Abstract and Introduction
Abstract
Ovine brucellosis is a very contagious zoonotic disease distributed worldwide and constitutes a very important zoosanitary and economic problem. The control of the disease includes animal vaccination and slaughter of infected flocks. However, the commercially available vaccine in most countries is based on the attenuated strain Brucella melitensis Rev 1, which presents important safety drawbacks. This review is focused on the most recent and promising acellular vaccine proposals.
Introduction
Brucellosis is considered a reemerging zoonosis, producing low mortality but high morbidity in both animals and humans. Most Brucella species affect domestic ruminants (Table 1) and, consequently, severely affect the economy of millions of people. Specifically, the costs associated with ovine brucellosis are related to losses in animal reproduction, infertility, abortion, delayed conception and reduced milk production. The causative agents of ovine brucellosis are Brucella ovis and Brucella melitensis.B. ovis is a nonzoonotic rough strain – it is responsible for contagious ram epididymitis and also alters the blood circulation of sheep placenta, implying the risk of placentitis, abortion as well as neonatal death. This species infects sheep, as well as farmed red deer (Odocoileus virginianus) in New Zealand. Experimental infections have been reported in goats and cattle, but there is no evidence that these species are infected in nature (Table 1). On the other hand, B. melitensis is a major worldwide zoonosis associated with significant economic losses and importance to public health. It is highly pathogenic to humans and responsible for classical testicular alterations in rams, reduced fertility and abortion. The infected females rarely clear the pathogen from their system and tend to shed through their next parturition. It may be venereally transmitted and shedding of the organism can occur for more than 4 years in rams. Semen quality deteriorates rapidly and inflammatory cells are often present. Once fibrous atrophy of the testes occurs, it is permanent. Thus, the development of new vaccines against B. melitensis and B. ovis would be of great benefit worldwide.
Different countries may require different strategies for its prevention and control in animals, depending on epidemiological and socioeconomic conditions. The general strategies proposed in 1998 by the WHO to control animal brucellosis were:
Prevention of spread between animals and monitoring of brucellosis-free flocks/herds and zones;
Elimination of infected animals by a test-and-slaughter program to obtain brucellosis-free flocks/herds and regions;
Vaccination to reduce the rate of infection. It is certain that vaccination is the most economic and effective measure to control infections.
According to the established guidelines by the WHO, an ideal vaccine should basically accomplish two goals – efficacy and innocuousness:
Innocuous for the vaccinated animals;
Not produce disease in the vaccinated animals;
Prevent infection, abortion and sterility;
Provide long-term protection against infection and abortion with a single vaccination (more than 90% of protection);
Must minimize long-term production of antibodies, which may interfere with serodiagnosis tests of field infections;
Noninvasive administration (mucosal administration preferably);
Not be transmitted to other animals if the vaccine strain establishes a long-term latent infection;
Biologically stable and free of reversion to virulence in vitro and in vivo;
Stable at room temperature;
Nonpathogenic for humans;
Not contaminate meat and milk products;
Culturable under large-scale conditions;
Should contain specific genetic or phenotypic markers that would make it easy to differentiate from field isolates.
Attenuated Vaccines
The classical live-attenuated vaccines are made of attenuated pathogens in order to diminish their virulence, while retaining their immunogenicity. However, several risks are associated with such vaccines including residual virulence or hazardous re-infection, which occur when the inactivation of the microorganism is incomplete. In this context, attenuated vaccines can regain their virulence in immunodeficient hosts, allowing pathogen release and spread into the environment. Thus, a careful and exhaustive evaluation of the potential impact of environmental release is required. This is the case for the currently best available immunoprophylaxis system against ovine brucellosis, the B. melitensis Rev 1 vaccine. Rev 1 was developed in 1950 by Elberg and Herzberg, derived from a virulent B. melitensis isolate that became streptomycin-dependent. Since its introduction into the marketplace in the 1960s, the benefit derived from its use is incalculable, protecting the ovine livestock and goats against B. melitensis and providing protection against B. ovis in ovine livestock. However, this vaccine is not free from virulence, and it presents important disadvantages, such as causing infertility and abortion in pregnant animals, and of being pathogenic for humans. Furthermore, it is resistant to streptomycin, the antibiotic that combined with doxycycline provides the most effective brucellosis therapy. Moreover, this strain possesses a smooth phenotype and, therefore, vaccinated animals produce persistent antibodies against the lipopolysaccharide (LPS) O-chain. As a consequence, the utilization of smooth vaccine strains makes the distinction between vaccinated and infected animals, including sheep, impossible. For all these reasons, the use of Rev 1 is prohibited in those countries where B. melitensis infection has been eradicated.
