The copy number of MIRU-VNTR loci in BCG Russia genome.
Background: The genome stability of attenuated live BCG vaccine preventing the acute forms of childhood tuberculosis is an important aspect of vaccine production. The purpose of our study was a whole genome comparative analysis of BCG sub-strains and identification of potential triggers of sub-strains’ transition.
- BCG sub-strains
- Mycobacterium bovis
- genome stability
- genome rearrangements
Tuberculosis (TB) is one of the top causes of death in the world. Currently, the only authorized vaccine for primary vaccination of children from TB remains BCG, first applied in 1921. It is broadly used in different countries as part of the national childhood immunization program. Despite the attempts of TB control through widespread introduction of vaccination it was estimated in 2014 worldwide that 9.6 million people have fallen ill with TB. Nevertheless, vaccination against TB reduced TB prevalence by 42% in 2015 compared to that in 1990 .
World Health Organization (WHO) controls BCG vaccine, and the WHO Expert Committee on Biological Standardization (ECBS) has developed the international requirements for the manufacture and control of BCG vaccine. In 2009, for BCG vaccines of three different sub-strains (Danish1331, Tokyo 172-1 and Russian BCG-I), WHO Reference Reagents were established by WHO ECBS. In addition, quality control requirements comprising molecular genetic characterization of final lots and working seeds of BCG vaccines were suggested . Russian research laboratories performed whole genome sequencing (WGS) of BCG Russia sub-strain genome as WHO and good manufacturing practice (GMP) recommended [3, 4, 5]. Currently, ten whole genome sequences of BCG sub-strains including BCG Russia are available in GenBank. It should be noted that since the 1920s, cultivation of the original strain BCG resulted in the emergence of numerous sub-strains that have evolved from it. So, now we could investigate the evolution of BCG sub-strains and the endpoints of this evolution could be assessed likewise in the study of Darwinian biological species evolution . The reason for BCG sub-strains’ transition remains unclear because the progenitor of BCG strains was lost. The comparative analyses of genome features of different BCG sub-strains can help in solving this problem.
The attention was focused on mobile elements of BCG sub-strain genomes especially on prophage sequences because of their contribution to the bacterial genome patterning. Following Brüssow et al. , 12 years later, we can reaffirm that there is a renaissance of phage research because now we have a lot of information about bacterial and phage genomes in the international databases. It was noticed that reintroduction of the fitness factor by phages usually influences the pathogenic factors of bacteria cells . Thus, phages are of great importance for bacterial short-term adaptation and our goal was to estimate a potential contribution of prophage sequences on the mosaic structure of vaccine BCG sub-strain formation.
2. BCG genome sequencing
M. bovis AF2122/97 (Accession Number NC_002945) was the first M. bovis strain, where complete genomic sequence was determined . The first BCG genomic sequence was performed for BCG Pasteur 1173P2 (NC_008769.1) . The sequences of these strains such as Tokyo 172 (NC_012207.1) and Moreau RDJ (NZ_AM412059.1) were generated from the small-insert libraries (1–4 kb) by using BigDye terminator chemistry on ABI377- or ABI3700-automated DNA sequencers [9, 10, 11, 12]. The large-insert library (40 bp) preparation method was used for Mexico BCG sub-strain (NC_016804.1) sequencing . Orduña et al.  were first who used for BCG genome analysis – the next generation sequencing (NGS) based on 454 technology and Sanger method. Late shotgun DNA libraries for strain sequencing were performed by commercial NGS kits. The strains were sequenced by the combination of 454 and Illumina platforms and the Sanger method, for example, BCG 3281 (NZ_CP008744.1) isolated from the human patient with TB and Korea 1168P (NC_020245.2) . Single-molecule real-time sequencing (SMRT) based on PacBio Systems was allowed to directly sequence DNA and achieve long sequencing reads (>10,000 bp) with uniform coverage . SMRT in combination with Illumina was used for M. bovis 1595 (NZ_CP012095.1) complete genomic sequence determination . The further development of SMRT technology was allowed to use this platform alone for complete genomic sequencing. Sequences of BCG sub-strain 26/ATCC 35735/Montreal (CP010331.1) and M. bovis 30 (CP010332.1) could be considered as the example of this approach . Whole genome sequence of BCG Russia sub-strain was performed using 454 and Sanger technology for short-gun and paired-end libraries. Whole genome map (WGM) creation was useful for the control of the repeat regions.
