Vaccine Potential and Diversity of the Putative Cell Binding Factor (CBF, NMB0345/NEIS1825) Protein of Neisseria meningitidis.
Vaccine Potential and Diversity of the Putative Cell Binding Factor (CBF, NMB0345/NEIS1825) Protein of Neisseria meningitidis.
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DOI：
10.1371/journal.pone.0160403
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Christodoulides M
中科院分区：
文献类型：
--
作者：
Humbert MV;Hung MC;Phillips R;Akoto C;Hill A;Tan WM;Heckels JE;Christodoulides M
将编码推定细胞结合因子（CBF，NMB0345/NEIS1825）蛋白的脑膜炎奈瑟氏球菌菌株 MC58 的 cbf 基因克隆到 pRSETA 系统中，并在大肠杆菌中表达约 36-kDa 的重组 (r)CBF 蛋白，并通过金属亲和层析纯化。使用 rCBF-盐水、rCBF-Al(OH)3、rCBF-脂质体或 rCBF-Zwittergent (Zw) 3-14 胶束（掺入或不掺入单磷酰脂质 A (MPLA) 佐剂）免疫后，在小鼠中诱导高滴度的 rCBF 抗体。抗 rCBF 血清在脑膜炎球菌裂解物的蛋白质印迹中与分子量约为 29.5 kDa 的单个蛋白带发生反应，表明成熟的 CBF 蛋白，但不与 Δnmb0345 突变体 (CBF-) 的裂解物反应，证明了小鼠免疫反应的特异性。迄今为止研究的所有脑膜炎球菌菌株均产生 CBF 蛋白，根据抗 rCBF 血清的 FACS 反应性判断，该蛋白存在于 MC58 (CBF+) 细菌的表面，但在 Δnmb0345 突变体 (CBF-) 细菌上不存在。对 pubmlst.org/Neisseria 数据库中具有确定等位基因的 6644 个脑膜炎奈瑟氏球菌分离株的 NEIS1825 氨基酸序列进行的分析表明，有 141 个 ST 类型，并且有 136 个不同的等位基因座编码 49 个非冗余蛋白质序列。只有 6/6644 (<0.1%) 脑膜炎奈瑟氏球菌分离株缺乏 nmb0345 基因。在全球 B 血清群分离株中，约 68% 和约 20% 分别表达由等位基因 1 和 18 编码的 CBF，这些蛋白质具有 >99% 的氨基酸同一性。使用幼兔血清补体和人血清补体 (h)SBA 检测，Zw 3-14 胶束中的 rCBF 鼠抗血清 + MPLA 可诱导针对同源等位基因 1 和异源等位基因 18 菌株的显着血清杀菌活性 (SBA)，但不会杀死表达由等位基因 2 或 3 编码的异源蛋白的菌株。此外，针对不同脑膜炎球菌的鼠抗血清诱导了不同的杀菌活性hSBA 测定中的菌株，这可能与 CBF 的可变表面暴露相关。无论如何，不同菌株之间氨基酸序列保守性和蛋白质表达的特性以及诱导交叉菌株杀菌抗体的能力表明rCBF可能是潜在的脑膜炎球菌疫苗抗原并值得进一步测试。
The cbf gene from Neisseria meningitidis strain MC58 encoding the putative Cell Binding Factor (CBF, NMB0345/NEIS1825) protein was cloned into the pRSETA system and a ~36-kDa recombinant (r)CBF protein expressed in Escherichia coli and purified by metal affinity chromatography. High titres of rCBF antibodies were induced in mice following immunization with rCBF-saline, rCBF-Al(OH)3, rCBF-Liposomes or rCBF-Zwittergent (Zw) 3–14 micelles, both with and without incorporated monophosphoryl lipid A (MPLA) adjuvant. Anti-rCBF sera reacted in western blots of meningococcal lysates with a single protein band of molecular mass ~29.5 kDa, indicative of mature CBF protein, but did not react with a lysate of a Δnmb0345 mutant (CBF-), demonstrating specificity of the murine immune responses. CBF protein was produced by all strains of meningococci studied thus far and the protein was present on the surface of MC58 (CBF+) bacteria, but absent on Δnmb0345 mutant (CBF-) bacteria, as judged by FACS reactivity of anti-rCBF sera. Analysis of the NEIS1825 amino acid sequences from 6644 N. meningitidis isolates with defined Alleles in the pubmlst.org/Neisseria database showed that there were 141 ST types represented and there were 136 different allelic loci encoding 49 non-redundant protein sequences. Only 6/6644 (<0.1%) of N. meningitidis isolates lacked the nmb0345 gene. Amongst serogroup B isolates worldwide, ~68% and ~20% expressed CBF encoded by Allele 1 and 18 respectively, with the proteins sharing >99% amino acid identity. Murine antisera to rCBF in Zw 3–14 micelles + MPLA induced significant serum bactericidal activity (SBA) against homologous Allele 1 and heterologous Allele 18 strains, using both baby rabbit serum complement and human serum complement (h)SBA assays, but did not kill strains expressing heterologous protein encoded by Alelle 2 or 3. Furthermore, variable bactericidal activity was induced by murine antisera against different meningococcal strains in the hSBA assay, which may correlate with variable surface exposure of CBF. Regardless, the attributes of amino acid sequence conservation and protein expression amongst different strains and the ability to induce cross-strain bactericidal antibodies indicates that rCBF could be a potential meningococcal vaccine antigen and merits further testing.