Abstract：
The ongoing antibiotic-resistance crisis is becoming a global problem affecting public health. Urgent efforts are required to design novel therapeutics against pathogenic bacterial species. Brucella melitensis is an etiological agent of brucellosis, which mostly affects sheep and goats but several cases have also been reported in cattle, water buffalo, yaks and dogs. Infected animals also represent the major source of infection for humans. Development of safer and effective vaccines for brucellosis remains a priority to support disease control and eradication in animals and to prevent infection to humans. In this research study, we designed an in-silico multi-epitopes vaccine for B. melitensis using computational approaches. The pathogen core proteome was screened for good vaccine candidates using subtractive proteomics, reverse vaccinology and immunoinformatic tools. In total, 10 proteins: catalase; siderophore ABC transporter substrate-binding protein; pyridoxamine 5\'-phosphate oxidase; superoxide dismutase; peptidylprolyl isomerase; superoxide dismutase family protein; septation protein A; hypothetical protein; binding-protein-dependent transport systems inner membrane component; and 4-hydroxy-2-oxoheptanedioate aldolase were selected for epitopes prediction. To induce cellular and antibody base immune responses, the vaccine must comprise both B and T-cells epitopes. The epitopes were next screened for antigenicity, allergic nature and water solubility and the probable antigenic, non-allergic, water-soluble and non-toxic nine epitopes were shortlisted for multi-epitopes vaccine construction. The designed vaccine construct comprises 274 amino acid long sequences having a molecular weight of 28.14 kDa and instability index of 27.62. The vaccine construct was further assessed for binding efficacy with immune cell receptors. Docking results revealed that the designed vaccine had good binding potency with selected immune cell receptors. Furthermore, vaccine-MHC-I, vaccine-MHC-II and vaccine-TLR-4 complexes were opted based on a least-binding energy score of -5.48 kcal/mol, 0.64 kcal/mol and -2.69 kcal/mol. Those selected were then energy refined and subjected to simulation studies to understand dynamic movements of the docked complexes. The docking results were further validated through MMPBSA and MMGBSA analyses. The MMPBSA calculated -235.18 kcal/mol, -206.79 kcal/mol, and -215.73 kcal/mol net binding free energy, while MMGBSA estimated -259.48 kcal/mol, -206.79 kcal/mol and -215.73 kcal/mol for TLR-4, MHC-I and MHC-II complexes, respectively. These findings were validated by water-swap and entropy calculations. Overall, the designed vaccine construct can evoke proper immune responses and the construct could be helpful for experimental researchers in formulation of a protective vaccine against the targeted pathogen for both animal and human use.
摘要：
持续的抗生素耐药性危机正在成为影响公共卫生的全球性问题。迫切需要设计针对病原细菌物种的新疗法。布鲁氏菌是布鲁氏菌病的病原体，主要影响绵羊和山羊，但也报道了一些牛的病例，水牛,牦牛和狗.受感染的动物也是人类感染的主要来源。开发更安全有效的布鲁氏菌病疫苗仍然是支持动物疾病控制和根除以及预防人类感染的优先事项。在这项研究中，我们使用计算方法设计了一种芯片多表位疫苗。使用减法蛋白质组学筛选病原体核心蛋白质组的良好疫苗候选物，反向疫苗学和免疫信息学工具。总的来说,10种蛋白质：过氧化氢酶；铁载体ABC转运蛋白底物结合蛋白；吡哆胺5-磷酸氧化酶；超氧化物歧化酶；肽基氨酰异构酶；超氧化物歧化酶家族蛋白；分离蛋白A；假设蛋白；结合蛋白依赖性转运系统内膜成分；和4-羟基-2-氧代庚二酸醛缩酶被选择用于表位预测。诱导细胞和抗体基础免疫反应，疫苗必须同时包含B和T细胞表位。接下来筛选表位的抗原性,过敏性和水溶性和可能的抗原性，不过敏,水溶性无毒9个表位入围多表位疫苗构建。设计的疫苗构建体包含274个氨基酸长的序列,具有28.14kDa的分子量和27.62的不稳定性指数。进一步评估疫苗构建体与免疫细胞受体的结合功效。对接结果表明，设计的疫苗与选定的免疫细胞受体具有良好的结合能力。此外，疫苗-MHC-I，基于-5.48kcal/mol的最小结合能评分选择疫苗-MHC-II和疫苗-TLR-4复合物，0.64kcal/mol和-2.69kcal/mol。然后对选定的对象进行能量精炼，并进行模拟研究，以了解对接复合体的动态运动。通过MMPBSA和MMGBSA分析进一步验证对接结果。MMPBSA计算的-235.18kcal/mol，-206.79千卡/摩尔，和-215.73kcal/mol净结合自由能，而MMGBSA估计为-259.48千卡/摩尔，-206.79kcal/mol和-215.73kcal/mol对于TLR-4，MHC-I和MHC-II复合物，分别。这些发现通过水交换和熵计算得到了验证。总的来说,设计的疫苗构建体可以引起适当的免疫反应，该构建体可能有助于实验研究人员配制针对动物和人类使用的目标病原体的保护性疫苗。
