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Open Access

Peer-reviewed

Research Article

Meta-Analysis and Advancement of Brucellosis Vaccinology

Abstract

Background/Objectives

In spite of all the research effort for developing new vaccines against brucellosis, it remains unclear whether these new vaccine technologies will in fact become widely used. The goal of this study was to perform a meta-analysis to identify parameters that influence vaccine efficacy as well as a descriptive analysis on how the field of Brucella vaccinology is advancing concerning type of vaccine, improvement of protection on animal models over time, and factors that may affect protection in the mouse model.

Methods

A total of 117 publications that met the criteria were selected for inclusion in this study, with a total of 782 individual experiments analyzed.

Results

Attenuated (n = 221), inactivated (n = 66) and mutant (n = 102) vaccines provided median protection index above 2, whereas subunit (n = 287), DNA (n = 68), and vectored (n = 38) vaccines provided protection indexes lower than 2. When all categories of experimental vaccines are analyzed together, the trend line clearly demonstrates that there was no improvement of the protection indexes over the past 30 years, with a low negative and non significant linear coefficient. A meta-regression model was developed including all vaccine categories (attenuated, DNA, inactivated, mutant, subunit, and vectored) considering the protection index as a dependent variable and the other parameters (mouse strain, route of vaccination, number of vaccinations, use of adjuvant, challenge Brucella species) as independent variables. Some of these variables influenced the expected protection index of experimental vaccines against Brucella spp. in the mouse model.

Conclusion

In spite of the large number of publication over the past 30 years, our results indicate that there is not clear trend to improve the protective potential of these experimental vaccines.

Citation: Carvalho TF, Haddad JPA, Paixão TA, Santos RL (2016) Meta-Analysis and Advancement of Brucellosis Vaccinology. PLoS ONE 11(11): e0166582. https://doi.org/10.1371/journal.pone.0166582

Editor: Roy Martin Roop II, East Carolina University Brody School of Medicine, UNITED STATES

Received: August 17, 2016; Accepted: October 31, 2016; Published: November 15, 2016

Copyright: © 2016 Carvalho et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: Work in RLS lab is supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil), FAPEMIG (Fundação de Amparo a Pesquisa do Estado de Minas Gerais, Brazil), and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil). RLS and JPAH have fellowships from CNPq. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Brucellosis is a zoonotic bacterial disease that accounts for approximately half a million new cases of human infections annually [1]. The disease is caused by different Brucella species, which are facultative intracellular Gram negative bacteria that belong to the α-2 Proteobacteriacea family [2,3]. Human patients with brucellosis develop nonspecific symptoms including undulating fever, and the disease may progress to endocarditis, arthritis, osteomyelitis, among other less common clinical manifestations [4]. In cattle, brucellosis is characterized by abortion and infertility [5–7]. Therefore, bovine brucellosis results in very significant economic losses [8,9].

Animal brucellosis control and prevention is largely based on vaccination. Therefore, over the past decades there has been an intensive research effort for developing safer and more efficacious vaccines against brucellosis [3,10–12]. Animal vaccination against brucellosis is based mostly on live attenuated vaccines [12], including Brucella abortus S19, Brucella abortus RB51, and Brucella melitensis Rev.1 [3,11,13], whereas Brucella abortus S19 is often considered a gold standard for vaccine development [14]. However, these live attenuated vaccine strains have some significant disadvantages including pathogenic potential for humans, induction of abortion in animals, shedding in the milk, and interference with serologic tests in the case of smooth LPS strains [3,15]. Furthermore, these traditional vaccine strains have their use restricted to ruminants, whereas pigs, camels, or wild life animals are not covered.

Traditionally, live attenuated vaccines have a much broader use and efficacy than inactivated vaccine formulations [12,16]. During the past few years, there have been an increasing number of studies on alternative approaches for immunization against brucellosis, including recombinant subunit vaccines using surface or intracellular proteins of Brucella spp. [17–20]. Several Brucella proteins have been used as immunogens for experimental subunit vaccine formulations, including outer membrane proteins, namely Omp16, Omp19, Omp31, Omp28, and Omp25 [21–24], ribosomal protein L7/L12 [17,25], Cu-Zn superoxide dismutase [26], a cytoplasmic protein p39 [27], lumazine synthase BLS [28], among others. In addition, experimental DNA vaccines [28,29] as well as vectored vaccines using deliver vectors such as Salmonella enterica serotype Typhimurium [30], Escherichia coli [31], Yersinia enterocolitica [32], Lactococcus lactis [33], and the influenza virus [34] have been increasingly studied. Overexpression of Brucella antigens in attenuated vaccine strains have also been experimentally evaluated [35]. However, up to date these new approaches have not resulted in the generation of commercially available vaccines.

Due to the limitations of experimental procedures involving the natural hosts, since it is expensive and time-consuming, the mouse has been largely used as an experimental model for vaccine development against brucellosis [15]. The mouse model is suitable for studying pathogenesis, host immune response, and vaccine protection [36,37]. However, experimental protocols for assessing vaccine efficacy using this animal model are not standardized, which generates results that are often not quite reproducible [38]. Balb/c is the most commonly used mouse strain, although other strains have also been used for vaccine experiments, namely CD1, C57BL/6, OF1, 129/Sv, Swiss, and, mixed/outbred [16]. Vaccine efficacy is assessed based on experimental challenge with a pathogenic wild type Brucella strain after immunization, and quantification of wild type bacteria in target organs, particularly the spleen [39].

In spite of all the research effort for developing new vaccines against brucellosis, it remains unclear whether these new vaccine technologies will in fact become widely used tools for preventing brucellosis. Therefore, the goal of this study was to perform a meta-analysis to identify parameters that influence vaccine efficacy as well as a descriptive analysis on how the field of Brucella vaccinology is advancing in regard to type of vaccine, improvement of protection on animal models over time, and factors that may affect protection in the mouse model.

Material and Methods

Data source

Data were retrieved from publications indexed in PubMed up to February 15th 2016, using the following combinations of terms: (i) “Brucella” and “vaccine”; (ii) “Brucella” and “vaccine” and “mice”; or (iii) “Brucella” and “vaccine” and “mice” and “challenge”. The list of publications were then manually disambiguated. Only papers using the mouse model were included in this study. Importantly, a criterion for inclusion was that the paper must indicate the protective index or provide original data that allowed us to calculate the index. By definition, protective index refers to the difference in the log of colony forming unit (CFU) numbers in the spleen of naive and vaccinated mice. Only papers published in English were included in this study. In addition, papers with insufficient data–i.e. absence of indication of number of mice per group, absence of CFU values with their standard deviation, and absence of non vaccinated controls–were not included in this study.

Data retrieval

This study was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses criteria (PRISMA) as detailed in S1 Table. Data were obtained from each individual experimental group in a given publication. These data were grouped according to the category of experimental vaccine being tested, including: (i) live attenuated strains, (ii) DNA vaccines; (iii) inactivated vaccines; (iv) mutant attenuated strains; (v) subunit vaccines; and (vi) vectored vaccines. Parameters extracted from each individual experiment and considered for analysis included: publication year, vaccine species (in the case of live vaccines), protection index, mouse strain, variables related to vaccination (route, dose, number of injections, and adjuvant), variables related to the challenge (challenge Brucella species and strain, route, and interval in days between challenge and sampling), vector species was considered in the case of vectored vaccines.

A linear regression analysis was performed considering the year of publication and protection index, for all experiments or grouped according to the category of vaccine. In addition, the influence of each parameter (category of vaccine, mouse strain, route of vaccination and challenge, number of vaccinations, adjuvant, challenge species, and interval between challenge and euthanasia) on the protective index.

Data transformation and meta-regression analysis

Arbitrary values were attributed to qualitative data. For instance, values from 0 to 5, being “0” for attenuated vaccines; “1” for DNA vaccines; “2” for inactivated vaccines; “3” for mutant vaccine strains; “4” for subunit vaccines; and “5” for vectored vaccines. Similarly, values were attributed to mouse strains, routes of vaccination and challenge, use of adjuvant, Brucella spp. species used for challenge, and number of vaccinations, applying the value zero to the reference and integral crescent values to the other categories. The interval between challenge and euthanasia was analyzed as linear quantitative data.

The coefficient of variation, standard error, and confidence intervals were calculated for each experiment included in this study.

Statistical analysis

The analysis was conducted initially a random effects meta-analysis estimation with a heterogeneity test. If the heterogeneity test is significant (p-value lower than 0.05), and probable would be significant because there are different types of study with different types of vaccines, it is necessary to work using a meta-regression in order to verify which factor has positive or negative effect over the protective index.

The conduction of the meta-regression would use first two independent variables, one always the type of vaccine with the objective of control the effect of the second independent variable. In this “controlled univariate meta-regression” will conduct checking the association of independent variables such as mouse strain, vaccination route, number of vaccinations, use of adjuvant, Brucella species used for challenge, route of challenge, interval between challenge and euthanasia; and the dependent variable Protective Index. The independent variables with over-all p-values lower than 0.200 will be selected to the next step of the multivariable meta-regression analysis. The multivariable meta-regression was conducted using Protective Index as dependent variable and all others, which selected in the controlled univariate as independent variable. The multivariable model was conducted in a backwards approach, but in this case the exclusion was done manually in order to understand how the removal of no significant variable would affect the other variables. The statistical package used was the Stata software (Statacorp, Texas, USA).

This meta-regression approach allowed for attributing a given weight for each individual experiment based on their standard error. Therefore, a multiple meta-regression analysis was performed, including all parameters together, generating a meta-regression final model. Values of p<0.05 were considered statistically significant and was retained in the final model.

Results

Literature search and study characteristics

A total of 117 articles and data from 782 individual experiments were included in this study. Criteria for inclusion in this study are detailed in Fig 1. A total of 117 publications that met the criteria were selected for inclusion in this study [14, 17, 18, 20–28, 32, 33, 38, 40–141]. Therefore, a total of 782 individual experiments were analyzed. Raw data extracted from all 117 publications and each individual experiment are provided in the S2 Table.

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Fig 1. Flow chart describing the selection of articles for inclusion in the meta-analysis.

https://doi.org/10.1371/journal.pone.0166582.g001

Protection against Brucella spp. induced by different categories of vaccines in mice—descriptive statistics

Currently, experimental subunit vaccines concentrate most of the research efforts in the field of Brucella vaccinology, since this category of vaccine accounted for 36.7%, followed by attenuated vaccine strains, which corresponded to 28.26% of all experiments. The others categories of experimental vaccines account for 13.04%, 8.69%, 8.43%, and 4.9%, in the case of mutant, DNA, inactivated, and vectored vaccines, respectively. Furthermore, the proteins that were more often used as subunit vaccines included: LPS fractions (n = 44), L7/L12 (n = 31), HS (n = 27), Omp19 (n = 22), Omp31 (n = 20), Omp16 (n = 17), Omp25 (n = 8), BLS (n = 8), SOD (n = 6), P39 (n = 6), BRF (n = 6), Omp28 (n = 5), and urease (n = 4).

