Introduction

Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in humans and various animals [1]. The genus Brucella comprises a growing number of species (at least 12) that infect a wide variety of mammals as primary hosts [2, 3]. Brucellosis is one of the most common zoonotic diseases worldwide and has become a serious concern in recent years [4]. At present, vaccination is the most effective approach to preventing and controlling brucellosis. Veterinary live attenuated vaccines have been widely used and play an important role in the control of brucellosis epidemics [5]. However, this bacterium remains a challenging vaccine target that due to some drawbacks shown by these live attenuated vaccines, including interference with diagnostic tests, pathogenicity for humans, potential to cause abortion in pregnant animals, among others [6]. Subunit vaccines have promising applications with the advantage of being safe, cost-effective and efficacious. During the past two decades, various antigens have been extracted from Brucella, such as Omp19, Omp25, L7/L12, P39, SodC, InpB, AsnC and TF [7,8,9,10,11,12,13,14,15,16]. These available antigens have been shown to provide protection against Brucella infection by reducing the organ bacterial load in mice. Whereas such findings are highly promising, subunit vaccines using known antigens cannot provide the levels of protection conferred by live attenuated vaccines [17]. Further investigation is needed to identify novel antigens, so as to increase vaccine efficacy. B. abortus, B. melitensis, and B. suis are considered the most highly pathogenic species, causing most cases of brucellosis in humans and domestic animals throughout Central Asia, Africa, South America, and the Mediterranean region [4]. It is of great importance to select broad-spectrum antigens that can simultaneously target various Brucella pathogens with a worldwide geographic distribution.

Reverse vaccinology (RV) has been proven to be a highly effective approach in which a rational vaccine design is used, with vaccine antigen prediction based on bioinformatics analysis of pathogen genomes [18, 19]. Several studies have used RV to screen potential vaccine candidates based on the protein coding genome of Brucella [

Materials and methods

Bioinformation and reverse vaccinology

The genome of B. abortus, B. melitensis, and B. suis strains with clear geographic characteristics were selected and downloaded from the NCBI website (as of March 2019) [26]. To identify the pan-core proteome, we used an ultra-fast computational pipeline Bacterial Pan Genome Analysis (BPGA) tool with default parameters [27]. The protein FASTA files of all strains were input for orthologous cluster analysis, with an 80% sequence identity cut-off value. The core/accessory/unique proteomes were defined as coverage > 95%, 95–15%, and  < 15%, respectively.

Proteins from the core proteome were first aligned with host (human/mouse) protein databases using the BLASTp tool [28]. Then, non-host-homology proteins were screened and scored according to a compositive strategy assigning six biological features: (1) subcellular localization (SCL), (2) antigen similarity, (3) antigenicity, (4) mature epitope density, (5) virulence, and (6) adhesion probability. Each of the six biological features was used to divide proteins into three levels of antigen probability: high probability (individual score = 1), moderate probability (individual score = 0.5), and low probability (individual score = 0). Then, all proteins were computed and ranked based on a predicted composite score of the six individual scores for further analysis. The remaining top-ranked (1% of the core proteome) proteins were considered potential vaccine candidates.

Briefly, the CELLO program was used to predict the most likely location for each protein and a subcellular localization individual score was obtained (extracellular/membrane, score = 1; periplasmic, score = 0.5; cytoplasmic, score = 0) [29]. For computation of antigen similarity, we made use of sequence similarity search programs in BLAST to identify similar sequences in the target of known protective antigens database (Protegen, exclude Brucella antigens) and obtained a similarity individual score (similarity ≥ 200, score = 1; 100 ≤ similarity < 200, score = 0.5; similarity < 100, score = 0) [

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Acknowledgements

We would like to thank Shuling Liu, Qingzhen Sun, Yujie Li, and Yue Zhang for their support in animal experiments and contribution to the reagents.

Funding

This research was funded by the National Natural Science Foundation of China, grant number “31800770” and the National Major Science and Technology Project of China, grant number “2016ZX10004001”.

Author information

Authors and Affiliations

  1. Laboratory of Vaccine and Antibody Engineering, Bei**g Institute of Biotechnology, Bei**g, China

    **aodong Zai, Ying Yin, Fengyu Guo, Qiaoling Yang, Ruihua Li, Yaohui Li, Jun Zhang, Junjie Xu & Wei Chen

Authors
  1. **aodong Zai
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  2. Ying Yin
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  3. Fengyu Guo
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  4. Qiaoling Yang
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  5. Ruihua Li
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  6. Yaohui Li
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  7. Jun Zhang
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  8. Junjie Xu
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  9. Wei Chen
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Contributions

Conceptualization, XZ, YY, JX, and WC; methodology, XZ, YY, QY and FG; formal analysis, XZ and YY; investigation, XZ, YY, QY and FG; resources, FG, RL, YL and JZ; data curation, XZ; writing—original draft preparation, XZ and YY; writing—review and editing, JX and WC; visualization, XZ; supervision, JX and WC; project administration, JX.; funding acquisition, XZ and JX. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Junjie Xu or Wei Chen.

Ethics declarations

Ethics approval and consent to participate

This study was carried out in strict accordance with the recommendations in the guidelines for the care and use of laboratory animals. All animal experiments were approved by the Bei**g Institute of Biotechnology, Bei**g, China (No. 20161101).

Competing interests

The authors declare that they no competing interests.

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Supplementary Information

Additional file 1: List of 213 pathogenic

Brucella spp. strains with clear genetic isolation information.

Additional file 2: The composite score for all non-host-homology

Brucella proteins using compositive reverse vaccinology methodology.

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Zai, X., Yin, Y., Guo, F. et al. Screening of potential vaccine candidates against pathogenic Brucella spp. using compositive reverse vaccinology. Vet Res 52, 75 (2021). https://doi.org/10.1186/s13567-021-00939-5

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  • DOI: https://doi.org/10.1186/s13567-021-00939-5

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