Genetic Composition of <i>Mycobacterium bovis</i> BCG Substrain Sofia

Abstract

Mycobacterium bovis BCG remains the most widely used vaccine in the world, with a range of substrains that are named after their locations of production. More than 40 million doses of the BCG substrain Sofia are distributed annually through the United Nations Children’s Fund (UNICEF) and the Pan American Health Organization (PAHO) to approximately 120 different countries. However, in spite of its widespread use BCG Sofia has never been investigated at the genetic level, raising questions as to its genetic stability and exact provenance. The Bulgarian BCG Laboratory was established in 1949 when Dr. Srebra Rodopska obtained the BCG strain from the Institute Pasteur. However, due to suppurative cervical lymphadenitis in about 1% of orally vaccinated newborns, a few years later it was replaced with the Russian BCG strain. During 1972 lot 222 was approved as a master seed lot, originating the BCG substrain Sofia SL222. A working seed lot was produced from the master in 1993 from which all commercial lots are currently produced. To investigate the genetic properties of BCG Sofia, including its position on the BCG genealogical tree developed by Behr and colleagues (1), we performed genotyping assays on the master and working seed lots and on commercial batches no. 906 and 909 of BCG Sofia. We initially concentrated on genomic loci that are prone to variation. Spoligotyping exploits a polymorphic direct repeat (DR) locus that is composed of multiple 36-bp DR copies interspersed by unique spacers, with strains varying in the presence or absence of spacers (4). No difference among any of the BCG Sofia lots tested was found, with all strains showing patterns identical to those of other BCGs. Variable number of tandem repeat (VNTR) typing targeted six alleles (A through F [2]) that vary in the length and number of repeats at each target; i.e., 7-5-5-4-3-3 would have 7 copies of allele A, 5 of B, etc. (2). The VNTR profile of BCG Sofia was determined to be 5-5-5-2-3-3.1, which is identical to the profiles of all other BCG strains tested. A whole-genome analysis of BCG Sofia was undertaken by using M. tuberculosis microarrays (5). This disclosed a range of deletions and a duplication that places BCG Sofia in the same lineage as BCG Russia, in accordance with the BCG genealogy. However, a novel 1.6-kb deletion that affects the Rv3697c and Rv3698 homologues was revealed. To determine whether this deletion had occurred during in vitro propagation of BCG Sofia, we screened for this region in other BCG strains. The region was also deleted in BCG Russia but not in any other strain. Hence, this deletion occurred prior to the in vitro cultivation of BCG Sofia. Public concern over the safety of various vaccines demands that they should be described in exquisite detail (3). Our analyses show that BCG Sofia and BCG Russia are indistinguishable, although this does not preclude the existence of single nucleotide changes between the strains. We have therefore confirmed the genetic identity and provenance of a BCG vaccine that is given to over 40 million individuals a year.