Some categories of vaccines were established earlier while other types of vaccines emerged over the time of this study (1986–2016) as demonstrated in Fig 2. By the end of 1980’s (1986–1990) there were only experiments with attenuated and subunit vaccines. Inactivated vaccines appear between 1991 and 1995, whereas more diverse vaccine approaches have been developed and studied beginning in 2001. The period between 2011 and 2016 included the largest number of experiments (n = 269) when compared to the other intervals, which clearly indicates an increasing investment of research time and resources for brucellosis vaccine development.

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Fig 2. Time line with the number and percentage of experiments for brucellosis vaccine development according to the type of vaccine.

Time intervals and corresponding number of experiments were: 1986–1990 (n = 73), 1991–1995 (n = 50), 1996–2000 (n = 13), 2001–2005 (n = 169), 2006–2010 (n = 208) e 2011–2016 (n = 269). The number of experiments for each data point is indicated in the graph.

https://doi.org/10.1371/journal.pone.0166582.g002

Data from 782 previously published experiments were grouped according to the category of experimental vaccines, namely naturally attenuated, mutant, inactivated, subunit, DNA, and vectored vaccines. Attenuated (n = 221), inactivated (n = 66) and mutant (n = 102) vaccines provided median protection index above 2, whereas subunit (n = 287), DNA (n = 68), and vectored (n = 38) vaccines provided protection indexes lower than 2 (Fig 3).

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Fig 3. Protection index provided by different categories of experimental vaccine candidates against Brucella spp. infection.

Values indicate the median, second and third quartiles (box), first and fourth quartiles (error bars). Outliers are indicated by dots. Median protection indexes were based on 782 independent experiments. The numbers of experimental groups per category are indicated between parentheses.

https://doi.org/10.1371/journal.pone.0166582.g003

Protection provided by experimental brucellosis vaccines over the past 30 years

In order to assess whether protection indexes have been improving over time, a correlation analysis was applied to protection indexes and the year of publication of each individual experiment over the past 30 years. When all categories of experimental vaccines are analyzed together, the trend line clearly demonstrates that there was no improvement of the protection indexes over the past 30 years, with a low negative and non significant linear coefficient (Fig 4). During this period of time, average protection indexes of experimental vaccines remained stable and close to 2 Log. A similar trend was observed when different vaccine categories were analyzed separately (Fig 5), with the exception of DNA vaccines that had a statistically significant positive correlation coefficient (Fig 5B). However, this trend to improving protection indexes over time in the case of DNA vaccines reflects the very low protection indexes of the early studies rather than high protection indexes since more recent studies have protection indexes that were in average below 2 Log (Fig 5).

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Fig 4. Linear regression of protection index over time for experimental vaccine candidates against Brucella spp. in the mouse model.

All experimental vaccine categories (attenuated strains, n = 221; attenuated mutant strains, n = 102; inactivated vaccines, n = 66; subunit vaccines, n = 287; DNA vaccines, n = 68; and vectored vaccines, n = 38) were included in this analysis, corresponding to 782 individual experiments (r = -0.0038; r2 = 0.09%; p = 0.4052).

https://doi.org/10.1371/journal.pone.0166582.g004

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Fig 5. Linear regression of protection index over time of different categories of experimental vaccines against Brucella spp. in the mouse model.

(A) attenuated strains (n = 221); (B) DNA vaccines (n = 68); (C) inactivated vaccines (n = 66); (D) attenuated mutant strains (n = 102); (E) subunit vaccines (n = 287); and (F) vectored vaccines (n = 38). Dots indicate each individual experiment, with solid trend lines and dotted lines indicating the confidence interval. Linear coefficients and p values are indicated in each graph.

https://doi.org/10.1371/journal.pone.0166582.g005

Parameters that influenced protection in the mouse model—descriptive statistics

A descriptive statistic analysis was performed considering the possible effect of several parameters, including mouse strain, vaccination routes, number of vaccinations, Brucella species used for experimental challenge, challenge route, and use of adjuvant, on protection indexes of experimental Brucella vaccines. Note this statistic descriptive does not take in account the weight of each experimental group, based in sample size and standard errors.

Protection indexes were evaluated according to mouse strains, including Balb/c, Swiss, C57BL/6 and others, used in each one of the 782 experiments. In average, the highest levels of protection were observed in experiments using Swiss mice and its variations, including albino Swiss and outbreed Swiss CD-1 (Fig 6A). Balb/c is the most commonly used mouse strain for Brucella vaccine experiments, corresponding to 88.75% (694/782) of all experiments included in this study. In average, this strain provided lower protection indexes (1.7076), when compared to Swiss mice (2.3791) or other strains (1.7293), but higher than C57BL/6, which provided the lowest protection indexes (1.296) (Fig 6), when all vaccine categories were grouped together. Protection indexes provided by each mouse strain according to the category of vaccine (attenuated, DNA, inactivated, mutant, subunit, and vectored) are described in S1 Fig.

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Fig 6. Protection indexes according to different parameters.

All experimental vaccine categories were analyzed together and grouped according to: (A) the mouse strains used in each individual experiment; (B) vaccination route; (C) number of vaccinations; (D) the Brucella spp. species used for experimental challenge; (E) challenge route; and (F) use of adjuvant. The number of experimental groups for each parameter is indicated between parentheses. Values indicate the median, second and third quartiles (box), first and fourth quartiles (error bars). Outliers are indicated by dots.

https://doi.org/10.1371/journal.pone.0166582.g006

Different vaccination routes, i.e. oral and intragastic (ORAL/IG), intramuscular (IM), intraperitoneal (IP), subcutaneous (SC), and others (intranasal, intraesplenic, etc) provided similar protection indexes when all vaccine categories were analyzed together (Fig 6B). Protection indexes provided by different vaccination routes according to the vaccine category are detailed in S2 Fig.

The effect of the number of vaccinations, i.e. single vs. multiple vaccinations (2, 3, 4, and 9 vaccinations) on protection indexes were compared grouping all vaccine categories together. Interestingly, single vaccinations provided the highest median protection index (Fig 6C). Protection indexes provided by single or multiple vaccinations according to each vaccine category are described in S3 Fig.

Post vaccination challenges with different Brucella spp. species, namely B. abortus, B. canis, B. melitensis, B. ovis, and B. suis, were compared. A marked variation in protection indexes were observed against these virulent challenge species, with nearly two logs of difference in protection indexes between the lower and higher protection indexes, and challenge with B. suis resulted in the highest median protection index, when all vaccine categories were analyzed together (Fig 6D). Protection indexes provided by different vaccine categories against different Brucella spp. is described in S4 Fig.

The effect of the route of challenge on the protection index was also evaluated after analyzing all vaccine categories together. The median protection indexes obtained with challenge through different routes, i.e. oral and intragastric (ORAL–IG), intraperitoneal (IP), other (intranasal, intraesplenic, etc) e intravenous (IV), were quite similar (Fig 6E). Protection indexes provided by different routes of challenge according to each vaccine category are described in S5 Fig.

When analyzing all vaccine categories together, protection indexes provided by experimental vaccines with or without adjuvant were similar (Fig 6F). Importantly the use of adjuvant is largely restricted to some categories of experimental vaccines, as detailed in S6 Fig.

Meta-analysis estimations

Random effects meta-analysis was conducted using 782 experimental groups from the 117 selected papers estimating the protraction index and testing for heterogeneity. This procedure was made for the experimental groups divided by type of vaccine as well. All estimations show high heterogeneity suggesting the necessity of use the meta-regression in order to access which factor is affecting the protection index. The results are displayed in the Table 1.

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Table 1. Random effect meta-analysis results.

https://doi.org/10.1371/journal.pone.0166582.t001

Bivariate analyses

In order to select variables to be included in the multivariate meta-regression model, a bivariate meta-regression analysis was performed considering each of the variables controlled by vaccine category, i.e. a bivariate analysis (Table 2). Variables studied included: vaccine category, mouse strain, vaccination route, number of vaccinations, use or adjuvant, Brucella species used for challenge, challenge route, and interval between challenge and euthanasia. Naturally attenuated vaccine strains with an average protection index of 2.079 were significantly more protective (p<0.001) than DNA, subunit and vectored vaccines, which had average protection indexes of 1.377, 1.369, and 1.180, respectively. In contrast, protection indexes provided by inactivated and mutant vaccine strains (2.758 and 2.527, respectively) were statistically similar to that of the naturally attenuated vaccine strains.

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Table 2. Bivariate meta-regression analysis of variables influencing the protection indexes of experimental brucellosis vaccines.

https://doi.org/10.1371/journal.pone.0166582.t002

Evaluation of mouse strains considering Balb/c as the reference strain, with a protection index of 2.058, indicated that it had significantly higher protection indexes when compared to C57BL/6 (p = 0.003) that had a median protection index of 1.43. Conversely, Swiss mice had protection indexes (2.478) that were significantly higher than those of Balb/c mice (p = 0.002), whereas no significant differences were observed among “other” strains of mice and Balb/c (Table 2).

Meta-regression analysis of vaccination routes, considering the oral/intragastric route as reference, demonstrated that this route, with a protection index of 1.726, was significantly less protective (p<0.001) than the subcutaneous route (2.205). Protection indexes provided by intramuscular, intraperitoneal, and others (2.083, 1.938, and 2.184, respectively) were similar to the oral/intragastric route (Table 2).

Considering one single vaccination as reference with a protection index of 2.059, two vaccinations with a protection index of 2.446 provided better protection (p = 0.002) than single vaccinations. Conversely, three, four or nine vaccinations, with protection indexes of 1.835, 1.795, and 2.576, respectively, were statistically similar (p>0.05) to single vaccinations (Table 2).

The use of adjuvant resulted in a significantly better (p = 0.002) protective index (2.359), when compared to vaccination without adjuvant that resulted in a protective index of 2.066 (Table 2).

The analysis of challenge species, considering B. abortus as the reference with a protection index of 1.954 demonstrated that protection indexes against B. melitensis, B. ovis, and B. suis (2.148, 2.774, and 2.762, respectively) were significantly higher when compared to B. abortus (Table 2). Conversely, the protection index against B. canis (2.357) was similar to that of B. abortus (p<0.05).

