*3.1. Construction of the BCG.HTI2auxo.int Vaccine Strain*

In the p2auxo.HTIint *E. coli*-mycobacterial shuttle vector, the heterologous open reading protein expression cassette is under the control of the *Mtb* α-antigen promoter, which is a weak promoter that has been shown to enhance protein stability [37]. The open reading frame of the heterologous protein is initiated by the mycobacterial 19-kDa protein signal sequence, which at its 5' end was fused to the HTI coding sequence. This enables the localization of the newly synthesized HTI polyprotein to the mycobacterial membrane, and subsequently its secretion, to prevent the internal accumulation of the heterologous protein and enhance protein immunogenicity (Figure 1A). The plasmid contains the *E. coli* origin of replication (*oriE*), attachment sites (*attP*), and the integrase (*int*) genes from the mycobacteriophage L5 [38], and integrates as a single copy into the *attB* region on the BCG chromosome. The plasmid also contains the wild-type glycine A-complementing gene (*glyA*) and lysine A-complementing gene (*lysA5*) for vector selection and maintenance in the auxotrophic *E. coli* and BCG strain, respectively [39,40]. The p2auxo.HTIint was obtained following the methodology previously described [33] and transformed into the glycine auxotrophic *E. coli* M15ΔglyA strain and the lysine auxotrophic BCG Pasteur strain (Δ*lysA5*) [41,42]. The positive recombinant *E. coli* colonies were selected through culture on Minimal M9-D agar plates and the BCG.HTI2auxo.int colonies on Middlebrook agar 7H10 medium without lysine supplementation. Integration of the p2auxo.HTIint plasmid DNA into the mycobacterial genome was assessed by PCR analysis of the *attR* and *attL* DNA insertion regions. The BCG.HTI2auxo.int was used as a template, and bands of 766 bp and 874 bp corresponding to the *attR* and *attL* DNA regions were detected (Figure 1B), demonstrating that p2auxo.HTIint had integrated at the *attB* genomic BCG DNA region. Expression of the full-size chimeric 19-kDa signal sequence-HTI protein was confirmed by Western blot analysis of BCG.HTI2auxo.int lysates (Figure 1C). The selected clones were preserved using the seed-lot system. Clone #3 was selected as the candidate, and Master Seed stocks and Working Vaccine Stocks were prepared for further molecular characterization, immunogenicity, and safety testing in mice.

**Figure 1.** *Cont*.

**Figure 1.** Construction of the recombinant Bacillus Calmette-Guérin HIVACAT T-cell immunogen (BCG.HTI2auxo.int) strain. (**A**) The HTI synthetic sequence was BCG codon-optimized and fused to the 19-kDa lipoprotein signal sequence and inserted into the integrative p2auxo.HTIint *E. coli*-mycobacterial shuttle plasmid. This vector contains P α-Ag, which is a *Mycobacterium tuberculosis* α-antigen promoter, PHSP60, which is a heat shock protein 60 gene promoter. The *glyA* and *LysA* complementing genes function as an antibiotic-free selection and maintenance system in the auxotrophic strains of *E. coli* M15Δ*glyA* and BCGΔ*Lys*, respectively; (**B**) PCR analysis of recombinant BCG clones transformed with p2auxo.HTIint for integration sites: "*attR*" (left panel; lanes 1–5; clones 1–5; lane 6: BCG.empty2auxo.int; lane 7: molecular weight marker; lane 8: p2auxo.HTIint) and "*attL"* (right panel; lanes 1–5; clones 1–5; lane 6: molecular weight marker; lane 7: p2auxo.HTIint; lane 8: BCG.empty2auxo.int), (**C**) PCR analysis using primers specific for *glyA* (left) and HTI (right) on BCG transformed with p2auxo.HTIint lanes 1–5; clones 1–5; lane 6: BCG.empty2auxo.int; lane 7: molecular weight marker; lane 8: p2auxo.HTIint. (**D**) Western blot of BCG.HTI2auxo.int lysates; lanes 1 and 7: Molecular weight marker; lanes 2 and 8: Master Seed of BCG.HTI2auxo.int clone 1; lanes 3 and 9: Master seed of BCG.HTI2auxoint clone 3; lanes 4 and 10: Working Vaccine Stock of BCG.HTI2auxo.int clone 3; lanes 5 and 11: BCGwt (negative control); lanes 6 and 12: purified recombinant HTI protein. The HTI proteins were detected using the n63 (left) and n69 (right) mAbs directed against the HTI protein (AELIX Therapeutics, Barcelona, Spain) followed by horseradish peroxidase-goat-anti-mouse and enhanced chemiluminescence (ECL) detection.
