057 ± 2779 and 91993 ± 5665, respectively). human brucellosis. The significant difference (P < 0.005) of these titres with the titres induced by LPS, LPS+IFA and LPS+CFA reveals that GBMOMV is the most efficient adjuvant among the three evaluat- REFERENCES ed adjuvants and LPS+GBMOMV is the most immunogenic compound that significantly induces the production of anti-B. abortus Adlimoghadam A., Hedayati M.H., Siadat S.D., Ahmadi H., Nejati S99 LPS IgG. Although there are a lot of proved efficient opti- M., Vandyousefi J., Norouzian D. (2008). 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