Bivariate meta-regression analysis also considered the route of challenge, with the oral/intragastric route with a protection index of 1.877 as the reference. The intravenous (IV) route, with a protection index of 2.318, was significantly more protective than the reference. Protection indexes provided by the intraperitoneal (IP) or “other” routes (2.044 and 1.833, respectively) were statistically similar to the reference (Table 2).

Importantly, considering that vaccine experiments are not standardized, we evaluated the effect of the interval between challenge and measurement of the protective index, and the number of days between challenge and euthanasia of experimental animals did not significantly influenced the protective index (Table 2).

Multivariate meta-regression

A meta-regression model was developed including all vaccine categories (attenuated, DNA, inactivated, mutant, subunit, and vectored) considering the protection index as a dependent variable and the other parameters (mouse strain, route of vaccination, number of vaccinations, use of adjuvant, challenge Brucella species) as independent variables (Table 3).

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Table 3. Multivariate meta-regression analysis of variables influencing the protection indexes of experimental brucellosis vaccines.

https://doi.org/10.1371/journal.pone.0166582.t003

Subunit and vectored vaccines provided significantly lower protection indexes when compared to attenuated vaccines (p<0.001), which was considered the reference vaccine category. Protection indexes provided by DNA, inactivated or mutant vaccines were statistically similar (p<0.05) to the reference (Table 3).

Regarding the mouse strain used in the experiment, C57BL/6 and Swiss mice resulted in protection indexes that were statistically similar to the reference Balb/c strain (Table 3). Interestingly, “other” mouse strains, which included mouse strains that were knockout for genes related to immunity on a 129/Sv background, resulted in a lower protection index (p = 0.021) when compared to the reference (Table 3).

With the exception of the intramuscular route of vaccination that provided lower protection index when compared to the oral/intragastric (p = 0.035), the other vaccination routes (intraperitoneal, subcutaneous, and others) provided similar protection when compared to the reference (Table 3). Two vaccinations performed better than the reference that was one single vaccination (p<0.001), whereas three, four or nine vaccination did not improve the protection index when compared to the reference (Table 3).

Experimental vaccines provided significantly higher protection indexes against B. melitensis, B. ovis, and B. suis when compared to the reference challenge with B. abortus (Table 3), whereas the use of adjuvant did not have significant effect on the protection index (Table 3).

Source of publications on brucellosis vaccinology

The data used in this study was obtained from 117 scientific articles, which were grouped according to the journal in which they were published. Frequencies of publications in different journals are detailed S3 Table.

Discussion

Brucellosis remains as one of the most important zoonotic diseases in the world, which justifies the large number of studies aiming to develop new and improved vaccines [10]. A meta-analysis based on brucellosis vaccine development experiments in the mouse model was performed in this study. A temporal analysis indicates that protection indexes remained stable over the past 30 years, which may indicate that the knowledge accumulated during the past decades did not necessarily translated into better protection when considered the mouse as a model. Another way to interpret this unexpected and disturbing finding is that the mouse model may have a limited range of protection when it comes to brucellosis, which may have resulted in stable protection indexes over time. Limited knowledge on protective immune resposes of mice and natural host species of Brucella spp. may also be a factor limiting advancement of this field. Furthermore, traditional vaccine strategies, particularly those based on the use of attenuated strains [79,88,142] provided better protection when compared to new strategies such as subunit, DNA, and vectored vaccines. In the case of live attenuated vaccine strains there is a clear correlation between results obtained in the mouse model and actual protection in the preferred host species [10,11,143,144]. Indeed, vaccine strains such as B. abortus S19 and B. melitensis Rev.1 are known to generate a robust immune response [11,143], and to induce significant levels of protection against B. abortus in cattle and B. melitensis in sheep and goats [10,11,144].

The mouse has been largely used as an experimental model for Brucella spp. infection [36]. This model allows for calculating the protection index that is based on the difference between the number of CFU (in Log) in the spleens of non vaccinated controls and vaccinated mice [39]. Thus, a higher protection index indicates a better protection provided by a given experimental vaccine. Experimentally, the protection index is very important for Brucella sp. vaccinology, which contrasts to other pathogens that are lethal, for which protection may be assessed by prevention of lethality in the mouse model [145]. Importantly, correlation between protection index in the mouse model and protection in the preferred host species is not clear for most of the recently developed experimental vaccines. For instance, we have recently developed a B. ovis attenuated mutant vaccine candidate strain that lacks an ABC transporter [36], which influences the virB-encoded Type IV secretion system [146] thus interfering with intracellular trafficking [147]. This vaccine strain provided only moderate protection in the mouse model, yielding a protection index of approximately 1.0 [120], whereas it surprisingly provided a very strong protection against experimental challenge in rams, preventing shedding of the wild type strain in the semen and urine, accumulation of inflammatory cells in the semen, and gross or microscopic lesions induced by wild type B. ovis, resulting in sterile immunity under experimental conditions [148]. This lack of a direct correlation between protection in the mouse and the preferred host species may also be related to the fact that protection indexes varied according to the wild type Brucella species used for challenging, which may indicate that optimal levels of protection indexes may vary among different Brucella species.

This study demonstrated that attenuated live vaccine strains tend to provide higher levels of protection. Considering that Brucella spp. is an intracellular pathogen, attenuated vaccines tend to provide superior protection because the vaccine strain remains with the same tissue and cell tropism as the wild type strain, thus mimicking a natural infection [149]. In fact, B. abortus S19 and B. melitensis Rev1 are largely used as vaccine strains worldwide. Although these vaccine strains generate high levels of protection against disease, there are considerable drawbacks since they both have residual virulence for their hosts, they cause human infections and disease, and they interfere with routine serological assays since they generate a an antibody response against smooth Brucella lipopolysaccharide (LPS). Additionally, the Rev 1 vaccine strain is resistant to streptomycin, one of the antibiotics used for brucellosis treatment in human patients [11,76]. Conversely, the B. abortus RB51 vaccine strain provides protection against the disease in cattle [150], and it has the advantage of not interfering with the standard serological tests since this strain has a rough LPS [119], but this strain is resistant to rifampicin, which is used for brucellosis treatment in human patients [11]. Mouse experiments demonstrated that RB51 protects against experimental challenge with several Brucella spp. species, including B. melitensis, B. ovis, B. abortus, and B. suis [88]. Thus, Brucella mutant strains carrying a rough LPS have been used in several vaccine experiments [11,15]. However, mutant rough strains provide lower levels of protection when compared to smooth attenuated vaccines such as Rev 1 [74,151].

Beginning in 2000, a large number of experiments evaluated mutant attenuated Brucella strains as vaccine candidates. For the same reasons discussed concerning naturally attenuated strains, these mutant strains tend to provide protection in the mouse model. A limiting factor for these vaccines is the fact that some of these mutants have poor persistence in the host, which may not allow enough time for exposure of the vaccine strain to the immune system, thus preventing appropriate levels of protection [152–154]. However, delivery systems that promote a slow delivery of the vaccine strain may overcome this limitation [120,148]. The mutagenesis in these cases usually targets genes that are required for virulence or survival in the host [93,153,155,156]. Mutant whose deleted genes are required during the early stages of infection are quickly eliminated by the host immune system [153] so they tend to generate insufficient protective immunity [157,158]. There is a great interest in the generation of mutant strains that carry a rough LPS, such as RB51, since these strains do not interfere with the most commonly used serologic diagnostic methods [11,101]. However, rough strains tend to be rapidly eliminated from the host, which results in lower levels of protection [101].

This study demonstrated that, in general, subunit vaccines provided lower levels of protection, which may be due to limitations to identify the most protective antigens, but it is reasonable to hypothesize that one single antigen may not be sufficient to trigger a strong protective immune response against Brucella spp. [159,160,161].

In this study, some parameters affected protection against experimental challenge in the mouse model. Balb/c is the most commonly used mouse strain for Brucella vaccine experiments [16]. Importantly, protection indexes are influenced by the mouse strain. Indeed, although C57BL/6 and Swiss mice provided protection indexes that were similar to those of Balb/c, other strains, which included knockout strains for immune genes, provided lower protection indexes. With the exception of the intramuscular route of vaccination, all other vaccination routes provided similar levels of protection, including the subcutaneous route that is one of the preferable routes for practical purposes. The efficacy of the subcutaneous route of vaccination is in agreement with previous studies [16]. Another parameter that may influence protection, particularly in the case of subunit or DNA vaccines is the number of vaccinations, with two vaccination providing better results than single vaccination.

This study associated descriptive statistics with a meta-regression analysis, which is a powerful tool for advancing research on animal health [162]. A previous meta-analysis study on Brucella vaccinology have identified factors that may influence experimental outcomes in experiments evaluating whole organism vaccine formulations [16]. This study was more inclusive, covering most of the relevant Brucella vaccine research performed using the mouse model over the past three decades. The identification of variables that significantly influence protection indexes in the mouse model, clearly indicates that more standardized experimental protocols are urgently required to generate data that is more reproducible and with higher prediction value for vaccine performance in the preferred host species. Comparing with a previous meta-analysis study, which was restricted to whole organism vaccines [16], we found variables that are equally significant for other kinds of vaccines. For instance, vaccine category, mouse strain, vaccination route, challenge pathogen strain, challenge route, and challenge-killing interval, influenced protection in the previous study [16] as well as in this more comprehensive meta-analysis. Therefore, this study largely expands the knowledge previously gained with meta-analysis on Brucella vaccinology [16].

A critical aspect of the mouse model for Brucella vaccine development is the lack of standardized experimental conditions, which has been previously reviewed [163]. Although the mouse is a well established model for Brucella infection and vaccinology [36, 163], and in spite of very specific recommendations by the World Organisation for Animal Health (OIE) for employing the mouse as a model for predicting protective potential against brucellosis in ruminants [39], there is a wide range of parameters in experimental protocols, including sex, age and strain of mice, vaccination and challenge routes, time elapsed between vaccination and challenge and/or between challenge and assessment of splenic bacterial loads, among others. This fact makes comparisons between studies and laboratories very unreliable.

Potential limitations of this study may be associated with restrictions of the original database, although PubMed covers the vast majority of relevant papers on the field of experimental Brucella vaccinology. Absence of publication of negative results may also have influenced the outcome of this study, although similar levels of negative results would be expected among different categories of experimental vaccines.

Conclusions

In conclusion, the importance of brucellosis as a threat for human health as well as due to economic losses for the animal industry [1,9], justifies the enormous scientific effort to develop better vaccines that lack residual pathogenic potential for animals and humans [19]. However, in spite of the large number of publication over the past 30 years, our results indicate that there is not clear trend to improve the protective potential of these experimental vaccines, which may at least in part explain why none of these new vaccine formulations or strategies has reached the market.

