The use of directed evolution to create a stable and immunogenic recombinant BCG expressing a modified HIV-1 Gag antigen

Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG) that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP). Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]). The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]). Mice primed with 10 7 CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/10 6 splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge

CTL responses measured in a ⁵¹Cr release assay.

<p>Splenocytes pooled from a group of 5 mice on day 68 after a prime with BCG[pWB206] or BCG[pHS207] (10⁷ CFU, 10⁵ CFU, 10³ CFU) or no prime and boost with MVA-Gag (10⁷ pfu) on day 56, were stimulated with the GagCD8 peptide for 6 days. Generated effector cells were used in a ⁵¹Cr release assay using p815 antigen presenting cells in the presence and absence of peptide. Data values indicate the mean net percentage Gag-peptide specific lysis ± the standard deviation (n = 3), calculated after the background lysis (<10%) in the absence of peptide has been subtracted. Net Gag-specific lysis was considered positive if >10% and are shown.</p

Maps and sequence of plasmids that express high levels of GFP, generated through mutation and selection.

<p>Position numbers are as for the parental plasmid pWB105.<b>A.</b> Potential ribosome binding site and start site utilized for P24-GFP expression in pWB106 transformants. <b>B.</b> Mutations found in transformants expressing high levels of p24. Mutation type symbols: X, 1 base pair deletion of an A nucleotide at position 4884 that allows expression of p24 from an internal start codon;Y, 1 bp insertion of an A nucleotide between positions 5539–5540; Z, 1 bp deletion of a C nucleotide at position 5706. Deletions (Δ) are shown with positions given to include the remaining portion. <b>C.</b> Map showing plasmids generated by deleting regions of plasmid pWB106 to determine whether p24-GFP is transcribed from the <i>hsp60</i> promoter. GFP fluorescence indicates whether <i>M. smegmatis</i> transformed with these plasmids showed GFP fluorescence.</p

Western blots of antigen expressed by recombinant BCG.

<p><b>A.</b> Detection with anti-GFP antibodies. <b>B.</b> Detection with anti-p24 antibodies: Lanes; 1, molecular size marker; 2, pWB105; 3, pWB106; 4, pWB206; 5, pHS200; 6, pHS207; 7, purified recombinant p24 protein.</p

rBCG prime and MVA-Gag boost immune responses.

<p>Mice were primed with BCG[pWB206] or BCG[pHS207] (intraperitoneal vaccination; doses of 10⁷ CFU, 10⁵ CFU, 10³ CFU) or left unprimed, then boosted on day 56 with MVA-Gag (intramuscular vaccination, 10⁷ pfu). Splenocytes pooled from a group of 5 mice were used on day 68 in <b>A.</b> an IFN-γ ELISPOT assay with the GFPCD8 peptide, GagCD8 peptide or GagCD4 peptide. Bars represent the average number of SFU/10⁶ splenocytes ± the standard deviation of triplicate reactions after subtraction of average background responses of not more than 20±10 SFU per 10⁶ splenocytes. <b>B.</b> Splenocytes were cultured (48 h) with the GFPCD8 peptide, GagCD8 peptide or GagCD4 peptide and cytokine levels in the culture supernatant were quantified using a Th1/Th2 cytokine bead array assay and flow cytometry. Only IFN-γ was detected in the culture fluid. Levels are expressed as pg/10⁶ splenocytes and are from a representative experiment.</p

GFP and Gag-Specific immune responses induced by rBCG vaccines.

<p>Mice were vaccinated with BCG[pHS207], [pWB105], [pWB106] and [pWB206] (intranasal, 10⁷ CFU). Splenocytes pooled from a group of 5 mice were used on day 56 in A. an IFN-γ ELISPOT assay with the GFPCD8 peptide, GagCD8 peptide or GagCD4 peptide. Bars represent the average number of SFU/10⁶ splenocytes ± the standard deviation of triplicate reactions after subtraction of average background responses of not more than 20±10 SFU per 10⁶ splenocytes. No response to the GagCD8 peptide was detected. B. Splenocytes were cultured (48 h) with the GFPCD8 peptide, GagCD8 peptide or GagCD4 peptide and cytokine levels in the culture supernatant were quantified using a Th1/Th2 cytokine bead array assay and flow cytometry. Only IFN-γ was detected in the culture fluid and for splencoytes stimulated with the GFPCD8 and GagCD4 peptides. Levels are expressed as pg/10⁶ splenocytes and are from a representative experiment.</p

Stability of recombinant <i>M. smegmatis</i>.

<p>Duplicate cultures of recombinant <i>M. smegmatis</i> were passaged daily for 40 generations in liquid media with <b>A.</b> or without <b>B.</b> antibiotic selection. Expression of p24 in cell-free lysates was measured by capture ELISA. <b>C.</b> The % CFU retaining antibiotic resistance when passaged without antibiotic selection was determined by plating suitable dilutions of the cultures on solid media with and without kanamycin.</p

Schematic map and details of the expression cassette and translation control signals of <i>E. coli-</i>mycobacterial shuttle vectors.

<p>A. Schematic map of plasmids pWB102 and pWB105: <i>hsp60</i>, the mycobacterial promoter; RBS, ribosome binding site; <i>tat</i> and <i>gag</i>, the translationally fused <i>tat</i> and <i>gag</i> genes; <i>rrnBt1 E. coli</i> transcription terminator; Linker sequence of A,L and S codons; <i>gfp</i>, gene encoding the green fluorescent protein. B. Details of translation control signals. The ribosome binding site, start codon and downsteam box are bold and underlined. Arrow indicates the beginning of the <i>tat</i> coding sequence. Nucleotide positions are as recorded in Genbank.</p

Expression of GFP by A. <i>M. smegmatis</i>[pWB105], B. <i>M. smegmatis</i>[pWB106. C. <i>M. smegmatis</i>[pWB107].

<p>Bar  = 20 µm.</p