3. Comparative genome analyses as proof of BCG Russia genome stability
In the vaccine manufacture, one of the important features of BCG sub-strain is the genome stability. So, BCG vaccines’ quality control and production now include characterization of BCG sub-strain genome. The importance of molecular genetic characterization is confirmed by the WHO requirements. According to these requirements, WGS of the last seed lot of BCG Russia (BCG Russia 368, 2006 year) was performed. Besides, two BCG Russia sub-strains from seed lots of 1963 and 1982 years (BCG Russia 311 and BCG Russia 977) were analyzed on the basis of WGS.
Comparative analyses of three BCG Russia sub-strains from different seed lots revealed only two differences. The first difference was the single-nucleotide polymorphism (SNP) in the position 3,175,301 (numeration according to reference strain BCG Tokyo) in the sub-strain BCG Russia 368. This SNP leads to the synonymous mutation in the uridylyltransferase gene. In the generation of 1963 and 1982, this mutation was not registered.
The second change in genome-affected glycerol-3-phosphate acyltransferase gene is shown in Figure 1. The mutation that occurred in this gene in the position 2,744,580 (an insertion of TGT bases instead of C base) truncated the protein. Nevertheless, the mutation was not concerned with the conservative domain of glycerol-3-phosphate acyltransferase and the protein could be functional. It should be noted that not all reads had the insertion of TGT. The changes were registered only for 14% reads of BCG Russia 311 and 54% reads of BCG Russia 977. In the last BCG Russia 368 generation, this mutation wasn’t found. So, the genome structures of three different BCG Russia seed lots remain stable. The last BCG Russia 368 generation that is discussed in the text later was deposited in GenBank with the Accession Number NZ_CP009243.1.
4. In silico genotyping of BCG-Russia sub-strain
Genomic feature of BCG Russia, as well as all BCG sub-strains, is a large deletion of the 10-kb genomic region of difference 1 (RD1) .
Spoligotyping profile is the second known characteristic of BCG sub-strain. This method is based on detection of Direct Repeats (DR) on the right and left sides of IS6110. DR loci are members of a universal family of sequences, designated as clustered regularly interspaced short palindromic repeats (CRISPR) sequence family.
Spoligotype profile of BCG Russia was typical for M. bovis with the absence of spacers 3, 9, 16, and 39–43, the in silico pattern corresponded to spoligo-international-type number (SIT) 482 according to the SPOLDB4 Database [19, 20].
The Mycobacterial interspersed repetitive unit (MIRU) profile of BCG Russia sub-strain based on 12 MIRU loci was 232,324,253,222 according to in silico genome analysis.
The whole data of BCG Russia MIRU-variable-number tandem repeats (VNTR) loci are summarized in Table 1. The repeat unit size (bp) and repeat number are indicated in the brackets, partial repeat size (bp) is outside. One discrepancy in polymerase chain reaction (PCR) and in silico results was revealed. The number of repeat units in MIRU_4/ETR_D was three in accordance with MIRU-VNTR analysis of BCG Russia genome data obtained in three different laboratories (NZ_CP009243.1, CP011455.1, CP013741.1). The number of repeat units in MIRU_4/ETR_D of BCG Tokyo was identical. However, PCR analyzes of MIRU-VNTR performed by Supply et al.  for BCG Russia sub-strain and by Mokrousov et al.  for the strains isolated from BCGitis patients revealed only two repeat units in MIRU_4/ETR_D. The discrepancy in PCR and in silico analyses of MIRU-VNTR could be explained by difficulties in the amplification of high GC genomes. The similar discrepancy in PCR and in silico results was described by Iwamoto et al.  for M. tuberculosis H37Rv. The repeat number in Mtub39 locus was five in PCR analysis but two copies were revealed in in silico genome investigation.