Supporting Information

S1 Fig. Protection index according to the mouse strain experimentally used for different categories of experimental vaccines against Brucella spp. infection.

Vaccine categories (attenuated strains, DNA vaccines, inactivated vaccines, attenuated mutant strains, subunit vaccines, and vectored vaccines were regrouped according to the mouse strain experimentally used (Balb/c, C57BL/6, Swiss, and others). Attenuated vaccines: Balb/c, n = 166; C57BL/6, n = 9; Swiss, n = 34; others, n = 12. DNA vaccines: Balb/c, n = 67; C57BL/6, n = 1. Inactivated vaccines: Balb/c, n = 60; Swiss, n = 6. Mutant vaccines: Balb/c, n = 89; C57BL/6, n = 6; Swiss, n = 4; others, n = 3. Subunit vaccines: Balb/c, n = 274; C57BL/6, n = 3; Swiss, n = 9; others, n = 1. Vectored vaccines: Balb/c, n = 38. Values indicate the median, second and third quartiles (box), first and fourth quartiles (error bars). Outliers are indicated by dots.

https://doi.org/10.1371/journal.pone.0166582.s001

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S2 Fig. Protection index according to the route of vaccination for different categories of Brucella spp. experimental vaccines.

Vaccine categories (attenuated strains, DNA vaccines, inactivated vaccines, attenuated mutant strains, subunit vaccines, and vectored vaccines) were regrouped according to the route of vaccination (intragastric and oral, n = 81; intramuscular, n = 90; intraperitoneal, n = 355; subcutaneous, n = 199; others, n = 9). Attenuated vaccines: intragastric and oral, n = 12; intraperitoneal, n = 119; subcutaneous, n = 48. DNA vaccines: intramuscular, n = 62; others, n = 4. Inactivated vaccines: oral, n = 20; intraperitoneal, n = 19; subcutaneous, n = 26; others, n = 1. Mutant vaccines: oral, n = 5; intraperitoneal, n = 79; subcutaneous, n = 14. Subunit vaccines: oral, n = 25; intramuscular, n = 28; intraperitoneal, n = 119, others, n = 4. Vectored vaccines: oral, n = 19; intraperitoneal, n = 19. Values indicate the median, second and third quartiles (box), first and fourth quartiles (error bars). Outliers are indicated by dots.

https://doi.org/10.1371/journal.pone.0166582.s002

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S3 Fig. Protection index according to the number of vaccinations for different categories of experimental vaccines against Brucella spp.

Vaccine categories (attenuated strains, DNA vaccines, inactivated vaccines, attenuated mutant strains, subunit vaccines, and vectored vaccines) were regrouped according to the number of vaccinations (1x, n = 394; 2x, n = 196; 3x, n = 111; 4x, n = 36; 9x, n = 2). Attenuated vaccines: 1x, n = 211; 2x, n = 6; 4x, n = 1). DNA vaccines: 1x, n = 6; 2x, n = 2; 3x, n = 34; 4x, n = 26). Inactivated vaccines: 1x, n = 4; 2x, n = 13; 3x, n = 6; 4x, n = 3. Mutant vaccines: 1x, n = 97; 3x, n = 1. Subunit vaccines: 1x, n = 32; 2x, n = 148; 3x, n = 68; 4x, n = 3. Vectored vaccines: 1x, n = 4; 2x, n = 27; 3x, n = 2; 4x, n = 3; 9x, n = 2. Values indicate the median, second and third quartiles (box), first and fourth quartiles (error bars). Outliers are indicated by dots.

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S4 Fig. Protection indexes according to the challenge Brucella spp. species for different categories of experimental vaccines.

Vaccine categories (attenuated strains, DNA vaccines, inactivated vaccines, attenuated mutant strains, subunit vaccines, and vectored vaccines) were regrouped according to the Brucella spp. species used for experimental challenge (B. abortus, B. canis, B. melitensis, B. ovis, and B. suis). Attenuated vaccines: B. abortus, n = 140; B. melitensis, n = 60; B. ovis, n = 12; B. suis, n = 9. DNA vaccines: B. abortus, n = 33; B. canis, n = 2; B. melitensis, n = 27; B. ovis, n = 6. Inactivated vaccines: B. abortus, n = 28; B. canis, n = 2; B. melitensis, n = 26; B. ovis, n = 7; B. suis, n = 3. Mutant vaccines: B. abortus, n = 40; B. canis, n = 4; B. melitensis, n = 47; B. ovis, n = 11. Subunit vaccines: B. abortus, n = 194; B. canis, n = 8; B. melitensis, n = 54; B. ovis, n = 31. Vectored vaccines: B. abortus, n = 29; B. melitensis, n = 9. Values indicate the median, second and third quartiles (box), first and fourth quartiles (error bars). Outliers are indicated by dots.

https://doi.org/10.1371/journal.pone.0166582.s004

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S5 Fig. Protection index according to the challenge route for different experimental vaccine categories against Brucella spp.

Vaccine categories (attenuated strains, DNA vaccines, inactivated vaccines, attenuated mutant strains, subunit vaccines, and vectored vaccines) were regrouped according to the route of challenge (oral and intragastric, n = 25; intraperitoneal, n = 587; others, n = 26; intravenous, n = 131). Attenuated vaccines: oral and intragastric, n = 5; intraperitoneal, n = 185; others, n = 4; intravenous, n = 23. DNA vaccines: oral and intragastric, n = 1; intraperitoneal, n = 48; intravenous, n = 15. Inactivated vaccines: intraperitoneal, n = 35; others, n = 14; intravenous, n = 17. Mutant vaccines: oral and intragastric, n = 1; intraperitoneal, n = 93; others, n = 4. Subunit vaccines: oral and intragastric, n = 16; intraperitoneal, n = 191; others, n = 4; intravenous, n = 75. Vectored vaccines: oral and intragastric, n = 2; intraperitoneal, n = 35; intravenous, n = 1. Values indicate the median, second and third quartiles (box), first and fourth quartiles (error bars). Outliers are indicated by dots.

https://doi.org/10.1371/journal.pone.0166582.s005

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S6 Fig. Protection index according to the use of adjuvant for different categories of experimental vaccines against Brucella spp.

Vaccine categories (attenuated strains, DNA vaccines, inactivated vaccines, attenuated mutant strains, subunit vaccines, and vectored vaccines) were regrouped according to the use or not of adjuvant (no, n = 528; yes, n = 253). Attenuated vaccines: no, n = 213; yes, n = 7. DNA vaccines: no, n = 61; yes, n = 7. Inactivated vaccines: no, n = 44; yes, n = 22. Mutant vaccines: no, n = 96; yes, n = 6. Subunit vaccines: no, n = 84; yes, n = 203. Vectored vaccines: no, n = 30; yes, n = 8. Values indicate the median, second and third quartiles (box), first and fourth quartiles (error bars). Outliers are indicated by dots.

https://doi.org/10.1371/journal.pone.0166582.s006

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S1 Table. PRISMA Checklist.

https://doi.org/10.1371/journal.pone.0166582.s007

(DOC)

S2 Table. Raw data extracted from all 117 publications and 782 individual experiments.

https://doi.org/10.1371/journal.pone.0166582.s008

(XLSX)

S3 Table. Distribution of the 117 articles included in this study according to the journal they were published.

https://doi.org/10.1371/journal.pone.0166582.s009

(DOC)

Acknowledgments

RLS, TAP and JPAH have fellowships from CNPq.

Author Contributions

  1. Conceptualization: TFC JPAH TAP RLS.
  2. Data curation: JPAH.
  3. Formal analysis: TFC JPAH TAP RLS.
  4. Funding acquisition: RLS.
  5. Investigation: TFC TAP RLS.
  6. Methodology: TFC JPAH TAP RLS.
  7. Project administration: RLS.
  8. Resources: JPAH.
  9. Software: JPAH.
  10. Supervision: RLS.
  11. Visualization: TFC JPAH TAP RLS.
  12. Writing – original draft: TFC RLS.
  13. Writing – review & editing: TFC JPAH TAP RLS.

