*3.2. Genetic Identification and Characterization of BCG.HTI2auxo.int*

To confirm that the identity of the BCG.HTI2auxo.int vaccine strains corresponded to the BCG Pasteur substrain, a multiplex PCR-based method was performed to analyze the BCG regions of difference such as RD1, 2, 8, 14, and 16 and the SenX3-RegX3 regions [35]. Different multiplex profiles obtained by this method allow the differentiation of BCG substrains. A PCR product of 196-bp length was generated using the primers ET1-3, indicating deletions of the RD1 region that were only found in BCG strains, not in the *Mycobacterium bovis* or *Mycobacterium tuberculosis* strains. The presence of the RD8 and RD16 regions was confirmed in the BCG.HTI2auxo.int and the BCG Pasteur substrain, which generated products of 472 bp and 401 bp, respectively. Products of 276 bp representing the SenX3-RegX3 region were also found. The molecular patterns of the BCG Danish, BCG Connaught, and BCG Pasteur substrains (Figure 2A) were consistent with previously published patterns [35].

**Figure 2.** Genetic characterization of the BCG.HTI2auxo.int strain. (**A**) Pasteur substrain identification of BCG.HTI2auxo.int strains by multiplex PCR assay. Lane 1: BCG.HTI2auxoint Working Vaccine Stock giving the bands of 472 bp, 401 bp, 276 bp, and 196 bp; lane 2: BCG Connaught giving the bands of 401 bp, 252 bp, and 196/199 bp; lane 3 BCG Danish with bands of 472 bp, 401 bp, 276 bp, 252 bp, and 196 bp, lane 4: BCG Pasteur with bands of 472 bp, 401 bp, 276 bp, and 196 bp; lane 5: negative control, distilled water; and lane 6: Molecular weight marker. (**B**) Enzymatic restriction analysis of p2auxo.HTIint plasmid DNA purified from transformed *E. coli* M15ΔglyA cultures (pre-BCG transformation). Lane 1: molecular weight marker; Lane 2: *Apa1* digestion of p2auxo.HTI.int; Lane 3: *BamHI* digestion of p2auxo.HTIint; Lane 4: *Bsal* digestion of p2auxo.HTIint; Lane 5: *StuI* digestion of p2auxo.HTI.int; Lane 6: *BamHI* digestion of p2auxo.emptyint; Lane 7: *NotI* digestion of p2auxo.emptyint. (**C**) PCR detection of the HTI gene in the BCG.HTI2auxo.int WVS; lane 1: H2O; lane 2: p2auxo.HTIint plasmid; lane 3: working vaccine stock of BCG.HTI2auxo.int; lane 4: BCG wt; lane 5: BCG transformed with empty 2auxo.int plasmid; lane 6: molecular weight marker. **(D)** PCR detection of the *GlyA* gene in the BCG.HTI2auxo.int working vaccine stock; lane 1: p2auxo.HTIint plasmid; lane 2: WVS of BCG.HTI2auxo.int; lane 3: BCGwt; lane 4: BCG transformed with empty 2auxo.int plasmid; lane 5: water; lane 6: molecular weight marker. PCR for right integration site, *attR* (**E,F**) left integration site, *attL* in the BCG.HTI2auxo.int WVS; lane 1 WVS of BCG.HTI2auxo.int; lane 2: BCGwt; lane 3: distilled water; lane 4: molecular weight marker; lane 5: positive control, BCG.HIVconsv12auxo.int Working Vaccine Stock (recombinant BCG expressing the HIVconsv1 immunogen [36,43]).

PCR and enzymatic restriction analysis were performed to characterize the p2auxo.HTIint plasmid DNA. Following transformation of the *E. coli* M15 Δ*glyA* strain, plasmid DNA was purified, and the enzymatic restriction analysis revealed results that were consistent with the predicted enzymatic pattern (Figure 2B): *ApaI* digestion (Lane 2; bands of 4005 bp, 1907 bp, 974 bp, 787 bp, and 498 bp), *BamHI* digestion (Lane 3; bands of 6280 bp and 2195 bp), *Bsal* digestion (lane 4; bands of 4648 bp, 1563 bp, and 686 bp), *Stul* digestion (Lane 5; bands of 4672 bp and 1931 bp). The empty plasmid p2auxo.∅INT restriction enzyme pattern was also consistent with the expected band pattern: *BamHI* (lane 6; 4672 bp and 2195 bp) and *Notl* (lane 7; bands of 3904 bp and 2963 bp). Next, we performed PCR analysis using specific primers for the HTI and E. coli *glyA* DNA coding sequences using the BCG.HTI2auxo.int Working Vaccine Stock as template. Bands of 1581 bp and 1760 bp corresponding to the expected size of HTI (Figure 2C) and the E. coli *glyA* DNA sequence (Figure 2D) were detected. Furthermore, PCR analysis using specific primers designed for 3' and 5' HTI ends was employed to confirm integration of the p2auxo.HTIint plasmid DNA into the parental BCGΔ*lysA* strain genome. The BCG.HTI2auxo.int Working Vaccine Stock was used as a template. Bands of 766 bp and 874 bp corresponding to the respective *attR* (Figure 2E) and *attL* (Figure 2F) attachment sites were detected in Working Vaccine Stocks, but not in BCG wt. The HTI DNA coding sequence was detected by PCR in the BCG.HTI2auxo.int after 35 bacterial generations (Appendix A Figure A1).
