Expression of Chlamydia psittaci- and human immunodeficiency virus-derived antigens on the cell surface of Lactobacillus fermentum BR11 as fusions to bspA

Abstract

The basic surface protein, BspA, has been used as a fusion partner to direct peptide antigens from the human immunodeficiency virus gp41 protein and the Chlamydia psittaci OmpA protein to the cell surface of Lactobacillus fermentum BR11. BspA has potential utility in the construction of live vaccines and diagnostic reagents.

FIG. 1

Proposed mechanism for integration of the pPNG301 and pPNG302 plasmids into the L. fermentum BR11 chromosome, downstream of the bspA promoter, via single-crossover homologous recombination. The bspA promoter is indicated as P ➞. The DNAs encoding the gp41(556–590) and OmpA(293–346) antigens are shown as gp41 and OmpA, respectively.

FIG. 2

Analysis of 5 M LiCl extracts from L. fermentum BR11, PNG301, and PNG302 cells. (A) On the left is Coomassie brilliant blue-stained SDS-PAGE of 5 M LiCl extracts from L. fermentum BR11 (lane 1) and PNG301 (lane 2). On the right is a Western blot of 5 M LiCl extracts from L. fermentum BR11 (lane 1) and PNG301 (lane 2) reacted with anti-C. psittaci GPIC serum. (B) On the left is Coomassie brilliant blue-stained SDS-PAGE of 5 M LiCl extracts from L. fermentum BR11 (lane 1) and PNG302 (lane 2). On the right is a Western blot of 5 M LiCl extracts from L. fermentum BR11 (lane 1) and PNG302 (lane 2) reacted with MAb-2A6. The numbers on the left are in kilodaltons.

FIG. 3

Colorimetric assays for the detection of cell surface-displayed gp41(556–590). (A) Dilutions of L. fermentum BR11 (BR11) and PNG302 cell suspensions were spotted onto duplicate nitrocellulose membranes (from left to right: undiluted, 1:2 diluted, and 1:4 diluted). Also, 5 M LiCl extracts from L. fermentum BR11 and PNG302 were spotted onto the bottom right of the membranes. The membranes were either reacted with MAb-2A6 or with anti-L. fermentum BR11 serum. (B) L. fermentum BR11 (BR11) and PNG302 cells were incubated with MAb-2A6, allowing it to bind to surface-displayed gp41(556–590) antigen. This binding was quantified by using a horseradish peroxidase-conjugated secondary antibody and a chromogenic substrate. The results are displayed as the means of the A₄₅₀ per OD₆₀₀ unit of the cells, and the standard deviations are shown by error bars.