Plasmidic versus insertional cloning of heterologous genes in Mycobacterium bovis BCG: impact on in vivo antigen persistence and immune responses

Abstract

Bivalent recombinant strains of Mycobacterium bovis BCG (rBCG) expressing the early regulatory nef and the structural gag(p26) genes from the simian immunodeficiency virus (SIV) SIVmac251 were engineered so that both genes were cotranscribed from a synthetic operon. The expression cassette was cloned into a multicopy-replicating vector, and the expression levels of both nef and gag in the bivalent rBCG(nef-gag) strain were found to be comparable to those of monovalent rBCG(nef) or rBCG(gag) strains. However, extrachromosomal cloning of the nef-gag operon into a replicative plasmid resulted in strains of low genetic stability that rapidly lost the plasmid in vivo. Thus, the nef-gag operon was inserted site specifically into the BCG chromosome by means of mycobacteriophage Ms6-derived vectors. The resulting integrative rBCG(nef-gag) strains showed very high genetic stability both in vitro and in vivo. The in vivo expression of the heterologous genes was much longer lived when the expression cassette was inserted into the BCG chromosome. In one of the strains obtained, integrative cloning did not reduce the expression levels of the genes even though a single copy was present. Accordingly, this strain induced cellular immune responses of the same magnitude as that of the replicative rBCG strain containing several copies of the genes.

FIG. 1.

(A) Construction of monovalent and bivalent rBCG strains expressing SIVmac251 nef and gag genes: nef (light dots) and gag (heavy dots) genes from SIVmac251 were organized as an operon under the control of the M. fortuitum pBlaF* promoter (striped arrow). A sequence derived from the Shine-Dalgarno consensus (MegaSD) indicated as a black box was inserted upstream from the ATG start codon of Nef and Gag to facilitate translation. STOP codons were inserted downstream from each gene. The pBlaF*-nef-gag operon was cloned into a replicative vector to give rise to pJLN29. Two other plasmids carrying a single nef (pJL17) or gag (pJL24) gene under the control of pBlaF* plus MegaSD were also obtained. Primers used for RT-PCR of transcribed nef and gag mRNAs are indicated as black arrows. (B) RT-PCR products obtained from rBCG-infected BMMP. Macrophages were infected either with bivalent rBCG(pJLN29) expressing nef and gag genes or with a mixture of monovalent rBCG(pJL17) and rBCG(pJL24) strains expressing nef or gag, respectively. Total RNA was harvested from infected macrophages 48 h later and reverse transcribed. PCR amplification was performed with N1-N2 primers amplifying a 375-bp product from nef mRNA, G1-G2 primers amplifying a 250-bp product from gag mRNA, or N3-G3 primers that could only amplify a 350-bp product from a bicistronic nef-gag mRNA. Lane 1, rBCG(pJLN29); lane 2, rBCG(pJL17) plus rBCG(pJL24) mixture. Lanes for negative controls: 3, wild-type BCG; 4, noninfected macrophages; 5, total RNA from pJLN29-infected macrophage with no RT; T−, no RNA in the reaction. Lane for positive control: T+, pJLN29 plasmid DNA. (C) Western blot analysis of monovalent Nef or Gag and bivalent Nef-Gag-producing rBCG strains. Total protein was extracted from rBCG strains cultured in vitro. Western blots were performed on 20 μg of total protein with monoclonal antibody against SIVmac251 Gag(p26) (left panel) or with a serum from an SIVmac251-infected macaque recognizing the SIVmac251 Nef protein (right panel). Lanes for Gag panel: 1, recombinant Gag(p26) (1 μg); 2, wild-type BCG; 3, rBCG(pJL24); 4, rBCG(pJLN29). Lanes for Nef panel: 1, extract of insect cells infected with recombinant SIVmac251 nef baculovirus; 2, wild-type BCG; 3, rBCG(pJL17); 4, rBCG(pJLN29).

FIG. 2.

