IAI Accepts, published online ahead of print on 3 November 2008 Infect. Immun. doi:10.1128/IAI.01151-08 Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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Immunization with recombinant Brucella spp. outer membrane
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proteins Omp16 or Omp19 in adjuvant induces specific CD4+ and
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CD8+ T cells as well as systemic and oral protection against
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Brucella abortus infection.
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Karina A. Pasquevich1,2, Silvia M. Estein3, Clara García Samartino1,2, Astrid
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Zwerdling1,2, Lorena M. Coria1,2, Paula Barrionuevo1,2, Carlos A. Fossati2,
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Guillermo H. Giambartolomei1,2 and Juliana Cassataro1,2*
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Laboratorio de Inmunogenética, Hospital de Clínicas “José de San Martín”, Facultad de
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Medicina. Universidad de Buenos Aires (UBA). Buenos Aires. Argentina1. Instituto de
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Estudios de la Inmunidad Humoral (IDEHU-CONICET), Facultad de Farmacia y Bioquímica, UBA, Buenos Aires, Argentina2. Laboratorio de Inmunología, Departamento de Sanidad Animal y Medicina Preventiva, Facultad de Ciencias Veterinarias, Universidad Nacional del
Centro de la Provincia de Buenos Aires, Tandil, Argentina3.
Running title: recombinant Brucella Omp16 or Omp19 protect against B. abortus infection.
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*Corresponding author. Mailing address: Laboratorio de Inmunogenética. Hospital de
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Clínicas “José de San Martín”. Facultad de Medicina, UBA. Córdoba 2351 3er Piso Sala 4
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(1120). Buenos Aires, Argentina. TEL:(54-11)5950-8755. FAX:(54-11)5950-8758.E-mail:
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[email protected]
2 ABSTRACT
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Available vaccines against Brucella spp. are live attenuated Brucella strains. In order
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to engineer a better vaccine to be used in animals and humans, in our laboratory we aim to
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develop an innocuous subunit vaccine. Particularly, we are interested in the outer membrane
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proteins (Omps) of B. abortus: Omp16 and Omp19. In this study, we assessed the use of these
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proteins as vaccines against Brucella in BALB/c mice. Immunization with lipidated (L)-
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Omp16 or L-Omp19 in incomplete Freund's adjuvant (IFA) conferred significant protection
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against B. abortus infection. Vaccination with unlipidated (U)-Omp16 or U-Omp19 in IFA
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induced a higher degree of protection than the respective lipidated versions. Moreover, the
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level of protection induced after U-Omp16 or U-Omp19 immunization in IFA was similar to
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the elicited by live B. abortus strain 19 (S19) immunization. Flow cytometric analysis showed
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that immunization with U-Omp16 or U-Omp19 induced antigen-specific CD4+ as well as
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CD8+ T cells producing interferon-γ. In vivo depletion of CD4+ or CD8+ T cells in U-Omp16-
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or U-Omp19- plus IFA-immunized mice resulted in a loss of the elicited protection, indicating that both cell types are mediating immune protection. U-Omp16 or U-Omp19 vaccination induced a T helper 1 response, systemic protection in Alum formulation and oral protection with cholera toxin adjuvant against B. abortus infection. Both immunization routes exhibited a similar degree of protection to attenuated Brucella vaccines (S19 and RB51 respectively).
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Overall these results indicate that U-Omp16 or U-Omp19 would be useful candidates for a
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subunit vaccine against human and animal brucellosis.
3 INTRODUCTION
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Brucellae Brucellae are facultative intracellular bacteria that infect animals provoking
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abortion and infertility, leading to important economic losses. The main pathogenic species
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for domestic animals are: Brucella abortus, responsible for bovine brucellosis; B. melitensis,
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the major etiologic agent of small ruminant brucellosis; and B. suis responsible for swine
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brucellosis (19). Brucellosis is also a human disease with minimal mortality, however, human
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brucellosis is one of the commonest zoonotic disease worldwide with more than 500,000 new
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cases annually (28) and it is a weakening disease that requires a prolonged antibiotic
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treatment often leaving permanent and disabling after-effects (27, 37). In association with
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animal infection, human brucellosis has been attributed to at least four of the six recognized
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Brucella species in terrestrial mammals, B. ovis and B. neotomae being the exceptions.
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Human brucellosis has also been attributed to some marine mammal strains recently (27).
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Control and eradication of brucellosis in domestic animals have important public health and
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economic implications. Test-and-slaughter programs and vaccination in conjunction are the most important methods of control of animal brucellosis. Thus, prevention of human brucellosis depends predominantly on the control of the disease in animals (19). Up to this moment, B. abortus strain 19 (S19) or B. abortus RB51 are used to
immunize cattle whereas B. melitensis Rev 1 strain is used to immunize goats and sheep. No
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other vaccines are licensed for other animals, and a human brucellosis vaccine does not exist
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(33).