References

  1. 1. Pappas G. The changing Brucella ecology: novel reservoirs, new threats. Int J Antimicrob Agents. 2010;36 (1):S8–S11. pmid:20696557
  2. 2. Cheers C. Pathogenesis and cellular immunity in experimental murine brucellosis. Dev Biol Stand 1984;56:237–46. pmid:6333362
  3. 3. Corbel MJ. Brucellosis in Humans and Animals. WHO Press, 2006; World Health Organization, Switzerland.
    • 4. Boschiroli ML, Foulongne V, O’Callaghan D. Brucellosis: a worldwide zoonosis. Curr Opin Microbiol 2001;4:58–64. pmid:11173035
    • 5. Xavier MN, Paixão TA, Poester FP, Lage AP, Santos RL. Pathological, immunohistochemical and bacteriological study of tissues and milk of cows and fetuses experimentally infected with Brucella abortus. J Comp Pathol 2009;140:149–57. pmid:19111839
    • 6. Carvalho Neta AV, Mol JPS, Xavier MN, Paixão TA, Lage AP, Santos RL. Pathogenesis of bovine brucellosis. Vet J 2010;184:146–55. pmid:19733101
    • 7. Poester FP, Samartino LE, Santos RL. Pathogenesis and pathobiology of brucellosis in livestock. Rev Sci Tech—OIE 2013;32:105–15.
    • 8. Cascio A, Bosilkovski M, Rodriguez-Morales AJ, Pappas G. The socio-ecology of zoonotic infections. Clin Microbiol Infect 2011;17:336–42. pmid:21175957
    • 9. Santos RL, Martins TM, Borges AM, Paixão TA. Economic losses due to bovine brucellosis in Brazil. Pesq Vet Bras 2013;33:759–64.
    • 10. Nicoletti P. Vaccination against Brucella. Adv Biotechnol Processes 1990;13:147–68. pmid:2185782
    • 11. Schurig GG, Sriranganathan N, Corbel MJ. Brucellosis vaccines: past, present and future. Vet Microbiol 2002;90:479–96. 10.1016/S0378-1135(02)00255-9 pmid:12414166
    • 12. Avila-Calderón ED, Lopez-Merino A, Sriranganathan N, Boyle SM, Contreras-Rodríguez A. A history of the development of Brucella vaccines. Biomed Res Int 2013;2013:743509. pmid:23862154
    • 13. Nicoletti P. Vaccination of cattle with Brucella abortus strain 19 administered by differing routes and doses. Vaccine 1984;2:133–5. pmid:6531956
    • 14. Iannino F, Herrmann CK, Roset MS, Briones G. Development of a dual vaccine for prevention of Brucella abortus infection and Escherichia coli O157:H7 intestinal colonization. Vaccine 2015;33:2248–53. pmid:25820069
    • 15. Moriyon I, Grillo MJ, Monreal D, Gonzalez D, Marin C, Lopez-Goni I, et al. Rough vaccines in animal brucellosis: structural and genetic basis and present status. Vet Res 2004;35:1–38. pmid:15099501
    • 16. Todd TE, Tibi O, Lin Y, Sayers S, Bronner DN, Xiang Z, et al. Meta-analysis of variables affecting mouse protection efficacy of whole organism Brucella vaccines and vaccine candidates. BMC Bioinformatics 2013;14:S3. pmid:23735014
    • 17. Oliveira SC, Splitter GA. Immunization of mice with recombinant L7/L12 ribosomal protein confers protection against Brucella abortus infection. Vaccine 1996;14:959–62. pmid:8873388
    • 18. Bhattacharjee AK, Izadjoo MJ, Zollinger WD, Nikolich MP, Hoover DL. Comparison of protective efficacy of subcutaneous versus intranasal immunization of mice with a Brucella melitensis lipopolysaccharide subunit vaccine. Infect Immun 2006;74:5820–25. pmid:16988260
    • 19. Olsen SC. Recent developments in livestock and wildlife brucellosis vaccination. Rev Sci Tech. 2013;32:207–17. pmid:23837378
    • 20. Velikovsky CA, Goldbaum FA, Cassataro J, Estein S, Bowden RA, Bruno L, et al. Brucella lumazine synthase elicits a mixed Th1-Th2 immune response and reduces infection in mice challenged with Brucella abortus 544 independently of the adjuvant formulation used. Infect Immun 2003;71:5750–55. pmid:14500496
    • 21. Pasquevich KA, Estein SM, Garcia Samartino C, Zwerdling A, Coria LM, Barrionuevo P, et al. Immunization with recombinant Brucella species outer membrane protein Omp16 or Omp19 in adjuvant induces specific CD4+ and CD8+ T cells as well as systemic and oral protection against Brucella abortus infection. Infect Immun 2009;77:436–45. pmid:18981242
    • 22. Kaushik P, Singh DK, Kumar SV, Tiwari AK, Shukla G, Dayal S, et al. Protection of mice against Brucella abortus 544 challenge by vaccination with recombinant OMP28 adjuvanted with CpG oligonucleotides. Vet Res Commun 2010;34:119–32. pmid:20013309
    • 23. Estein SM, Cassataro J, Vizcaíno N, Zygmunt MS, Cloeckaert A, Bowden AR. The recombinant Omp31 from Brucella melitensis alone or associated with rough lipopolysaccharide induces protection against Brucella ovis infection in BALB/c mice. Microbes Infect 2003;5:85–93. pmid:12650766
    • 24. Goel D, Bhatnagar R. Intradermal immunization with outer membrane protein 25 protects Balb/c mice from virulent B. abortus 544. Mol Immunol 2012;51:159–68. pmid:22464098
    • 25. Golshani M, Rafati S, Dashti A, Gholami E, Siadat SD, Oloomi M, et al. Vaccination with recombinant L7/L12-truncated Omp31 protein induces protection against Brucella infection in BALB/c mice. Mol Immunol 2015;65:287–92. pmid:25723468
    • 26. Singha H, Mallick AI, Jana C, Fatima N, Owais M, Chaudhuri P. Co-immunization with interlukin-18 enhances the protective efficacy of liposomes encapsulated recombinant Cu–Zn superoxide dismutase protein against Brucella abortus. Vaccine 2011;29:4720–27. pmid:21565241
    • 27. Al-Mariri A, Tibor A, Mertens P, DeBolle X, Michel P, Godfroid J, et al. Induction of immune response in BALB/c mice with a DNA vaccine encoding bacterioferritin or P39 of Brucella spp. Infect Immun 2001;69:6264–70. pmid:11553569
    • 28. Cassataro J, Pasquevich KA, Estein SM, Laplagne DA, Zwerdling A, Barrera S, et al. A DNA vaccine coding for the chimera BLSOmp31 induced a better degree of protection against B. ovis and a similar degree of protection against B. melitensis than Rev. 1 vaccination. Vaccine 2007;25:5958–67. pmid:17600596
    • 29. Liljeqvist S, Ståhl S. Production of recombinant subunit vaccines: protein immunogens, live delivery systems and nucleic acid vaccines. J Biotechnol 1999;73:1–33. pmid:10483112
    • 30. Onate AA, Vemulapalli R, Andrews E, Schurig GG, Boyle S, Folch H. Vaccination with live Escherichia coli expressing Brucella abortus Cu/Zn superoxide dismutase protects mice against virulent B. abortus. Infect Immun 1999;67:986–88. pmid:9916121
    • 31. Harms JS, Durward MA, Magnani DM, Splitter GA. Evaluation of recombinant invasive, non-pathogenic Eschericia coli as a vaccine vector against the intracellular pathogen, Brucella. J Immune Based Ther Vaccines 2009;7:1. pmid:19126207
    • 32. Al-Mariri A, Tibor A, Lestrate P, Mertens P, DeBolle X, Letesson JJ. Yersinia enterocolitica as a vehicle for a Naked DNA vaccine encoding Brucella abortus bacterioferritin or p39 antigen. Infect Immun 2002;70:1915–23. pmid:11895955
    • 33. Saez D, Fernandez P, Rivera A, Andrews E, Onate A. Oral immunization of mice with recombinant Lactococcus lactis expressing Cu, Zn superoxide dismutase of Brucella abortus triggers protective immunity. Vaccine 2012;30:1283–90. pmid:22222868
    • 34. Tabynov K, Kydyrbayev Z, Ryskeldinova S, Yespembetov B, Zinina N, Assanzhanova N, et al. Novel influenza virus vectors expressing Brucella L7/L12 or Omp16 proteins in cattle induced a strong T-cell immune response, as well as high protectiveness against B. abortus infection. Vaccine 2014;32(18):2034–41. pmid:24598723
    • 35. Vemulapalli R, He Y, Cravero S, Sriranganathan N, Boyle SM, Schurig GG. Overexpression of protective antigen as a novel approach to enhance vaccine efficacy of Brucella abortus strain RB51. Infect Immun 2000;68:3286–89. pmid:10816475
    • 36. Silva TMA, Costa EA, Paixão TA, Tsolis RM, Santos RL. Laboratory animal models for brucellosis research. J Biomed Biotechnol 2011;2011:1–9. pmid:21403904
    • 37. Baldwin CL, Parent M. Fundamentals of host immune response against Brucella abortus: what the mouse model has revealed about control of infection. Vet Microbiol 2002;90:367–82. pmid:12414157
    • 38. Miranda KL, Dorneles EMS, Pauletti RB, Poester FP, Lage AP. Brucella abortus S19 and RB51 vaccine immunogenicity test: Evaluation of three mice (BALB/c, Swiss and CD-1®) and two challenge strains (544 and 2308). Vaccine 2015;33:507–11. pmid:25498211
    • 39. OIE Biological Standards Commission, Manual of diagnostic tests and vaccines for terrestrial animals. 6ed. Paris: World Organisation for Animal Health, 2008.
      • 40. Abkar M, Amani J, Sahebghadam Lotfi A, Nikbakht Brujeni G, Alamian S, Kamali M. Subcutaneous immunization with a novel immunogenic candidate (urease) confers protection against Brucella abortus and Brucella melitensis infections. APMIS 2015;123:667–75. pmid:25939375
      • 41. Abkar M, Lotfi AS, Amani J, Eskandari K, Ramandi MF, Salimian J, et al. Survey of Omp19 immunogenicity against Brucella abortus and Brucella melitensis: influence of nanoparticulation versus traditional immunization. Vet Res Commun 2015;39:217–28. pmid:26395469
      • 42. Adone R, Ciuchini F, Marianelli C, Tarantino M, Pistoia C, Marcon G, et al. Protective properties of rifampin-resistant rough mutants of Brucella melitensis. Infect Immun 2005;73:4198–204. pmid:15972510
      • 43. Adone R, Francia M, Ciuchini F. Evaluation of Brucella melitensis B115 as rough-phenotype vaccine against B. melitensis and B. ovis infections. Vaccine 2008;26:4913–17. pmid:18675869
      • 44. Adone R, Francia M, Pistoia C, Pesciaroli M, Pasquali P. B. melitensis rough strain B115 is protective against heterologous Brucella spp. infections. Vaccine 2011;29:2523–29. pmid:21300102
      • 45. Al-Mariri A, Abbady AQ. Evaluation of the immunogenicity and the protective efficacy in mice of a DNA vaccine encoding SP41 from Brucella melitensis. J Infect Dev Ctries 2013;7:329–37. pmid:23592643
