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. 1999 Oct;67(10):5100-5.
doi: 10.1128/IAI.67.10.5100-5105.1999.

Humoral and cellular immune responses in mice immunized with recombinant Mycobacterium bovis Bacillus Calmette-Guérin producing a pertussis toxin-tetanus toxin hybrid protein

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Humoral and cellular immune responses in mice immunized with recombinant Mycobacterium bovis Bacillus Calmette-Guérin producing a pertussis toxin-tetanus toxin hybrid protein

B Abomoelak et al. Infect Immun. 1999 Oct.

Abstract

The development of combined vaccines constitutes one of the priorities in modern vaccine research. One of the most successful combined vaccines in use is the diphtheria-pertussis-tetanus vaccine. However, concerns about the safety of the pertussis arm have led to decreased acceptance of the vaccine but also to the development of new, safer, and effective acellular vaccines against pertussis. Unfortunately, the production cost of these new vaccines is significantly higher than that of previous vaccines. Here, we explore the potential of live recombinant Mycobacterium bovis BCG producing the hybrid protein S1-TTC, which contains the S1 subunit of pertussis toxin fused to fragment C of tetanus toxin, as an alternative to the acellular vaccines. S1-TTC was produced in two different expression systems. In the first system its production was under the control of the 85A antigen promoter and signal peptide, and in the second system it was under the control of the hsp60 promoter. Although expression of the hybrid antigen was obtained in both cases, only the second expression system yielded a recombinant BCG strain able to induce both a specific humoral immune response and a specific cellular immune response. The antibodies generated were directed against the TTC part and neutralized toxin activity in an in vivo challenge model, whereas interleukin-2 production was specific for both parts of the molecule. Since protection against tetanus is antibody mediated and protection against pertussis may be cell mediated, this constitutes a first promising step towards the development of a cost-effective, protective, and safe combined vaccine against pertussis, tetanus, and tuberculosis.

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Figures

FIG. 1
FIG. 1
Schematic representation of pEN004 and pBMX. The following relevant features of pEN004 (A) and pBMX (B) are shown. The mycobacterial and E. coli origins of replication are indicated by the black boxes labelled ori myco and ColE1, respectively. The Kanr gene is indicated by the stippled arrow labelled KmR. The black boxes labelled hsp60 and 85A denote the hsp60 promoter and the antigen 85A promoter and signal peptide coding sequence, respectively. The grey boxes correspond to the S1-TTC-coding sequence.
FIG. 2
FIG. 2
Immunoblot analysis of recombinant BCG(pEN004). Recombinant BCG(pEN004) (lanes 3 to 5) was analyzed with anti-S1 monoclonal antibody 1B7 (lanes 1 to 4) and anti-TTC polyclonal antibodies (lane 5). Both whole-cell extracts (lanes 4 and 5) and culture supernatant (lane 3) were analyzed. Lane 1 contains a whole-cell extract, and lane 2 contains culture supernatant of nonrecombinant BCG as a control. The sizes of the molecular mass markers are given in the right margin.
FIG. 3
FIG. 3
Immunoblot analysis of recombinant BCG(pBMX). Recombinant BCG(pBMX) (lanes 2 and 3) was analyzed with anti-S1 monoclonal antibody 1B7 (lane 3) and anti-TTC polyclonal antibodies (lane 2). Lane 1 contains a whole-cell extract of nonrecombinant BCG as a control. The sizes of the molecular mass markers are given in the right margin.
FIG. 4
FIG. 4
Immunoblot analysis of sera of mice immunized with BCG(pBMX). Ten weeks after the first immunization, the sera of mice immunized with BCG(pBMX) (lanes 2 and 3) or with nonrecombinant BCG (lane 1) was analyzed by immunoblotting with purified S1-TTC (lanes 1 and 3) or recombinant S1d (lane 2) as an antigen. Lane 4 contains the molecular mass markers, the sizes of which are given in the right margin.
FIG. 5
FIG. 5
Kinetics of anti-S1-TTC antibody titers of mice immunized with BCG(pBMX). The total anti-S1-TTC IgG titers of mice immunized with BCG(pBMX) were determined at 0, 10, 14, 18, and 26 weeks after the first immunization, as indicated. The titers correspond to the reciprocals of the highest serum dilutions giving optical density values that are at least threefold over background levels.
FIG. 6
FIG. 6
Isotype profile of anti-S1-TTC antibodies of mice immunized with BCG(pBMX). Pooled sera of mice immunized with BCG(pBMX) were analyzed for their anti-S1-TTC IgG1, IgG2a, IgG2b, and IgG3 titers. The titers were estimated 18 weeks after the first immunization and correspond to the reciprocals of the highest serum dilutions giving optical density values that are at least threefold over background levels.
FIG. 7
FIG. 7
Antigen-specific IL-2 production in mice immunized with BCG(pBMX). The IL-2 contents in culture supernatants of splenocytes isolated from mice immunized with nonrecombinant BCG (left) or BCG(pBMX) (right) were estimated as described in Materials and Methods either after stimulation with purified S1-TTC (A) or with PTX (B). The error bars indicate the standard deviations.

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