Owing to the unacceptable levels of antibodies interfering with diagnostic tests, attempts were made to circumvent this problem. Blasco and colleagues found that conjunctival vaccination with Rev 1 did not induce such long-lasting serological responses in rams after vaccination. Moreover, conjunctival vaccination of rams was safer than subcutaneous vaccination, reducing the number of abortions. However, there were still a significant proportion of vaccinated animals excreting Rev 1 bacteria.
Another approach to avoid the interference of the O-chain with disease control surveillance is based on the use of rough vaccine strains. The rifampicin-resistant mutant B. abortus strain 2308, denominated B. abortus RB51, is a genetically stable, rough morphology mutant that lacks the polysaccharide O-side chains on the surface of the bacteria responsible for the development of the diagnostic antibody responses of an animal to brucellosis infection. However, in sheep, it has been clearly demonstrated that RB51 does not confer enough protection against B. melitensis or B. ovis infections. Moreover, even though the risks are low, human infections due to RB51 have also been described, and being resistant to rifampin is problematic since this antibiotic combined with doxycycline is widely used for treating brucellosis in humans.
B. melitensis B115 is a natural Brucella rough strain, according to the absence of agglutination with monospecific sera to A and M antigens of Brucella; however, it has a cytoplasmic O-chain. Adone et al. have shown that B.melitensis is highly protective against B. melitensis and B. ovis infections in mice, without inducing interfering antibodies, suggesting its potential usefulness to control brucellosis, but it must be tested in the natural host.
A further approach is the construction of recombinant strains with deletion in the genes encoding relevant diagnostic proteins. Thus, a Rev 1 vaccine strain with a deletion in the gene coding for Bp26 periplasmic protein resulted in the same protective efficacy as Rev 1 against B. ovis in sheep, but the performance of the Bp26-based differential diagnostic test was only very limited.
The elucidation of the genome of Brucella is now providing new paths towards the development of safer attenuated vaccines against brucellosis. Thus, using a defective mutant B. melitensis ΔmucR against intraperitoneal and aerosol B. melitensis challenge, Arenas-Gamboa et al. obtained significant protection in BALB/c mice. MucR is a transcriptional regulator that regulates exopolysaccharide biosynthesis, iron sequestration and storage, nitrogen metabolism and stress response mechanisms.
Bacterins
The use of inactivated whole bacteria as a vaccine (bacterin) was introduced as a safer alternative. A commercial killed B. ovis vaccine is used in New Zealand, but its efficacy is limited and it may display inactivation problems. Several new vaccination approaches have emerged with significant advantages over traditional former approaches. Magnani et al. have proposed the use of B. melitensis inactivated by γ-irradiation in order to inhibit its replication but retaining metabolic and transcriptional activity. These inactivated bacteria trigger danger signals that allow efficient processing and presentation of antigens, generate antigen-specific cytotoxic T cells and, most importantly, protected mice against virulent bacterial challenge without signs of residual virulence. This may be a promising strategy for a safe vaccination that should be tested in the natural host.
Subunit Vaccines
The greatest challenge for vaccine development against bacterial diseases is the development of vaccines against intracellular pathogens, such as Brucella spp. The ideal brucellosis vaccine, which will provide effective and safe protection against all species of Brucella in all animals, has not been developed yet.
To overcome the limitations of the rough vaccine strains, residual virulence and diagnosis interference, interesting approaches to develop a new generation of 'ideal vaccines' are being investigated. It is probable that some of these vaccines will become serious candidates to replace the available classical vaccines in the near future.
Concerning the quantitative costs and benefits of a vaccine, we should project not only the economic costs derived from investigation, development and production, but also the costs derived from the negative effects such as the residual virulence in the host. In the case of brucellosis, the cost–benefit ratio is not favorable for live-attenuated vaccines, such as Rev 1. In fact, acellular extracts could be effective for infections caused by smooth and rough species, with the advantage that they avoid the problem of obtaining seropositive reactions against the smooth LPS, making the differentiation between the infected animals and those vaccinated with Rev 1 possible, as well avoiding the risk of infecting humans, especially veterinarians or those who manipulate existing vaccines.
