Parenteral adjuvant potential of recombinant B subunit of Escherichia coli heat-labile enterotoxin

Abstract

Background: The B subunit of Escherichia coli heat-labile enterotoxin (LTB) is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant.

Objectives: We aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead.

Methods: BALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA) were assessed by enzyme-linked immunosorbent assay (ELISA). The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response.

Findings: Western blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide.

Main conclusions: The results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.

Fig. 1. antigenic characterisation of rLTB and rR1 by SDS-PAGE and western blot. (A) SDS-PAGE (15%) of rLTBR1 and rR1 after nickel-affinity chromatography purification. (B) Western blot analysis of rLTBR1 and rR1 with F1B6 monoclonal antibody against R1. In panels A and B, lane 1 is purified rLTBR1, and lane 2 is purified rR1. Benchmarker™ Pre-stained Protein Ladder was used as the molecular weight marker (lane M of panels A and B). (C) The capacity of rLTBR1 to bind to GM1 ganglioside determined by enzyme-linked immunosorbent assay (ELISA). The asterisk indicates significant statistical differences (p < 0.01).

Fig. 2. seroconversion of total immunoglobulins against rR1. Indirect enzyme-linked immunosorbent assay (ELISA) was used to assess levels of total immunoglobulins from animals inoculated with either rLTBR1, rR1, or phosphate-buffered saline (PBS). There was a significant statistical difference between animals vaccinated with rLTB or aluminium hydroxide used as adjuvants (represented by an asterisk at different time points, p < 0.05). Seven animals were used per group. Data are shown as mean values with standard deviations from triplicate experiments.

Fig. 3. determination of immunoglobulin isotypes in animals vaccinated with either rLTB or aluminium hydroxide as adjuvants. The levels of specific anti rR1 IgG1 (A), IgG2a (B), and IgA (C) were determined by enzyme-linked immunosorbent assay (ELISA) in each experimental group (seven mice per group). Asterisks represent significant statistical differences between groups at the given time points (p < 0.05). Data are shown as mean values with standard deviations from triplicate experiments.

Fig. 4. stratification of antibody isotypes by vaccination group. Levels of specific anti-rR1 IgG1 and IgG2a antibody isotypes were compared in animals inoculated with rLTBR1 (A) or rR1 adsorbed to aluminium hydroxide (B) to determine the type of immune response generated by each adjuvant. Seven animals were used per group. Data are shown as mean values with standard deviations from triplicate experiments.