|Locus||Copy number of locus, bp*||Locus||Copy number of locus, bp|
|MIRU_2||(53 × 2) + 8||MIRU_23||(53 × 4) + 5|
|Mtub04||(51 × 0) + 30||MIRU_24||(54 × 2) + 30|
|ETR_C||(58 × 4) + 37||MIRU_26||(51 × 5) + 13|
|MIRU_4/ETR_D||(77 × 3) + 4||MIRU_27/QUB-5||(53 × 3) + 25|
|MIRU_40||(54 × 2) + 19||Mtub34||(54 × 2) + 51|
|MIRU_10||(53 × 1) + 51||MIRU_31/ETR_E||(53 × 2) + 3|
|MIRU_16||(53 × 3) + 18||Mtub39||(58 × 2)|
|Mtub21||(57 × 0) + 34||QUB-26||(111 × 4) + 24|
|MIRU_20||(77 × 2) + 11||QUB-4156||(32 + (59 × 0) + 19)|
|QUB-11b||(69 × 3) + 10||MIRU_39||(53 × 1) + 29|
|ETR_A||(75 × 5) + 20||QUB-3232||(56 × 5) + 48|
|Mtub29||(13 + (57 × 1) + 35)||VNTR-3820||(59 × 5) + 47|
|Mtub30||(58 × 1) + 53||VNTR-4120||(57 × 2) + 23|
|ETR_B||(57 × 5) + 8|
5. Is the original BCG Russia sub-strain recA a mutant?
High degree of genomic stability of BCG Russia sub-strain is seen as an inexplicable fact by some scientists. One of the explanations of this fact proposed by Keller et al. is in the highly cited paper . They postulated recA gene inactivation in BCG Russia sub-strain. RecA is a multifunctional and ubiquitous recombinase protein involved both in general recombination and in DNA repair. RecA-dependent recombination mediates genetic rearrangements resulting in increased genetic instability, while RecA-mediated DNA repair mechanisms have been shown to be essential for intracellular survival and persistence .
Among the mechanisms of bacterial evolution, the leading role belongs to recombination events. The large-scale rearrangements, deletions and duplications were revealed during comparative genomics analyses in M. leprae , M. tuberculosis  and M. bovis BCG . Gene duplication has led to the origin of the half tubercle bacillus proteins . Tandem duplications and homologous recombination also make a significant contribution to the diversity of mycobacteria. As an example, recombination between adjacent repeats of IS6110 elements resulted in deletions of several genome regions in M. tuberculosis H37Rv ,
Keller et al. detected the single-nucleotide insertion of “C” at the 5′ end of the recA gene of BCG Russia sub-strain received from TD Allergen . As a result, the stop codon was formed and recombinase A synthesis was absent. These data have not been confirmed by the genome analysis of the original BCG Russia sub-strain. Whole genome sequence (NZ_CP009243), obtained in our laboratory, and other sequences (CP011455.1, CP013741.1), did not have the single nucleotide insertion in the recA gene. So the complete reading frame for recombinase A was annotated. Thus, the original BCG Russia sub-strain is not a recA mutant and the stability of the BCG Russia genome cannot be associated with recA inactivation.
Keller et al. findings may indicate that the sub-strain used by authors was not original or has been changed during cultivation. The last one is possible. According to our data whole and truncated variants of glycerol-3-phosphate acyltransferase gene was identified in one of the BCG Russia generations (Figure 1).
6. The “early” sub-strain genomes comparison
Genome sequences were compared using BCG sub-strain Tokyo 172 genome, a member of the “early” sub-strains group as reference. First, among this group, BCG Tokyo sub-strain is closest to BCG Russia sub-strain as regards the time of its provision by the Pasteur Institute to Tokyo (in 1925). Second, it was lyophilized in the 1940s and used later as a freeze-dried vaccine, as BCG Russia sub-strain. Then, in 1960, the 172nd transfer on bile-potato medium was freeze dried and adopted as a primary seed lot . Finally, one of the first BCG genomes that were accurately sequenced, assembled, and submitted to GenBank was the genome of this seed lot .
We observed no significant diversity in the sequences of the BCG Russia 368 and BCG Tokyo 172 genomes. The revealed genomic differences were summarized in Table 2 and could be subdivided into three groups: region of differences (RDs), ins/del and SNP. Only two RDs were detected between the “early” sub-strains. First, a 22 bp insertion was found in the TetR family transcriptional regulator gene of BCG Russia 368 genome. One variant of Japan BCG vaccine (Type I), submitted in GenBank, included this deletion (RD16). The RD16 band identical to those of other BCG sub-strains was found in the Type II strain . A 1602 bp deletion in BCG Russia 368 genome was the second RD, corresponding to the region from 4,110,452 to 4,112,053 bp in BCG Tokyo 172, beginning in JTY_RS19265 (ribonuclease gene), including JTY_RS19270 (antitoxin VapB48 gene) and finishing inside JTY_RS19275 (glutamate-cysteine ligase gene).