      • 46. Al-Mariri A, Akel R, Abbady AQ. A DNA vaccine encoding p39 and sp41 of Brucella melitensis induces protective immunity in BALB/c mice. Arch Med Vet 2014;46:53–62.
      • 47. Al-Mariri A, Mahmoud NH, Hammoud R. Efficacy evaluation of live Escherichia coli expression Brucella P39 protein combined with CpG oligodeoxynucleotides vaccine against Brucella melitensis 16M, in BALB/c mice. Biologicals 2012;40:140–45. pmid:22296786
      • 48. Al-Mariri A, Tibor A, Mertens P, Bolle X, Michel P, Godefroid J, et al. Protection of BALB/c mice against Brucella abortus 544 challenge by vaccination with bacterioferritin or P39 recombinant proteins with CpG oligodeoxynucleotides as adjuvant. Infect Immun 2001;69:4816–22. pmid:11447155
      • 49. Al-Mariri A. Protection of BALB/c mice against Brucella melitensis 16M infection induced by vaccination with live Escherchia coli expression Brucella P39 protein. Vaccine 2010;28:1766–70. pmid:20036752
      • 50. Bosseray N, Plommet M. Brucella suis S2, Brucella melitensis Rev.1 and Brucella abortus S19 living vaccines: residual virulence and immunity induced against three Brucella species challenge strains in mice. Vaccine 1990;8:462–68. pmid:2123586
      • 51. Briones G, Iannino NI, Roset M, Vigliocco A, Paulo PS, Ugalde RA. Brucella abortus cyclic beta-1,2-glucan mutants have reduced virulence in mice and are defective in intracellular replication in HeLa cells. Infect Immun 2001;69:4528–35. pmid:11401996
      • 52. Cabrera A, Sáez D, Céspedes S, Andrews E, Oñate A. Vaccination with recombinant Semliki Forest virus particles expressing translation initiation factor 3 of Brucella abortus induces protective immunity in BALB/c mice. Immunobiology 2009;214:467–74. pmid:19150742
      • 53. Cassataro J, Estein SM, Pasquevich KA, Velikovsky CA, Barrera S, Bowden R, et al. Vaccination with the recombinant Brucella outer membrane protein 31 or a derived 27-amino-acid synthetic peptide elicits a CD4+T helper 1 response that protects against Brucella melitensis infection. Infect Immun 2005;73:8079–88. pmid:16299302
      • 54. Cassataro J, Pasquevich KA, Estein SM, Laplagne DA, Velikovsky CA, Barrera S, et al. A recombinant subunit vaccine based on the insertion of 27 amino acids from Omp31 to the N-terminus of BLS induced a similar degree of protection against B. ovis than Rev.1 vaccination. Vaccine 2007;25:4437–46. pmid:17442465
      • 55. Cassataro J, Velikovsky CA, Barrera S, Estein SM, Bruno L, Bowden R, et al. A DNA vaccine coding for the Brucella outer membrane protein 31 confers protection against B. melitensis and B. ovis infection by eliciting a specific cytotoxic response. Infect Immun 2005;73:6537–46. pmid:16177328
      • 56. Cassataro J, Velikovsky CA, Bruno L, Estein SM, Barrera S, Bowden R, et al. Improved immunogenicity of a vaccination regimen combining a DNA vaccine encoding Brucella melitensis outer membrane protein 31 (Omp31) and recombinant Omp31 boosting. Clin Vaccine Immunol 2007;14:869–74. pmid:17428946
      • 57. Cassataro J, Velikovsky CA, Giambartolomei GH, Estein S, Bruno L, Cloeckaert A, et al. Immunogenicity of the Brucella melitensis recombinant ribosome recycling factor-homologous protein and its cDNA. Vaccine 2002;20:1660–69. pmid:11858876
      • 58. Cespedes S, Andrews E, Folch H, Oñate A. Identification and partial characterisation of a new protective antigen of Brucella abortus. J Med Microbiol 2000;49:165–70. pmid:10670567
      • 59. Clapp B, Skyberg JA, Yang X, Thornburg T, Walters N, Pascual DW. Protective live oral brucellosis vaccines stimulate Th1 and Th17 cell responses. Infect Immun 2011;79:4165–74. pmid:21768283
      • 60. Clausse M, Díaz AG, Ghersi G, Zylberman V, Cassataro J, Giambartolomei GH, et al. The vaccine candidate BLSOmp31 protects mice against Brucella canis infection. Vaccine 2013;31:6129–35. pmid:23906889
      • 61. Clausse M, Díaz AG, Ibañez AE, Cassataro J, Giambartolomei GH, Estein SM. Evaluation of the efficacy of outer membrane protein 31 vaccine formulations for protection against Brucella canis in BALB/c mice. Clin Vaccine Immunol 2014;21:1689–94. pmid:25339409
      • 62. Cloeckaert A, Jacques I, Grilló MJ, Marín CM, Grayon M, Blasco JM, et al. Development and evaluation as vaccines in mice of Brucella melitensis Rev.1 single and double deletion mutants of the bp26 and omp31 genes coding for antigens of diagnostic significance in ovine brucellosis. Vaccine 2004;22:2827–35. pmid:15246618
      • 63. Commander NJ, Brewer JM, Wren BW, Spencer SA, MacMillan AP, Stack JA. Liposomal delivery of p-ialB and p-omp25 DNA vaccines improves immunogenicity but fails to provide full protection against B. melitensis challenge. Genet Vaccines Ther 2010;8:1–12. pmid:20637091
      • 64. Commander NJ, Spencer SA, Wren BW, MacMillan AP. The identification of two protective DNA vaccines from a panel of five plasmid constructs encoding Brucella melitensis 16M genes. Vaccine 2007;25:43–54. pmid:17049676
      • 65. Dabral N, Moreno-Lafont M, Sriranganathan N, Vemulapalli R. Oral immunization of mice with gamma-irradiated Brucella neotomae induces protection against intraperitoneal and intranasal challenge with virulent B. abortus 2308. PlosOne 2014;9:e107180. pmid:25225910
      • 66. Delpino MV, Estein SM, Fossati CA, Baldi PC, Cassataro J. Vaccination with Brucella recombinant DnaK and SurA proteins induces protection against Brucella abortus infection in BALB/c mice. Vaccine 2007;25:6721–29. pmid:17686554
      • 67. Estevan M, Gamazo C, Grilló MJ, Barrio GG, Blasco JM, Irache JM. Experiments on a sub-unit vaccine encapsulated in microparticles and its efficacy against Brucella melitensis in mice. Vaccine 2006;24:4179–87. pmid:16481077
      • 68. Fu S, Xu J, Li X, Xie Y, Qiu Y, Du X, et al. Immunization of mice with recombinant protein CobB or AsnC confers protection against Brucella abortus infection. PlosOne 2012;7:e29552. pmid:22383953
      • 69. Ghasemi A, Jeddi-Tehrani M, Mautner J, Salari MH, Zarnani AH. Immunization of mice with a novel recombinant molecular chaperon confers protection against Brucella melitensis infection. Vaccine 2014;32:6659–66. pmid:25240754
      • 70. Ghasemi A, Zarnani AH, Ghoodjani A, Rezania S, Salari MH, Jeddi-Tehrani M. Identification of a new immunogenic candidate conferring protection against Brucella melitensis infection in mice. Mol Immunol 2014;62:142–49. pmid:24995396
      • 71. Ghasemi A, Jeddi-Tehrani M, Mautner J, Salari MH, Zarnani AH. Simultaneous immunization of mice with Omp31 and TF provides protection against Brucella melitensis infection. Vaccine 2015;33:5532–8. pmid:26384448
      • 72. Goel D, Rajendran V, Ghosh PC, Bhatnagar R. Cell mediated immune response after challenge in Omp25 liposome immunized mice contributes to protection against virulent Brucella abortus 544. Vaccine 2013;31:1231–37. pmid:23273966
      • 73. Golshani M, Rafati S, Siadat SD, Nejati-Moheimani M, Shahcheraghi F, Arsang A, et al. Improved immunogenicity and protective efficacy of a divalent DNA vaccine encoding Brucella L7/L12-truncated Omp31 fusion protein by a DNA priming and protein boosting regimen. Mol Immunol 2015;66:384–91. pmid:25968974
      • 74. González D, Grilló MJ, Miguel MJ, Ali T, Arce-Gorvel V, Delrue RM, et al. Brucellosis vaccines: assessment of Brucella melitensis lipopolysaccharide rough mutants defective in core and O-polysaccharide synthesis and export. PlosOne 2008;3:e2760. pmid:18648644
      • 75. Gonzalez-Smith A, Vemulapalli R, Andrews E, Onate A. Evaluation of Brucella abortus DNA vaccine by expression of Cu-Zn superoxide dismutase antigen fused to IL-2. Immunobiology 2006;211:65–74. pmid:16446171
      • 76. Grilló MJ, Manterola L, Miguel MJ, Muñoz PM, Blasco JM, Moriyón I, et al. Increases of efficacy as vaccine against Brucella abortus infection in mice by simultaneous inoculation with avirulent smooth bvrS/bvrR and rough wbkA mutants. Vaccine 2006;24:2910–16. pmid:16439039
      • 77. Guilloteau LA, Laroucau K, Vizcaíno N, Jacques I, Dubray G. Immunogenicity of recombinant Escherichia coli expressing the omp31 gene of Brucella melitensis in BALB/c mice. Vaccine 1999;17:353–61. pmid:9987174
      • 78. Gupta VK, Rout PK, Vihan VS. Induction of immune response in mice with a DNA vaccine encoding outer membrane protein (omp31) of Brucella melitensis 16M. Res Vet Sci 2007;82:305–13. pmid:17014873
      • 79. Hamdy MER, El-Gibaly SM, Montasser AM. Comparison between immune responses and resistance induced in BALB/c mice vaccinated with RB51 and Rev. 1 vaccines and challenged with Brucella melitensis bv. 3. Vet Microbiol 2002;88:85–94. pmid:12119140
      • 80. He Y, Vemulapalli R, Schurig GG. Recombinant Ochrobactrum anthropi expressing Brucella abortus Cu,Zn superoxide dismutase protects mice against B. abortus infection only after switching of immune responses to Th1 type. Infect Immun 2002;70:2535–43. pmid:11953393
      • 81. Hu XD, Chen ST, Li JY, Yu DH, Zhang Y, Cai H. An IL-15 adjuvant enhances the efficacy of a combined DNA vaccine against Brucella by increasing the CD8+ cytotoxic T cell response. Vaccine 2010;28:2408–15. pmid:20064480
      • 82. Izadjoo MJ, Bhattacharjee AK, Paranavitana CM, Hadfield TL, Hoover DL. Oral vaccination with Brucella melitensis WR201 protects mice against intranasal challenge with virulent Brucella melitensis 16M. Infect Immun 2004;72:4031–39. pmid:15213148
      • 83. Jain L, Rawat M, Prajapati A, Tiwari AK, Kumar B, Chaturvedi VK, et al. Protective immune-response of aluminium hydroxide gel adjuvanted phage lysate of Brucella abortus S19 in mice against direct virulent challenge with B. abortus 544. Biologicals 2015;43:369–76. pmid:26156404
      • 84. Jain S, Afley P, Dohre SK, Saxena N, Kumar S. Evaluation of immunogenicity and protective efficacy of a plasmid DNA vaccine encoding ribosomal protein L9 of Brucella abortus in BALB/c mice. Vaccine 2014;32:4537–42. pmid:24950353