One of the alternative approaches is the development of acellular vaccines, specifically subcellular fractions able to stimulate an adequate Brucella spp. immune response. The success of a subunit vaccine is strongly associated with its composition. Under this approach, the use of highly conserved immunogenic antigens is required. Various authors have challenged either rams or mice with acellular extracts of Brucella spp., recombinant proteins, synthetic peptides, DNA vaccines or anti-idiotypic antibodies that mimic the O-chain, among others, from either smooth or rough strains, as potential protective antigens. Thus, the outer membrane of Brucella contains the main immunodominant antigens involved in the host-specific immune response. Similar to other Gram-negative bacteria, the outer membrane of rough Brucella spp. is composed of phospholipids, outer membrane proteins (OMPs) and rough LPS, which are the immunodominant antigens. B. ovis is a stable rough form which lacks the OPS chains characteristic of the smooth strains, but contains an OMP profile similar to other members of the genus.
In the case of the rough strains, the most immunogenic antigens are the OMPs, mostly because these are the most exposed antigens on the cellular surface (owing to the lack of O-chains). Omp31 appears to be the immunodominant antigen during the course of infection of rams, and Omp25 has been shown to be involved in the virulence of B. melitensis, B. abortus and B. ovis. In previous studies it was demonstrated that the OMP pattern obtained for B. melitensis and B. ovis strains from different geographical origins were very similar. Considering that 97% of their amino acids are conserved, along with their strong immunogenicity, an acellular vaccine containing an outer membrane extract from B. ovis might be effective and protective against infections caused by both rough B. ovis and smooth B. melitensis species. In fact, as previously mentioned, the smooth vaccine strain B. melitensis Rev 1 protects against the rough-type B. ovis.
The enzyme lumazine synthase from Brucella spp. is a highly immunogenic decameric protein that activates dendritic cells (DCs) via Toll-like receptor 4 (TLR4), with adjuvant properties when a foreign antigen is attached covalently. Given the fact that Omp31 and lumazine synthase have been implicated in the generation of protective cellular and humoral immune responses, authors have generated chimera vaccines of either the recombinant protein or DNA. Coadministration with incomplete Freund's adjuvant has been shown to confer some degree of protection against B. abortus in mice and B. ovis in rams, and similar protection to B. melitensis Rev 1 against B. ovis infection in BALB/c mice.
Immunoadjuvants
The main limitation of subunit vaccines is the low immunogenicity usually achieved, thus requiring booster doses in order to induce protection. Moreover, immunity against Brucella requires cell-mediated mechanisms, in particular Th1 immune responses, characterized by IFN-γ production. Therefore, it is necessary to combine the subcellular components with suitable adjuvants in order to reach the appropriate immunity. The selection of the correct adjuvant to tailor the adequate immune mechanisms requires a good knowledge of its potential and characteristics, an understanding of the pathogenesis and the molecular basis of the disease, as well as the role of the immune system.
For standard prophylactic immunization, only an adjuvant that induces minimal adverse toxic effects will be accepted. In addition, practical issues that are considered important for adjuvant development include biodegradability, stability, ease of manufacture, cost and applicability. Despite the recognition of many different types of adjuvant, the events triggered by them are poorly understood. However, two main mechanisms, which are not independent, were suggested and comprehensively reviewed. The first is based on the depot effect induced by the adjuvant, and the second related to the role of the APCs. The adjuvant may be able to present the antigen directly to the competent cells (i.e., macrophages and DCs) – these are categorized as 'vacine delivery systems', and are generally emulsions, nano- or micro-particles, immune stimulating complexes and liposomes. APC adjuvant-induced enhancement of an immune response may improve the delivery of the vaccine antigen into the draining lymph nodes. This can be achieved by facilitating antigen delivery to APCs, in particular to DCs, which are known to be the most prominent T-cell activators. The results increase the provision of antigen-loaded APCs for cognate naive T cells, promoting upregulation of costimulatory signals or MHC expression, inducing cytokine release and thus enhancing the magnitude and duration of the immune response.