(A) Construction of integrative rBCG(pBlaF*nef-gag) strains by means of mycobacteriophage Ms6-derived vector pAV6950. The pBlaF*-nef-gag operon was isolated from pJLN29 and was cloned into the pAV6950 vector carrying the attP site and integrase gene from the Ms6 cassette. This gave rise to pOIP4 or pOIP12, according to the orientation of the expression cassette compared to the Ms6 int gene. In pOIP33, the pBlaF*-nef-gag operon was flanked by two T4 transcription terminators to prevent potential readthrough from outside promoters. (B) Western blot analysis of integrative rBCG pBlaF*-nef-gag strains. Western blotting was performed on 20 μg of total protein from rBCG strains cultured in vitro with anti-Gag(p26) monoclonal antibody (left panel) or with a serum from an SIVmac251-infected macaque reacting against the SIVmac251 Nef protein (right panel). On both gels positive controls were loaded on lane 1 and were either recombinant purified Gag(p26) (1 μg) or 10 μl of extract of insect cells infected with recombinant SIVmac251 nef baculovirus. Lanes for left and right panels: 2, rBCG(pJN30) (negative control); 3, rBCG(pJLN29); 4, rBCG::pOIP4; 5, rBCG::pOIP12; 6, rBCG::pOIP33. Comparable levels of Gag and Nef were produced by rBCG(pJLN29) and rBCG::pOIP4 strains. Approximately 10 times less Nef and Gag were produced by rBCG::pOIP33 than by rBCG::pOIP4. Nef and Gag proteins were barely detectable in rBCG::pOIP12.

FIG. 3.

(A) In vitro stability of replicative and integrative bivalent rBCG strains containing the pBlaF*-nef-gag operon. One colony of rBCG strains transformed either with plasmid pJLN29 or integration-proficient vectors pOIP4 and pOIP33 was inoculated into liquid medium without antibiotic and grown for 100 days. At different time points, aliquots of each culture were sampled and serially diluted before plating onto selective and nonselective medium. Ratios of kanamycin-resistant (rBCG) versus kanamycin-sensitive (total BCG) colonies were calculated for each of the three strains over cell divisions. Generations equal the number of days of culture, since the generation time for BCG is approximately 24 h. The most representative of two experiments is shown. (B) In vivo stability of replicative and integrative rBCG(pBlaF*-nef-gag) strains. On day 0 three groups of 25 BALB/c mice were inoculated i.v. with 5 × 10⁶ CFU of rBCG(pJLN29), rBCG::pOIP4, or rBCG::pOIP33. At different times after initial inoculation, CFU recovered from spleen, liver, or lungs from five individual mice per group were numbered onto medium supplemented (rBCG colonies) or not supplemented (total BCG colonies) with kanamycin. Graph represents mean ratios of rBCG colonies/total BCG colonies ± standard deviations for the three mice groups.

FIG. 4.

In vivo expression of the SIVmac251 gag gene in spleen of infected mice. BALB/c mice were inoculated i.v. with 5 × 10⁶ CFU of wild-type BCG, rBCG(pJLN29), rBCG::pOIP4, or rBCG::pOIP33. Two animals were sacrificed on day 14 (A to D and J) and day 77 (E to H and K) postinoculation. Labeling of the spleen paraffin section was performed with anti-Gag polyclonal antibody (A to I) or anti-BCG polyclonal antibody (J and K), followed by secondary horseradish peroxidase-conjugated anti-rabbit IgG. Immunoperoxidase activity was revealed by redness, and cells were counterstained with hematoxylin. Spleen of nonimmunized control mouse (I) stained with anti-Gag is also shown.

FIG. 5.

IFN-γ production by CD4⁺ T cells from replicative rBCG- and integrative rBCG-inoculated mice. BALB/c mice were inoculated once i.v. with 5 × 10⁶ CFU of three rBCG strains; one group received the same dose of wild-type BCG (1173P2), and a last group was injected with PBS (Naives). Eight weeks later, all groups received 5 μg of recombinant Gag(p26) by the same route. On day 41 (white bars) or day 63—i.e., 1 week after boost (dotted bars)—splenocytes from two mice per group were pooled and CD4⁺ T cells were positively selected on magnetic beads. CD4⁺ T cells (10⁶) were cocultured with 2.5 × 10⁴ syngeneic BMDC (ratio, 40 to 1) that had been pulsed 18 h before with 10 μg of purified Gag protein ml⁻¹ (left panel) or 10 μg of BCG CFA ml⁻¹ (right panel). Culture supernatant was harvested 72 h later, and IFN-γ produced was monitored by ELISA.

FIG. 6.

Antibody responses induced by rBCG strains. Groups of five BALB/c mice were inoculated i.p. with 10⁶ CFU of rBCG(pJLN29), rBCG::pOIP4, rBCG::pOIP33, wild-type BCG1173P2, or PBS. A boost of the same dose of rBCG (two thicker arrows under x axis) was administered 4 weeks later by the same route. On week 8, all mice received 5 μg of purified Gag protein (light arrows) i.p. Total serum anti-IgG was followed by ELISA on each individual mouse. Results given in titers represent means for five individual animals.