|Type of differences||Number of differences|
|BCG Russia368/BCG Tokyo 172||BCG Tokyo 172/BCGPasteur1173P2 |
|Region of Differences (more than 20 bp)||2||20|
|Insertions/deletions <20 bp (1–9 bp)||10||20|
|SNP in total||52||68|
|nonsynonymous SNP (without nonsense)||31|
|Nonsense SNP as variant of nonsynonymous||2|
The sub-strains used for vaccine production in Bulgaria (BCG Sofia) and India were obtained from BCG Russia. Nowadays, UNICEF uses four variants of BCG vaccine on behalf of the Global Alliance for Vaccines and immunization. The Statens Serum Institute in Denmark produces BCG-Denmark; Bulbio (BBNCIPD) in Bulgaria; and the Serum Institute in India produces BCG-Russia (genetically identical to BCG-Bulgaria) and the Japan BCG laboratory produces BCG-Japan .
We could trace the genome characteristics of BCG Russia daughter sub-strains using published data. Stefanova et al. analyzed the BCG sub-strain used for production in Bulgaria (named Sofia SL222) with M. tuberculosis microarrays. They detected a 1.6-kb deletion that affects Rv3697c and Rv3698 homologs. The deletion of this region was also noted in BCG Russia but not in any other strains . The authors concluded that RD 1602 bp is an old deletion, because BCG Pasteur was replaced with BCG Russia in Bulgaria BCG laboratory in the 1950s.
According to Seki M. et al. differences between the “early” sub-strain Tokyo and the “late” sub-strain Pasteur were more significant and the number of RD increased tenfold .
Less ins/del differences were found between BCG Russia and BCG Tokyo genomes, then between BCG Tokyo and BCG Pasteur genomes. The size of ins/del differences was small: only 1–9 bp.
However, the number of SNPs was nearly the same in the two pairs of the genomes. Non-synonymous SNP in BCG Russia 368 amounted to 60%, but most of them were associated with conservative substitutions in the proteins. Only seven proteins had radical substitutions, though three of them were from the PE-PGRS/PPE family. This finding has emphasized the significance of these proteins for BCG sub-strain adaptation.
7. A whole genome restriction map analysis
The large array of published literature accentuated the important role of RD in BCG sub-strains differentiation. For checking these statements the methods by OpGen Incorporated Company was used. So, first of all, the assembly of DU2 region and the number of tandem duplications in this region in BCG Russia 368 genome were performed by the Argus™ Optical Mapping System. WGM of the sub-strain BCG Russia 368 was created by the laboratory of OpGen Incorporated Company (Maryland, the USA), according to the Argus™ Optical Mapping System user manual . DNA was digested with NheI. Map Solver software version 3.2 was employed for creating the final circular WGM; the whole genome map of BCG Russia 368 is represented in Figure 2. The separate comparison of DU2 regions (Figure 3) has shown that genomes BCG Russia 368 and BCG Tokyo 172 are identical in this region, unlike from the BCG Pasteur optical map, which can be confirmed by presence of three copies (triple tandem duplications) in the DU2 region of BCG Russia 368.
The cluster construction based on map similarity of the six references of BCG sub-strains is shown in Figure 4. As you can see, the cluster was split into two groups: BCG Tice (ATCC 35743) was attributed to the group of the “early”, while BCG Mexico to the “late” group of sub-strains in accordance with the NheI restriction fragments.
8. Genome map construction and the analyses of repetitive elements of BCG Russia 368
The whole genome gap-less BCG Russia 368 chromosome after the verification of the number of DU2 repeats was visualized in GeneWiz  (Figure 5). Genome atlas option of GeneWiz primarily GC Skew was selected as an appropriate instrument for verifying the accuracy of genome assemblies and OriC detection. The place of the change in GC Skew agreed with the OriC and the first nucleotide position in the BCG Russia 368 genome. Other DNA properties, intrinsic curvature, stacking energy, position preference, global direct repeats, global inverted repeats, and AT-content, were essential for genome structure description.
|PPE protein gene||66|
|PE protein gene||33|
|PE_PGRS protein gene||69|
The insertion sequences (IS) elements and affiliated resolvases, transposases, and integrases genes were predicted by ISfinder. Classification of IS elements and determination of inverted repeats flanking IS were made by the use of ISfinder database [36, 37]. Clustered regularly interspaced short palindromic repeats (CRISPR) and RISPR-associated Cas and Csm family proteins were predicted by CRISPRfinder [38, 39].
