Mapping the phylogenyand lineage history of geographically distinct BCG vaccine strains. Mapping the phylogenyand lineage history of geographically distinct BCG vaccine strains

Introduction: The Bacillus Calmette–Guérin (BCG) vaccine has been in use forprevention of tuberculosis for overa centuryand remains the only widely available tuberculosisvaccineand its protective efficacy has varied across geographical regions. Since it was developed, the BCG strain has been shared acrossdifferent laboratories around the world, where use of differing culture methodsresulted ingenetically distinctstrainsover time. Hypothesis/gap statement: Whilst differing BCG vaccine efficacy around the worldis well documented, and whilst the reasons for this may be multifactorial, it has been hypothesised that genetic differencesin BCG vaccine strains contribute to this variation. Methods: Isolates from an historic archive of lyophilised BCG strainswereregrown,DNA was extractedandthen whole genome sequenced usingOxford Nanopore Technologies. The resulting whole genome data were plotted on a dendrogramand analysed to identify presence or absence of regions of difference and SNPs relating to virulence, growth and cell wall structure.Results: Of 50strainsavailable, 36 were revived in culture and 38 were sequenced. Morphology differed between the strains distributedbefore and after 1934. There was phylogenetic association amongst certain geographically classifiedstrains, most notably BCG-Russia, BCG-Japan and BCG-Danish. RD2, RD171 and RD713 deletions were associated with late strains(seeded after 1927). When mapped to BCG-Pasteur 1172, SNPs in sigK, plaA, mmaA3and eccC5were associated with early strains.Discussion:WhilstBCG-Russia, BCG-Japan and BCG-Danish showed strong geographicalisolate clustering, the late strains distributed after 1934, includingBCG-Pasteur, showed more variation.Awide range of SNPs were seen within geographically classifiedstrains, and as much intra-strain variation as between strainvariationwas seen. Thedate of distribution from the original Pasteur laboratory(earlypre-1927 or latepost-1927) gave the strongest association withgenetic differences in regions of difference and virulence-related SNPs, suggesting early strains may be responsible for better vaccine efficacy.

引言：卡介苗（Bacillus Calmette–Guérin, BCG）用于预防结核病已有百余年历史，仍是目前唯一广泛可用的结核病疫苗，但其保护效力在不同地理区域存在显著差异。自研发以来，全球不同实验室间不断共享BCG菌株，由于培养方法存在差异，随着时间推移逐渐形成了遗传特征各异的菌株群体。
假说与研究空白：尽管全球范围内BCG疫苗保护效力存在差异这一现象已有充分文献记录，且其成因可能为多因素共同作用，但已有假说提出BCG疫苗菌株的遗传差异是导致该效力差异的潜在原因之一。
方法：对冻干BCG菌株历史存档中的分离株进行复培养殖，提取基因组DNA后采用牛津纳米孔科技（Oxford Nanopore Technologies）开展全基因组测序。将所得全基因组数据绘制于系统发育树状图，并分析差异区域的有无以及与毒力、生长特性和细胞壁结构相关的单核苷酸多态性（Single Nucleotide Polymorphism, SNP）。
结果：现有50株菌株中，36株成功复培养殖，38株完成全基因组测序。1934年前后分发的菌株形态存在明显差异。部分按地理来源分类的菌株呈现出显著的系统发育相关性，其中尤以BCG-Russia、BCG-Japan及BCG-Danish菌株最为典型。RD2、RD171与RD713基因缺失与1927年后分发的晚代菌株相关。以BCG-Pasteur 1172为参考基因组进行比对时，sigK、plaA、mmaA3及eccC5基因上的单核苷酸多态性与早代菌株显著相关。
讨论：尽管BCG-Russia、BCG-Japan及BCG-Danish菌株呈现出强烈的地理来源分离株聚类特征，但1934年后分发的晚代菌株（包括BCG-Pasteur菌株）则表现出更高的遗传变异度。按地理来源分类的菌株内部存在广泛的单核苷酸多态性，且菌株内变异程度与菌株间变异程度相当。源自巴斯德原始实验室的菌株分发时间（1927年前的早代或1927年后的晚代）与差异区域及毒力相关单核苷酸多态性的遗传差异关联最强，这提示早代菌株可能具备更优的疫苗保护效力。