Adjuvants can also be used to promote the induction of mucosal immune responses, critical against brucellosis as these are pathogens that initiate infection and colonize at mucosal surfaces. This can be achieved by the mucosal delivery of the vaccine, since administration by injection generally stimulates poor mucosal immune responses. In this context, the classical vaccine adjuvants (aluminum hydroxide, aluminum phosphate or calcium phosphate, and MF59 emulsion) do not elicit an effective mucosal immune response, as demonstrated when administered by the oral or nasal route. In fact, the development of adjuvants for mucosal immunization is an important current area of vaccine development. New mucosal adjuvants need to consider the ability to stimulate the APCs throughout the various mucosal routes. In this context, several adjuvants has been described including monophosphoryl lipid A (MPL®, GlaxoSmithKline Biologicals), CpG oligonucleotides, cholera toxin and Escherichia coli heat-labile enterotoxin.
Omp19 is a lipoprotein broadly expressed within the Brucella genus that when administered with the mucosal adjuvant cholera toxin confers protection against oral challenge with virulent Brucella. Recently, in order to avoid the toxicity due to the cholera toxin adjuvant, Omp19 was expressed in Nicotiana benthamina and the transgenic leaf material was used as an edible plant-made vaccine against brucellosis, demonstrating its protective capacity when orally administered without the need of additional adjuvants.
Nowadays, new adjuvants under investigation are the particulate polymeric systems. These particulate systems can act as vaccine adjuvants or/and as immunomodulators, increasing the response to the antigen with which it is jointly administered. Biodegradable and biocompatible polymeric particles are highly useful and many antigens, regardless of their structure and water solubility, can be loaded into these systems by the use of different manufacturing techniques. Microparticles are defined as relatively solid spherical particles, with diameters of between 1 and 1000 µm that form a continuous network or matrix system composed of one or more polymeric substances, in which the antigen is either dispersed in the molecular form or through solid dispersion. According to their structure, microparticles can be classified into microcapsules and microspheres. Thus, microcapsules are vesicular systems in which the active substance is confined to a cavity and is surrounded by a unique polymeric membrane; and microspheres are matricial systems in which the active substance is dispersed. The many advantages that these systems offer in biomedicine applications include:
Increased stability of the antigens incorporated;
Protection against chemical and enzymatic inactivation in the environmental conditions of the organism;
Improved antigen transport to areas of the body in which it can produce its beneficial action, including the ability to interact with systemic immune cells (phagocytic cells, DCs and macrophages) that efficiently present carried antigens to T and B cells;
Prolonged time of residence of the drug in the organism.
Microparticles can be prepared with natural (albumin, collagen, gelatine and polysaccharides) or synthetic materials (hyaluronic acid, alginic acid, chitosan, polyesters, polyorthoesters, polyalkylcarbonates, polyaminoacids, polyanhydrides, polyacrylamides and polyalkylcyanoacrylates). Poly(ε-caprolactone) (PEC) is a biocompatible and biodegradable polyester polymer that degrades slowly and does not generate an acid environment, unlike the poly(lactic-co-glycolic acid) copolymers. Other advantages of PEC include its hydrophobicity, stability and low cost. Several experiments have demonstrated that the outer membrane complex of B. ovis (HS complex) incorporated into PEC-microparticles (HS-PEC) induces an adequate immune response and protection against experimental brucellosis in mice and rams.
The mucosal route is one of the preferred routes of antigen delivery, not only because it represents one of the safer and more comfortable routes of administration, but also, in the case of Brucella spp., because mucosal vaccination imitates the infection pathway. The delivery of antigens of Brucella to mucosal surfaces is of remarkable interest as has been shown by the ocular administration of Rev 1 vaccine. Arenas-Gamboa et al. investigated the possibility of delivering the currently licensed vaccine against bovine brucellosis, B. abortus S19 strain, in a controlled microencapsulated format consisting of alginate microspheres. In a challenge study using red deer (Cervus elaphus elaphus) the encapsulated strain vaccine delivered by oral route conferred protection against an experimental challenge.
Finally, the enzyme lumazine synthase from Brucella spp. has been shown to confer protection against B. abortus and it has been described that an antigen delivery system effectively induces oral immunity. Thus, virus-like particles comprised of repeating units of lumazine synthase decorated with Omp31 provided protection against B. ovis in mice similar to that of the control vaccine Rev 1.