Locations of IS elements, repeats, prophage sequences, and PE, PPE, and PE_PGRS genes in BCG Russia 368 genome are visualized in Figure 6. Most of the repetitive elements of BCG Russia 368 genome, including some prophage sequences, are coinciding, overlapping, or interconnecting. So, it is hard to annotate some fragments of BCG Russia 368 genome. Special difficulties have arisen in the differentiation of bacterial and phage genes during PE/PGRS genes characterization.
9. The phages predicted in M. bovis genomes
Along with other mobile elements, the variability of predicted prophages may be the best indexes for characterization of mosaic structure genome. All of the prophages described in M. bovis genome were computed us by PHAge Search Tool (PHAST) [40, 41]. GenVision Plug-In of the DNASTAR Lasergene program package was selected for the visualization of prophage sequences. According to PHAST data, all of the predicted phages could be shared into three groups (see Figure 7). So, the first group composed of the common ones for M. bovis and M. bovis BCG prophages. A 7.5-kb prophage was revealed in most of the BCG genomes and in three M. bovis; exceptions include– BCG sub-strains Tice and Montreal. A 20.3-kb prophage in “early” sub-strain genomes (BCG Tokyo, Moreau, Russia) was replaced by an 11.2-kb one but was lost in “late” sub-strain genomes. The second group represented six BCG Montreal prophages, the third represented 15 BCG Tice sub-strain prophages. The prophages in the second and third groups were unique and did not coincide with the prophages of other sub-strains (see circle B in Figure 6). Like most of phage ORFs in the common ones, M. bovis prophages were annotated as genes belonging to the order Caudovirales (Myoviridae, Siphoviridae, and Podoviridae family), while most of phage ORF in BCG Tice or BCG Montreal prophages were similar to the genes of various Herpesviruses (Human, Bovine, Macaci, Alcela, Anguil).
The mosaic BCG genome structure has been verified by comparative prophage analyses. A partial similarity of BCG Tice/BCG Montreal prophage fragments has been identified after pair-wise alignment of BCG Montreal and BCG Tice phage sequences with BCG Russia 368 whole genome. The regions of similarity defined as the purple (BCG Tice) and blue (BCG Montreal) blocks on circle B are phage sequences discovered in different M. bovis genomes (Figure 6). In BCG Russia 368 genome, Tice-specific prophages (13.4 and 13.9 kb) were split into five and three parts, respectively. Fragments which are homologous to BCG Montreal-specific prophages represented in the genome of BCG Russia 368 as a sequence with multiple gaps ranging from 14 to 128 bp.
In turn, the 7.5-kb BCG Russia 368 prophage was split on 0.9- and 6.6-kb fragments located in different regions of BCG Tice/Montreal genomes. Moreover, these fragments lacked the transposase gene, which was specific to 7.5-kb BCG Russia 368 prophage.
Also, the 7.5-kb BCG Russia 368 prophages had 922-bp repeats in BCG Russia 368 genome (red bar in Figure 6, circle B) and was located near ISMt1 insertion element. Interestingly, besides prophage fragments, two intact prophages associated with the insertion elements have been predicted by PHAST in BCG Russia 368 genome. Thus, 11-kb and 7.5-kb prophage sequences were linked with IS6110 and IS1560 elements, respectively. So, the connection between prophage sequences and the IS elements has a considerable impact on the BCG genome evolution.
10. Phylogeny reconstruction
Phylogeny reconstruction was made using the genome sequences of analyzed M. bovis strains and BCG sub-strains. The full-genome comparison and phylogeny reconstruction were based on BLAST alignment and neighbor-joining algorithm  used in NCBI BLAST. The trees were represented by MEGA 6.0 . Taking into account the prophage profile data, the congruence of obtained phylogenetic tree and vaccine sub-strains genealogy based on the DU2 region [10, 44] has been evaluated. M. bovis strains and BCG sub-strains formed different clusters on the tree (see Figure 8). As was expected, the “early” (Russia, Tokyo) and the “late” (Pasteur 1173P2, Korea 1168P, Mexico) BCG sub-strains formed separate but closely related groups. The basal branch in the BCG cluster was represented by BCG Moreau sub-strain. The unexpected position of the BCG Tice and the BCG Montreal has been revealed. The BCG Montreal showed some relationship with the “early” sub-strains. The BCG Tice has been placed in the most divergent basal position on the tree. In turn, BCG 3281, isolated from a pulmonary TB, patient had the relationship with the “late” sub-strains. Remarkably, all phylogenetic groups of the tree were characterized by specific sets of prophage sequences. The DU2 region genealogy only partially correlated with the whole genome phylogeny obtained in this study. If the DU2-I “early” sub-strain group showed common origin but the DU2-IV group split apart (see Figure 8). The BCG 3281 that represented DU2-III group  took an intermediate position. One of the possible explanations for this discrepancy may be numerous prophage-associated genome rearrangements.