      • 85. Jain S, Afley P, Kumar S. Immunological responses to recombinant cysteine synthase A of Brucella abortus in BALB/c mice. World J Microbiol Biotechnol 2013;29:907–13. pmid:23269507
      • 86. Jain-Gupta N, Contreras-Rodriguez A, Vemulapalli R, Witonsky SG, Boyle SM, Sriranganathan N. Pluronic P85 enhances the efficacy of outer membrane vesicles as a subunit vaccine against Brucella melitensis challenge in mice. FEMS Immunol Med Microbiol 2012;66:436–44. pmid:23163875
      • 87. Jiménez de Bagüés MP, Elzer PH, Blasco JM, Marín CM, Gamazo C, Winter AJ. Protective immunity to Brucella ovis in BALB/c mice following recovery from primary infection or immunization with subcellular vaccines. Infect Immun 1994;62:632–38. pmid:8300219
      • 88. Jiménez de Bagués MP, Elzer PH, Jones SM, Blasco JM, Enright FM, Schurig GG, et al. Vaccination with Brucella abortus rough mutant RB51 protects BALB/c mice against virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis. Infect Immun 1994;62:4990–96. pmid:7927779
      • 89. Lacerda TLS, Cardoso PG, Almeida LA, Camargo ILBC, Afonso DAF, Trant CC, et al. Inactivation of formyl transferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice. Vaccine 2010;28:5627–34. pmid:20580469
      • 90. Lalsiamthara J, Gogia N, Goswami TK, Singh RK, Chaudhuri P. Intermediate rough Brucella abortus S19Δper mutant is DIVA enable, safe to pregnant guinea pigs and confers protection to mice. Vaccine 2015;33:2577–83. pmid:25869887
      • 91. Leclerq S, Harms JS, Rosinha GMS, Azevedo V, Oliveira SC. Induction of a Th1-type of immune response but not protective immunity by intramuscular DNA immunisation with Brucella abortus GroEL heat-shock gene. J Med Microbiol 2002;51:20–6. pmid:11803949
      • 92. Li X, Xu J, Xie Y, Qiu Y, Fu S, Yuan X, et al. Vaccination with recombinant flagellar proteins FlgJ and FliN induce protection against Brucella abortus 544 infection in BALB/c mice. Vet Microbiol 2012;161:137–44. pmid:22854331
      • 93. Li Z, Gui D, Zhang J, Zhang W, Zhang H, Guo F, et al. Immunization of BALB/c mice with Brucella abortus 2308ΔwbkA confers protection against wild-type infection. J Vet Sci 2015;16:467–73. pmid:26040616
      • 94. Li ZQ, Shi JX, Fu WD, Zhang Y, Zhang J, Wang Z, et al. A Brucella melitensis M5-90 wboA deletion strain is attenuated and enhances vaccine efficacy. Mol Immunol 2015;66:276–83. pmid:25899866
      • 95. Lim JJ, Kim DH, Lee JJ, Kim DG, Min W, Lee HJ, et al. Protective effects of recombinant Brucella abortus Omp28 against infection with a virulent strain of Brucella abortus 544 in mice. J Vet Sci 2012;13:287–92. pmid:23000585
      • 96. Liu J, Li Y, Sun Y, Ji X, Zhu L, Guo X, et al. Immune responses and protection induced by Brucella suis S2 bacterial ghosts in mice. Vet Immunol Immunopathol 2015;166:138–44. pmid:26022514
      • 97. Luo D, Ni B, Li P, Shi W, Zhang S, Han Y, et al. Protective immunity elicited by a divalent DNA vaccine encoding both the L7/L12 and Omp16 genes of Brucella abortus in BALB/c Mice. Infect Immun 2006;74:2734–41. pmid:16622210
      • 98. Mallick AI, Singha H, Khan S, Anwar T, Ansari MA, Khalid R, et al. Escheriosome-mediated delivery of recombinant ribosomal L7/L12 protein confers protection against murine brucellosis. Vaccine 2007;25:7873–84. pmid:17931756
      • 99. Mancilla M, Grilló MJ, Miguel MJ, López-Goñi I, San-Román B, Zabalza-Baranguá A, et al. Deletion of the GI-2 integrase and the wbkA flanking transposase improves the stability of Brucella melitensis Rev 1 vaccine. BMC Vet Res 2013;44:105. pmid:24176078
      • 100. Martins RC, Gamazo C, Sánchez-Martínez M, Barberán M, Peñuelas I, Irache JM. Conjunctival vaccination against Brucella ovis in mice with mannosylated nanoparticles. J Control Release 2012;162:553–60. pmid:22846987
      • 101. Monreal D, Grilló MJ, González D, Marín CM, De Miguel MJ, López-Goñi I, et al. Characterization of Brucella abortus O-polysaccharide and core lipopolysaccharide mutants and demonstration that a complete core is required for rough vaccines to be efficient against Brucella abortus and Brucella ovis in the mouse model. Infect Immun 2003;71: 3261–71. pmid:12761107
      • 102. Montaraz JA, Winter AJ. Comparison of living and nonliving vaccines for Brucella abortus in BALB/c mice. Infect Immun 1986;53:245–51. pmid:3089933
      • 103. Moustafa D, Garg VK, Jain N, Sriranganathan N, Vemulapalli R. Immunization of mice with gamma-irradiated Brucella neotomae and its recombinant strains induces protection against virulent B. abortus, B. melitensis, and B. suis challenge. Vaccine 2011;29:784–94. pmid:21109033
      • 104. Muñoz-Montesino C, Andrews E, Rivers R, González-Smith A, Moraga-Cid G, Folch H, et al. Intraspleen delivery of a DNA vaccine coding for superoxide dismutase (SOD) of Brucella abortus induces SOD-specific CD4 and CD8 T Cells. Infect Immun 2004;72:2081–87. pmid:15039330
      • 105. Murillo M, Grilló MJ, Reñé J, Marín CM, Barberán M, Goñi MM, et al. A Brucella ovis antigenic complex bearing poly-ε-caprolactone microparticles confer protection against experimental brucellosis in mice. Vaccine 2001;19:4099–106. pmid:11457533
      • 106. Oñate AA, Céspedes S, Cabrera A, Rivers R, González A, Muñoz C, et al. A DNA vaccine encoding Cu,Zn superoxide dismutase of Brucella abortus induces protective immunity in BALB/c mice. Infect Immun 2003;71:4857–61. pmid:12933826
      • 107. Oñate AA, Donoso G, Moraga-Cid G, Folch H, Céspedes S, Andrews E. An RNA vaccine based on recombinant Semliki Forest Virus Particles Expressing the Cu,Zn superoxide dismutase protein of Brucella abortus induces protective immunity in BALB/c mice. Infect Immun 2005;73:3294–300. pmid:15908354
      • 108. Oñate AA, Vemulapalli R, Andrews E, Schurig GG, Boyle S, Folch H. Vaccination with live Escherichia coli expressing Brucella abortus Cu/Zn superoxide dismutase protects mice against virulent B. abortus. Infect Immun 1999;67:986–88. pmid:9916121
      • 109. Palomares-Resendiz E, Arellano-Reynoso B, Hernández-Castro R, Tenorio-Gutiérrez V, Salas-Téllez E, Suárez-Güemes F, et al. Immunogenic response of Brucella canis virB10 and virB11 mutants in a murine model. Front Cell Infect Microbiol 2012;2:35. pmid:22919627
      • 110. Pasquevich KA, Garcia Samartino C, Coria LM, Estein SM, Zwerdling A, Ibañez AE, et al. The protein moiety of Brucella abortus outer membrane protein 16 is a new bacterial pathogen-associated molecular pattern that activates dendritic cells in vivo, induces a Th1 immune response, and is a promising self-adjuvanting vaccine against systemic and oral acquired brucellosis. J Immunol 2010;184:5200–12. pmid:20351187
      • 111. Pasquevich KA, Ibañez AE, Coria LM, Samartino CG, Estein SM, Zwerdling A, et al. An oral vaccine based on U-Omp19 induces protection against B. abortus mucosal challenge by inducing an adaptive IL-17 immune response in mice. PlosOne 2011;6:e16203. pmid:21264260
      • 112. Pollak CN, Wanke MM, Estein SM, Delpino MV, Monachesi NE, Comercio EA, et al. Immunization with Brucella VirB proteins reduces organ colonization in mice through a Th1-type immune response and elicits a similar immune response in dogs. Clin Vaccine Immunol 2015;22:274–81. pmid:25540276
      • 113. Pugh GW Jr, Phillips M, Tabatabai LB, McDonald TJ. Unresponsiveness of vaccinated BALB/c mice to a second inoculation of lipopolysaccharide from Brucella abortus strain 2308. Vet Microbiol 1991;26:167–77. pmid:1902610
      • 114. Retamal-Díaz A, Riquelme-Neira R, Sáez D, Rivera A, Fernández P, Cabrera A, et al. Use of S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol as an adjuvant improved protective immunity associated with a DNA vaccine encoding Cu,Zn superoxide dismutase of Brucella abortus in mice. Clin Vaccine Immunol 2014;21:1474–80. pmid:25165025
      • 115. Riquelme-Neira R, Retamal-Díaz A, Acuña F, Riquelme P, Rivera A, Sáez D, et al. Protective effect of a DNA vaccine containing an open reading frame with homology to an ABC-type transporter present in the genomic island 3 of Brucella abortus in BALB/c mice. Vaccine 2013;31:3663–67. pmid:23834811
      • 116. Rosinha GMS, Freitas DA, Miyoshi A, Azevedo V, Campos E, Cravero SL, et al. Identification and characterization of a Brucella abortus ATP-binding cassette transporter homolog to Rhizobium meliloti ExsA and its role in virulence and protection in mice. Infect Immun 2002;70:5036–44. pmid:12183550
      • 117. Rosinha GMS, Myioshi A, Azevedo V, Splitter GA, Oliveira SC. Molecular and immunological characterisation of recombinant Brucella abortus glyceraldehyde-3- phosphate-dehydrogenase, a T- and B-cell reactive protein that induces partial protection when co-administered with an interleukin-12-expressing plasmid in a DNA vaccine formulation. J Med Microbiol 2002;51:661–71. pmid:12171297
      • 118. Sanakkayala N, Sokolovska A, Gulani J, HogenEsch H, Sriranganathan N, Boyle SM, et al. Induction of antigen-specific Th1-Type immune responses by gamma-irradiated recombinant Brucella abortus RB51. Clin Diagn Lab Immunol 2005;12:1429–36. pmid:16339067
      • 119. Schurig GG, Roop RM, Bagchi T, Boyle S, Buhrman D, Sriranganathan N. Biological properties of RB51; a stable rough strain of Brucella abortus. Vet Microbiol 1991;28:171–88. pmid:1908158
      • 120. Silva AP, Macêdo AA, Silva TM, Ximenes LC, Brandão HM, Paixão TA, et al. Protection provided by an encapsulated live attenuated ΔabcBA strain of Brucella ovis against experimental challenge in a murine model. Clin Vaccine Immunol 2015;22:789–97. pmid:25947146