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In general the use of live attenuated organisms as vaccines, though a tried and true
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approach, has some troubles in terms of safety during vaccine production (e.g.: the potential
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of the organism to revert to its original virulent condition and the shed of the organism into
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the environment with the danger to immunocompromised recipients) (10, 22). In fact,
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attenuated Brucella vaccines have many disadvantages (26, 33). For these reasons, different
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strategies are being sought that provide safe, non-replicating vaccines that are easy to
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reproduce for quality assurance (10, 22). Related to this concern, in our laboratory we have
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been working on the development of a subunit vaccine against Brucella. In particular we aim
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to develop a recombinant subunit vaccine that can be more versatile, this is: i) to be applicable
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to any host, ii) to elicit immunity at different sites of infection by different application
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methods and iii) to be protective against any species of Brucella. We envision that this
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vaccine would have to be made of different Brucella proteins.
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In the search of novel vaccine targets we have focus on the outer membrane proteins
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(Omps) of B. abortus Omp16 and Omp19. It has been established by molecular cloning and
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sequencing
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Physicochemical and functional analysis have verified that Omp16 and Omp19 are indeed
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lipoproteins and that they are surface-exposed (34). It has also been reported that these
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lipoproteins are present in all six Brucella species and all their biovars (18). In addition, it has
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that
these
Omps
have
structural
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features
of
bacterial
lipoproteins.
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been demonstrated that monoclonal antibodies (mAbs) against Omp16 or Omp19 can protect mice against a B. ovis challenge (9). We cloned, expressed in E. coli and purified recombinant Omp16 and Omp19 and used them as model stimulants. We have shown that they are important mediators of the pro-inflammatory response elicited by heat-killed B. abortus (18). Therefore, vaccines based upon recombinant Omp16 and Omp19 lipoproteins could
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probably elicit a cellular immune response and provide the host protection against Brucella
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infection. We test this hypothesis in the present work.
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5 MATERIALS AND METHODS
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Mice. Female BALB/c mice (8 to 9 weeks old) obtained from the University of La Plata,
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Argentina were acclimated and randomly distributed into experimental groups. The mice were
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kept in conventional animal facilities and received water and food ad libitum. After
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inoculation with B. abortus mice were kept in biosafety level 2 animal facilities.
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Bacterial strains. B. abortus 544, B. abortus 2308 (wild type, smooth, virulent strains), B.
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abortus S19 (vaccine strain, smooth), B. abortus RB51 (vaccine strain, rough), B. ovis REO
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198 (wild type, rough, virulent strain) and B. melitensis H38 (wild type, smooth, virulent
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strain) were obtained from our own laboratory collection (12, 15, 35). Bacteria were grown
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and inocula were prepared as described (12, 15, 21, 35). Brucella strains manipulations were
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performed in biosafety level 3 facilities.
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Antigen (Ag) production. Methods for cloning, expression in E. coli and purification of
recombinant lipidated (L-) and unlipidated (U-) Omp16 and Omp19 from B. abortus have
been previously described (18). Briefly, the complete sequence information of both proteins was previously reported by Tibor et al. (34). Specific primers for the entire sequence of L-
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Omp16 and of L-Omp19 were designed. The unlipidated versions of Omp16 and Omp19 (U-
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Omp16 and U-Omp19) were cloned using different forward primers, flanking the complete
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gene of the protein avoiding the putative signal peptide for lipidation. B. abortus 544 genomic
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DNA was used as template for PCR. The products were cloned into the pET 22b+ vector
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(Novagen, Madison, WI) resulting the plasmids pET-L-Omp16, pET-U-Omp16, pET-L-
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Omp19, pET-U-Omp19 containing the genes with a COOH-terminal 6x histidine tag. The
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recombinant Omps were successfully expressed in E. coli BL21(DE3). Recombinant lipidated
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L-Omp16 and L-Omp19 were isolated from bacterial membranes by sonication and selective
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extraction by phase partitioning with 2% Triton X-114. U-Omp16 and U-Omp19 were
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isolated from bacterial cytoplasm by sonication. These preparations were further purified by
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affinity chromatography with a Ni-NTA resin (Qiagen, Dorking, U.K.). Expression and
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purification of the recombinant proteins were monitored by SDS-PAGE, followed by silver
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staining. Identity of Omps was confirmed by Western blot with anti-Omp16 or anti-Omp19
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mAbs as described (18). To eliminate LPS contamination, Omps were adsorbed with
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Sepharose-polymyxin B (Sigma–Aldrich, Saint Louis,MO). They contained