Numerous comparative genomics investigations of BCG sub-strains confirmed significant genomic polymorphism of BCG sub-strains which arose from one progenitor. RDs, indels and SNPs are real evidences still going on in the in vitro evolution of BCG sub-strains.
Here we supposed that genomic evolution and the BCG sub-strains diversity is a direct consequence of prophage-associated genome rearrangements. It is well known that 10–20% of bacterial genomes represented prophage sequences. Most of the prophages are damaged and mutated. Nevertheless, recombinant events between homologous prophage sequences are possible. Moreover, some genes of defective prophages can be still working . So, prophage genome content is an important biological driver/trigger of genomic rearrangements and evolution. Extensive contribution of prophages to bacterial fitness was supposed as a result of unexpected evolutionary prophage patterns . Suggesting our assumption, outstanding differences between prophage profiles have been revealed in our comparative genome analysis of nine BCG sub-strains and three M. bovis strains. Big differences in the number and composition of prophages in the genomes of the late strains Tice and Montreal were discovered. According to the Brosch et al., both BCG Tice and BCG Montreal or Frappier were taken from the Pasteur Institute after 1934. They had close phylogenetic relations because they fall in one phylogenetic group, “DU2 IV, Δint” . Dr. Rosenthal, who received the first Tice sub-strain from the Pasteur Institute, demonstrated heterogeneity of the “late” BCG sub-strain. It was a progenitor of at least six different daughter BCG sub-strains: H, K, E, L, LH, and BL. The sub-strain BL was strongly attenuated in laboratory studies. In 1952, BL was mixed with a new routine ‘P’ strain, received from the Pasteur Institute in 1951, in the ratio 3:1. This new sub-strain was called BLP. Since 1953 only freeze-dried BCG vaccine from this mixed strain has been produced . The history of BCG Montreal sub-strain is also well known. Three times these BCG sub-strains were sent to Canada from the Pasteur Institute . Significant changes of BCG genomes have been reflected in the appearance of new prophage profiles in BCG Tice and BCG Montreal sub-strains. It could also impact on vaccine properties of the sub-strains. According to Zhang et al. , BCG Tice, BCG Montreal/Frappier along with BCG Prague, and BCG Phipps sub-strains have lost the largest number of T-cell epitopes, defining its vaccine properties. In contrast, BCG Russia and BCG Tokyo sub-strains still have the largest number of T-cell epitopes among other BCGs. So an extended genomic sequencing is very important to identify prophages as potential markers of genomic rearrangement. Prophage studies could enhance our understanding of the genetic features of various BCG sub-strains and may also be useful for checking the genetic stability of the seed-lot sub-strain.
People migration from regions with a high incidence of TB and the growth of the number of HIV-infected individuals last decade resulted in the necessity of TB vaccination not only among children but also among adolescents and adults. In 2015, 15 vaccine candidates were considered in clinical trials. BCG vaccine replacement or vaccine boosting for the protection of adolescents and adults were considered. Recombinant BCGs, recombinant viral-vectored platforms, protein/adjuvant combinations, attenuated M. tuberculosis strains and mycobacterial extracts were included in the list . A subunit vaccine developed in N.F. Gamaleya Research Center was based on the fusion of mycobacterial proteins with cellulose-binding domain . On the other hand, new areas of BCG vaccine application have been proposed. As most humans are born in bacteriological environments characterized by a low microbial diversity, the effects of BCG vaccine administrated immediately after birth, as a modulator of Th-1/Th-2 responses, is very important and should be analyzed . In this situation, the control of BCG genome stability is the important task, which will continue to be relevant.