      • 121. Singh D, Goel D, Bhatnagar R. Recombinant L7/L12 protein entrapping PLGA (poly lactide-co-glycolide) micro particles protect BALB/c mice against the virulent B. abortus 544 infection. Vaccine 2015;33:2786–92. pmid:25930114
      • 122. Singha H, Mallick AI, Jana C, Isore DP, Goswami TK, Srivastava SK, et al. Escheriosomes entrapped DNA vaccine co-expressing Cu-Zn superoxide dismutase and IL-18 confers protection against Brucella abortus. Microbes Infect 2008;10:1089–96. pmid:18602490
      • 123. Singh D, Somani VK, Aggarwal S, Bhatnagar R. PLGA (85:15) nanoparticle based delivery of rL7/L12 ribosomal protein in mice protects against Brucella abortus 544 infection: A promising alternate to traditional adjuvants. Mol Immunol 2015;68:272–9. pmid:26442664
      • 124. Sislema-Egas F, Céspedes S, Fernández P, Retamal-Díaz A, Sáez D, Oñate A. Evaluation of protective effect of DNA vaccines encoding the BAB1_0263 and BAB1_0278 open reading frames of Brucella abortus in BALB/c mice. Vaccine 2012;30:7286–91. pmid:23026687
      • 125. Soler-Lloréns P, Gil-Ramírez Y, Zabalza-Baranguá A, Iriarte M, Conde-Álvarez R, Zúñiga-Ripa A, et al. Mutants in the lipopolysaccharide of Brucella ovis are attenuated and protect against B. ovis infection in mice. BMC Vet Res 2014;45:2–11. pmid:25029920
      • 126. Souza Filho JA, Martins VP, Campos PC, Alves-Silva J, Santos NV, Oliveira FS, et al. Mutant Brucella abortus membrane fusogenic protein (BMFP) is highly attenuated and induces protection against challenge infection in mice. Infect Immun 2015;83:1458–64.
      • 127. Tabatabai LB, Pugh GW Jr. Modulation of immune responses in Balb/c mice vaccinated with Brucella abortus Cu-Zn superoxide dismutase synthetic peptide vaccine. Vaccine 1994;12:919–24. pmid:7526568
      • 128. Trant CGMC, Lacerda TLS, Carvalho NB, Azevedo V, Rosinha GMS, Salcedo SP, et al. The Brucella abortus phosphoglycerate kinase mutant is highly attenuated and induces protection superior to that of vaccine strain 19 in immunocompromised and immunocompetent mice. Infect Immun 2010; 78:2283–91. pmid:20194591
      • 129. Truong QL, Cho Y, Barate AK, Kim S, Hahn TW. Characterization and protective property of Brucella abortus cydC and looP mutants. Clin Vaccine Immunol 2014;21:1573–80. pmid:25253663
      • 130. Ugalde JE, Comerci DJ, Leguizamón MS, Ugalde RA. Evaluation of Brucella abortus phosphoglucomutase (pgm) mutant as a new live rough-phenotype vaccine. Infect Immun 2003;71:6264–69. pmid:14573645
      • 131. Velikovsky CA, Cassataro J, Giambartolomei GH, Goldbaum FA, Estein S, Bowden RA, et al. A DNA vaccine encoding lumazine synthase from Brucella abortus induces protective immunity in BALB/c mice. Infect Immun 2002;70:2507–11. pmid:11953389
      • 132. Verma SK, Jain S, Kumar S. Immunogenicity and protective potential of a bacterially expressed recombinant dihydrolipoamide succinyltransferase (rE2o) of Brucella abortus in BALB/c mice. World J Microbiol Biotechnol 2012;28:2487–95. pmid:22806154
      • 133. Wang X, An C, Yang M, Li X, Ke Y, Lei S, et al. Immunization with individual proteins of the Lrp/AsnC family induces protection against Brucella melitensis 16M challenges in mice. Front Microbiol 2015;6:1193. pmid:26579099
      • 134. Wang Z, Niu J, Wang S, Lv Y, Wu Q. In vivo differences in the virulence, pathogenicity, and induced protective immunity of wboA mutants from genetically different parent Brucella spp. Clin Vaccine Immunol 2013;20:174–80. pmid:23239800
      • 135. Winter AJ, Rowe GE, Duncan JR, Eis MJ, Widom J, Ganem B, et al. Effectiveness of natural and synthetic complexes of porin and O polysaccharide as vaccines against Brucella abortus in mice. Infect Immun 1988;56:2808–17. pmid:2844673
      • 136. Yang Y, Wang L, Yin J, Wang X, Cheng S, Lang X, et al. Immunoproteomic analysis of Brucella melitensis and identification of a new immunogenic candidate protein for the development of brucellosis subunit vaccine. Mol Immunol 2011;49:175–84. pmid:21943783
      • 137. Yang Y, Yin J, Guo D, Lang X, Wang X. Immunization of mice with recombinant S-adenosyl-L-homocysteine hydrolase protein confers protection against Brucella melitensis infection. FEMS Immunol Med Microbiol 2011;61:159–67. pmid:21166726
      • 138. Yu DH, Hu XD, Cai H. A combined DNA vaccine encoding BCSP31, SOD, and L7/L12 confers high protection against Brucella abortus 2308 by inducing specific CTL responses. DNA Cell Biol 2007;26:435–43. pmid:17570767
      • 139. Zhang J, Guo F, Chen C, Li Z, Zhang H, Wang Y, et al. Brucella melitensis 16MΔhfq attenuation confers protection against wild‐type challenge in BALB/c mice. Microbiol Immunol 2013;57:502–10. pmid:23647412
      • 140. Zhang J, Yin S, Guo F, Meng R, Chen C, Zhang H, et al. A potent Brucella abortus 2308 Δery live vaccine allows for the differentiation between natural and vaccinated infection. J Microbiol 2014;52:681–88. pmid:24994009
      • 141. Zhao Z, Li M, Luo D, Xing L, Wu S, Duan Y, et al. Protection of mice from Brucella infection by immunization with attenuated Salmonella enterica serovar typhimurium expressing A L7/L12 and BLS fusion antigen of Brucella. Vaccine 2009;27:5214–19. pmid:19596411
      • 142. Pasquali P, Rosanna A, Pistoia C, Petrucci P, Ciuchini F. Brucella abortus RB51 induces protection in mice orally infected with the virulent strain B. abortus 2308. Infect Immun 2003;71:2326–30. pmid:12704101
      • 143. Deqiu S, Donglou X, Jiming Y. Epidemiology and control of brucellosis in China. Vet Microbiol 2002;90:165–82. pmid:12414142
      • 144. Blasco JM. A review of the use of Brucella melitensis Rev. 1 vaccine in adult sheepand goats. Prev Vet Med 1997;31:275–83. pmid:9234451
      • 145. Tamura S, Hasegawa H, Kurata T. Estimation of the effective doses of nasal-inactivated influenza vaccine in humans from mouse-model experiments. Jpn J Infect Dis 2010, 63:8–15. pmid:20093755
      • 146. Silva TMA, Mol JPS, Winter MG, Atluri VL, Xavier MN, Pires SF, et al. The predicted ABC transporter AbcEDCBA is required for type IV secretion system expression and lysosomal evasion by Brucella ovis. PLoS One 2014;9:e114532. pmid:25474545
      • 147. Macêdo AA, Silva APC, Mol JPS, Costa LF, Garcia LNN, Araújo MSS, et al. The abcEDCBA-encoded ABC transporter and the virB operon-encoded type IV secretion system of Brucella ovis are critical for intracellular trafficking and survival in ovine monocyte-derived macrophages. PLoS One 2015;10(9):e0138131. pmid:26366863
      • 148. Silva APC, Macêdo AA, Costa LF, Rocha CE, Garcia LNN, Farias JRD, et al. Encapsulated Brucella ovis lacking a putative ATP-binding cassette transporter (ΔabcBA) protects against wild type Brucella ovis in Rams. PLoS One 2015b;10:e0136865. pmid:26317399
      • 149. Ficht TA, Kahl-McDonagh MM, Arenas-Gamboa AM, Rice-Ficht AC. Brucellosis: the case for live, attenuated vaccines. Vaccine 2009, 27(Suppl. 4):D40–3. pmid:19837284
      • 150. Poester FP, Gonçalves VSP, Paixão TA, Santos RL, Olsen SC, Schurig GG, et al. Efficacy of strain RB51 vaccine in heifers against experimental brucellosis. Vaccine 2006;24:5327–34. pmid:16713034
      • 151. Barrio MB, Grillo MJ, Munoz PM, Jacques I, Gonzalez D, Miguel MJ, et al. Rough mutants defective in core and O-polysaccharide synthesis and export induce antibodies reacting in an indirect ELISA with smooth lipopolysaccharide and are less effective than Rev 1 vaccine against Brucella melitensis infection of sheep. Vaccine 2009;27:1741e9. pmid:19186196
      • 152. Young EJ, Gomez CI, Yawn DH, Musher DM. Comparison of Brucella abortus and Brucella melitensis infections of mice and their effect on acquired cellular resistance. Infect Immun 1979;26:680–5. pmid:121113
      • 153. Hong PC, Tsolis RM, Ficht TA. Identification of genes required for chronic persistence of Brucella abortus in mice. Infect Immun 2000;68:4102–07. pmid:10858227
      • 154. Kahl-McDonagh MM, Ficht TA. Evaluation of protection afforded by Brucella abortus and Brucella melitensis unmarked deletion mutants exhibiting different rates of clearance in BALB/c mice. Infect Immun 2006;74:4048–57. pmid:16790778
      • 155. Allen CA, Adams LG, Ficht TA. Transposon-derived Brucella abortus rough mutants are attenuated and exhibit reduced intracellular survival. Infect Immun 1998;66:1008–16. pmid:9488389
      • 156. Ficht TA. Discovery of Brucella virulence mechanisms using mutational analysis. Vet Microbiol 2002;90:311–15. pmid:12414151
      • 157. Edmonds M, Booth N, Hagius S, Walker J, Enright F, Martin Roop RM II, et al. Attenuation and immunogenicity of a Brucella abortus htrA cycL double mutant in cattle. Vet Microbiol 2000;76:81–90. pmid:10925044
      • 158. Fiorentino MA, Campos E, Cravero SL, Arese AI, Paolicchi F, Campero C, et al. Protection levels in vaccinated heifers with experimental vaccines Brucella abortus M1-luc and INTA 2. Vet Microbiol 2008;132:302–11. pmid:18565697
      • 159. Titball RW. Vaccines against intracellular bacterial pathogens. Drug Discov Today 2008;13:596–600. pmid:18598915
      • 160. Plotkin SA. Correlates of protection induced by vaccination. Clin Vaccine Immunol 2010;17;1055–65. pmid:20463105
      • 161. Gomez G, Adams LG, Rice-Ficht A, Ficht TA. Host-Brucella interactions and the Brucella genome as tools for subunit antigen discovery and immunization against brucellosis. Front Cell Infect Microbiol 2013;3:17. pmid:23720712
      • 162. Lean IJ, Rabiee AR, Duffield TF, Dohoo IR. Invited review: Use of meta-analysis in animal health and reproduction: methods and applications. J Dairy Sci 2009;92:3545–65. pmid:19620636
      • 163. Grilló MJ, Blasco JM, Gorvel JP, Moriyón I, Moreno E. What have we learned from brucellosis in the mouse model? Vet Res 2012;43:29. pmid:22500859